重组表达载体系统、重组工程菌及其制备方法和用途
文献发布时间:2023-06-19 10:25:58
技术领域
本发明涉及一种生物工程技术,特别是涉及一种重组表达载体系统、重组工程菌及其制备方法和用途。
背景技术
人乳头瘤病毒(Human papillomavirus,HPV)主要感染人上皮细胞,引起上皮细胞损伤,从而导致包括生殖器疣、宫颈癌等一系列疾病,近年来,越来越多的研究表明HPV感染还可以引起其他肛门与生殖器相关癌症,如阴道癌、肛门癌、阴茎癌等,甚至还与口咽癌有关。其中关注度最高、HPV致病机理最清楚的是宫颈癌,作为世界第三大女性癌症,宫颈癌每年夺走25万人的生命,而99%以上的宫颈癌都是由于HPV感染引起的。
HPV的主要传播途径为性传播,其进入人体的主要机理与上皮细胞的微损伤有关。
目前已发现至少两百多种基因型的HPV,根据其感染后是否具有致癌性,可分为高危型和低危型两种,其中高危型HPV主要与各种恶性肿瘤相关,低危型HPV主要引起尖锐湿疣、呼吸系统刺瘤等良性肿瘤。虽然在不同的国家和地区的HPV流行病学数据有一定的差异,但总体来讲,目前世界范围内流行程度最广的高危型HPV型别为HPV16和HPV18,这两种型别与70%的宫颈癌有关,其他流行程度较高的的高危型HPV型别为HPV31、HPV33、HPV45、HPV52、HPV58等。低危型HPV的主要流行型别为HPV6和HPV11。基于以上的流行病学趋势,目前已有三种HPV预防性疫苗上市,分别是葛兰素史克的
HPV属双链DNA病毒,环状基因组全长约7900~8000bp,编码7个非结构蛋白E1~E7和2个衣壳蛋白L1、L2。其中L1蛋白为主要衣壳蛋白,360拷贝的L1蛋白组成72个五聚体,形成T=7的病毒粒子;L2蛋白为次要衣壳蛋白,不承担结构骨架的功能,但与受体结合后的病毒DNA释放有关。HPV主要的中和表位存在于L1蛋白上,基于这些中和表位的中和抗体可有效预防HPV的感染。
基于多种重组表达系统的研究已证明单独表达L1蛋白即可形成构象接近于天然病毒粒子的病毒样颗粒(Virus-like particles,VLPs),这些HPV-VLP保留了天然HPV的中和表位,同时缺少病毒复制必须的DNA,其安全性和可靠性已通过基于这些VLP的HPV预防性疫苗
发明内容
为实现上述目的及其他相关目的,本发明提供一种重组表达载体系统、重组工程菌及其制备方法和用途,用于解决现有技术中制备HPV抗体工艺复杂、繁琐、成本高、可控性差的问题。
本发明的第一方面提供了一种重组表达载体系统,所述重组表达载体系统至少包含两个重组表达载体,每个所述重组表达载体上含有一个或多个核酸表达盒,每个核酸表达盒包含目的基因,以及与目的基因可操作地连接的启动子,每个所述目的基因分别独立选自HPV6 L1、HPV11 L1、HPV16 L1、HPV18 L1、HPV31 L1、HPV33 L1、HPV35 L1、HPV39 L1、HPV45 L1、HPV51 L1、HPV52 L1、HPV53 L1、HPV56 L1、HPV58 L1、HPV59 L1、HPV66 L1或HPV68 L1中的一种,且所有目的基因都互不相同。
所述多个可以是两个。所述多个可以是三个。所述多个可以是四个。
一种实施方式中,所述HPV6 L1如SEQ ID NO:1所示。
一种实施方式中,所述HPV11 L1如SEQ ID NO:2所示。
一种实施方式中,所述HPV16 L1如SEQ ID NO:3所示。
一种实施方式中,所述HPV18 L1如SEQ ID NO:4所示。
一种实施方式中,所述HPV31 L1的序列为AEI60965.1。
一种实施方式中,所述HPV33 L1的序列为AAA46964.1。
一种实施方式中,所述HPV35 L1的序列为ARQ82609.1。
一种实施方式中,所述HPV39 L1的序列为AGU90477.1。
一种实施方式中,所述HPV45 L1的序列为CAA52578.1。
一种实施方式中,所述HPV51 L1的序列为AMK51256.1。
一种实施方式中,所述HPV52 L1的序列为ACX32362.1。
一种实施方式中,所述HPV53 L1的序列为ABU54083.1。
一种实施方式中,所述HPV56 L1的序列为ABO76795.1。
一种实施方式中,所述HPV58 L1的序列为ACL12325.1。
一种实施方式中,所述HPV59 L1的序列为ALT54908.1。
一种实施方式中,所述HPV66 L1的序列为ABO76872.1。
一种实施方式中,所述HPV68L1的序列为AGU90680.1。
HPV6 L1 DNA编码序列(SEQ ID NO:1)
atgtggagaccatctgactcgactgtctacgttcctccacctaacccagtctctaaggttgtggctaccgacgcctacgtgactagaaccaacatcttctaccacgcatcgtcctctagattgctggccgttggacatccttacttctccatcaagagagccaacaaaaccgtcgtgccaaaggtttctggctaccagtatagagtcttcaaggtggttctgcctgaccctaacaagtttgccctgccagactcgtccttgttcgatcctacgactcaaagactcgtttgggcctgcactggtcttgaggttggcagaggacagccactcggagtcggcgtttctggtcatcctttcctgaacaagtacgacgatgttgagaactctggttccggaggcaatcctggacaggacaacagagtcaatgttggcatggactacaagcaaactcagctctgtatggtgggatgcgcacctccactgggtgagcattggggaaaaggcaagcagtgcaccaacactccagttcaggcaggtgactgccctccattggagctgattacctcggtcatccaggacggtgatatggttgacaccggattcggtgccatgaacttcgctgacctgcagaccaacaagtccgatgtgccaatcgacatctgcggaacgacctgtaaataccctgactacttgcagatggcagccgatccatacggcgacagactcttcttctttctgagaaaggagcagatgtttgctagacactttttcaacagagccggagaggtcggtgagcctgttccagacaccctgatcattaagggctcgggaaacagaacttctgtcggctcctcgatctacgtgaacaccccttccggttcgttggtgtcgtccgaggctcaactgttcaacaagccatactggctccagaaagcccaaggtcacaacaatggcatctgctggggtaaccagctgttcgttactgtcgtggacaccacgagatcgaccaacatgactctgtgtgcctccgttaccacatcttcgacttacaccaactccgactataaggagtacatgagacacgttgaagagtacgacttgcaattcatctttcagctctgctcgattaccctgtccgcagaagtcatggcctacatccacactatgaatccatcggtgcttgaggactggaactttggcttgtccccacctccaaacggaactctggaagacacctatagatacgtgcagtcgcaagccatcacgtgccagaagccaacccctgagaaggaaaagccagatccttacaagaacctgtccttctgggaggtcaatctcaaggagaagttctcgtccgaactggaccagtacccattgggtagaaagttcctgctccagtctggataccgtggtagatcgtccatcagaaccggagtgaagagaccagcagtttccaaggcttctgccgcacctaagcgtaaaagagccaagaccaagagataatag
HPV11 L1 DNA编码序列(SEQ ID NO:2)
atgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatag
HPV16 L1 DNA编码序列(SEQ ID NO:3)
atgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatag
HPV18 L1 DNA编码序列(SEQ ID NO:4)
atggctctgtggagaccatctgacaacaccgtctacttgccacctccatctgttgccagagtcgttaacaccgacgattacgtcactagaacatccatcttctaccacgctggttcctctagattgcttactgttggtaatccatacttcagagttccagctggtggaggtaataagcaagacattcctaaggtttctgcttaccaatatagagtctttagagttcagttgcctgacccaaacaagttcggtttgcctgatacttctatctacaacccagagactcaaagattggtttgggcctgtgctggtgtcgagatcggtagaggacaacctttgggagttggtctttctggacacccattctacaacaagttggatgacactgaatcttcacatgctgccacttccaatgtttctgaggatgttcgtgacaacgtctctgttgattacaagcaaactcagttgtgtatccttggatgtgcccctgctattggtgaacactgggctaaaggtactgcctgtaagtccagaccattgtctcaaggtgactgcccacctttggagcttaagaacactgttttggaagatggtgacatggttgatactggatatggtgctatggacttctctaccctgcaggatactaagtgtgaggttccattggacatctgtcaatctatttgtaagtaccctgattatcttcagatgtctgctgacccatacggtgattctatgttcttttgcttgagacgtgagcaacttttcgctagacacttttggaacagagcaggtactatgggtgacaccgttccacaatctttgtatatcaaaggaactggtatgagagcttctcctggatcctgtgtttactcaccatctccttccggttctattgttacctcagactctcaattgtttaacaagccatactggttacacaaggctcagggtcataacaatggtgtttgctggcacaaccaattgttcgttactgtcgttgacaccactagatccaccaatttgacaatttgtgcttctactcagtcccctgttccaggtcaatatgatgctaccaaattcaagcaatactctagacacgttgaggaatatgacttgcagtttatcttccaattgtgtactattaccttgactgccgatgttatgtcttacattcattccatgaactcttcaatcttggaggattggaactttggtgttccaccacctccaacaacttctttggttgacacctacagattcgtccaatctgttgctattacctgtcagaaggatgccgcaccagctgaaaacaaggatccatacgacaagttgaagttttggaatgtcgatttgaaggagaagttctcccttgacttggatcaatacccacttggtagaaagttcttggttcaggctggtttgagacgtaagccaactatcggtccaagaaaacgttctgctccatcagccactacctcttccaaacctgctaagagagttcgtgtcagagctagaaagtaatag
进一步地,每个所述核酸表达盒内的启动子选自MOX。所述MOX的序列如SEQ IDNO:5所示。
进一步地,每个所述核酸表达盒内的终止序列选自AOXTT。所述AOXTT的序列如SEQID NO:6所示。
MOX DNA序列(SEQ ID NO:5)
gtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaa
AOXTT DNA序列(SEQ ID NO:6)
gtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatc
进一步地,每个所述重组表达载体还包含一个或者多个含有选择物标记基因的核酸表达盒。
进一步地,所述选择物标记基因选自抗生素抗性基因。所述抗生素抗性基因可以是Zeocin抗性基因、G418抗性基因或者其他目前常用的抗生素抗性基因。
进一步地,所述启动子选自酵母表达片段。所述启动子基因可选自MOX、GAPDH、DAS或者其他酵母可用的启动子。
进一步地,所述重组表达载体通过将目的基因插入质粒获得。例如质粒可以是pPICZB。
进一步地,所述重组表达载体系统包含两个重组表达载体,每个重组表达载体上包含两个核酸表达盒,两个核酸表达盒的目的基因分别独立的选自HPV6 L1、HPV11 L1、HPV16 L1、HPV18 L1、HPV31 L1、HPV33 L1、HPV35 L1、HPV39 L1、HPV45 L1、HPV51 L1、HPV52L1、HPV53 L1、HPV56 L1、HPV58 L1、HPV59 L1、HPV66 L1或HPV68 L1中的一种,且所选目的基因都互不相同。
本发明的第二方面提供了一种重组工程菌,其包含如上所述的重组表达载体系统。
进一步地,所述重组工程菌是酵母。
进一步地,所述酵母选自酿酒酵母(Saccharomycescerevisiae)、多形汉逊酵母(Hansenula polymorpha)、巴斯德毕赤酵母(Pichiapastoris)、脆壁克鲁维氏酵母(Kluyveromyces fragilis)、乳酸克鲁维氏酵母(Kluyveromyces lactis),或栗酒裂殖酵母(Schizosaccharomyces pombe)中的任一种或多种。
本发明的第三方面提供了一种上述重组表达载体系统的制备方法,所述方法为通过改造原始质粒获得,具体包括以下步骤:将目的基因的启动子和终止序列分别替换两个原始质粒的启动子和终止序列;再将目的基因分别插入启动子和终止序列之间。
进一步地,所述方法为:将质粒pPICZB改造获得,具体包括以下步骤:
(1)质粒pMMZ的构建:采用第一启动子MOX插入片段和第一终止序列AOXTT分别替换质粒pPICZB中的启动子和终止序列,获得质粒pMOXZ-1;采用第二启动子MOX插入片段和第二终止序列AOXTT分别替换质粒pPICZB中的启动子和终止序列,获得质粒pMOXZ-2;连接质粒pMOXZ-1和质粒pMOXZ-2;
(2)替换质粒上的选择标记物基因,获得与步骤(1)中的质粒具有不同选择标记物基因的质粒;
(3)将目的基因分别插入步骤(1)和步骤(2)构建的质粒中获得重组表达载体。
进一步地,所述目的基因分别独立选自HPV6 L1、HPV11 L1、HPV16 L1、HPV18 L1、HPV31 L1、HPV33 L1、HPV35 L1、HPV39 L1、HPV45 L1、HPV51 L1、HPV52 L1、HPV53L1、HPV58L1、HPV59 L1、HPV66 L1或HPV68 L1中的至少一种。
进一步地,所述第一启动子MOX插入片段为采用SEQ ID NO:7、SEQ ID NO:8所示序列为引物,汉逊酵母基因组DNA为模板,扩增得到。
进一步地,所述第一终止序列AOXTT采用SEQ ID NO:10、SEQ ID NO:11所示序列为引物,pPICZB质粒为模板,扩增得到。
更进一步地,质粒pMOXZ-1为采用BglII+NdeI联合酶切处理MOX启动子插入片段,NdeI+BamHI处理AOXTT终止序列片段,BglII+BamHI处理pPICZB,再用小牛肠碱性磷酸酶处理pPICZB-BglII+BamHI片段,三片段连接后得到。
进一步地,所述第二启动子MOX插入片段为采用SEQ ID NO:7、SEQ ID NO:9所示序列为引物,汉逊酵母基因组DNA为模板,扩增得到。
进一步地,所述第二终止序列AOXTT为采用SEQ ID NO:11、SEQ ID NO:12所示序列为引物,pPICZB质粒为模板,扩增得到。
更进一步地,质粒pMOXZ-2为采用BglII+HindIII联合酶切处理MOX启动子插入片段,HindIII+BamHI处理AOXTT终止序列片段,BglII+BamHI处理pPICZB,再用小牛肠碱性磷酸酶处理pPICZB-BglII+BamHI片段,三片段连接后得到。
本发明的第四方面提供了一种重组工程菌的制备方法,所述方法为将上述重组表达载体系统转化至工程菌中获得。
本发明的第五方面提供了上述重组表达载体系统或重组工程菌在制备HPVL1蛋白中的用途。
本发明的第六方面提供了一种制备HPVL1蛋白的方法,所述选自以下两种的任意一种:
a.将如上所述的重组表达载体系统转化至重组工程菌中,利用重组工程菌表达HPVL1蛋白;
b.利用如上所述的重组工程菌表达HPVL1蛋白获得。
利用上述方法获得的HPVL1蛋白至少有两种亚型。
本发明的第七方面提供了一种蛋白,所述蛋白采用如上所述的制备HPVL1蛋白的方法制备获得。
如上所述,本发明的多基因重组表达片段、表达载体系统、重组工程菌及其制备方法和用途,具有以下有益效果:
本发明制备的重组工程菌可以用来制备HPV疫苗。重组工程菌可以同时表达多种HPVL1蛋白。实验结果显示,Quadri-L1免疫后血清可诱导HPV抗体,抗体滴度水平相近,具有一定的量效关系。
附图说明
图1显示为本发明酵母双表达质粒pMMZ结构示意图。
图2显示为本发明酵母双表达质粒pMMG结构示意图。
图3显示为本发明HPV6 L1/HPV11 L1双表达质粒结构示意图。
图4显示为本发明HPV16 L1/HPV18 L1双表达质粒结构示意图。
图5显示为本发明6L1-11L1-pMMG、16L1-18L1-pMMZ酶切鉴定结构示意图。
其中1.6L1-11L1-pMMG未酶切;2.6L1-11L1-pMMG NheI+NotI酶切鉴定;3.6L1-11L1-pMMG BstBI+SbfI酶切鉴定;4.16L1-18L1-pMMZ未酶切;5.16L1-18L1-pMMZ NheI+NotI酶切鉴定;6.16L1-18L1-pMMZ BstBI+SbfI酶切鉴定;7.250bp DNAMarker(Takara).
图6显示为本发明型别特异性中和单抗检测HPV16 L1/HPV18 L1双表达菌株破菌上清。
A1~A6、B1~B2:H16.V5检测16L1-18L1-CBS4732-1~8#;B3:H16.V5检测阳性对照(HPV16 L1标准品);C1~C6、D1~D2:H18.J4检测16L1-18L1-CBS4732-1~8#;D3:H18.J4检测阳性对照(HPV18 L1标准品)。
图7显示为本发明型别特异性中和单抗检测HPV6 L1/HPV11 L1/HPV16 L1/HPV18L1四表达菌株破菌上清。
A1~A10、B7:H6.M48检测6L1-11L1-16L1-18L1-CBS4732-1~11#;B1、B8:H6.M48检测阳性对照(HPV6 L1标准品);
C1~C10、D7:H11.H3检测6L1-11L1-16L1-18L1-CBS4732-1~11#;D1、D8:H11.H3检测阳性对照(HPV11 L1标准品);
E1~E10、F7:H16.V5检测6L1-11L1-16L1-18L1-CBS4732-1~11#;F1、F8:H16.V5检测阳性对照(HPV16 L1标准品);
A1~A10、H7:H18.J4检测6L1-11L1-16L1-18L1-CBS4732-1~11#;H1、H8:H18.J4检测阳性对照(HPV18 L1标准品);
图8显示为本发明Quadri-L1-VLP透射电子显微镜图。
图9显示为本发明Quadri-L1鼠血清几何抗体平均滴度(GMT)。
具体实施方式
术语“多核苷酸”和“核酸”在本文中可互换使用,并且通常是指基本上由核苷酸,例如脱氧核糖核苷酸和/或核糖核苷酸组成的任何长度的聚合物。核酸可包含嘌呤和/或嘧啶碱基,和/或其他天然的、化学或生物化学修饰的(例如甲基化的)、非天然的或衍生化的核苷酸碱基。核酸的主链可以包含糖和磷酸基团,如通常可以在RNA或DNA中发现的,和/或一种或多种修饰的或取代的(例如,2'-O-烷基化,例如,2'-O-甲基化或2'-O-乙基化;或2'-O,4'-C-炔基化,例如2'-O,4'-C-乙基化)糖或一个或多个修饰或取代的磷酸基团。
术语“核酸表达盒”指包含一种或多种转录控制元件(例如但不限于启动子、增强子、多腺苷酸化序列和内含子)的核酸分子,所述转录控制元件指导与它们可操作地连接的(转)基因表达。
术语“可操作地连接”是指各种核酸分子元件相对于每一种的排列,使得元件功能性连接并且能够在基因表达的背景下彼此相互作用。此类元件可以包括但不限于启动子、增强子、多腺苷酸化序列、一个或多个内含子、以及待表达的感兴趣基因的编码序列(例如目的基因)。当适当取向或可操作地连接时,核酸序列元件一起起作用以确保或调节编码序列的表达。调节是指增加、降低或维持特定元件的活性水平。每个元件相对于其他元件的位置可以以每个元件的5'末端和3'末端而言表示,并且任何特定元件之间的距离可以通过元件之间的居间核苷酸或碱基对的数目来表示。
术语“目的基因”是指要在导入有核酸序列的宿主细胞中表达的编码多肽或多肽的一部分的特定核酸序列。如何将核酸序列导入宿主细胞中不是必需的,例如它可以通过整合到基因组中或作为附加型质粒。
术语“启动子”是指能够结合RNA聚合酶并且启动与其可操作连接的一个或多个核酸编码序列(例如目的基因)的转录的核酸序列。启动子通常位于同一链上的基因的转录起始位点附近和核苷酸编码序列的上游(有义链中的5')。启动子可以单独发挥功能以调节转录或可以通过一个或多个调节序列(例如增强子或沉默子)进一步调节。
术语“转录终止序列”包括转录单元末端的控制序列,其表示转录的3'加工和终止。
术语“选择标志物基因”包括赋予表达它的宿主细胞表型以促进鉴定和/或选择用转基因转染或转化的宿主细胞的任何基因。
术语“载体”是指可以接受多核苷酸插入或克隆的多核苷酸分子,优选源自例如质粒、噬菌体或植物病毒的DNA分子。载体优选含有一个或多个独特的限制性内切酶位点,并且可以能够在确定的宿主细胞中自主复制,或者可以整合在确定的宿主的基因组内,使得克隆的序列是可复制的。载体的选择通常取决于载体与要导入载体的宿主细胞的相容性。
术语“重组工程菌”是指用于转化的那些细胞,即用于表达目的基因的细胞。重组工程菌可以是分离的细胞或培养物中培养的细胞系,或者是存在于活组织或生物体中的细胞。在本发明的上下文中,宿主细胞优选是能够在培养物中生长的细胞。
术语“转化”是指将外源核酸导入生物体中,使得核酸可作为染色体外元件或通过染色体整合而复制。
本文提供的表达片段包含至少第一核酸表达盒和第二核酸表达盒,其中所述第一核酸表达盒和所述第二核酸表达盒不是彼此的拷贝,并且其中每个表达盒包含可操作地连接到一个或更多个目的基因的启动子。每个表达盒上连接在每个目的基因上的启动子可以相同也可以不同。在优选的实施例中为采用相同的启动子。
其他序列可以掺入根据本发明的核酸表达盒中。更具体地,核酸表达盒包括进一步增加或稳定转基因产物表达的序列(例如内含子和/或转录终止序列)。
优选地,本文涵盖的表达片段包含选择标志物基因。所述选择标志物基因优选不包含在表达片段的核酸表达盒中。因此,在实施方案中,本文涵盖的表达片段还包含一个或多个包含选择标志物基因的核酸表达盒。选择标志物基因的表达可以指示宿主细胞的核酸表达盒已经转化,因此,允许选择经转化的宿主细胞。选择标志物基因盒通常还包括启动子和转录终止子序列,其可操作地连接到选择标志物基因。
合适的标志物可以选自赋予抗生素抗性、视觉标志物或互补宿主细胞营养缺陷的标志物。例如,选择标志物可以赋予对抗生素如潮霉素B(如hph基因)、zeocin/腐草霉素(如ble基因)、卡那霉素或G418(如nptII或aphVIII基因)、壮观霉素(如aadA基因)、新霉素(如aphVIII基因)、杀稻瘟素(如bsd基因)、诺尔丝菌素(如natR基因)、嘌呤霉素(如pac基因)和巴龙霉素(如aphVIII基因)的抗性。也可以使用视觉标志物并包括例如β-葡糖醛酸酶(GUS)、萤光素酶和荧光蛋白,如绿色荧光蛋白(GFP)、黄色荧光蛋白等。营养缺陷的两个突出实例是氨基酸亮氨酸缺陷(例如LEU2基因)或尿嘧啶缺陷(例如URA3基因)。乳清苷-5'-磷酸脱羧酶阴性(ura3-)的细胞不能在缺乏尿嘧啶的培养基上生长。因此,功能性URA3基因可以在具有尿嘧啶缺陷的宿主细胞上用作选择标志物,并且可以在缺乏尿嘧啶的培养基上选择成功的转化体。仅用功能性URA3基因转化的细胞能够合成尿嘧啶并在此类培养基上生长。如果野生型菌株不具有尿嘧啶缺陷,则必须制备具有缺陷的营养缺陷型突变体,以便使用URA3作为菌株的选择标志物。实现这点的方法是本领域公知的。
本文涵盖的表达盒可以原样使用,或者通常,它们可以是核酸载体的一部分(即导入核酸载体)。本文公开的表达片段的核酸表达盒可以位于相同载体上或在不同载体上。本发明特别涵盖包含至少两个载体的载体系统,其中每个载体至少包括两个核酸表达盒。本文涵盖的载体系统的载体可以相同或不同。
本文教导的载体可以进一步含有用于线性化或片段化的各种类型的限制性位点。
许多载体是本领域技术人员已知的,并且可以使用任何此类载体。选择合适的载体是一个选择问题。载体可以是非病毒载体或病毒载体。非病毒载体包括但不限于质粒、阳离子脂质、脂质体、纳米颗粒、PEG、PEI等。病毒载体源自病毒,包括但不限于:逆转录病毒、慢病毒、腺相关病毒、腺病毒、疱疹病毒、肝炎病毒等。
本文用于转化重组工程菌的方法是技术人员所公知的。例如,可以使用如本领域已知的电穿孔和/或化学(例如基于氯化钙或乙酸锂的)转化方法或根癌土壤杆菌(Agrobacterium tumefaciens)介导的转化方法。
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围;在本发明说明书和权利要求书中,除非文中另外明确指出,单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
实施例1酵母双表达质粒的构建与改造
为同时在酵母中表达HPV6 L1、HPV11 L1、HPV16 L1和HPV 18L1,需要改造得到两种可同时表达两种蛋白的表达质粒,每一种质粒都含有两个独立的表达盒,同时两种质粒含有的筛选标记不同,以便于通过两次转化、筛选的方法将两种不同的双表达质粒导入酵母。具体的质粒构建方案如下:
1.1酵母双表达质粒pMMZ的构建
通过PCR方式,采用SEQ ID NO:7-8所示序列为引物,汉逊酵母全基因组DNA为模板,扩增得到MOX启动子插入片段(SEQ ID NO:26);采用SEQ ID NO:10,SEQ ID NO:12所示序列为引物,pPICZB质粒为模板,扩增得到含AOXTT终止序列(SEQ ID NO:27)的片段。BglII+NdeI联合酶切处理MOX启动子插入片段,NdeI+BamHI处理AOXTT终止序列片段,BglII+BamHI处理pPICZB,再用小牛肠碱性磷酸酶处理pPICZB-BglII+BamHI片段,三片段连接后得到质粒pMOXZ-1(SEQ ID NO:28)。
引物序列(SEQ ID NO:7)
5’-accaaaagatctgtcgacgcggag-3;
引物序列(SEQ ID NO:8)
5’-cttggctacatatgatcgatcgtgtacacgtttcgaatttgtttttgtacttt-3’;
引物序列(SEQ ID NO:10)
5’-gcccgatcatatgcctgcaggcagctttctagaacaa-3’
引物序列(SEQ ID NO:12)
5’-tgggggatccgcacaaacgaa-3’
PCR序列(SEQ ID NO:26)
accaaaagatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgtagccaag
PCR序列(SEQ ID NO:27)
gcccgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatccccca
pMOXZ-1序列(SEQ ID NO:28)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
通过PCR方式,采用SEQ ID NO:7,9所示序列为引物,汉逊酵母基因组DNA为模板,扩增得到MOX启动子插入片段(SEQ ID NO:29);采用SEQ ID NO:11-12所示序列为引物,pPICZB质粒为模板,扩增得到含AOXTT终止序列(SEQ ID NO:30)的片段。BglII+HindIII联合酶切处理MOX启动子插入片段,HindIII+BamHI处理AOXTT终止序列片段,BglII+BamHI处理pPICZB,再用小牛肠碱性磷酸酶处理pPICZB-BglII+BamHI片段,三片段连接后得到质粒pMOXZ-2(SEQ ID NO:31)。
引物序列(SEQ ID NO:9)
5’-tcaagtcaagcttcacgtggctagctttgtttttgtacttt-3’;
引物序列(SEQ ID NO:11)
5’-aaacgtgaagcttgaattcgcggccgccagctttct-3’;
PCR序列(SEQ ID NO:29)
accaaaagatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgacttga
PCR序列(SEQ ID NO:30)
aaacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatccccca
pMOXZ-2序列(SEQ ID NO:31)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
将pMOXZ-2质粒表达盒通过BglII和BamHI酶切后连接的方式(参见InvitrogenpPICZB操作手册提到的体外多拷贝质粒构建方案)插入BglII处理的pMOXZ-1质粒,得到带有Zeocin抗性的汉逊酵母双表达质粒pMMZ(SEQ ID NO:32)(图1)。
pMMZ序列(SEQ ID NO:32)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
1.2酵母双表达质粒pMMG的构建
通过PCR方式,采用SEQ ID NO:13-14所示序列为引物,pPICZB质粒为模板,扩增得到TEF启动子+EM7启动子片段(SEQ ID NO:33);采用SEQ ID NO:15-16所示序列为引物,pPIC9K质粒为模板,扩增得到G418抗性基因片段(SEQ ID NO:34);BglII+AflII联合酶切处理TEF启动子+EM7启动子片段,AflII+SacII联合酶切处理G418抗性基因片段,BglII+SacII联合酶切处理pMOXZ-1质粒,三片段连接后得到pMOXZG质粒(SEQ ID NO:35);
引物序列(SEQ ID NO:13)
5’-cccttagatctcccacacaccatagct-3’
引物序列(SEQ ID NO:14)
5’-tgcggtcttaaggtttagttcctcacc-3’
引物序列(SEQ ID NO:15)
5’-tattggcttaagatgagccatattcaac-3’
引物序列(SEQ ID NO:16)
5’-gggtatcgtcaccgcggccggctacagcacttgaagtcggacag-3’
PCR序列(SEQ ID NO:33)
cccttagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagaccgca
PCR序列(SEQ ID NO:34)
gggtatcgtcaccgcggccggctacagcacttgaagtcggacagtgagtgtagtcttgagaaattctgaagccgtatttttattatcagtgagtcagtcatcaggagatcctctacgccggacgcatcgtggccgacctgcagggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcatcttaagccaata
pMOXZG序列(SEQ ID NO:35)
agatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctcagcgccccccccccccctgcaggtcggccacgatgcgtccggcgtagaggatctcctgatgactgactcactgataataaaaatacggcttcagaatttctcaagactacactcactgtccgacttcaagtgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
通过PCR方式,采用SEQ ID NO:17-18所示序列为引物,pMOXZG为模板,扩增得到pUC复制子+TEF1启动子+EM7启动子+G418抗性基因片段(SEQ ID NO:36);BamHI酶切处理pUC复制子+TEF启动子+EM7启动子+G418抗性基因片段,BamHI+BglII联合酶切处理pMMZ质粒(剔除原pMMZ质粒的Zeocin抗性表达盒和pUC复制子),两片段连接后得到带有G418抗性的汉逊酵母双表达质粒pMMG(SEQ ID NO:37)(图2)。
引物序列(SEQ ID NO:17)
5’-gttggatcctcgatcacatgtgagc-3’
引物序列(SEQ ID NO:18)
5’-aaaggatcctgaggtctgcctcgt-3’
PCR序列(SEQ ID NO:36)
gttggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctcaggatccttt
pMMG序列(SEQ ID NO:37)
ggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgtgtacacgatcgatcatatgcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctca
实施例2构建6L1-11L1-pMMG双表达质粒和16L1-18L1-pMMZ双表达质粒
2.1HPVL1双表达质粒构建
编码HPV6 L1、HPV11 L1、HPV16 L1、HPV18 L1的DNA序列如SEQ ID NO:1-4,由苏州金唯智公司合成,密码子使用频率参考酵母偏爱性密码子,连接在金唯智公司提供的通用克隆质粒pUC57上,分别得到HPV6L1-pUC57、HPV11L1-pUC57、HPV16L1-pUC57、HPV18L1-pUC57。分别将HPV6 L1、HPV11 L1插入pMMG质粒,将HPV16 L1、HPV18 L1基因插入pMMZ质粒。
2.2 HPV6 L1/HPV11 L1双表达质粒构建
通过PCR方式,采用SEQ ID NO:19-20所示序列为引物,HPV11L1-pUC57质粒为模板,扩增得到HPV11 L1片段(SEQ ID NO:38);BstBI+SbfI联合酶切处理HPV11 L1片段和pMMG质粒,两片段连接后得到11L1-pMMG(SEQ ID NO:39)。
通过PCR方式,采用SEQ ID NO:21-22所示序列为引物,HPV6L1-pUC57质粒为模板,扩增得到HPV6 L1片段(SEQ ID NO:40),NheI+NotI联合酶切处理HPV6 L1片段和11L1-pMMG质粒,两片段连接后得到6L1-11L1-pMMG(SEQ ID NO:41)(图3)。
2.3 HPV16 L1/HPV18 L1双表达质粒构建
通过PCR方式,采用SEQ ID NO:19、23所示序列为引物,HPV16L1-pUC57质粒为模板,扩增得到HPV16 L1片段(SEQ ID NO:42);BstBI+SbfI联合酶切处理HPV16 L1片段和pMMZ质粒,两片段连接后得到16L1-pMMZ(SEQ ID NO:43)。
通过PCR方式,采用SEQ ID NO:24-25所示序列为引物,HPV18L1-pUC57质粒为模板,扩增得到HPV18 L1片段(SEQ ID NO:44),NheI+NotI联合酶切处理HPV18 L1片段和16L1-pMMZ质粒,两片段连接后得到16L1-18L1-pMMZ(SEQ ID NO:45)(图4)。
引物序列(SEQ ID NO:19)
5’-cggacaaattcgaaacgatgt-3’
引物序列(SEQ ID NO:20)
5’-acagaaacctgcaggctattactttttggtc-3’
引物序列(SEQ ID NO:21)
5’-aatcgctagcatgtggagaccatctg-3’
引物序列(SEQ ID NO:22)
5’-ttggtaatgcggccgcctattatctcttggtc-3’
引物序列(SEQ ID NO:23)
5’-tgcttcctgcaggctattacagttttctc-3’
引物序列(SEQ ID NO:24)
5’-attcgctagcatggctctgtggagac-3’
引物序列(SEQ ID NO:25)
5’-ttccttatgcggccgctattactttctagct-3’
PCR序列(SEQ ID NO:38)
cggacaaattcgaaacgatgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatagcctgcaggtttctgt
11L1-pMMG序列(SEQ ID NO:39)
ggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctca
PCR序列(SEQ ID NO:40)
aatcgctagcatgtggagaccatctgactcgactgtctacgttcctccacctaacccagtctctaaggttgtggctaccgacgcctacgtgactagaaccaacatcttctaccacgcatcgtcctctagattgctggccgttggacatccttacttctccatcaagagagccaacaaaaccgtcgtgccaaaggtttctggctaccagtatagagtcttcaaggtggttctgcctgaccctaacaagtttgccctgccagactcgtccttgttcgatcctacgactcaaagactcgtttgggcctgcactggtcttgaggttggcagaggacagccactcggagtcggcgtttctggtcatcctttcctgaacaagtacgacgatgttgagaactctggttccggaggcaatcctggacaggacaacagagtcaatgttggcatggactacaagcaaactcagctctgtatggtgggatgcgcacctccactgggtgagcattggggaaaaggcaagcagtgcaccaacactccagttcaggcaggtgactgccctccattggagctgattacctcggtcatccaggacggtgatatggttgacaccggattcggtgccatgaacttcgctgacctgcagaccaacaagtccgatgtgccaatcgacatctgcggaacgacctgtaaataccctgactacttgcagatggcagccgatccatacggcgacagactcttcttctttctgagaaaggagcagatgtttgctagacactttttcaacagagccggagaggtcggtgagcctgttccagacaccctgatcattaagggctcgggaaacagaacttctgtcggctcctcgatctacgtgaacaccccttccggttcgttggtgtcgtccgaggctcaactgttcaacaagccatactggctccagaaagcccaaggtcacaacaatggcatctgctggggtaaccagctgttcgttactgtcgtggacaccacgagatcgaccaacatgactctgtgtgcctccgttaccacatcttcgacttacaccaactccgactataaggagtacatgagacacgttgaagagtacgacttgcaattcatctttcagctctgctcgattaccctgtccgcagaagtcatggcctacatccacactatgaatccatcggtgcttgaggactggaactttggcttgtccccacctccaaacggaactctggaagacacctatagatacgtgcagtcgcaagccatcacgtgccagaagccaacccctgagaaggaaaagccagatccttacaagaacctgtccttctgggaggtcaatctcaaggagaagttctcgtccgaactggaccagtacccattgggtagaaagttcctgctccagtctggataccgtggtagatcgtccatcagaaccggagtgaagagaccagcagtttccaaggcttctgccgcacctaagcgtaaaagagccaagaccaagagataataggcggccgcattaccaa
6L1-11L1-pMMG序列(SEQ ID NO:41)
ggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagcatgtggagaccatctgactcgactgtctacgttcctccacctaacccagtctctaaggttgtggctaccgacgcctacgtgactagaaccaacatcttctaccacgcatcgtcctctagattgctggccgttggacatccttacttctccatcaagagagccaacaaaaccgtcgtgccaaaggtttctggctaccagtatagagtcttcaaggtggttctgcctgaccctaacaagtttgccctgccagactcgtccttgttcgatcctacgactcaaagactcgtttgggcctgcactggtcttgaggttggcagaggacagccactcggagtcggcgtttctggtcatcctttcctgaacaagtacgacgatgttgagaactctggttccggaggcaatcctggacaggacaacagagtcaatgttggcatggactacaagcaaactcagctctgtatggtgggatgcgcacctccactgggtgagcattggggaaaaggcaagcagtgcaccaacactccagttcaggcaggtgactgccctccattggagctgattacctcggtcatccaggacggtgatatggttgacaccggattcggtgccatgaacttcgctgacctgcagaccaacaagtccgatgtgccaatcgacatctgcggaacgacctgtaaataccctgactacttgcagatggcagccgatccatacggcgacagactcttcttctttctgagaaaggagcagatgtttgctagacactttttcaacagagccggagaggtcggtgagcctgttccagacaccctgatcattaagggctcgggaaacagaacttctgtcggctcctcgatctacgtgaacaccccttccggttcgttggtgtcgtccgaggctcaactgttcaacaagccatactggctccagaaagcccaaggtcacaacaatggcatctgctggggtaaccagctgttcgttactgtcgtggacaccacgagatcgaccaacatgactctgtgtgcctccgttaccacatcttcgacttacaccaactccgactataaggagtacatgagacacgttgaagagtacgacttgcaattcatctttcagctctgctcgattaccctgtccgcagaagtcatggcctacatccacactatgaatccatcggtgcttgaggactggaactttggcttgtccccacctccaaacggaactctggaagacacctatagatacgtgcagtcgcaagccatcacgtgccagaagccaacccctgagaaggaaaagccagatccttacaagaacctgtccttctgggaggtcaatctcaaggagaagttctcgtccgaactggaccagtacccattgggtagaaagttcctgctccagtctggataccgtggtagatcgtccatcagaaccggagtgaagagaccagcagtttccaaggcttctgccgcacctaagcgtaaaagagccaagaccaagagataataggcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtggagaccatctgactcgaccgtctacgttcctccacctaacccagtctccaaagtggttgctactgatgcctacgtcaagagaaccaacatcttctaccacgcctcttcgtccagattgctggcagttggacatccttactattccatcaagaaagttaacaagaccgtggtccctaaggtttcgggctatcagtacagagtgttcaaggtcgttctgccagaccctaacaagtttgccttgccagattcctcgctgttcgaccctaccactcaaagactggtttgggcttgcacgggtttggaagtgggcagaggacaaccactgggtgtcggagtttccggccatccattgctgaacaagtacgacgatgttgagaactcgggtggatacggtggcaatccaggacaagacaacagagtgaatgtcggcatggactacaagcaaacccagctgtgcatggttggttgtgcaccacctctgggtgaacattggggaaagggcactcagtgctccaacacctctgttcagaatggtgactgtcctccactggagctgatcacctcggttattcaggatggagacatggtggacactggcttcggtgcaatgaactttgccgacttgcagaccaacaagtccgatgttccattggacatttgcggcactgtgtgcaaatatcctgactacctgcagatggctgcagatccatacggtgacagactcttcttctacctgagaaaggagcagatgttcgccagacacttctttaacagagctggcaccgttggtgagcctgttccagatgacctgttggtgaagggtggaaacaatagatcctctgttgcctcgtccatctacgtccacaccccatcgggctctctggtgtcctcggaggcccaactgttcaacaagccttactggctccagaaggcccaaggacacaacaatggcatctgctggggaaaccacctgttcgtcaccgttgtggacacgactagatccaccaacatgactttgtgtgcctctgtttccaagtcggctacctacacgaactcggactataaggagtacatgagacatgttgaagagttcgacctgcagtttatcttccagctctgctccattaccttgtcggcagaagtgatggcctacatccacacgatgaacccatccgtcttggaagactggaacttcggtctgtctccacctccaaatggcaccctggaagacacgtacagatatgtgcagtcgcaagccatcacctgtcagaaaccaactcctgaaaaggagaaacaggacccatacaaggatatgtcgttctgggaggtcaacctgaaggagaagttctcttcggaactggaccagttcccattgggaagaaagttcctgctccagtctggttatagaggaagaacctccgcaagaactggcatcaagagacctgccgtgtcgaagccatccaccgctcctaagagaaagagaacgaagaccaaaaagtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcctcgatcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatcagatctcccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccttaagatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagaattggttaattggttgtaacactggcagagcattacgctgacttgacgggacggcggctttgttgaataaatcgaacttttgctgagttgaaggatcagatcacgcatcttcccgacaacgcagaccgttccgtggcaaagcaaaagttcaaaatcaccaactggtccacctacaacaaagctctcatcaaccgtggctccctcactttctggctggatgatggggcgattcaggcctggtatgagtcagcaacaccttcttcacgaggcagacctca
PCR序列(SEQ ID NO:42)
cggacaaattcgaaacgatgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatagcctgcaggaagca
16L1-pMMZ序列(SEQ ID NO:43)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagccacgtgaagcttgaattcgcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
PCR序列(SEQ ID NO:44)
attcgctagcatggctctgtggagaccatctgacaacaccgtctacttgccacctccatctgttgccagagtcgttaacaccgacgattacgtcactagaacatccatcttctaccacgctggttcctctagattgcttactgttggtaatccatacttcagagttccagctggtggaggtaataagcaagacattcctaaggtttctgcttaccaatatagagtctttagagttcagttgcctgacccaaacaagttcggtttgcctgatacttctatctacaacccagagactcaaagattggtttgggcctgtgctggtgtcgagatcggtagaggacaacctttgggagttggtctttctggacacccattctacaacaagttggatgacactgaatcttcacatgctgccacttccaatgtttctgaggatgttcgtgacaacgtctctgttgattacaagcaaactcagttgtgtatccttggatgtgcccctgctattggtgaacactgggctaaaggtactgcctgtaagtccagaccattgtctcaaggtgactgcccacctttggagcttaagaacactgttttggaagatggtgacatggttgatactggatatggtgctatggacttctctaccctgcaggatactaagtgtgaggttccattggacatctgtcaatctatttgtaagtaccctgattatcttcagatgtctgctgacccatacggtgattctatgttcttttgcttgagacgtgagcaacttttcgctagacacttttggaacagagcaggtactatgggtgacaccgttccacaatctttgtatatcaaaggaactggtatgagagcttctcctggatcctgtgtttactcaccatctccttccggttctattgttacctcagactctcaattgtttaacaagccatactggttacacaaggctcagggtcataacaatggtgtttgctggcacaaccaattgttcgttactgtcgttgacaccactagatccaccaatttgacaatttgtgcttctactcagtcccctgttccaggtcaatatgatgctaccaaattcaagcaatactctagacacgttgaggaatatgacttgcagtttatcttccaattgtgtactattaccttgactgccgatgttatgtcttacattcattccatgaactcttcaatcttggaggattggaactttggtgttccaccacctccaacaacttctttggttgacacctacagattcgtccaatctgttgctattacctgtcagaaggatgccgcaccagctgaaaacaaggatccatacgacaagttgaagttttggaatgtcgatttgaaggagaagttctcccttgacttggatcaatacccacttggtagaaagttcttggttcaggctggtttgagacgtaagccaactatcggtccaagaaaacgttctgctccatcagccactacctcttccaaacctgctaagagagttcgtgtcagagctagaaagtaatagcggccgcataaggaa
16L1-18L1-pMMZ序列(SEQ ID NO:45)
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaagctagcatggctctgtggagaccatctgacaacaccgtctacttgccacctccatctgttgccagagtcgttaacaccgacgattacgtcactagaacatccatcttctaccacgctggttcctctagattgcttactgttggtaatccatacttcagagttccagctggtggaggtaataagcaagacattcctaaggtttctgcttaccaatatagagtctttagagttcagttgcctgacccaaacaagttcggtttgcctgatacttctatctacaacccagagactcaaagattggtttgggcctgtgctggtgtcgagatcggtagaggacaacctttgggagttggtctttctggacacccattctacaacaagttggatgacactgaatcttcacatgctgccacttccaatgtttctgaggatgttcgtgacaacgtctctgttgattacaagcaaactcagttgtgtatccttggatgtgcccctgctattggtgaacactgggctaaaggtactgcctgtaagtccagaccattgtctcaaggtgactgcccacctttggagcttaagaacactgttttggaagatggtgacatggttgatactggatatggtgctatggacttctctaccctgcaggatactaagtgtgaggttccattggacatctgtcaatctatttgtaagtaccctgattatcttcagatgtctgctgacccatacggtgattctatgttcttttgcttgagacgtgagcaacttttcgctagacacttttggaacagagcaggtactatgggtgacaccgttccacaatctttgtatatcaaaggaactggtatgagagcttctcctggatcctgtgtttactcaccatctccttccggttctattgttacctcagactctcaattgtttaacaagccatactggttacacaaggctcagggtcataacaatggtgtttgctggcacaaccaattgttcgttactgtcgttgacaccactagatccaccaatttgacaatttgtgcttctactcagtcccctgttccaggtcaatatgatgctaccaaattcaagcaatactctagacacgttgaggaatatgacttgcagtttatcttccaattgtgtactattaccttgactgccgatgttatgtcttacattcattccatgaactcttcaatcttggaggattggaactttggtgttccaccacctccaacaacttctttggttgacacctacagattcgtccaatctgttgctattacctgtcagaaggatgccgcaccagctgaaaacaaggatccatacgacaagttgaagttttggaatgtcgatttgaaggagaagttctcccttgacttggatcaatacccacttggtagaaagttcttggttcaggctggtttgagacgtaagccaactatcggtccaagaaaacgttctgctccatcagccactacctcttccaaacctgctaagagagttcgtgtcagagctagaaagtaatagcggccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacggcacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctgcgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttcgcgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtagaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatgttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggacgaacggcgtggccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctggatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctggatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagagggccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagtgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtccccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatgtctctttggttgccatccgaggctactgtttacttgccacctgtcccagtttctaaggtcgtgtcaactgatgaatacgttgctagaaccaacatctattaccacgctggaacttctagattgcttgctgttggtcatccatacttccctatcaagaaacctaacaataacaagatccttgttccaaaggtctctggattgcagtacagagttttcagaatccacttgcctgacccaaacaagttcggttttcctgacacctccttctacaacccagatactcaaagattggtgtgggcctgtgttggtgtcgaggttggacgtggtcaaccattgggagttggtatttctggacatcctttgcttaacaagttggatgacactgaaaatgcttctgcctacgcagccaacgctggtgttgacaacagagagtgtatctctatggattacaaacagacccaattgtgtcttattggttgcaagccacctattggtgaacactggggtaagggatctccatgtaccaacgttgctgtcaatccaggtgattgtcctccattggagcttatcaacactgttattcaagatggtgacatggttgatactggatttggtgctatggacttcactaccttgcaagctaacaagtccgaggttccattggacatttgtacttctatctgcaagtatccagactacattaagatggtctctgaaccatacggtgactccttgtttttctaccttcgtagagagcaaatgtttgttagacacttgttcaacagagctggtgccgttggtgagaatgtcccagacgatttgtacatcaagggatctggttccactgcaaacttggcctcttccaactacttcccaactccttcaggttctatggttacctccgatgctcaaatcttcaacaagccttactggttgcaaagagcacagggtcacaacaatggaatttgttggggtaaccaacttttcgttactgtcgttgataccacaagatccaccaacatgtccttgtgtgctgccatttctacttcagaaaccacatacaagaatactaacttcaaggagtaccttagacatggtgaggaatacgacttgcagttcatctttcaattgtgcaagattaccttgactgctgacgttatgacctacatccactctatgaactccactatccttgaagattggaacttcggattgcaaccacctccaggtggaaccttggaagatacttacagatttgtcacatcccaagccattgcttgtcagaagcacactcctccagctcctaaggaagatccacttaagaaatacaccttctgggaagtcaacttgaaagagaagttctctgctgaccttgatcagtttcctttgggtagaaagttcttacttcaagctggattgaaggccaaaccaaagttcaccttgggtaaaagaaaggctactccaaccacatcatctacttccacaaccgccaaacgtaagaagagaaaactgtaatagcctgcaggcagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttgggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatttgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtataggattttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgcagctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttcttggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
6L1-11L1-pMMG与16L1-18L1-pMMZ酶切鉴定结果见图5,从结果可知,6L1-11L1-pMMG经NheI+NotI酶切处理后可得到约7719bp+1513bp片段,经BstBI+SbfI酶切处理后可得到约7710bp+1522bp片段;16L1-18L1-pMMZ经NheI+NotI酶切处理后可得到约7322bp+1533bp片段,经BstBI+SbfI酶切处理后可得到约4496+2825+1534bp片段。酶切结果与理论条带相符。
实施例3四表达菌株构建与筛选
基于实施例2中构建的6L1-11L1-pMMG双表达质粒和16L1-18L1-pMMZ双表达质粒,通过两次转化、两次筛选(两种筛选标记)的方法将两种双表达质粒整合于汉逊酵母宿主菌CBS4732(ATCC 34438),得到同时表达HPV6 L1、HPV11 L1、HPV16 L1、HPV18 L1的重组工程酵母菌株,具体步骤如下:
3.1 HPV16 L1/HPV18 L1双表达菌株构建与筛选
将重组构建的16L1-18L1-pMMZ质粒由BglII酶线性化,电转汉逊酵母CBS4732,电转条件为1500V,120Ω,50μF。电转后菌液涂布YPD平板(200μg/ml Zeocin),37℃过夜倒置培养。挑取部分克隆划线YPD平板(1500μg/ml Zeocin),37℃倒置培养2天。挑取生长情况较好的数株接种于YPD液体培养基,37℃振荡培养一天,离心收集菌体后弃去YPD培养基,加入BMMY培养基(0.5%甲醇),37℃振荡诱导培养48小时。收集菌体,玻璃珠剧烈振荡破碎后收集破菌上清,通过斑点杂交法检测分别检测HPV16 L1和HPV18 L1表达情况(图6),检测用抗体分别为HPV16 L1型别特异性中和单抗H16.V5和HPV18 L1型别特异性中和单抗H18.J4中和单抗(宾夕法尼亚州立大学Neil Christensen实验室提供)。挑取HPV16 L1和HPV18 L1表达情况均较理想的一株保存并用于进一步构建,该菌株命名为Dual-L1-HP。
3.2 HPV6 L1/HPV11 L1/HPV16 L1/HPV18 L1四表达菌株构建与筛选
将重组构建的6L1-11L1-pMMG质粒由BglII酶线性化,电转实施例3.1构建得到的Dual-L1-HP,电转条件为1500V,120Ω,50μF。电转后菌液涂布YPD平板(200μg/ml G418),37℃过夜倒置培养。挑取部分克隆划线YPD平板(1200μg/ml G418),37℃倒置培养2天。挑取生长情况较好的数株接种于YPD液体培养基,37℃振荡培养一天,离心收集菌体后弃去YPD培养基,加入BMMY培养基(0.5%甲醇),37℃振荡诱导培养48小时。收集菌体,玻璃珠剧烈振荡破碎后收集破菌上清,通过斑点杂交法检测分别检测HPV6 L1、HPV11 L1、HPV16 L1、HPV18L1表达情况(图7),检测用抗体分别为HPV6 L1型别特异性中和单抗H6.M48、HPV11 L1型别特异性中和单抗H11.H3中和单抗、HPV16 L1型别特异性中和单抗H16.V5中和单抗、HPV18L1型别特异性中和单抗H18.J4中和单抗。挑取HPV6 L1、HPV11L1、HPV16 L1、HPV18 L1表达情况均较理想的一株保存,该菌株命名为Quadri-L1-HP。
实施例4 HPV6 L1/HPV11 L1/HPV16 L1/HPV18 L1抗原制备
将Quadri-L1-HP接种于500ml YPD培养基,37℃振荡培养16~20小时,培养后菌液作为发酵种子菌液以1:15的比例接种于BIOTECH-7JG-7000A发酵罐(保兴生物),培养基为基础盐BSM。发酵初始温度设定37℃,初始pH 5~6,转速300rpm,通气量0.5vvm,DO值100%,添加PTM1痕量盐类。初始增殖阶段培养20~24小时左右,通过调节搅拌转速、空气流量、罐压和补加纯氧维持溶氧值20~40%。当菌体湿重达到60~90g/L时,补加50%甘油或葡萄糖溶液。维持溶氧值20~40%,补加4~8小时。检测菌体湿重增加到100~200g/L时,停止补料。将温度设定在30~37℃,pH值控制调为6~7,开始加入甲醇诱导,甲醇的补加速度为50~100mL/min。维持溶氧值>20%,温度设定在30~37℃,pH值控制为6~7,诱导40小时发酵结束。4℃,8000rpm,离心10分钟收集菌泥,-20℃冻存。按1:3加入破菌缓冲液,1200bar,高压匀浆破碎4次。破碎后悬浊液以4℃,8000rpm,离心30min收集上清液。破菌上清液使用20%~50%蔗糖梯度超离,4℃,40000rpm,超离4小时,收集40%梯度样品,超滤去除蔗糖,得到Quadri-L1纯化样品。
实施例5 HPV6 L1/HPV11 L1/HPV16 L1/HPV18 L1抗原含量检测
夹心法ELISA检测Quadri-L1中HPV6 L1、HPV11 L1、HPV16 L1和HPV18 L1含量,包被抗体为自制兔多抗,检测抗体为型别特异性中和单抗:使用HPV6 L1兔多抗和H6.M48中和单抗对HPV6 L1含量进行定量;使用HPV11 L1兔多抗(和H11.H3中和单抗对HPV11 L1含量进行定量;使用HPV16 L1兔多抗和H16.V5中和单抗对HPV16 L1含量进行定量;使用HPV18 L1兔多抗和H18.J4中和单抗对HPV18 L1含量进行定量。
操作步骤简述如下:向96孔酶标板中分别加入不同型别的自制兔多抗,4℃包被过夜;弃去包被液,洗涤,37℃封闭2小时;加入Quadri-L1纯化样品和对应的HPV6 L1、HPV11L1、HPV16 L1和HPV18 L1标准品(对倍稀释6~12个梯度),37℃放置1小时;对应兔多抗型别,分别加入不同的型别特异性中和单抗,37℃放置1小时;加入HRP标记的羊抗鼠IgG,37℃1小时;加入TMB显色液,37℃放置5~20分钟;加入终止液终止显色反应,酶标仪测定450nm吸收值。根据HPV6 L1、HPV11 L1、HPV16 L1和HPV18 L1标准品的读数制作标准曲线,确定Quadri-L1纯化样品中相应的抗原含量(表1)。
表1Quadri-L1纯化样品中HPV6 L1、HPV11 L1、HPV16 L1和HPV18 L1抗原含量检测(夹心法ELISA)
实施例6 HPV6 L1/HPV11 L1/HPV16 L1/HPV18 L1颗粒结构确证
透射电子显微镜(TEM)观察Quadri-L1纯化样品,结果如图8,Quadri-L1纯化样品形成了颗粒直径约55~70nm的VLP。
实施例7 HPV6 L1/HPV11 L1/HPV16 L1/HPV18 L1疫苗制备
将Quadri-L1纯化样品吸附磷酸铝佐剂,制备得到Quadri-L1疫苗。
实施例8 HPV6 L1/HPV11 L1/HPV16 L1/HPV18 L1疫苗免疫原性测定
取6~8周龄的BALB/c小鼠(北京维通利华实验动物技术有限公司,SCXK(京)2016-0011),每组8只,分为6组,分别对应5μg、2μg和0.5μg HPV16 L1的Quadri-L1,另取1组免疫未吸附抗原蛋白的空白氢氧化铝佐剂。免疫程序:0天、14天、28天三次免疫(皮下注射),42天处死小鼠,眼球取血,4000g离心10分钟,取血清-20℃冻存。
ELISA检测鼠血清转阳率,方法简述如下:向96孔酶标板中分别加入HPV6 L1、HPV11L1、HPV16 L1和HPV18 L1标准品,4℃包被过夜;弃去包被液,洗涤,37℃封闭2小时;加入对倍稀释的鼠血清,37℃放置1小时;弃去鼠血清,洗涤,加入HRP标记的羊抗鼠IgG,37℃1小时;加入TMB显色液,37℃放置5~20分钟;加入终止液终止显色反应,酶标仪测定450nm吸收值,以8个阴性对照血清(免疫铝佐剂的鼠血清)的450nm吸收值的平均值加上3倍标准差(SD)设定为Cutoff值。稀释后待检血清的OD450值大于Cutoff值的为阳性,否则为阴性。取所有待检血清最高的阳性稀释倍数作为血清抗体滴度值,分别计算各组的抗体几何平均滴度(GMT),结果如图9。
结果显示,Quadri-L1免疫后血清可诱导HPV6 L1、HPV11 L1、HPV16 L1和HPV18 L1抗体,抗体滴度水平相近,具有一定的量效关系。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法、组合物的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
序列表
<110> 重庆博唯佰泰生物制药有限公司
上海博唯生物科技有限公司
<120> 重组表达载体系统、重组工程菌及其制备方法和用途
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1506
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgtggagac catctgactc gactgtctac gttcctccac ctaacccagt ctctaaggtt 60
gtggctaccg acgcctacgt gactagaacc aacatcttct accacgcatc gtcctctaga 120
ttgctggccg ttggacatcc ttacttctcc atcaagagag ccaacaaaac cgtcgtgcca 180
aaggtttctg gctaccagta tagagtcttc aaggtggttc tgcctgaccc taacaagttt 240
gccctgccag actcgtcctt gttcgatcct acgactcaaa gactcgtttg ggcctgcact 300
ggtcttgagg ttggcagagg acagccactc ggagtcggcg tttctggtca tcctttcctg 360
aacaagtacg acgatgttga gaactctggt tccggaggca atcctggaca ggacaacaga 420
gtcaatgttg gcatggacta caagcaaact cagctctgta tggtgggatg cgcacctcca 480
ctgggtgagc attggggaaa aggcaagcag tgcaccaaca ctccagttca ggcaggtgac 540
tgccctccat tggagctgat tacctcggtc atccaggacg gtgatatggt tgacaccgga 600
ttcggtgcca tgaacttcgc tgacctgcag accaacaagt ccgatgtgcc aatcgacatc 660
tgcggaacga cctgtaaata ccctgactac ttgcagatgg cagccgatcc atacggcgac 720
agactcttct tctttctgag aaaggagcag atgtttgcta gacacttttt caacagagcc 780
ggagaggtcg gtgagcctgt tccagacacc ctgatcatta agggctcggg aaacagaact 840
tctgtcggct cctcgatcta cgtgaacacc ccttccggtt cgttggtgtc gtccgaggct 900
caactgttca acaagccata ctggctccag aaagcccaag gtcacaacaa tggcatctgc 960
tggggtaacc agctgttcgt tactgtcgtg gacaccacga gatcgaccaa catgactctg 1020
tgtgcctccg ttaccacatc ttcgacttac accaactccg actataagga gtacatgaga 1080
cacgttgaag agtacgactt gcaattcatc tttcagctct gctcgattac cctgtccgca 1140
gaagtcatgg cctacatcca cactatgaat ccatcggtgc ttgaggactg gaactttggc 1200
ttgtccccac ctccaaacgg aactctggaa gacacctata gatacgtgca gtcgcaagcc 1260
atcacgtgcc agaagccaac ccctgagaag gaaaagccag atccttacaa gaacctgtcc 1320
ttctgggagg tcaatctcaa ggagaagttc tcgtccgaac tggaccagta cccattgggt 1380
agaaagttcc tgctccagtc tggataccgt ggtagatcgt ccatcagaac cggagtgaag 1440
agaccagcag tttccaaggc ttctgccgca cctaagcgta aaagagccaa gaccaagaga 1500
taatag 1506
<210> 2
<211> 1509
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtggagac catctgactc gaccgtctac gttcctccac ctaacccagt ctccaaagtg 60
gttgctactg atgcctacgt caagagaacc aacatcttct accacgcctc ttcgtccaga 120
ttgctggcag ttggacatcc ttactattcc atcaagaaag ttaacaagac cgtggtccct 180
aaggtttcgg gctatcagta cagagtgttc aaggtcgttc tgccagaccc taacaagttt 240
gccttgccag attcctcgct gttcgaccct accactcaaa gactggtttg ggcttgcacg 300
ggtttggaag tgggcagagg acaaccactg ggtgtcggag tttccggcca tccattgctg 360
aacaagtacg acgatgttga gaactcgggt ggatacggtg gcaatccagg acaagacaac 420
agagtgaatg tcggcatgga ctacaagcaa acccagctgt gcatggttgg ttgtgcacca 480
cctctgggtg aacattgggg aaagggcact cagtgctcca acacctctgt tcagaatggt 540
gactgtcctc cactggagct gatcacctcg gttattcagg atggagacat ggtggacact 600
ggcttcggtg caatgaactt tgccgacttg cagaccaaca agtccgatgt tccattggac 660
atttgcggca ctgtgtgcaa atatcctgac tacctgcaga tggctgcaga tccatacggt 720
gacagactct tcttctacct gagaaaggag cagatgttcg ccagacactt ctttaacaga 780
gctggcaccg ttggtgagcc tgttccagat gacctgttgg tgaagggtgg aaacaataga 840
tcctctgttg cctcgtccat ctacgtccac accccatcgg gctctctggt gtcctcggag 900
gcccaactgt tcaacaagcc ttactggctc cagaaggccc aaggacacaa caatggcatc 960
tgctggggaa accacctgtt cgtcaccgtt gtggacacga ctagatccac caacatgact 1020
ttgtgtgcct ctgtttccaa gtcggctacc tacacgaact cggactataa ggagtacatg 1080
agacatgttg aagagttcga cctgcagttt atcttccagc tctgctccat taccttgtcg 1140
gcagaagtga tggcctacat ccacacgatg aacccatccg tcttggaaga ctggaacttc 1200
ggtctgtctc cacctccaaa tggcaccctg gaagacacgt acagatatgt gcagtcgcaa 1260
gccatcacct gtcagaaacc aactcctgaa aaggagaaac aggacccata caaggatatg 1320
tcgttctggg aggtcaacct gaaggagaag ttctcttcgg aactggacca gttcccattg 1380
ggaagaaagt tcctgctcca gtctggttat agaggaagaa cctccgcaag aactggcatc 1440
aagagacctg ccgtgtcgaa gccatccacc gctcctaaga gaaagagaac gaagaccaaa 1500
aagtaatag 1509
<210> 3
<211> 1521
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtctcttt ggttgccatc cgaggctact gtttacttgc cacctgtccc agtttctaag 60
gtcgtgtcaa ctgatgaata cgttgctaga accaacatct attaccacgc tggaacttct 120
agattgcttg ctgttggtca tccatacttc cctatcaaga aacctaacaa taacaagatc 180
cttgttccaa aggtctctgg attgcagtac agagttttca gaatccactt gcctgaccca 240
aacaagttcg gttttcctga cacctccttc tacaacccag atactcaaag attggtgtgg 300
gcctgtgttg gtgtcgaggt tggacgtggt caaccattgg gagttggtat ttctggacat 360
cctttgctta acaagttgga tgacactgaa aatgcttctg cctacgcagc caacgctggt 420
gttgacaaca gagagtgtat ctctatggat tacaaacaga cccaattgtg tcttattggt 480
tgcaagccac ctattggtga acactggggt aagggatctc catgtaccaa cgttgctgtc 540
aatccaggtg attgtcctcc attggagctt atcaacactg ttattcaaga tggtgacatg 600
gttgatactg gatttggtgc tatggacttc actaccttgc aagctaacaa gtccgaggtt 660
ccattggaca tttgtacttc tatctgcaag tatccagact acattaagat ggtctctgaa 720
ccatacggtg actccttgtt tttctacctt cgtagagagc aaatgtttgt tagacacttg 780
ttcaacagag ctggtgccgt tggtgagaat gtcccagacg atttgtacat caagggatct 840
ggttccactg caaacttggc ctcttccaac tacttcccaa ctccttcagg ttctatggtt 900
acctccgatg ctcaaatctt caacaagcct tactggttgc aaagagcaca gggtcacaac 960
aatggaattt gttggggtaa ccaacttttc gttactgtcg ttgataccac aagatccacc 1020
aacatgtcct tgtgtgctgc catttctact tcagaaacca catacaagaa tactaacttc 1080
aaggagtacc ttagacatgg tgaggaatac gacttgcagt tcatctttca attgtgcaag 1140
attaccttga ctgctgacgt tatgacctac atccactcta tgaactccac tatccttgaa 1200
gattggaact tcggattgca accacctcca ggtggaacct tggaagatac ttacagattt 1260
gtcacatccc aagccattgc ttgtcagaag cacactcctc cagctcctaa ggaagatcca 1320
cttaagaaat acaccttctg ggaagtcaac ttgaaagaga agttctctgc tgaccttgat 1380
cagtttcctt tgggtagaaa gttcttactt caagctggat tgaaggccaa accaaagttc 1440
accttgggta aaagaaaggc tactccaacc acatcatcta cttccacaac cgccaaacgt 1500
aagaagagaa aactgtaata g 1521
<210> 4
<211> 1527
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggctctgt ggagaccatc tgacaacacc gtctacttgc cacctccatc tgttgccaga 60
gtcgttaaca ccgacgatta cgtcactaga acatccatct tctaccacgc tggttcctct 120
agattgctta ctgttggtaa tccatacttc agagttccag ctggtggagg taataagcaa 180
gacattccta aggtttctgc ttaccaatat agagtcttta gagttcagtt gcctgaccca 240
aacaagttcg gtttgcctga tacttctatc tacaacccag agactcaaag attggtttgg 300
gcctgtgctg gtgtcgagat cggtagagga caacctttgg gagttggtct ttctggacac 360
ccattctaca acaagttgga tgacactgaa tcttcacatg ctgccacttc caatgtttct 420
gaggatgttc gtgacaacgt ctctgttgat tacaagcaaa ctcagttgtg tatccttgga 480
tgtgcccctg ctattggtga acactgggct aaaggtactg cctgtaagtc cagaccattg 540
tctcaaggtg actgcccacc tttggagctt aagaacactg ttttggaaga tggtgacatg 600
gttgatactg gatatggtgc tatggacttc tctaccctgc aggatactaa gtgtgaggtt 660
ccattggaca tctgtcaatc tatttgtaag taccctgatt atcttcagat gtctgctgac 720
ccatacggtg attctatgtt cttttgcttg agacgtgagc aacttttcgc tagacacttt 780
tggaacagag caggtactat gggtgacacc gttccacaat ctttgtatat caaaggaact 840
ggtatgagag cttctcctgg atcctgtgtt tactcaccat ctccttccgg ttctattgtt 900
acctcagact ctcaattgtt taacaagcca tactggttac acaaggctca gggtcataac 960
aatggtgttt gctggcacaa ccaattgttc gttactgtcg ttgacaccac tagatccacc 1020
aatttgacaa tttgtgcttc tactcagtcc cctgttccag gtcaatatga tgctaccaaa 1080
ttcaagcaat actctagaca cgttgaggaa tatgacttgc agtttatctt ccaattgtgt 1140
actattacct tgactgccga tgttatgtct tacattcatt ccatgaactc ttcaatcttg 1200
gaggattgga actttggtgt tccaccacct ccaacaactt ctttggttga cacctacaga 1260
ttcgtccaat ctgttgctat tacctgtcag aaggatgccg caccagctga aaacaaggat 1320
ccatacgaca agttgaagtt ttggaatgtc gatttgaagg agaagttctc ccttgacttg 1380
gatcaatacc cacttggtag aaagttcttg gttcaggctg gtttgagacg taagccaact 1440
atcggtccaa gaaaacgttc tgctccatca gccactacct cttccaaacc tgctaagaga 1500
gttcgtgtca gagctagaaa gtaatag 1527
<210> 5
<211> 1512
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gtcgacgcgg agaacgatct cctcgagctg ctcgcggatc agcttgtggc ccggtaatgg 60
aaccaggccg acggcacgct ccttgcggac cacggtggct ggcgagccca gtttgtgaac 120
gaggtcgttt agaacgtcct gcgcaaagtc cagtgtcaga tgaatgtcct cctcggacca 180
attcagcatg ttctcgagca gccatctgtc tttggagtag aagcgtaatc tctgctcctc 240
gttactgtac cggaagaggt agtttgcctc gccgcccata atgaacaggt tctctttctg 300
gtggcctgtg agcagcgggg acgtctggac ggcgtcgatg aggcccttga ggcgctcgta 360
gtacttgttc gcgtcgctgt agccggccgc ggtgacgata cccacataga ggtccttggc 420
cattagtttg atgaggtggg gcaggatggg cgactcggca tcgaaatttt tgccgtcgtc 480
gtacagtgtg atgtcaccat cgaatgtaat gagctgcagc ttgcgatctc ggatggtttt 540
ggaatggaag aaccgcgaca tctccaacag ctgggccgtg ttgagaatga gccggacgtc 600
gttgaacgag ggggccacaa gccggcgttt gctgatggcg cggcgctcgt cctcgatgta 660
gaaggccttt tccagaggca gtctcgtgaa gaagctgcca acgctcggaa ccagctgcac 720
gagccgagac aattcggggg tgccggcttt ggtcatttca atgttgtcgt cgatgaggag 780
ttcgaggtcg tggaagattt ccgcgtagcg gcgttttgcc tcagagttta ccatgaggtc 840
gtccactgca gagatgccgt tgctcttcac cgcgtacagg acgaacggcg tggccagcag 900
gcccttgatc cattctatga ggccatctcg acggtgttcc ttgagtgcgt actccactct 960
gtagcgactg gacatctcga gactgggctt gctgtgctgg atgcaccaat taattgttgc 1020
cgcatgcatc cttgcaccgc aagtttttaa aacccactcg ctttagccgt cgcgtaaaac 1080
ttgtgaatct ggcaactgag ggggttctgc agccgcaacc gaacttttcg cttcgaggac 1140
gcagctggat ggtgtcatgt gaggctctgt ttgctggcgt agcctacaac gtgaccttgc 1200
ctaaccggac ggcgctaccc actgctgtct gtgcctgcta ccagaaaatc accagagcag 1260
cagagggccg atgtggcaac tggtggggtg tcggacaggc tgtttctcca cagtgcaaat 1320
gcgggtgaac cggccagaaa gtaaattctt atgctaccgt gcagtgactc cgacatcccc 1380
agtttttgcc ctacttgatc acagatgggg tcagcgctgc cgctaagtgt acccaaccgt 1440
ccccacacgg tccatctata aatactgctg ccagtgcacg gtggtgacat caatctaaag 1500
tacaaaaaca aa 1512
<210> 6
<211> 341
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtttgtagcc ttagacatga ctgttcctca gttcaagttg ggcacttacg agaagaccgg 60
tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag atgcaggctt 120
catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt gtcattttgt 180
ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa tatcttgtgg 240
taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc actcctcttc 300
agagtacaga agattaagtg agaccttcgt ttgtgcggat c 341
<210> 7
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
accaaaagat ctgtcgacgc ggag 24
<210> 8
<211> 53
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cttggctaca tatgatcgat cgtgtacacg tttcgaattt gtttttgtac ttt 53
<210> 9
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tcaagtcaag cttcacgtgg ctagctttgt ttttgtactt t 41
<210> 10
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gcccgatcat atgcctgcag gcagctttct agaacaa 37
<210> 11
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aaacgtgaag cttgaattcg cggccgccag ctttct 36
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgggggatcc gcacaaacga a 21
<210> 13
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cccttagatc tcccacacac catagct 27
<210> 14
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tgcggtctta aggtttagtt cctcacc 27
<210> 15
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tattggctta agatgagcca tattcaac 28
<210> 16
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gggtatcgtc accgcggccg gctacagcac ttgaagtcgg acag 44
<210> 17
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gttggatcct cgatcacatg tgagc 25
<210> 18
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aaaggatcct gaggtctgcc tcgt 24
<210> 19
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cggacaaatt cgaaacgatg t 21
<210> 20
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
acagaaacct gcaggctatt actttttggt c 31
<210> 21
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aatcgctagc atgtggagac catctg 26
<210> 22
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ttggtaatgc ggccgcctat tatctcttgg tc 32
<210> 23
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgcttcctgc aggctattac agttttctc 29
<210> 24
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
attcgctagc atggctctgt ggagac 26
<210> 25
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ttccttatgc ggccgctatt actttctagc t 31
<210> 26
<211> 1561
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
accaaaagat ctgtcgacgc ggagaacgat ctcctcgagc tgctcgcgga tcagcttgtg 60
gcccggtaat ggaaccaggc cgacggcacg ctccttgcgg accacggtgg ctggcgagcc 120
cagtttgtga acgaggtcgt ttagaacgtc ctgcgcaaag tccagtgtca gatgaatgtc 180
ctcctcggac caattcagca tgttctcgag cagccatctg tctttggagt agaagcgtaa 240
tctctgctcc tcgttactgt accggaagag gtagtttgcc tcgccgccca taatgaacag 300
gttctctttc tggtggcctg tgagcagcgg ggacgtctgg acggcgtcga tgaggccctt 360
gaggcgctcg tagtacttgt tcgcgtcgct gtagccggcc gcggtgacga tacccacata 420
gaggtccttg gccattagtt tgatgaggtg gggcaggatg ggcgactcgg catcgaaatt 480
tttgccgtcg tcgtacagtg tgatgtcacc atcgaatgta atgagctgca gcttgcgatc 540
tcggatggtt ttggaatgga agaaccgcga catctccaac agctgggccg tgttgagaat 600
gagccggacg tcgttgaacg agggggccac aagccggcgt ttgctgatgg cgcggcgctc 660
gtcctcgatg tagaaggcct tttccagagg cagtctcgtg aagaagctgc caacgctcgg 720
aaccagctgc acgagccgag acaattcggg ggtgccggct ttggtcattt caatgttgtc 780
gtcgatgagg agttcgaggt cgtggaagat ttccgcgtag cggcgttttg cctcagagtt 840
taccatgagg tcgtccactg cagagatgcc gttgctcttc accgcgtaca ggacgaacgg 900
cgtggccagc aggcccttga tccattctat gaggccatct cgacggtgtt ccttgagtgc 960
gtactccact ctgtagcgac tggacatctc gagactgggc ttgctgtgct ggatgcacca 1020
attaattgtt gccgcatgca tccttgcacc gcaagttttt aaaacccact cgctttagcc 1080
gtcgcgtaaa acttgtgaat ctggcaactg agggggttct gcagccgcaa ccgaactttt 1140
cgcttcgagg acgcagctgg atggtgtcat gtgaggctct gtttgctggc gtagcctaca 1200
acgtgacctt gcctaaccgg acggcgctac ccactgctgt ctgtgcctgc taccagaaaa 1260
tcaccagagc agcagagggc cgatgtggca actggtgggg tgtcggacag gctgtttctc 1320
cacagtgcaa atgcgggtga accggccaga aagtaaattc ttatgctacc gtgcagtgac 1380
tccgacatcc ccagtttttg ccctacttga tcacagatgg ggtcagcgct gccgctaagt 1440
gtacccaacc gtccccacac ggtccatcta taaatactgc tgccagtgca cggtggtgac 1500
atcaatctaa agtacaaaaa caaattcgaa acgtgtacac gatcgatcat atgtagccaa 1560
g 1561
<210> 27
<211> 443
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gcccgatcat atgcctgcag gcagctttct agaacaaaaa ctcatctcag aagaggatct 60
gaatagcgcc gtcgaccatc atcatcatca tcattgagtt tgtagcctta gacatgactg 120
ttcctcagtt caagttgggc acttacgaga agaccggtct tgctagattc taatcaagag 180
gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg 240
taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc 300
tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg 360
agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga 420
ccttcgtttg tgcggatccc cca 443
<210> 28
<211> 3884
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaatt cgaaacgtgt acacgatcga tcatatgcct gcaggcagct 1560
ttctagaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcatc 1620
atcatcattg agtttgtagc cttagacatg actgttcctc agttcaagtt gggcacttac 1680
gagaagaccg gtcttgctag attctaatca agaggatgtc agaatgccat ttgcctgaga 1740
gatgcaggct tcatttttga tactttttta tttgtaacct atatagtata ggattttttt 1800
tgtcattttg tttcttctcg tacgagcttg ctcctgatca gcctatctcg cagctgatga 1860
atatcttgtg gtaggggttt gggaaaatca ttcgagtttg atgtttttct tggtatttcc 1920
cactcctctt cagagtacag aagattaagt gagaccttcg tttgtgcgga tcccccacac 1980
accatagctt caaaatgttt ctactccttt tttactcttc cagattttct cggactccgc 2040
gcatcgccgt accacttcaa aacacccaag cacagcatac taaattttcc ctctttcttc 2100
ctctagggtg tcgttaatta cccgtactaa aggtttggaa aagaaaaaag agaccgcctc 2160
gtttcttttt cttcgtcgaa aaaggcaata aaaattttta tcacgtttct ttttcttgaa 2220
attttttttt ttagtttttt tctctttcag tgacctccat tgatatttaa gttaataaac 2280
ggtcttcaat ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact 2340
tcttgttcat tagaaagaaa gcatagcaat ctaatctaag gggcggtgtt gacaattaat 2400
catcggcata gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca 2460
agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct 2520
ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc 2580
gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc 2640
tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt 2700
ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg 2760
ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc 2820
aggactgaca cgtccgacgg cggcccacgg gtcccaggcc tcggagatcc gtcccccttt 2880
tcctttgtcg atatcatgta attagttatg tcacgcttac attcacgccc tccccccaca 2940
tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt 3000
tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 3060
tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga 3120
cgctcgaagg ctttaatttg caagctggag accaacatgt gagcaaaagg ccagcaaaag 3180
gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 3240
gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 3300
taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 3360
accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc 3420
tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 3480
cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 3540
agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 3600
gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 3660
gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 3720
tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 3780
acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 3840
cagtggaacg aaaactcacg ttaagggatt ttggtcatga gatc 3884
<210> 29
<211> 1549
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
accaaaagat ctgtcgacgc ggagaacgat ctcctcgagc tgctcgcgga tcagcttgtg 60
gcccggtaat ggaaccaggc cgacggcacg ctccttgcgg accacggtgg ctggcgagcc 120
cagtttgtga acgaggtcgt ttagaacgtc ctgcgcaaag tccagtgtca gatgaatgtc 180
ctcctcggac caattcagca tgttctcgag cagccatctg tctttggagt agaagcgtaa 240
tctctgctcc tcgttactgt accggaagag gtagtttgcc tcgccgccca taatgaacag 300
gttctctttc tggtggcctg tgagcagcgg ggacgtctgg acggcgtcga tgaggccctt 360
gaggcgctcg tagtacttgt tcgcgtcgct gtagccggcc gcggtgacga tacccacata 420
gaggtccttg gccattagtt tgatgaggtg gggcaggatg ggcgactcgg catcgaaatt 480
tttgccgtcg tcgtacagtg tgatgtcacc atcgaatgta atgagctgca gcttgcgatc 540
tcggatggtt ttggaatgga agaaccgcga catctccaac agctgggccg tgttgagaat 600
gagccggacg tcgttgaacg agggggccac aagccggcgt ttgctgatgg cgcggcgctc 660
gtcctcgatg tagaaggcct tttccagagg cagtctcgtg aagaagctgc caacgctcgg 720
aaccagctgc acgagccgag acaattcggg ggtgccggct ttggtcattt caatgttgtc 780
gtcgatgagg agttcgaggt cgtggaagat ttccgcgtag cggcgttttg cctcagagtt 840
taccatgagg tcgtccactg cagagatgcc gttgctcttc accgcgtaca ggacgaacgg 900
cgtggccagc aggcccttga tccattctat gaggccatct cgacggtgtt ccttgagtgc 960
gtactccact ctgtagcgac tggacatctc gagactgggc ttgctgtgct ggatgcacca 1020
attaattgtt gccgcatgca tccttgcacc gcaagttttt aaaacccact cgctttagcc 1080
gtcgcgtaaa acttgtgaat ctggcaactg agggggttct gcagccgcaa ccgaactttt 1140
cgcttcgagg acgcagctgg atggtgtcat gtgaggctct gtttgctggc gtagcctaca 1200
acgtgacctt gcctaaccgg acggcgctac ccactgctgt ctgtgcctgc taccagaaaa 1260
tcaccagagc agcagagggc cgatgtggca actggtgggg tgtcggacag gctgtttctc 1320
cacagtgcaa atgcgggtga accggccaga aagtaaattc ttatgctacc gtgcagtgac 1380
tccgacatcc ccagtttttg ccctacttga tcacagatgg ggtcagcgct gccgctaagt 1440
gtacccaacc gtccccacac ggtccatcta taaatactgc tgccagtgca cggtggtgac 1500
atcaatctaa agtacaaaaa caaagctagc cacgtgaagc ttgacttga 1549
<210> 30
<211> 449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
aaacgtgaag cttgaattcg cggccgccag ctttctagaa caaaaactca tctcagaaga 60
ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta gccttagaca 120
tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct agattctaat 180
caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt gatacttttt 240
tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct cgtacgagct 300
tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt ttgggaaaat 360
cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac agaagattaa 420
gtgagacctt cgtttgtgcg gatccccca 449
<210> 31
<211> 3879
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat 1980
agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc 2040
gccgtaccac ttcaaaacac ccaagcacag catactaaat tttccctctt tcttcctcta 2100
gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc 2160
tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaatttt 2220
tttttttagt ttttttctct ttcagtgacc tccattgata tttaagttaa taaacggtct 2280
tcaatttctc aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg 2340
ttcattagaa agaaagcata gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg 2400
gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg 2460
accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc 2520
gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac 2580
gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc 2640
tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg 2700
aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg 2760
gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac 2820
tgacacgtcc gacggcggcc cacgggtccc aggcctcgga gatccgtccc ccttttcctt 2880
tgtcgatatc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc 2940
tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat 3000
agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag 3060
acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc 3120
gaaggcttta atttgcaagc tggagaccaa catgtgagca aaaggccagc aaaaggccag 3180
gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca 3240
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca 3300
ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg 3360
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag 3420
gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt 3480
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca 3540
cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg 3600
cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt 3660
tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc 3720
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg 3780
cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg 3840
gaacgaaaac tcacgttaag ggattttggt catgagatc 3879
<210> 32
<211> 5846
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacgt gtacacgatc gatcatatgc ctgcaggcag ctttctagaa caaaaactca 3540
tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta 3600
gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct 3660
agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt 3720
gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct 3780
cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt 3840
ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac 3900
agaagattaa gtgagacctt cgtttgtgcg gatcccccac acaccatagc ttcaaaatgt 3960
ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc gtaccacttc 4020
aaaacaccca agcacagcat actaaatttt ccctctttct tcctctaggg tgtcgttaat 4080
tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt ttcttcgtcg 4140
aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaattttttt ttttagtttt 4200
tttctctttc agtgacctcc attgatattt aagttaataa acggtcttca atttctcaag 4260
tttcagtttc atttttcttg ttctattaca acttttttta cttcttgttc attagaaaga 4320
aagcatagca atctaatcta aggggcggtg ttgacaatta atcatcggca tagtatatcg 4380
gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc 4440
cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt 4500
tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt 4560
tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc 4620
gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg 4680
cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc 4740
gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtccgac 4800
ggcggcccac gggtcccagg cctcggagat ccgtccccct tttcctttgt cgatatcatg 4860
taattagtta tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag 4920
gaaggagtta gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta 4980
ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca 5040
tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt 5100
tgcaagctgg agaccaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 5160
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 5220
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 5280
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 5340
tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 5400
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 5460
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 5520
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 5580
ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 5640
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 5700
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 5760
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 5820
cgttaaggga ttttggtcat gagatc 5846
<210> 33
<211> 502
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
cccttagatc tcccacacac catagcttca aaatgtttct actccttttt tactcttcca 60
gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta 120
aattttccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa 180
gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc 240
acgtttcttt ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg 300
atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct 360
attacaactt tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg 420
gcggtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga 480
ggaactaaac cttaagaccg ca 502
<210> 34
<211> 1269
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gggtatcgtc accgcggccg gctacagcac ttgaagtcgg acagtgagtg tagtcttgag 60
aaattctgaa gccgtatttt tattatcagt gagtcagtca tcaggagatc ctctacgccg 120
gacgcatcgt ggccgacctg cagggggggg gggggcgctg aggtctgcct cgtgaagaag 180
gtgttgctga ctcataccag gcctgaatcg ccccatcatc cagccagaaa gtgagggagc 240
cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac ttttgctttg 300
ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac tcagcaaaag 360
ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct gccagtgtta 420
caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt 480
attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga 540
aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac 600
tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga 660
gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt 720
ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa 780
accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg 840
acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat 900
attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc 960
agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg 1020
cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct 1080
acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat 1140
tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc 1200
catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat ggctcatctt 1260
aagccaata 1269
<210> 35
<211> 5223
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
agatctccca cacaccatag cttcaaaatg tttctactcc ttttttactc ttccagattt 60
tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt 120
tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa 180
aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt 240
tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc cattgatatt 300
taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt gttctattac 360
aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct aaggggcggt 420
gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac 480
taaaccttaa gatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt 540
ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag 600
gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg 660
gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg 720
aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac 780
tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat cctgattcag 840
gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt 900
gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga 960
ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac 1020
aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg 1080
gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg 1140
ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg 1200
gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg 1260
atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta 1320
attggttgta acactggcag agcattacgc tgacttgacg ggacggcggc tttgttgaat 1380
aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg 1440
ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat 1500
caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggcct ggtatgagtc 1560
agcaacacct tcttcacgag gcagacctca gcgccccccc cccccctgca ggtcggccac 1620
gatgcgtccg gcgtagagga tctcctgatg actgactcac tgataataaa aatacggctt 1680
cagaatttct caagactaca ctcactgtcc gacttcaagt gctgtagccg gccgcggtga 1740
cgatacccac atagaggtcc ttggccatta gtttgatgag gtggggcagg atgggcgact 1800
cggcatcgaa atttttgccg tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct 1860
gcagcttgcg atctcggatg gttttggaat ggaagaaccg cgacatctcc aacagctggg 1920
ccgtgttgag aatgagccgg acgtcgttga acgagggggc cacaagccgg cgtttgctga 1980
tggcgcggcg ctcgtcctcg atgtagaagg ccttttccag aggcagtctc gtgaagaagc 2040
tgccaacgct cggaaccagc tgcacgagcc gagacaattc gggggtgccg gctttggtca 2100
tttcaatgtt gtcgtcgatg aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt 2160
ttgcctcaga gtttaccatg aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt 2220
acaggacgaa cggcgtggcc agcaggccct tgatccattc tatgaggcca tctcgacggt 2280
gttccttgag tgcgtactcc actctgtagc gactggacat ctcgagactg ggcttgctgt 2340
gctggatgca ccaattaatt gttgccgcat gcatccttgc accgcaagtt tttaaaaccc 2400
actcgcttta gccgtcgcgt aaaacttgtg aatctggcaa ctgagggggt tctgcagccg 2460
caaccgaact tttcgcttcg aggacgcagc tggatggtgt catgtgaggc tctgtttgct 2520
ggcgtagcct acaacgtgac cttgcctaac cggacggcgc tacccactgc tgtctgtgcc 2580
tgctaccaga aaatcaccag agcagcagag ggccgatgtg gcaactggtg gggtgtcgga 2640
caggctgttt ctccacagtg caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct 2700
accgtgcagt gactccgaca tccccagttt ttgccctact tgatcacaga tggggtcagc 2760
gctgccgcta agtgtaccca accgtcccca cacggtccat ctataaatac tgctgccagt 2820
gcacggtggt gacatcaatc taaagtacaa aaacaaattc gaaacgtgta cacgatcgat 2880
catatgcctg caggcagctt tctagaacaa aaactcatct cagaagagga tctgaatagc 2940
gccgtcgacc atcatcatca tcatcattga gtttgtagcc ttagacatga ctgttcctca 3000
gttcaagttg ggcacttacg agaagaccgg tcttgctaga ttctaatcaa gaggatgtca 3060
gaatgccatt tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta 3120
tatagtatag gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag 3180
cctatctcgc agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga 3240
tgtttttctt ggtatttccc actcctcttc agagtacaga agattaagtg agaccttcgt 3300
ttgtgcggat cccccacaca ccatagcttc aaaatgtttc tactcctttt ttactcttcc 3360
agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc acagcatact 3420
aaattttccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa ggtttggaaa 3480
agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa aaatttttat 3540
cacgtttctt tttcttgaaa tttttttttt tagttttttt ctctttcagt gacctccatt 3600
gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt tttcttgttc 3660
tattacaact ttttttactt cttgttcatt agaaagaaag catagcaatc taatctaagg 3720
ggcggtgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg 3780
aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc 3840
gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac 3900
gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag 3960
gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc 4020
gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag 4080
atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg 4140
cacttcgtgg ccgaggagca ggactgacac gtccgacggc ggcccacggg tcccaggcct 4200
cggagatccg tccccctttt cctttgtcga tatcatgtaa ttagttatgt cacgcttaca 4260
ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt 4320
ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat ttatatttca 4380
aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt 4440
gcttgagaag gttttgggac gctcgaaggc tttaatttgc aagctggaga ccaacatgtg 4500
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4560
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4620
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4680
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4740
gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4800
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4860
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4920
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4980
cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 5040
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 5100
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 5160
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 5220
atc 5223
<210> 36
<211> 2344
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gttggatcct cgatcacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 60
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 120
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 180
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 240
ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg 300
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 360
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 420
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 480
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 540
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 600
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 660
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 720
gttaagggat tttggtcatg agatcagatc tcccacacac catagcttca aaatgtttct 780
actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa 840
cacccaagca cagcatacta aattttccct ctttcttcct ctagggtgtc gttaattacc 900
cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa 960
aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt agtttttttc 1020
tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc 1080
agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta gaaagaaagc 1140
atagcaatct aatctaaggg gcggtgttga caattaatca tcggcatagt atatcggcat 1200
agtataatac gacaaggtga ggaactaaac cttaagatga gccatattca acgggaaacg 1260
tcttgctcga ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg 1320
gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat 1380
gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag 1440
atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc 1500
cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag 1560
gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg 1620
cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt 1680
ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac 1740
gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc 1800
tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag 1860
gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat 1920
cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt 1980
caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat 2040
gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat tacgctgact 2100
tgacgggacg gcggctttgt tgaataaatc gaacttttgc tgagttgaag gatcagatca 2160
cgcatcttcc cgacaacgca gaccgttccg tggcaaagca aaagttcaaa atcaccaact 2220
ggtccaccta caacaaagct ctcatcaacc gtggctccct cactttctgg ctggatgatg 2280
gggcgattca ggcctggtat gagtcagcaa caccttcttc acgaggcaga cctcaggatc 2340
cttt 2344
<210> 37
<211> 6261
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ggatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacgt gtacacgatc gatcatatgc ctgcaggcag ctttctagaa caaaaactca 3540
tctcagaaga ggatctgaat agcgccgtcg accatcatca tcatcatcat tgagtttgta 3600
gccttagaca tgactgttcc tcagttcaag ttgggcactt acgagaagac cggtcttgct 3660
agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg cttcattttt 3720
gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt tgtttcttct 3780
cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg tggtaggggt 3840
ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc ttcagagtac 3900
agaagattaa gtgagacctt cgtttgtgcg gatcctcgat cacatgtgag caaaaggcca 3960
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 4020
ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4080
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4140
gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg 4200
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4260
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4320
cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4380
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4440
aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 4500
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 4560
gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 4620
tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat cagatctccc 4680
acacaccata gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact 4740
ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc atactaaatt ttccctcttt 4800
cttcctctag ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg 4860
cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct 4920
tgaaattttt ttttttagtt tttttctctt tcagtgacct ccattgatat ttaagttaat 4980
aaacggtctt caatttctca agtttcagtt tcatttttct tgttctatta caactttttt 5040
tacttcttgt tcattagaaa gaaagcatag caatctaatc taaggggcgg tgttgacaat 5100
taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaacctta 5160
agatgagcca tattcaacgg gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg 5220
atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa 5280
tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta 5340
gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc 5400
ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg 5460
cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata 5520
ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc 5580
cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt 5640
tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga 5700
aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct 5760
cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag 5820
tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt 5880
ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata 5940
aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt aattggttgt 6000
aacactggca gagcattacg ctgacttgac gggacggcgg ctttgttgaa taaatcgaac 6060
ttttgctgag ttgaaggatc agatcacgca tcttcccgac aacgcagacc gttccgtggc 6120
aaagcaaaag ttcaaaatca ccaactggtc cacctacaac aaagctctca tcaaccgtgg 6180
ctccctcact ttctggctgg atgatggggc gattcaggcc tggtatgagt cagcaacacc 6240
ttcttcacga ggcagacctc a 6261
<210> 38
<211> 1541
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
cggacaaatt cgaaacgatg tggagaccat ctgactcgac cgtctacgtt cctccaccta 60
acccagtctc caaagtggtt gctactgatg cctacgtcaa gagaaccaac atcttctacc 120
acgcctcttc gtccagattg ctggcagttg gacatcctta ctattccatc aagaaagtta 180
acaagaccgt ggtccctaag gtttcgggct atcagtacag agtgttcaag gtcgttctgc 240
cagaccctaa caagtttgcc ttgccagatt cctcgctgtt cgaccctacc actcaaagac 300
tggtttgggc ttgcacgggt ttggaagtgg gcagaggaca accactgggt gtcggagttt 360
ccggccatcc attgctgaac aagtacgacg atgttgagaa ctcgggtgga tacggtggca 420
atccaggaca agacaacaga gtgaatgtcg gcatggacta caagcaaacc cagctgtgca 480
tggttggttg tgcaccacct ctgggtgaac attggggaaa gggcactcag tgctccaaca 540
cctctgttca gaatggtgac tgtcctccac tggagctgat cacctcggtt attcaggatg 600
gagacatggt ggacactggc ttcggtgcaa tgaactttgc cgacttgcag accaacaagt 660
ccgatgttcc attggacatt tgcggcactg tgtgcaaata tcctgactac ctgcagatgg 720
ctgcagatcc atacggtgac agactcttct tctacctgag aaaggagcag atgttcgcca 780
gacacttctt taacagagct ggcaccgttg gtgagcctgt tccagatgac ctgttggtga 840
agggtggaaa caatagatcc tctgttgcct cgtccatcta cgtccacacc ccatcgggct 900
ctctggtgtc ctcggaggcc caactgttca acaagcctta ctggctccag aaggcccaag 960
gacacaacaa tggcatctgc tggggaaacc acctgttcgt caccgttgtg gacacgacta 1020
gatccaccaa catgactttg tgtgcctctg tttccaagtc ggctacctac acgaactcgg 1080
actataagga gtacatgaga catgttgaag agttcgacct gcagtttatc ttccagctct 1140
gctccattac cttgtcggca gaagtgatgg cctacatcca cacgatgaac ccatccgtct 1200
tggaagactg gaacttcggt ctgtctccac ctccaaatgg caccctggaa gacacgtaca 1260
gatatgtgca gtcgcaagcc atcacctgtc agaaaccaac tcctgaaaag gagaaacagg 1320
acccatacaa ggatatgtcg ttctgggagg tcaacctgaa ggagaagttc tcttcggaac 1380
tggaccagtt cccattggga agaaagttcc tgctccagtc tggttataga ggaagaacct 1440
ccgcaagaac tggcatcaag agacctgccg tgtcgaagcc atccaccgct cctaagagaa 1500
agagaacgaa gaccaaaaag taatagcctg caggtttctg t 1541
<210> 39
<211> 7750
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ggatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacga tgtggagacc atctgactcg accgtctacg ttcctccacc taacccagtc 3540
tccaaagtgg ttgctactga tgcctacgtc aagagaacca acatcttcta ccacgcctct 3600
tcgtccagat tgctggcagt tggacatcct tactattcca tcaagaaagt taacaagacc 3660
gtggtcccta aggtttcggg ctatcagtac agagtgttca aggtcgttct gccagaccct 3720
aacaagtttg ccttgccaga ttcctcgctg ttcgacccta ccactcaaag actggtttgg 3780
gcttgcacgg gtttggaagt gggcagagga caaccactgg gtgtcggagt ttccggccat 3840
ccattgctga acaagtacga cgatgttgag aactcgggtg gatacggtgg caatccagga 3900
caagacaaca gagtgaatgt cggcatggac tacaagcaaa cccagctgtg catggttggt 3960
tgtgcaccac ctctgggtga acattgggga aagggcactc agtgctccaa cacctctgtt 4020
cagaatggtg actgtcctcc actggagctg atcacctcgg ttattcagga tggagacatg 4080
gtggacactg gcttcggtgc aatgaacttt gccgacttgc agaccaacaa gtccgatgtt 4140
ccattggaca tttgcggcac tgtgtgcaaa tatcctgact acctgcagat ggctgcagat 4200
ccatacggtg acagactctt cttctacctg agaaaggagc agatgttcgc cagacacttc 4260
tttaacagag ctggcaccgt tggtgagcct gttccagatg acctgttggt gaagggtgga 4320
aacaatagat cctctgttgc ctcgtccatc tacgtccaca ccccatcggg ctctctggtg 4380
tcctcggagg cccaactgtt caacaagcct tactggctcc agaaggccca aggacacaac 4440
aatggcatct gctggggaaa ccacctgttc gtcaccgttg tggacacgac tagatccacc 4500
aacatgactt tgtgtgcctc tgtttccaag tcggctacct acacgaactc ggactataag 4560
gagtacatga gacatgttga agagttcgac ctgcagttta tcttccagct ctgctccatt 4620
accttgtcgg cagaagtgat ggcctacatc cacacgatga acccatccgt cttggaagac 4680
tggaacttcg gtctgtctcc acctccaaat ggcaccctgg aagacacgta cagatatgtg 4740
cagtcgcaag ccatcacctg tcagaaacca actcctgaaa aggagaaaca ggacccatac 4800
aaggatatgt cgttctggga ggtcaacctg aaggagaagt tctcttcgga actggaccag 4860
ttcccattgg gaagaaagtt cctgctccag tctggttata gaggaagaac ctccgcaaga 4920
actggcatca agagacctgc cgtgtcgaag ccatccaccg ctcctaagag aaagagaacg 4980
aagaccaaaa agtaatagcc tgcaggcagc tttctagaac aaaaactcat ctcagaagag 5040
gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttgtag ccttagacat 5100
gactgttcct cagttcaagt tgggcactta cgagaagacc ggtcttgcta gattctaatc 5160
aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg atactttttt 5220
atttgtaacc tatatagtat aggatttttt ttgtcatttt gtttcttctc gtacgagctt 5280
gctcctgatc agcctatctc gcagctgatg aatatcttgt ggtaggggtt tgggaaaatc 5340
attcgagttt gatgtttttc ttggtatttc ccactcctct tcagagtaca gaagattaag 5400
tgagaccttc gtttgtgcgg atcctcgatc acatgtgagc aaaaggccag caaaaggcca 5460
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5520
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5580
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5640
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5700
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5760
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5820
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5880
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 5940
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6000
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6060
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6120
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc agatctccca cacaccatag 6180
cttcaaaatg tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc 6240
cgtaccactt caaaacaccc aagcacagca tactaaattt tccctctttc ttcctctagg 6300
gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt 6360
tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaatttttt 6420
tttttagttt ttttctcttt cagtgacctc cattgatatt taagttaata aacggtcttc 6480
aatttctcaa gtttcagttt catttttctt gttctattac aacttttttt acttcttgtt 6540
cattagaaag aaagcatagc aatctaatct aaggggcggt gttgacaatt aatcatcggc 6600
atagtatatc ggcatagtat aatacgacaa ggtgaggaac taaaccttaa gatgagccat 6660
attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta 6720
tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg 6780
tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat 6840
gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc 6900
atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccggg 6960
aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg 7020
ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc 7080
gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg 7140
agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat 7200
aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac 7260
cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca 7320
gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta 7380
cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt 7440
catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag 7500
agcattacgc tgacttgacg ggacggcggc tttgttgaat aaatcgaact tttgctgagt 7560
tgaaggatca gatcacgcat cttcccgaca acgcagaccg ttccgtggca aagcaaaagt 7620
tcaaaatcac caactggtcc acctacaaca aagctctcat caaccgtggc tccctcactt 7680
tctggctgga tgatggggcg attcaggcct ggtatgagtc agcaacacct tcttcacgag 7740
gcagacctca 7750
<210> 40
<211> 1532
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
aatcgctagc atgtggagac catctgactc gactgtctac gttcctccac ctaacccagt 60
ctctaaggtt gtggctaccg acgcctacgt gactagaacc aacatcttct accacgcatc 120
gtcctctaga ttgctggccg ttggacatcc ttacttctcc atcaagagag ccaacaaaac 180
cgtcgtgcca aaggtttctg gctaccagta tagagtcttc aaggtggttc tgcctgaccc 240
taacaagttt gccctgccag actcgtcctt gttcgatcct acgactcaaa gactcgtttg 300
ggcctgcact ggtcttgagg ttggcagagg acagccactc ggagtcggcg tttctggtca 360
tcctttcctg aacaagtacg acgatgttga gaactctggt tccggaggca atcctggaca 420
ggacaacaga gtcaatgttg gcatggacta caagcaaact cagctctgta tggtgggatg 480
cgcacctcca ctgggtgagc attggggaaa aggcaagcag tgcaccaaca ctccagttca 540
ggcaggtgac tgccctccat tggagctgat tacctcggtc atccaggacg gtgatatggt 600
tgacaccgga ttcggtgcca tgaacttcgc tgacctgcag accaacaagt ccgatgtgcc 660
aatcgacatc tgcggaacga cctgtaaata ccctgactac ttgcagatgg cagccgatcc 720
atacggcgac agactcttct tctttctgag aaaggagcag atgtttgcta gacacttttt 780
caacagagcc ggagaggtcg gtgagcctgt tccagacacc ctgatcatta agggctcggg 840
aaacagaact tctgtcggct cctcgatcta cgtgaacacc ccttccggtt cgttggtgtc 900
gtccgaggct caactgttca acaagccata ctggctccag aaagcccaag gtcacaacaa 960
tggcatctgc tggggtaacc agctgttcgt tactgtcgtg gacaccacga gatcgaccaa 1020
catgactctg tgtgcctccg ttaccacatc ttcgacttac accaactccg actataagga 1080
gtacatgaga cacgttgaag agtacgactt gcaattcatc tttcagctct gctcgattac 1140
cctgtccgca gaagtcatgg cctacatcca cactatgaat ccatcggtgc ttgaggactg 1200
gaactttggc ttgtccccac ctccaaacgg aactctggaa gacacctata gatacgtgca 1260
gtcgcaagcc atcacgtgcc agaagccaac ccctgagaag gaaaagccag atccttacaa 1320
gaacctgtcc ttctgggagg tcaatctcaa ggagaagttc tcgtccgaac tggaccagta 1380
cccattgggt agaaagttcc tgctccagtc tggataccgt ggtagatcgt ccatcagaac 1440
cggagtgaag agaccagcag tttccaaggc ttctgccgca cctaagcgta aaagagccaa 1500
gaccaagaga taataggcgg ccgcattacc aa 1532
<210> 41
<211> 9238
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ggatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagcatgtgg agaccatctg actcgactgt ctacgttcct 1560
ccacctaacc cagtctctaa ggttgtggct accgacgcct acgtgactag aaccaacatc 1620
ttctaccacg catcgtcctc tagattgctg gccgttggac atccttactt ctccatcaag 1680
agagccaaca aaaccgtcgt gccaaaggtt tctggctacc agtatagagt cttcaaggtg 1740
gttctgcctg accctaacaa gtttgccctg ccagactcgt ccttgttcga tcctacgact 1800
caaagactcg tttgggcctg cactggtctt gaggttggca gaggacagcc actcggagtc 1860
ggcgtttctg gtcatccttt cctgaacaag tacgacgatg ttgagaactc tggttccgga 1920
ggcaatcctg gacaggacaa cagagtcaat gttggcatgg actacaagca aactcagctc 1980
tgtatggtgg gatgcgcacc tccactgggt gagcattggg gaaaaggcaa gcagtgcacc 2040
aacactccag ttcaggcagg tgactgccct ccattggagc tgattacctc ggtcatccag 2100
gacggtgata tggttgacac cggattcggt gccatgaact tcgctgacct gcagaccaac 2160
aagtccgatg tgccaatcga catctgcgga acgacctgta aataccctga ctacttgcag 2220
atggcagccg atccatacgg cgacagactc ttcttctttc tgagaaagga gcagatgttt 2280
gctagacact ttttcaacag agccggagag gtcggtgagc ctgttccaga caccctgatc 2340
attaagggct cgggaaacag aacttctgtc ggctcctcga tctacgtgaa caccccttcc 2400
ggttcgttgg tgtcgtccga ggctcaactg ttcaacaagc catactggct ccagaaagcc 2460
caaggtcaca acaatggcat ctgctggggt aaccagctgt tcgttactgt cgtggacacc 2520
acgagatcga ccaacatgac tctgtgtgcc tccgttacca catcttcgac ttacaccaac 2580
tccgactata aggagtacat gagacacgtt gaagagtacg acttgcaatt catctttcag 2640
ctctgctcga ttaccctgtc cgcagaagtc atggcctaca tccacactat gaatccatcg 2700
gtgcttgagg actggaactt tggcttgtcc ccacctccaa acggaactct ggaagacacc 2760
tatagatacg tgcagtcgca agccatcacg tgccagaagc caacccctga gaaggaaaag 2820
ccagatcctt acaagaacct gtccttctgg gaggtcaatc tcaaggagaa gttctcgtcc 2880
gaactggacc agtacccatt gggtagaaag ttcctgctcc agtctggata ccgtggtaga 2940
tcgtccatca gaaccggagt gaagagacca gcagtttcca aggcttctgc cgcacctaag 3000
cgtaaaagag ccaagaccaa gagataatag gcggccgcca gctttctaga acaaaaactc 3060
atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttgt 3120
agccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc 3180
tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt 3240
tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc 3300
tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg 3360
tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta 3420
cagaagatta agtgagacct tcgtttgtgc ggatctgtcg acgcggagaa cgatctcctc 3480
gagctgctcg cggatcagct tgtggcccgg taatggaacc aggccgacgg cacgctcctt 3540
gcggaccacg gtggctggcg agcccagttt gtgaacgagg tcgtttagaa cgtcctgcgc 3600
aaagtccagt gtcagatgaa tgtcctcctc ggaccaattc agcatgttct cgagcagcca 3660
tctgtctttg gagtagaagc gtaatctctg ctcctcgtta ctgtaccgga agaggtagtt 3720
tgcctcgccg cccataatga acaggttctc tttctggtgg cctgtgagca gcggggacgt 3780
ctggacggcg tcgatgaggc ccttgaggcg ctcgtagtac ttgttcgcgt cgctgtagcc 3840
ggccgcggtg acgataccca catagaggtc cttggccatt agtttgatga ggtggggcag 3900
gatgggcgac tcggcatcga aatttttgcc gtcgtcgtac agtgtgatgt caccatcgaa 3960
tgtaatgagc tgcagcttgc gatctcggat ggttttggaa tggaagaacc gcgacatctc 4020
caacagctgg gccgtgttga gaatgagccg gacgtcgttg aacgaggggg ccacaagccg 4080
gcgtttgctg atggcgcggc gctcgtcctc gatgtagaag gccttttcca gaggcagtct 4140
cgtgaagaag ctgccaacgc tcggaaccag ctgcacgagc cgagacaatt cgggggtgcc 4200
ggctttggtc atttcaatgt tgtcgtcgat gaggagttcg aggtcgtgga agatttccgc 4260
gtagcggcgt tttgcctcag agtttaccat gaggtcgtcc actgcagaga tgccgttgct 4320
cttcaccgcg tacaggacga acggcgtggc cagcaggccc ttgatccatt ctatgaggcc 4380
atctcgacgg tgttccttga gtgcgtactc cactctgtag cgactggaca tctcgagact 4440
gggcttgctg tgctggatgc accaattaat tgttgccgca tgcatccttg caccgcaagt 4500
ttttaaaacc cactcgcttt agccgtcgcg taaaacttgt gaatctggca actgaggggg 4560
ttctgcagcc gcaaccgaac ttttcgcttc gaggacgcag ctggatggtg tcatgtgagg 4620
ctctgtttgc tggcgtagcc tacaacgtga ccttgcctaa ccggacggcg ctacccactg 4680
ctgtctgtgc ctgctaccag aaaatcacca gagcagcaga gggccgatgt ggcaactggt 4740
ggggtgtcgg acaggctgtt tctccacagt gcaaatgcgg gtgaaccggc cagaaagtaa 4800
attcttatgc taccgtgcag tgactccgac atccccagtt tttgccctac ttgatcacag 4860
atggggtcag cgctgccgct aagtgtaccc aaccgtcccc acacggtcca tctataaata 4920
ctgctgccag tgcacggtgg tgacatcaat ctaaagtaca aaaacaaatt cgaaacgatg 4980
tggagaccat ctgactcgac cgtctacgtt cctccaccta acccagtctc caaagtggtt 5040
gctactgatg cctacgtcaa gagaaccaac atcttctacc acgcctcttc gtccagattg 5100
ctggcagttg gacatcctta ctattccatc aagaaagtta acaagaccgt ggtccctaag 5160
gtttcgggct atcagtacag agtgttcaag gtcgttctgc cagaccctaa caagtttgcc 5220
ttgccagatt cctcgctgtt cgaccctacc actcaaagac tggtttgggc ttgcacgggt 5280
ttggaagtgg gcagaggaca accactgggt gtcggagttt ccggccatcc attgctgaac 5340
aagtacgacg atgttgagaa ctcgggtgga tacggtggca atccaggaca agacaacaga 5400
gtgaatgtcg gcatggacta caagcaaacc cagctgtgca tggttggttg tgcaccacct 5460
ctgggtgaac attggggaaa gggcactcag tgctccaaca cctctgttca gaatggtgac 5520
tgtcctccac tggagctgat cacctcggtt attcaggatg gagacatggt ggacactggc 5580
ttcggtgcaa tgaactttgc cgacttgcag accaacaagt ccgatgttcc attggacatt 5640
tgcggcactg tgtgcaaata tcctgactac ctgcagatgg ctgcagatcc atacggtgac 5700
agactcttct tctacctgag aaaggagcag atgttcgcca gacacttctt taacagagct 5760
ggcaccgttg gtgagcctgt tccagatgac ctgttggtga agggtggaaa caatagatcc 5820
tctgttgcct cgtccatcta cgtccacacc ccatcgggct ctctggtgtc ctcggaggcc 5880
caactgttca acaagcctta ctggctccag aaggcccaag gacacaacaa tggcatctgc 5940
tggggaaacc acctgttcgt caccgttgtg gacacgacta gatccaccaa catgactttg 6000
tgtgcctctg tttccaagtc ggctacctac acgaactcgg actataagga gtacatgaga 6060
catgttgaag agttcgacct gcagtttatc ttccagctct gctccattac cttgtcggca 6120
gaagtgatgg cctacatcca cacgatgaac ccatccgtct tggaagactg gaacttcggt 6180
ctgtctccac ctccaaatgg caccctggaa gacacgtaca gatatgtgca gtcgcaagcc 6240
atcacctgtc agaaaccaac tcctgaaaag gagaaacagg acccatacaa ggatatgtcg 6300
ttctgggagg tcaacctgaa ggagaagttc tcttcggaac tggaccagtt cccattggga 6360
agaaagttcc tgctccagtc tggttataga ggaagaacct ccgcaagaac tggcatcaag 6420
agacctgccg tgtcgaagcc atccaccgct cctaagagaa agagaacgaa gaccaaaaag 6480
taatagcctg caggcagctt tctagaacaa aaactcatct cagaagagga tctgaatagc 6540
gccgtcgacc atcatcatca tcatcattga gtttgtagcc ttagacatga ctgttcctca 6600
gttcaagttg ggcacttacg agaagaccgg tcttgctaga ttctaatcaa gaggatgtca 6660
gaatgccatt tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta 6720
tatagtatag gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag 6780
cctatctcgc agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga 6840
tgtttttctt ggtatttccc actcctcttc agagtacaga agattaagtg agaccttcgt 6900
ttgtgcggat cctcgatcac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6960
aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 7020
gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 7080
ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 7140
cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt 7200
cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 7260
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 7320
cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 7380
agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 7440
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 7500
ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 7560
gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 7620
cacgttaagg gattttggtc atgagatcag atctcccaca caccatagct tcaaaatgtt 7680
tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg taccacttca 7740
aaacacccaa gcacagcata ctaaattttc cctctttctt cctctagggt gtcgttaatt 7800
acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga 7860
aaaaggcaat aaaaattttt atcacgtttc tttttcttga aatttttttt tttagttttt 7920
ttctctttca gtgacctcca ttgatattta agttaataaa cggtcttcaa tttctcaagt 7980
ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgttca ttagaaagaa 8040
agcatagcaa tctaatctaa ggggcggtgt tgacaattaa tcatcggcat agtatatcgg 8100
catagtataa tacgacaagg tgaggaacta aaccttaaga tgagccatat tcaacgggaa 8160
acgtcttgct cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa 8220
tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc 8280
gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 8340
gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt 8400
atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc 8460
caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc 8520
ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt 8580
cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat 8640
gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca 8700
ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac 8760
gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag 8820
gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 8880
tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc 8940
gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag cattacgctg 9000
acttgacggg acggcggctt tgttgaataa atcgaacttt tgctgagttg aaggatcaga 9060
tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc aaaatcacca 9120
actggtccac ctacaacaaa gctctcatca accgtggctc cctcactttc tggctggatg 9180
atggggcgat tcaggcctgg tatgagtcag caacaccttc ttcacgaggc agacctca 9238
<210> 42
<211> 1551
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
cggacaaatt cgaaacgatg tctctttggt tgccatccga ggctactgtt tacttgccac 60
ctgtcccagt ttctaaggtc gtgtcaactg atgaatacgt tgctagaacc aacatctatt 120
accacgctgg aacttctaga ttgcttgctg ttggtcatcc atacttccct atcaagaaac 180
ctaacaataa caagatcctt gttccaaagg tctctggatt gcagtacaga gttttcagaa 240
tccacttgcc tgacccaaac aagttcggtt ttcctgacac ctccttctac aacccagata 300
ctcaaagatt ggtgtgggcc tgtgttggtg tcgaggttgg acgtggtcaa ccattgggag 360
ttggtatttc tggacatcct ttgcttaaca agttggatga cactgaaaat gcttctgcct 420
acgcagccaa cgctggtgtt gacaacagag agtgtatctc tatggattac aaacagaccc 480
aattgtgtct tattggttgc aagccaccta ttggtgaaca ctggggtaag ggatctccat 540
gtaccaacgt tgctgtcaat ccaggtgatt gtcctccatt ggagcttatc aacactgtta 600
ttcaagatgg tgacatggtt gatactggat ttggtgctat ggacttcact accttgcaag 660
ctaacaagtc cgaggttcca ttggacattt gtacttctat ctgcaagtat ccagactaca 720
ttaagatggt ctctgaacca tacggtgact ccttgttttt ctaccttcgt agagagcaaa 780
tgtttgttag acacttgttc aacagagctg gtgccgttgg tgagaatgtc ccagacgatt 840
tgtacatcaa gggatctggt tccactgcaa acttggcctc ttccaactac ttcccaactc 900
cttcaggttc tatggttacc tccgatgctc aaatcttcaa caagccttac tggttgcaaa 960
gagcacaggg tcacaacaat ggaatttgtt ggggtaacca acttttcgtt actgtcgttg 1020
ataccacaag atccaccaac atgtccttgt gtgctgccat ttctacttca gaaaccacat 1080
acaagaatac taacttcaag gagtacctta gacatggtga ggaatacgac ttgcagttca 1140
tctttcaatt gtgcaagatt accttgactg ctgacgttat gacctacatc cactctatga 1200
actccactat ccttgaagat tggaacttcg gattgcaacc acctccaggt ggaaccttgg 1260
aagatactta cagatttgtc acatcccaag ccattgcttg tcagaagcac actcctccag 1320
ctcctaagga agatccactt aagaaataca ccttctggga agtcaacttg aaagagaagt 1380
tctctgctga ccttgatcag tttcctttgg gtagaaagtt cttacttcaa gctggattga 1440
aggccaaacc aaagttcacc ttgggtaaaa gaaaggctac tccaaccaca tcatctactt 1500
ccacaaccgc caaacgtaag aagagaaaac tgtaatagcc tgcaggaagc a 1551
<210> 43
<211> 7347
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagccacgtg aagcttgaat tcgcggccgc cagctttcta 1560
gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat 1620
cattgagttt gtagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa 1680
gaccggtctt gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc 1740
aggcttcatt tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca 1800
ttttgtttct tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc 1860
ttgtggtagg ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc 1920
ctcttcagag tacagaagat taagtgagac cttcgtttgt gcggatctgt cgacgcggag 1980
aacgatctcc tcgagctgct cgcggatcag cttgtggccc ggtaatggaa ccaggccgac 2040
ggcacgctcc ttgcggacca cggtggctgg cgagcccagt ttgtgaacga ggtcgtttag 2100
aacgtcctgc gcaaagtcca gtgtcagatg aatgtcctcc tcggaccaat tcagcatgtt 2160
ctcgagcagc catctgtctt tggagtagaa gcgtaatctc tgctcctcgt tactgtaccg 2220
gaagaggtag tttgcctcgc cgcccataat gaacaggttc tctttctggt ggcctgtgag 2280
cagcggggac gtctggacgg cgtcgatgag gcccttgagg cgctcgtagt acttgttcgc 2340
gtcgctgtag ccggccgcgg tgacgatacc cacatagagg tccttggcca ttagtttgat 2400
gaggtggggc aggatgggcg actcggcatc gaaatttttg ccgtcgtcgt acagtgtgat 2460
gtcaccatcg aatgtaatga gctgcagctt gcgatctcgg atggttttgg aatggaagaa 2520
ccgcgacatc tccaacagct gggccgtgtt gagaatgagc cggacgtcgt tgaacgaggg 2580
ggccacaagc cggcgtttgc tgatggcgcg gcgctcgtcc tcgatgtaga aggccttttc 2640
cagaggcagt ctcgtgaaga agctgccaac gctcggaacc agctgcacga gccgagacaa 2700
ttcgggggtg ccggctttgg tcatttcaat gttgtcgtcg atgaggagtt cgaggtcgtg 2760
gaagatttcc gcgtagcggc gttttgcctc agagtttacc atgaggtcgt ccactgcaga 2820
gatgccgttg ctcttcaccg cgtacaggac gaacggcgtg gccagcaggc ccttgatcca 2880
ttctatgagg ccatctcgac ggtgttcctt gagtgcgtac tccactctgt agcgactgga 2940
catctcgaga ctgggcttgc tgtgctggat gcaccaatta attgttgccg catgcatcct 3000
tgcaccgcaa gtttttaaaa cccactcgct ttagccgtcg cgtaaaactt gtgaatctgg 3060
caactgaggg ggttctgcag ccgcaaccga acttttcgct tcgaggacgc agctggatgg 3120
tgtcatgtga ggctctgttt gctggcgtag cctacaacgt gaccttgcct aaccggacgg 3180
cgctacccac tgctgtctgt gcctgctacc agaaaatcac cagagcagca gagggccgat 3240
gtggcaactg gtggggtgtc ggacaggctg tttctccaca gtgcaaatgc gggtgaaccg 3300
gccagaaagt aaattcttat gctaccgtgc agtgactccg acatccccag tttttgccct 3360
acttgatcac agatggggtc agcgctgccg ctaagtgtac ccaaccgtcc ccacacggtc 3420
catctataaa tactgctgcc agtgcacggt ggtgacatca atctaaagta caaaaacaaa 3480
ttcgaaacga tgtctctttg gttgccatcc gaggctactg tttacttgcc acctgtccca 3540
gtttctaagg tcgtgtcaac tgatgaatac gttgctagaa ccaacatcta ttaccacgct 3600
ggaacttcta gattgcttgc tgttggtcat ccatacttcc ctatcaagaa acctaacaat 3660
aacaagatcc ttgttccaaa ggtctctgga ttgcagtaca gagttttcag aatccacttg 3720
cctgacccaa acaagttcgg ttttcctgac acctccttct acaacccaga tactcaaaga 3780
ttggtgtggg cctgtgttgg tgtcgaggtt ggacgtggtc aaccattggg agttggtatt 3840
tctggacatc ctttgcttaa caagttggat gacactgaaa atgcttctgc ctacgcagcc 3900
aacgctggtg ttgacaacag agagtgtatc tctatggatt acaaacagac ccaattgtgt 3960
cttattggtt gcaagccacc tattggtgaa cactggggta agggatctcc atgtaccaac 4020
gttgctgtca atccaggtga ttgtcctcca ttggagctta tcaacactgt tattcaagat 4080
ggtgacatgg ttgatactgg atttggtgct atggacttca ctaccttgca agctaacaag 4140
tccgaggttc cattggacat ttgtacttct atctgcaagt atccagacta cattaagatg 4200
gtctctgaac catacggtga ctccttgttt ttctaccttc gtagagagca aatgtttgtt 4260
agacacttgt tcaacagagc tggtgccgtt ggtgagaatg tcccagacga tttgtacatc 4320
aagggatctg gttccactgc aaacttggcc tcttccaact acttcccaac tccttcaggt 4380
tctatggtta cctccgatgc tcaaatcttc aacaagcctt actggttgca aagagcacag 4440
ggtcacaaca atggaatttg ttggggtaac caacttttcg ttactgtcgt tgataccaca 4500
agatccacca acatgtcctt gtgtgctgcc atttctactt cagaaaccac atacaagaat 4560
actaacttca aggagtacct tagacatggt gaggaatacg acttgcagtt catctttcaa 4620
ttgtgcaaga ttaccttgac tgctgacgtt atgacctaca tccactctat gaactccact 4680
atccttgaag attggaactt cggattgcaa ccacctccag gtggaacctt ggaagatact 4740
tacagatttg tcacatccca agccattgct tgtcagaagc acactcctcc agctcctaag 4800
gaagatccac ttaagaaata caccttctgg gaagtcaact tgaaagagaa gttctctgct 4860
gaccttgatc agtttccttt gggtagaaag ttcttacttc aagctggatt gaaggccaaa 4920
ccaaagttca ccttgggtaa aagaaaggct actccaacca catcatctac ttccacaacc 4980
gccaaacgta agaagagaaa actgtaatag cctgcaggca gctttctaga acaaaaactc 5040
atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttgt 5100
agccttagac atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc 5160
tagattctaa tcaagaggat gtcagaatgc catttgcctg agagatgcag gcttcatttt 5220
tgatactttt ttatttgtaa cctatatagt ataggatttt ttttgtcatt ttgtttcttc 5280
tcgtacgagc ttgctcctga tcagcctatc tcgcagctga tgaatatctt gtggtagggg 5340
tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt tcccactcct cttcagagta 5400
cagaagatta agtgagacct tcgtttgtgc ggatccccca cacaccatag cttcaaaatg 5460
tttctactcc ttttttactc ttccagattt tctcggactc cgcgcatcgc cgtaccactt 5520
caaaacaccc aagcacagca tactaaattt tccctctttc ttcctctagg gtgtcgttaa 5580
ttacccgtac taaaggtttg gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc 5640
gaaaaaggca ataaaaattt ttatcacgtt tctttttctt gaaatttttt tttttagttt 5700
ttttctcttt cagtgacctc cattgatatt taagttaata aacggtcttc aatttctcaa 5760
gtttcagttt catttttctt gttctattac aacttttttt acttcttgtt cattagaaag 5820
aaagcatagc aatctaatct aaggggcggt gttgacaatt aatcatcggc atagtatatc 5880
ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt 5940
ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 6000
ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 6060
ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 6120
cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 6180
gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 6240
cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtccga 6300
cggcggccca cgggtcccag gcctcggaga tccgtccccc ttttcctttg tcgatatcat 6360
gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa 6420
ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt 6480
attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc 6540
atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat 6600
ttgcaagctg gagaccaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 6660
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 6720
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 6780
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 6840
ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 6900
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 6960
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 7020
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 7080
gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 7140
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 7200
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 7260
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 7320
acgttaaggg attttggtca tgagatc 7347
<210> 44
<211> 1552
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
attcgctagc atggctctgt ggagaccatc tgacaacacc gtctacttgc cacctccatc 60
tgttgccaga gtcgttaaca ccgacgatta cgtcactaga acatccatct tctaccacgc 120
tggttcctct agattgctta ctgttggtaa tccatacttc agagttccag ctggtggagg 180
taataagcaa gacattccta aggtttctgc ttaccaatat agagtcttta gagttcagtt 240
gcctgaccca aacaagttcg gtttgcctga tacttctatc tacaacccag agactcaaag 300
attggtttgg gcctgtgctg gtgtcgagat cggtagagga caacctttgg gagttggtct 360
ttctggacac ccattctaca acaagttgga tgacactgaa tcttcacatg ctgccacttc 420
caatgtttct gaggatgttc gtgacaacgt ctctgttgat tacaagcaaa ctcagttgtg 480
tatccttgga tgtgcccctg ctattggtga acactgggct aaaggtactg cctgtaagtc 540
cagaccattg tctcaaggtg actgcccacc tttggagctt aagaacactg ttttggaaga 600
tggtgacatg gttgatactg gatatggtgc tatggacttc tctaccctgc aggatactaa 660
gtgtgaggtt ccattggaca tctgtcaatc tatttgtaag taccctgatt atcttcagat 720
gtctgctgac ccatacggtg attctatgtt cttttgcttg agacgtgagc aacttttcgc 780
tagacacttt tggaacagag caggtactat gggtgacacc gttccacaat ctttgtatat 840
caaaggaact ggtatgagag cttctcctgg atcctgtgtt tactcaccat ctccttccgg 900
ttctattgtt acctcagact ctcaattgtt taacaagcca tactggttac acaaggctca 960
gggtcataac aatggtgttt gctggcacaa ccaattgttc gttactgtcg ttgacaccac 1020
tagatccacc aatttgacaa tttgtgcttc tactcagtcc cctgttccag gtcaatatga 1080
tgctaccaaa ttcaagcaat actctagaca cgttgaggaa tatgacttgc agtttatctt 1140
ccaattgtgt actattacct tgactgccga tgttatgtct tacattcatt ccatgaactc 1200
ttcaatcttg gaggattgga actttggtgt tccaccacct ccaacaactt ctttggttga 1260
cacctacaga ttcgtccaat ctgttgctat tacctgtcag aaggatgccg caccagctga 1320
aaacaaggat ccatacgaca agttgaagtt ttggaatgtc gatttgaagg agaagttctc 1380
ccttgacttg gatcaatacc cacttggtag aaagttcttg gttcaggctg gtttgagacg 1440
taagccaact atcggtccaa gaaaacgttc tgctccatca gccactacct cttccaaacc 1500
tgctaagaga gttcgtgtca gagctagaaa gtaatagcgg ccgcataagg aa 1552
<210> 45
<211> 8855
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc 420
cttggccatt agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc 480
gtcgtcgtac agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat 540
ggttttggaa tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg 600
gacgtcgttg aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc 660
gatgtagaag gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag 720
ctgcacgagc cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat 780
gaggagttcg aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat 840
gaggtcgtcc actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc 900
cagcaggccc ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc 960
cactctgtag cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat 1020
tgttgccgca tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg 1080
taaaacttgt gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc 1140
gaggacgcag ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga 1200
ccttgcctaa ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca 1260
gagcagcaga gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt 1320
gcaaatgcgg gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac 1380
atccccagtt tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc 1440
aaccgtcccc acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat 1500
ctaaagtaca aaaacaaagc tagcatggct ctgtggagac catctgacaa caccgtctac 1560
ttgccacctc catctgttgc cagagtcgtt aacaccgacg attacgtcac tagaacatcc 1620
atcttctacc acgctggttc ctctagattg cttactgttg gtaatccata cttcagagtt 1680
ccagctggtg gaggtaataa gcaagacatt cctaaggttt ctgcttacca atatagagtc 1740
tttagagttc agttgcctga cccaaacaag ttcggtttgc ctgatacttc tatctacaac 1800
ccagagactc aaagattggt ttgggcctgt gctggtgtcg agatcggtag aggacaacct 1860
ttgggagttg gtctttctgg acacccattc tacaacaagt tggatgacac tgaatcttca 1920
catgctgcca cttccaatgt ttctgaggat gttcgtgaca acgtctctgt tgattacaag 1980
caaactcagt tgtgtatcct tggatgtgcc cctgctattg gtgaacactg ggctaaaggt 2040
actgcctgta agtccagacc attgtctcaa ggtgactgcc cacctttgga gcttaagaac 2100
actgttttgg aagatggtga catggttgat actggatatg gtgctatgga cttctctacc 2160
ctgcaggata ctaagtgtga ggttccattg gacatctgtc aatctatttg taagtaccct 2220
gattatcttc agatgtctgc tgacccatac ggtgattcta tgttcttttg cttgagacgt 2280
gagcaacttt tcgctagaca cttttggaac agagcaggta ctatgggtga caccgttcca 2340
caatctttgt atatcaaagg aactggtatg agagcttctc ctggatcctg tgtttactca 2400
ccatctcctt ccggttctat tgttacctca gactctcaat tgtttaacaa gccatactgg 2460
ttacacaagg ctcagggtca taacaatggt gtttgctggc acaaccaatt gttcgttact 2520
gtcgttgaca ccactagatc caccaatttg acaatttgtg cttctactca gtcccctgtt 2580
ccaggtcaat atgatgctac caaattcaag caatactcta gacacgttga ggaatatgac 2640
ttgcagttta tcttccaatt gtgtactatt accttgactg ccgatgttat gtcttacatt 2700
cattccatga actcttcaat cttggaggat tggaactttg gtgttccacc acctccaaca 2760
acttctttgg ttgacaccta cagattcgtc caatctgttg ctattacctg tcagaaggat 2820
gccgcaccag ctgaaaacaa ggatccatac gacaagttga agttttggaa tgtcgatttg 2880
aaggagaagt tctcccttga cttggatcaa tacccacttg gtagaaagtt cttggttcag 2940
gctggtttga gacgtaagcc aactatcggt ccaagaaaac gttctgctcc atcagccact 3000
acctcttcca aacctgctaa gagagttcgt gtcagagcta gaaagtaata gcggccgcca 3060
gctttctaga acaaaaactc atctcagaag aggatctgaa tagcgccgtc gaccatcatc 3120
atcatcatca ttgagtttgt agccttagac atgactgttc ctcagttcaa gttgggcact 3180
tacgagaaga ccggtcttgc tagattctaa tcaagaggat gtcagaatgc catttgcctg 3240
agagatgcag gcttcatttt tgatactttt ttatttgtaa cctatatagt ataggatttt 3300
ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga tcagcctatc tcgcagctga 3360
tgaatatctt gtggtagggg tttgggaaaa tcattcgagt ttgatgtttt tcttggtatt 3420
tcccactcct cttcagagta cagaagatta agtgagacct tcgtttgtgc ggatctgtcg 3480
acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg taatggaacc 3540
aggccgacgg cacgctcctt gcggaccacg gtggctggcg agcccagttt gtgaacgagg 3600
tcgtttagaa cgtcctgcgc aaagtccagt gtcagatgaa tgtcctcctc ggaccaattc 3660
agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg ctcctcgtta 3720
ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc tttctggtgg 3780
cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg ctcgtagtac 3840
ttgttcgcgt cgctgtagcc ggccgcggtg acgataccca catagaggtc cttggccatt 3900
agtttgatga ggtggggcag gatgggcgac tcggcatcga aatttttgcc gtcgtcgtac 3960
agtgtgatgt caccatcgaa tgtaatgagc tgcagcttgc gatctcggat ggttttggaa 4020
tggaagaacc gcgacatctc caacagctgg gccgtgttga gaatgagccg gacgtcgttg 4080
aacgaggggg ccacaagccg gcgtttgctg atggcgcggc gctcgtcctc gatgtagaag 4140
gccttttcca gaggcagtct cgtgaagaag ctgccaacgc tcggaaccag ctgcacgagc 4200
cgagacaatt cgggggtgcc ggctttggtc atttcaatgt tgtcgtcgat gaggagttcg 4260
aggtcgtgga agatttccgc gtagcggcgt tttgcctcag agtttaccat gaggtcgtcc 4320
actgcagaga tgccgttgct cttcaccgcg tacaggacga acggcgtggc cagcaggccc 4380
ttgatccatt ctatgaggcc atctcgacgg tgttccttga gtgcgtactc cactctgtag 4440
cgactggaca tctcgagact gggcttgctg tgctggatgc accaattaat tgttgccgca 4500
tgcatccttg caccgcaagt ttttaaaacc cactcgcttt agccgtcgcg taaaacttgt 4560
gaatctggca actgaggggg ttctgcagcc gcaaccgaac ttttcgcttc gaggacgcag 4620
ctggatggtg tcatgtgagg ctctgtttgc tggcgtagcc tacaacgtga ccttgcctaa 4680
ccggacggcg ctacccactg ctgtctgtgc ctgctaccag aaaatcacca gagcagcaga 4740
gggccgatgt ggcaactggt ggggtgtcgg acaggctgtt tctccacagt gcaaatgcgg 4800
gtgaaccggc cagaaagtaa attcttatgc taccgtgcag tgactccgac atccccagtt 4860
tttgccctac ttgatcacag atggggtcag cgctgccgct aagtgtaccc aaccgtcccc 4920
acacggtcca tctataaata ctgctgccag tgcacggtgg tgacatcaat ctaaagtaca 4980
aaaacaaatt cgaaacgatg tctctttggt tgccatccga ggctactgtt tacttgccac 5040
ctgtcccagt ttctaaggtc gtgtcaactg atgaatacgt tgctagaacc aacatctatt 5100
accacgctgg aacttctaga ttgcttgctg ttggtcatcc atacttccct atcaagaaac 5160
ctaacaataa caagatcctt gttccaaagg tctctggatt gcagtacaga gttttcagaa 5220
tccacttgcc tgacccaaac aagttcggtt ttcctgacac ctccttctac aacccagata 5280
ctcaaagatt ggtgtgggcc tgtgttggtg tcgaggttgg acgtggtcaa ccattgggag 5340
ttggtatttc tggacatcct ttgcttaaca agttggatga cactgaaaat gcttctgcct 5400
acgcagccaa cgctggtgtt gacaacagag agtgtatctc tatggattac aaacagaccc 5460
aattgtgtct tattggttgc aagccaccta ttggtgaaca ctggggtaag ggatctccat 5520
gtaccaacgt tgctgtcaat ccaggtgatt gtcctccatt ggagcttatc aacactgtta 5580
ttcaagatgg tgacatggtt gatactggat ttggtgctat ggacttcact accttgcaag 5640
ctaacaagtc cgaggttcca ttggacattt gtacttctat ctgcaagtat ccagactaca 5700
ttaagatggt ctctgaacca tacggtgact ccttgttttt ctaccttcgt agagagcaaa 5760
tgtttgttag acacttgttc aacagagctg gtgccgttgg tgagaatgtc ccagacgatt 5820
tgtacatcaa gggatctggt tccactgcaa acttggcctc ttccaactac ttcccaactc 5880
cttcaggttc tatggttacc tccgatgctc aaatcttcaa caagccttac tggttgcaaa 5940
gagcacaggg tcacaacaat ggaatttgtt ggggtaacca acttttcgtt actgtcgttg 6000
ataccacaag atccaccaac atgtccttgt gtgctgccat ttctacttca gaaaccacat 6060
acaagaatac taacttcaag gagtacctta gacatggtga ggaatacgac ttgcagttca 6120
tctttcaatt gtgcaagatt accttgactg ctgacgttat gacctacatc cactctatga 6180
actccactat ccttgaagat tggaacttcg gattgcaacc acctccaggt ggaaccttgg 6240
aagatactta cagatttgtc acatcccaag ccattgcttg tcagaagcac actcctccag 6300
ctcctaagga agatccactt aagaaataca ccttctggga agtcaacttg aaagagaagt 6360
tctctgctga ccttgatcag tttcctttgg gtagaaagtt cttacttcaa gctggattga 6420
aggccaaacc aaagttcacc ttgggtaaaa gaaaggctac tccaaccaca tcatctactt 6480
ccacaaccgc caaacgtaag aagagaaaac tgtaatagcc tgcaggcagc tttctagaac 6540
aaaaactcat ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt 6600
gagtttgtag ccttagacat gactgttcct cagttcaagt tgggcactta cgagaagacc 6660
ggtcttgcta gattctaatc aagaggatgt cagaatgcca tttgcctgag agatgcaggc 6720
ttcatttttg atactttttt atttgtaacc tatatagtat aggatttttt ttgtcatttt 6780
gtttcttctc gtacgagctt gctcctgatc agcctatctc gcagctgatg aatatcttgt 6840
ggtaggggtt tgggaaaatc attcgagttt gatgtttttc ttggtatttc ccactcctct 6900
tcagagtaca gaagattaag tgagaccttc gtttgtgcgg atcccccaca caccatagct 6960
tcaaaatgtt tctactcctt ttttactctt ccagattttc tcggactccg cgcatcgccg 7020
taccacttca aaacacccaa gcacagcata ctaaattttc cctctttctt cctctagggt 7080
gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa gagaccgcct cgtttctttt 7140
tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc tttttcttga aatttttttt 7200
tttagttttt ttctctttca gtgacctcca ttgatattta agttaataaa cggtcttcaa 7260
tttctcaagt ttcagtttca tttttcttgt tctattacaa ctttttttac ttcttgttca 7320
ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt tgacaattaa tcatcggcat 7380
agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca 7440
gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc 7500
ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg 7560
tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg 7620
tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact 7680
tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt 7740
tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac 7800
acgtccgacg gcggcccacg ggtcccaggc ctcggagatc cgtccccctt ttcctttgtc 7860
gatatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta 7920
accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt 7980
atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc 8040
gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag 8100
gctttaattt gcaagctgga gaccaacatg tgagcaaaag gccagcaaaa ggccaggaac 8160
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 8220
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 8280
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 8340
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat 8400
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 8460
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 8520
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 8580
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 8640
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 8700
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 8760
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 8820
gaaaactcac gttaagggat tttggtcatg agatc 8855
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