基于C2c1核酸酶的基因组编辑系统和方法

本申请为申请日为2018年11月2日、申请号为201811300251.6、发明名称为“基于C2c1核酸酶的基因组编辑系统和方法”的发明专利申请的分案申请。

技术领域

本发明涉及基因工程领域。具体而言，本发明涉及基于C2c1核酸酶的基因组编辑系统和方法。本发明还涉及可与不同C2c1核酸酶组合用于基因组编辑的人工向导RNA。

发明背景

随着CRISPR-Cas(

V-B型CRISPR-C2c1系统是一种新兴的具有前景的基因工程技术。然而，可用于哺乳动物基因组编辑的C2c1却很少，大大限制了其应用。本领域仍然需要新的可用于哺乳动物基因组编辑的基于C2c1核酸酶的基因组编辑系统。

发明简述

在一方面，本发明提供了一种用于对细胞基因组中的靶序列进行定点修饰的基因组编辑系统，其包含以下i)至v)中至少一项：

i)C2c1蛋白或其变体，和向导RNA；

ii)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和向导RNA；

iii)C2c1蛋白或其变体，和包含编码向导RNA的核苷酸序列的表达构建体；

iv)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；

v)包含编码C2c1蛋白或其变体的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；

其中所述向导RNA能够与所述C2c1蛋白或其变体形成复合物，将所述C2c1蛋白直系同源物或其变体靶向所述细胞基因组中的靶序列。

在一些实施方案中，所述C2c1蛋白是来自Alicyclobacillus acidiphilus的AaC2c1蛋白、来自Alicyclobacillus kakegawensis的AkC2c1蛋白、来自Alicyclobacillusmacrosporangiidus的AmC2c1蛋白、来自Bacillus hisashii的BhC2c1蛋白、来自Bacillus属的BsC2c1蛋白、来自Bacillus属的Bs3C2c1蛋白、来自Desulfovibrio inopinatus的DiC2c1蛋白、来自Laceyella sediminis的LsC2c1蛋白、来自Spirochaetes bacterium的SbC2c1蛋白、来自Tuberibacillus calidus的TcC2c1蛋白。例如，所述C2c1蛋白是来自Alicyclobacillus acidiphilus NBRC 100859的AaC2c1蛋白、来自Alicyclobacilluskakegawensis NBRC 103104的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidusstrain DSM 17980的AmC2c1蛋白、来自Bacillus hisashii strain C4的BhC2c1蛋白、来自Bacillus属NSP2.1的BsC2c1蛋白、来自Bacillus属V3-13 contig_40的Bs3C2c1蛋白、来自Desulfovibrio inopinatus DSM 10711的DiC2c1蛋白、来自Laceyella sediminis strainRHA1的LsC2c1蛋白、来自Spirochaetes bacterium GWB1_27_13的SbC2c1蛋白、来自Tuberibacillus calidus DSM 17572的TcC2c1蛋白。

在第二方面，本发明提供了一种对细胞基因组中的靶序列进行定点修饰的方法，包括将本发明的基因组编辑系统导入所述细胞。

在第三方面，本发明提供了一种治疗有需要的对象中的疾病的方法，包括向所述对象递送有效量的本发明的基因组编辑系统以修饰所述对象中与所述疾病相关的基因。

在第四方面，本发明提供了本发明的基因组编辑系统在制备用于治疗有需要的对象中的疾病的药物组合物中的用途，其中所述基因组编辑系统用于修饰所述对象中与所述疾病相关的基因。

在第五方面，本发明提供了用于本发明的方法的试剂盒，该试剂盒包括本发明的基因组编辑系统，以及使用说明。

在第六方面，本发明提供了一种用于治疗有需要的对象中的疾病的药物组合物，其包含本发明的基因组编辑系统和药学可接受的载体，其中所述基因组编辑系统用于修饰所述对象中与所述疾病相关的基因。

附图简述

图1.选择用于基因组编辑测试的非冗余C2c1直系同源物的系统发生树及其基因座。

(a)邻接系统发生树，显示测试的C2c1直系同源物的进化关系。(b)对应于(a)中突出显示的8种C2c1蛋白的细菌基因座图谱crRNA DR和推定的tracrRNA的模拟共折叠显示出稳定的二级结构。DR，直接重复。每个细菌基因组间隔区(spacer)的数目在其CRISPR阵列的上方或下方表示。

图2.C2c1直系同源物的蛋白质比对：测试的10种C2c1直系同源物的氨基酸序列的多序列比对。保守的残基用红色背景突出显示，保守突变用轮廓和红色字体突出显示。

图3.人293T细胞中C2c1直系同源物介导的基因组靶向。

(a)T7EI测定结果表明在人类基因组中与其同源sgRNA结合的八种C2c1蛋白的基因组靶向活性。三角形表示切割的条带。(b)T7EI测定结果表明在人293T细胞中由与其同源sgRNA(Bs3sgRNA)结合的Bs3C2c1介导的同时多重基因组靶向。(c)Sanger测序显示由与Bs3sgRNA结合的Bs3C2c1诱导的代表性插入缺失(indel)。PAM和原间隔区序列分别用红色和蓝色着色。插入缺失和插入分别用紫色破折号和绿色小写字符表示。

图4.用于RNA指导的基因组编辑的C2c1蛋白。

(a)本发明中测试的10种C2c1直系同源物的图形概述。示出其大小(氨基酸数目)。(b)T7EI测定结果表明在人293T细胞中由其同源sgRNA指导的八种C2c1直系同源物的基因组靶向活性。三角形表示切割的条带。(c-d)T7EI测定结果表明在人293T细胞中由AasgRNA(c)和AksgRNA(d)指导的八种C2c1直系同源物的基因组靶向活性。三角形表示切割的条带。

图5.C2c1的sgRNA的DNA比对：测试衍生自10个C2c1基因座的8种sgRNA的DNA序列的多序列比对。

图6.不同C2c1直系同源物与sgRNA之间的可互换性。

T7EI测定结果表明在人293T细胞中由AasgRNA(a)、AksgRNA(b)、AmsgRNA(c)、Bs3sgRNA(d)和LssgRNA(e)指导的八种C2c1直系同源物的基因组靶向活性。红色三角形表示切割的条带。

图7.人工sgRNA介导的多重基因组靶向。

(a)对应于DiC2c1和TcC2c1的细菌基因座的图谱。两个C2c1基因座没有CRISPR阵列。(b-c)T7EI测定结果表明在人293T细胞中由AasgRNA(b)和AksgRNA(c)指导的AaC2c1、DiC2c1和TcC2c1的基因组靶向活性。三角形表示切割的条带。(d)T7EI测定结果表明在人293T细胞中由与AksgRNA结合的TcC2c1介导的同时多重基因组靶向。(e)示意图说明人工sgRNA支架13(artgRNA13)的二级结构。(f)T7EI测定结果表明在人293T细胞中由与artgRNA13结合的TcC2c1介导的同时多重基因组靶向。

图8.不同sgRNA指导C2c1进行基因组编辑。

T7EI测定结果表明在人293T细胞中由AasgRNA(a)、AksgRNA(b)、AmsgRNA(c)、Bs3sgRNA(d)和LssgRNA(e)指导的AaC2c1、DiC2c1和TcC2c1的基因组靶向活性。三角形表示切割的条带。

图9.TcC2c1介导的多重基因组编辑。

(a)T7EI测定结果表明在人293T细胞中由与AmsgRNA结合的TcC2c1介导的同时多重基因组靶向。(b-c)Sanger测序显示由与AksgRNA(b)和AmsgRNA(c)结合的TcC2c1诱导的代表性插入缺失。PAM和原间隔区序列分别用红色和蓝色着色。插入缺失和插入分别用紫色破折号和绿色小写字符表示。

图10.人工sgRNA指导TcC2c1进行基因组编辑。

(a)示意图说明36种人工sgRNA(artgRNA)支架(支架：1-12和14-37)的二级结构。(b)T7EI测定结果表明在人293T细胞中artsgRNA指导的TcC2c1的基因组靶向活性。三角形表示切割的条带。(c)T7EI测定结果表明在人293T细胞中由与artgRNA13结合的AaC2c1介导的同时多重基因组靶向。

发明详述

在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。例如，本发明中使用的标准重组DNA和分子克隆技术为本领域技术人员熟知，并且在如下文献中有更全面的描述：Sambrook，J.，Fritsch，E.F.和Maniatis，T.，MolecularCloning：A Laboratory Manual；Cold Spring Harbor Laboratory Press：Cold SpringHarbor，1989(下文称为“Sambrook”)。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。

在一方面，本发明提供了一种用于对细胞基因组中的靶序列进行定点修饰的基因组编辑系统，其包含以下i)至v)中至少一项：

i)C2c1蛋白或其变体，和向导RNA；

ii)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和向导RNA；

iii)C2c1蛋白或其变体，和包含编码向导RNA的核苷酸序列的表达构建体；

iv)包含编码C2c1蛋白或其变体的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；

v)包含编码C2c1蛋白或其变体的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体；

其中所述向导RNA能够与所述C2c1蛋白或其变体形成复合物，将所述C2c1蛋白或其变体靶向所述细胞基因组中的靶序列。在一些实施方案中，所述靶向导致所述靶序列中的一或多个核苷酸的取代、缺失和/或添加。

“基因组”如本文所用不仅涵盖存在于细胞核中的染色体DNA，而且还包括存在于细胞的亚细胞组分(如线粒体、质体)中的细胞器DNA。

“C2c1核酸酶”、“C2c1蛋白”和“C2c1”在本文中可互换使用，指的是包括C2c1蛋白或其片段的RNA指导的核酸酶。C2c1具有向导RNA介导的DNA结合活性以及DNA切割活性，能在向导RNA的指导下靶向并切割DNA靶序列形成DNA双链断裂(DSB)。DSB能够激活细胞内固有的修复机制非同源末端连接(Non-homologous end joining,NHEJ)和同源重组(Homologous recombination,HR)对细胞中的DNA损伤进行修复，在修复过程中，对该特定的DNA序列进行定点编辑。

在一些实施方案中，所述C2c1蛋白是来自Alicyclobacillus acidiphilus的AaC2c1蛋白、来自Alicyclobacillus kakegawensis的AkC2c1蛋白、来自Alicyclobacillusmacrosporangiidus的AmC2c1蛋白、来自Bacillus hisashii的BhC2c1蛋白、来自Bacillus属的BsC2c1蛋白、来自Bacillus属的Bs3C2c1蛋白、来自Desulfovibrio inopinatus的DiC2c1蛋白、来自Laceyella sediminis的LsC2c1蛋白、来自Spirochaetes bacterium的SbC2c1蛋白、来自Tuberibacillus calidus的TcC2c1蛋白。

例如，所述C2c1蛋白是来自Alicyclobacillus acidiphilus NBRC 100859的AaC2c1蛋白、来自Alicyclobacillus kakegawensis NBRC 103104的AkC2c1蛋白、来自Alicyclobacillus macrosporangiidus strain DSM 17980的AmC2c1蛋白、来自Bacillushisashii strain C4的BhC2c1蛋白、来自Bacillus属NSP2.1的BsC2c1蛋白、来自Bacillus属V3-13 contig_40的Bs3C2c1蛋白、来自Desulfovibrio inopinatus DSM 10711的DiC2c1蛋白、来自Laceyella sediminis strain RHA1的LsC2c1蛋白、来自Spirochaetesbacterium GWB1_27_13的SbC2c1蛋白、来自Tuberibacillus calidus DSM 17572的TcC2c1蛋白。

在本发明一些实施方式中，所述C2c1蛋白是其天然基因座不具有CRISPR阵列的C2c1蛋白。在一些实施方式中，所述天然基因座不具有CRISPR阵列的C2c1蛋白是DiC2c1或TcC2c1蛋白。

在一些实施方案中，所述C2c1蛋白包含SEQ ID NO:1-10中任一所示的氨基酸序列。例如，所述AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白分别包含SEQ ID NO:1-10所示氨基酸序列。

在一些实施方案中，所述C2c1蛋白的变体分别包含与野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1或TcC2c1蛋白)具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列，并且分别具有野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)的基因组编辑和/靶向活性。

在一些实施方案中，所述C2c1蛋白的变体分别包含相对于野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)具有一或多个氨基酸残基取代、缺失或添加的氨基酸序列，并且分别具有野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)的基因组编辑和/或靶向活性。例如，所述C2c1蛋白的变体分别包含相对于野生型C2c1蛋白(如野生型AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白)具有1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸残基取代、缺失或添加的氨基酸序列。在一些实施方案中，所述氨基酸取代是保守型取代。

“多肽”、“肽”、和“蛋白质”在本发明中可互换使用，指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的人工化学类似物的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物。术语“多肽”、“肽”、“氨基酸序列”和“蛋白质”还可包括修饰形式，包括但不限于糖基化、脂质连接、硫酸盐化、谷氨酸残基的γ羧化、羟化和ADP-核糖基化。

序列“相同性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。(参见，例如：Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988；Biocomputing:Informatics and GenomeProjects,Smith,D.W.,ed.,Academic Press,New York,1993；Computer Analysis ofSequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,NewJersey,1994；Sequence Analysis in Molecular Biology,von Heinje,G.,AcademicPress,1987；and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,MStockton Press,New York,1991)。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J AppliedMath 48:1073(1988))。

在肽或蛋白中，合适的保守型氨基酸取代是本领域技术人员已知的，并且一般可以进行而不改变所得分子的生物活性。通常，本领域技术人员认识到多肽的非必需区中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson et al.,Molecular Biologyof the Gene,4th Edition,1987,The Benjamin/Cummings Pub.co.,p.224)。

在一些实施方案中，所述C2c1蛋白的变体包含核酸酶死亡的C2c1蛋白(dC2c1)。核酸酶死亡的C2c1蛋白指的是保留向导RNA介导的DNA结合活性但是不具备双链DNA切割活性的C2c1蛋白。在一些实施方案中，所述核酸酶死亡的C2c1蛋白涵盖C2c1切口酶，其只切割双链靶DNA的一条链。

在一些实施方案中，所述C2c1蛋白的变体是核酸酶死亡的C2c1蛋白与脱氨酶的融合蛋白。例如，所述融合蛋白中的核酸酶死亡的C2c1蛋白与脱氨酶可以通过接头例如肽接头连接。

如本发明所用，“脱氨酶”是指催化脱氨基反应的酶。在本发明一些实施方式中，所述脱氨酶指的是胞嘧啶脱氨酶，其能够接受单链DNA作为底物并能够催化胞苷或脱氧胞苷分别脱氨化为尿嘧啶或脱氧尿嘧啶。在本发明一些实施方式中，所述脱氨酶指的是腺嘌呤脱氨酶，其能够接受单链DNA作为底物并能够催化腺苷或脱氧腺苷(A)形成肌苷(I)。通过使用核酸酶死亡的C2c1蛋白与脱氨酶的融合蛋白，可以实现靶DNA序列中的碱基编辑，例如C至T的转换或A至G的转换。

在本发明的一些实施方案中，本发明的基因组编辑系统中的C2c1蛋白或其变体还可以包含核定位序列(NLS)。一般而言，所述C2c1蛋白或其变体中的一个或多个NLS应具有足够的强度，以便在细胞核中驱动所述C2c1蛋白或其变体以可实现其基因编辑功能的量积聚。一般而言，核定位活性的强度由所述C2c1蛋白或其变体中NLS的数目、位置、所使用的一个或多个特定的NLS、或这些因素的组合决定。

在本发明的一些实施方案中，本发明的基因组编辑系统中的C2c1蛋白或其变体的NLS可以位于N端和/或C端。在一些实施方案中，所述C2c1蛋白或其变体包含约1、2、3、4、5、6、7、8、9、10个或更多个NLS。在一些实施方案中，所述C2c1蛋白或其变体包含在或接近于N端的约1、2、3、4、5、6、7、8、9、10个或更多个NLS。在一些实施方案中，所述C2c1蛋白或其变体包含在或接近于C端约1、2、3、4、5、6、7、8、9、10个或更多个NLS。在一些实施方案中，所述C2c1蛋白或其变体包含这些的组合，如包含在N端的一个或多个NLS以及在C端的一个或多个NLS。当存在多于一个NLS时，每一个可以被选择为不依赖于其他NLS。在本发明的一些实施方式中，所述C2c1蛋白或其变体包含2个NLS，例如所述2个NLS分别位于N端和C端。

一般而言，NLS由暴露于蛋白表面上的带正电的赖氨酸或精氨酸的一个或多个短序列组成，但其他类型的NLS也是已知的。NLS的非限制性实例包括：KKRKV、PKKKRKV，或SGGSPKKKRKV。

此外，根据所需要编辑的DNA位置，本发明的C2c1蛋白或其变体还可以包括其他的定位序列，例如细胞质定位序列、叶绿体定位序列、线粒体定位序列等。

在本发明的一些实施方案中，所述靶序列长度为18-35个核苷酸，优选20个核苷酸。在本发明的一些实施方案中，所述靶序列在其5’端侧翼为选自：5’TTTN-3’、5’ATTN-3’、5’GTTN-3’、5’CTTN-3’、5’TTC-3’、5’TTG-3’、5’TTA-3’、5’TTT-3’、5’TAN-3’、5’TGN-3’、5’TCN-3’和5’ATC-3’的PAM(前间区邻近基序)序列，其中N选自A、G、C和T。

在本发明中，待进行修饰的靶序列可以位于基因组的任何位置，例如位于功能基因如蛋白编码基因内，或者例如可以位于基因表达调控区如启动子区或增强子区，从而实现对所述基因功能修饰或对基因表达的修饰。可以通过T7EI、PCR/RE或测序方法检测基因组靶序列中的取代、缺失和/或添加。

“向导RNA”和“gRNA”在本文中可互换使用，通常由部分互补形成复合物的crRNA和tracrRNA分子构成，其中crRNA包含与靶序列具有足够相同性以便与靶序列的互补序列杂交并且指导CRISPR复合物(C2c1+crRNA+tracrRNA)与该靶序列以序列特异性方式结合的序列。然而，可以设计并使用单向导RNA(sgRNA)，其同时包含crRNA和tracrRNA的特征。

在本发明的一些实施方案中，所述向导RNA是sgRNA。在一些具体实施方案中，所述sgRNA由选自以下之一的核酸序列编码：

5’-GTCTAAAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAACTTCTCAAAAAGAACGCTCGCTCAGTGTTCTGACGTCGGATCACTGAGCGAGCGATCTGAGAAGTGGCAC-N

5’-tcgtctataGGACGGCGAGGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGCTTCAAGATGACCGCTCGCTCAGCGATCTGACAACGGATCGCTGAGCGAGCGGTCTGAGAAGTGGCAC-N

5’-ggaattgccgatctaTAGGACGGCAGATTCAACGGGATGTGCCAATGCACTCTTTCCAGGAGTGAACACCCCGTTGGCTTCAACATGATCGCCCGCTCAACGGTCCGATGTCGGATCGTTGAGCGGGCGATCTGAGAAGTGGCAC-N

5’-GAGGTTCTGTCTTTTGGTCAGGACAACCGTCTAGCTATAAGTGCTGCAGGGTGTGAGAAACTCCTATTGCTGGACGATGTCTCTTTTATTTCTTTTTTCTTGGATGTCCAAGAAAAAAGAAATGATACGAGGCATTAGCAC-N

5’-CCATAAGTCGACTTACATATCCGTGCGTGTGCATTATGGGCCCATCCACAGGTCTATTCCCACGGATAATCACGACTTTCCACTAAGCTTTCGAATGTTCGAAAGCTTAGTGGAAAGCTTCGTGGTTAGCAC-N

5’-GGTGACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGGATTATCTTATTTCTGCTAAGTGTTTAGTTGCCTGAATACTTAGCAGAAATAATGATGATTGGCAC-N

5’-GGCAAAGAATACTGTGCGTGTGCTAAGGATGGAAAAAATCCATTCAACCACAGGATTACATTATTTATCTAATCACTTAAATCTTTAAGTGATTAGATGAATTAAATGTGATTAGCAC-N

5’-GTCTTAGGGTATATCCCAAATTTGTCTTAGTATGTGCATTGCTTACAGCGACAACTAAGGTTTGTTTATCTTTTTTTTACATTGTAAGATGTTTTACATTATAAAAAGAAGATAATCTTATTGCAC-N

其中Nx表示X个连续的核苷酸组成的核苷酸序列(spacer序列)，N各自独立地选自A、G、C和T；X为18≤X≤35的整数。优选地，X＝20。在一些实施方案中，序列Nx(spacer序列)能够与靶序列的互补序列特异性杂交。所述sgRNA中除Nx之外的序列为sgRNA的支架(scaffold)序列。在一些实施方案中，所述sgRNA包含由SEQ ID NO:31-38中任一项的核苷酸序列编码的支架序列。

本发明令人惊奇地发现，不同的C2c1系统中的C2c1蛋白以及向导RNA可以互换使用，从而使得可以人工设计通用的向导RNA。

因此在另一方面，本发明提供一种人工sgRNA，其由选自以下的核苷酸序列编码：

5’-GGTCTAAAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAACTTCAAGCGAAGTGGCAC-N

5’-GGTCTAAAGGACAGAAGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGACTTCAAGCGAAGTGGCAC-N

5’-GGTCTAAAGGACAGAAAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGACTTCAAGCGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAACTTCAAGCGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGACTTCAAGCGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGACTTCAAGCGAAGTGGCAC-N

5’-GGTGACCTATAGGGTCAATGTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAACTTCAAGCGAAGTGGCAC-N

5’-GGTGACCTATAGGGTCAATGGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGACTTCAAGCGAAGTGGCAC-N

5’-GGTGACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGACTTCAAGCGAAGTGGCAC-N

5’-GGTCTAAAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGCTTCAAAGAAGTGGCAC-N

5’-GGTCTAAAGGACAGAAGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGCTTCAAAGAAGTGGCAC-N

5’-GGTCTAAAGGACAGAAAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGGCTTCAAAGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGCTTCAAAGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGCTTCAAAGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGGCTTCAAAGAAGTGGCAC-N

5’-GGTGACCTATAGGGTCAATGTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGCTTCAAAGAAGTGGCAC-N

5’-GGTGACCTATAGGGTCAATGGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGCTTCAAAGAAGTGGCAC-N

5’-GGTGACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGGCTTCAAAGAAGTGGCAC-N

5’-GGTCTAAAGGACAGAATTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGATTATCTATGATGATTGGCAC-N

5’-GGTCTAAAGGACAGAAGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGATTATCTATGATGATTGGCAC-N

5’-GGTCTAAAGGACAGAAAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGGATTATCTATGATGATTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGATTATCTATGATGATTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGATTATCTATGATGATTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGGATTATCTATGATGATTGGCAC-N

5’-GGTGACCTATAGGGTCAATGTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGATTATCTATGATGATTGGCAC-N

5’-GGTGACCTATAGGGTCAATGGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGATTATCTATGATGATTGGCAC-N

5’-GGTGACCTATAGGGTCAATGAATCTGTGCGTGTGCCATAAGTAATTAAAAATTACCCACCACAGGATTATCTATGATGATTGGCAC-N

5’-GGTCTAAAGGACAGAACAACGGGATGTGCCAATGCACTCTTTCCAGGAGTGAACACCCCGTTGACTTCAAGCGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGCAACGGGATGTGCCAATGCACTCTTTCCAGGAGTGAACACCCCGTTGACTTCAAGCGAAGTGGCAC-N

5’-GGAATTGCCGATCTATAGGACGGCAGATTTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAACTTCAAGCGAAGTGGCAC-N

5’-GGAATTGCCGATCTATAGGACGGCAGATTGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGACTTCAAGCGAAGTGGCAC-N

5’-GGAATTGCCGATCTATAGGACGGCAGATTCAACGGGATGTGCCAATGCACTCTTTCCAGGAGTGAACACCCCGTTGACTTCAAGCGAAGTGGCAC-N

5’-GGTCTAAAGGACAGAACAACGGGATGTGCCAATGCACTCTTTCCAGGAGTGAACACCCCGTTGGCTTCAAAGAAGTGGCAC-N

5’-GGTCGTCTATAGGACGGCGAGCAACGGGATGTGCCAATGCACTCTTTCCAGGAGTGAACACCCCGTTGGCTTCAAAGAAGTGGCAC-N

5’-GGAATTGCCGATCTATAGGACGGCAGATTTTTTTCAACGGGTGTGCCAATGGCCACTTTCCAGGTGGCAAAGCCCGTTGAGCTTCAAAGAAGTGGCAC-N

5’-GGAATTGCCGATCTATAGGACGGCAGATTGACAACGGGAAGTGCCAATGTGCTCTTTCCAAGAGCAAACACCCCGTTGGCTTCAAAGAAGTGGCAC-N

5’-GGAATTGCCGATCTATAGGACGGCAGATTCAACGGGATGTGCCAATGCACTCTTTCCAGGAGTGAACACCCCGTTGGCTTCAAAGAAGTGGCAC-N

其中Nx表示X个连续的核苷酸组成的核苷酸序列(spacer序列)，N各自独立地选自A、G、C和T；X为18≤X≤35的整数。优选地，X＝20。在一些实施方案中，序列Nx(spacer序列)能够与靶序列的互补序列特异性杂交。所述sgRNA中除Nx之外的序列为sgRNA的支架(scaffold)序列。

在一些实施方案中，所述人工sgRNA包含由SEQ ID NO:39-75中任一项的核苷酸序列编码的支架序列。

在一些实施方案中，本发明的基因组编辑系统中的向导RNA是本发明的人工sgRNA。

为了在靶细胞中获得有效表达，在本发明的一些实施方式中，所述编码C2c1蛋白或其变体的核苷酸序列针对待进行基因组编辑的细胞所来自的生物体进行密码子优化。

密码子优化是指通过用在宿主细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子(例如约或多于约1、2、3、4、5、10、15、20、25、50个或更多个密码子同时维持该天然氨基酸序列而修饰核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关，而该翻译效率则被认为依赖于被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此，可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得，例如在www.kazusa.orjp/codon/上可获得的密码子使用数据库(“Codon Usage Database”)中，并且这些表可以通过不同的方式调整适用。参见，Nakamura Y.等，“Codon usage tabulatedfrom the international DNA sequence databases:status for theyear2000.Nucl.Acids Res.，28:292(2000)。

可通过本发明的系统进行基因组编辑的细胞所来自的生物体优选是真核生物，包括但不限于，哺乳动物如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅；植物包括单子叶植物和双子叶植物，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等。

在本发明的一些具体实施方式中，所述编码C2c1蛋白或其变体的核苷酸序列针对人进行密码子优化。在一些具体实施方式中，所述密码子优化的编码AaC2c1、AkC2c1、AmC2c1、BhC2c1、BsC2c1、Bs3C2c1、DiC2c1、LsC2c1、SbC2c1、TcC2c1蛋白的核苷酸序列分别选自SEQ ID NO:11-20。

根据本发明的一些实施方式，本发明所述系统的表达构建体中所述编码C2c1蛋白或其变体的核苷酸序列和/或所述编码向导RNA的核苷酸序列与表达调控元件如启动子可操作地连接。

如本发明所用，“表达构建体”是指适于感兴趣的核苷酸序列在生物体中表达的载体如重组载体。“表达”指功能产物的产生。例如，核苷酸序列的表达可指核苷酸序列的转录(如转录生成mRNA或功能RNA)和/或RNA翻译成前体或成熟蛋白质。本发明的“表达构建体”可以是线性的核酸片段、环状质粒、病毒载体，或者，在一些实施方式中，可以是能够翻译的RNA(如mRNA)。

本发明的“表达构建体”可包含不同来源的调控序列和感兴趣的核苷酸序列，或相同来源但以不同于通常天然存在的方式排列的调控序列和感兴趣的核苷酸序列。

“调控序列”和“调控元件”可互换使用，指位于编码序列的上游(5'非编码序列)、中间或下游(3'非编码序列)，并且影响相关编码序列的转录、RNA加工或稳定性或者翻译的核苷酸序列。调控序列可包括但不限于启动子、翻译前导序列、内含子和多腺苷酸化识别序列。

“启动子”指能够控制另一核酸片段转录的核酸片段。在本发明的一些实施方案中，启动子是能够控制细胞中基因转录的启动子，无论其是否来源于所述细胞。启动子可以是组成型启动子或组织特异性启动子或发育调控启动子或诱导型启动子。

“组成型启动子”指一般将引起基因在多数细胞类型中在多数情况下表达的启动子。“组织特异性启动子”和“组织优选启动子”可互换使用，并且指主要但非必须专一地在一种组织或器官中表达，而且也可在一种特定细胞或细胞型中表达的启动子。“发育调控启动子”指其活性由发育事件决定的启动子。“诱导型启动子”响应内源性或外源性刺激(环境、激素、化学信号等)而选择性表达可操纵连接的DNA序列。

如本文中所用，术语“可操作地连接”指调控元件(例如但不限于，启动子序列、转录终止序列等)与核酸序列(例如，编码序列或开放读码框)连接，使得核苷酸序列的转录被所述转录调控元件控制和调节。用于将调控元件区域可操作地连接于核酸分子的技术为本领域已知的。

本发明可使用的启动子的实例包括但不限于聚合酶(pol)I、pol II或pol III启动子。pol I启动子的实例包括鸡RNApol I启动子。pol II启动子的实例包括但不限于巨细胞病毒立即早期(CMV)启动子、劳斯肉瘤病毒长末端重复(RSV-LTR)启动子和猿猴病毒40(SV40)立即早期启动子。pol III启动子的实例包括U6和H1启动子。可以使用诱导型启动子如金属硫蛋白启动子。启动子的其他实例包括T7噬菌体启动子、T3噬菌体启动子、β-半乳糖苷酶启动子和Sp6噬菌体启动子。当用于植物时，启动子可以是花椰菜花叶病毒35S启动子、玉米Ubi-1启动子、小麦U6启动子、水稻U3启动子、玉米U3启动子、水稻肌动蛋白启动子。

可通过本发明的系统进行基因组编辑的细胞优选是真核生物细胞，包括但不限于，哺乳动物细胞如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅的细胞；植物细胞包括单子叶植物细胞和双子叶植物细胞，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等的细胞。在本发明的一些实施方案中，所述细胞是真核生物细胞，优选哺乳动物细胞，更优选是人细胞。

在另一方面，本发明提供了一种修饰细胞基因组中靶序列的方法，包括将本发明的基因组编辑系统导入所述细胞，由此所述向导RNA将所述C2c1蛋白或其变体靶向所述细胞基因组中的靶序列。在一些实施方案中，所述靶向导致所述靶序列中的一或多个核苷酸的取代、缺失和/或添加。

将本发明的基因组编辑系统的核酸分子(例如质粒、线性核酸片段、RNA等)或蛋白质“导入”细胞是指用所述核酸或蛋白质转化细胞，使得所述核酸或蛋白质在细胞中能够发挥功能。本发明所用的“转化”包括稳定转化和瞬时转化。“稳定转化”指将外源核苷酸序列导入基因组中，导致外源基因稳定遗传。一旦稳定转化，外源核酸序列稳定地整合进所述生物体和其任何连续世代的基因组中。“瞬时转化”指将核酸分子或蛋白质导入细胞中，执行功能而没有外源基因稳定遗传。瞬时转化中，外源核酸序列不整合进基因组中。

可用于将本发明的基因组编辑系统导入细胞的方法包括但不限于：磷酸钙转染、原生质融合、电穿孔、脂质体转染、微注射、病毒感染(如杆状病毒、痘苗病毒、腺病毒、腺相关病毒、慢病毒和其他病毒)、基因枪法、PEG介导的原生质体转化、土壤农杆菌介导的转化。

在一些实施方式中，所述方法在体外进行。例如，所述细胞是分离的细胞。在一些实施方式中，所述细胞是CAR-T细胞。在一些实施方式中，所述细胞是诱导的胚胎干细胞。

在另一些实施方式中，所述方法还可以在体内进行。例如，所述细胞是生物体内的细胞，可以通过例如病毒介导的方法将本发明的系统体内导入所述细胞。例如，所述细胞可以是患者体内的肿瘤细胞。

在另一方面，本发明还提一种产生经遗传修饰的细胞的方法，包括将本发明的基因组编辑系统导入细胞中，由此所述向导RNA将所述C2c1蛋白或其变体靶向所述细胞基因组中的靶序列。在一些实施方式中，所述靶向导致所述靶序列中的一或多个核苷酸取代、缺失和/或添加。

在另一方面，本发明还提供经遗传修饰的生物体，其包含通过本发明的方法产生的经遗传修饰的细胞或其后代。

如本文所用，“生物体”包括适于基因组编辑的任何生物体，优选真核生物。生物体的实例包括但不限于，哺乳动物如人、小鼠、大鼠、猴、犬、猪、羊、牛、猫；家禽如鸡、鸭、鹅；植物包括单子叶植物和双子叶植物，例如水稻、玉米、小麦、高粱、大麦、大豆、花生、拟南芥等。在本发明的一些实施方案中，所述生物体是真核生物，优选哺乳动物，更优选人。

如本文所用，“经遗传修饰的生物体”或“经遗传修饰的细胞”意指在其基因组内包含外源多核苷酸或修饰的基因或表达调控序列的生物体或细胞。例如外源多核苷酸能够稳定地整合进生物体或细胞的基因组中，并遗传连续的世代。外源多核苷酸可单独地或作为重组DNA构建体的部分整合进基因组中。修饰的基因或表达调控序列为在生物体或细胞基因组中所述序列包含单个或多个脱氧核苷酸取代、缺失和添加。针对序列而言的“外源”意指来自外来物种的序列，或者如果来自相同物种，则指通过蓄意的人为干预而从其天然形式发生了组成和/或基因座的显著改变的序列。

在另一方面，本发明提供了一种基因表达调控系统，其基于本发明的核酸酶死亡的C2c1蛋白。此系统尽管并没有改变靶基因的序列，在本文范围内也定义为基因组编辑系统。

在一些实施方案中，本发明的基因表达调控系统是基因抑制或沉默系统，其可以包含以下之一：

i)核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白，和向导RNA；

ii)包含编码核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白的核苷酸序列的表达构建体，和向导RNA；

iii)核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白，和包含编码向导RNA的核苷酸序列的表达构建体；

iv)包含编码核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；或

v)包含编码核酸酶死亡的C2c1蛋白或其与转录阻遏蛋白的融合蛋白的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体。

所述核酸酶死亡的C2c1蛋白或向导RNA的定义如上所述。所述转录阻遏蛋白的选择属于本领域技术人员的技能范围。

如本文所用，基因抑制或沉默是指基因表达水平的下调或消除，优选在转录水平。

然而，本发明的基因表达调控系统还可以使用核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白。在此种情况下，所述基因表达调控系统是基因表达激活系统。例如，本发明的基因表达激活系统可以包含以下之一：

i)核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白，和向导RNA；

ii)包含编码核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白的核苷酸序列的表达构建体，和向导RNA；

iii)核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白，和包含编码向导RNA的核苷酸序列的表达构建体；

iv)包含编码核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白的核苷酸序列的表达构建体，和包含编码向导RNA的核苷酸序列的表达构建体；或

v)包含编码核酸酶死亡的C2c1蛋白和转录激活蛋白的融合蛋白的核苷酸序列和编码向导RNA的核苷酸序列的表达构建体。

所述核酸酶死亡的C2c1蛋白或向导RNA的定义如上所述。所述转录激活蛋白的选择属于本领域技术人员的技能范围。

如本文所用，基因激活是指基因表达水平的上调，优选在转录水平。

在另一方面，本发明还涵盖本发明的基因组编辑系统在疾病治疗中的应用。

通过本发明的基因组编辑系统对疾病相关基因进行修饰，可以实现疾病相关基因的上调、下调、失活、激活或者突变纠正等，从而实现疾病的预防和/或治疗。例如，本发明中靶序列可以位于疾病相关基因的蛋白编码区内，或者例如可以位于基因表达调控区如启动子区或增强子区，从而可以实现对所述疾病相关基因功能修饰或对疾病相关基因表达的修饰。

“疾病相关”基因是指与非疾病对照的组织或细胞相比，在来源于疾病影响的组织的细胞中以异常水平或以异常形式产生转录或翻译产物的任何基因。在改变的表达与疾病的出现和/或进展相关的情况下，它可以是以异常高的水平被表达的基因；它可以是以异常低的水平被表达的基因。疾病相关基因还指具有一个或多个突变或直接负责或与一个或多个负责疾病的病因学的基因连锁不平衡的遗传变异的基因。转录的或翻译的产物可以是已知的或未知的，并且可以处于正常或异常水平。

因此，本发明还提供治疗有需要的对象中的疾病的方法，包括向所述对象递送有效量的本发明的基因组编辑系统以修饰与所述疾病相关的基因。

本发明还提供本发明的基因组编辑系统在制备用于治疗有需要的对象中的疾病的药物组合物中的用途，其中所述基因组编辑系统用于修饰与所述疾病相关的基因。

本发明还提供用于治疗有需要的对象中的疾病的药物组合物，其包含本发明的基因组编辑系统和药学可接受的载体，其中所述基因组编辑系统用于修饰与所述疾病相关的基因。

在一些实施方式中，所述对象是哺乳动物，例如人。

所述疾病的实例包括但不限于肿瘤、炎症、帕金森病、心血管疾病、阿尔茨海默病、自闭症、药物成瘾、年龄相关性黄斑变性、精神分裂症、遗传性疾病等。

在仍另一方面，本发明的范围内还包括用于本发明的方法的试剂盒，该试剂盒包括本发明的基因组编辑系统，以及使用说明。试剂盒一般包括表明试剂盒内容物的预期用途和/或使用方法的标签。术语标签包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。

实施例

为了便于理解本发明，下面将参照相关具体实施例及附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。

材料与方法

根据Molecular Cloning:A Laboratory Manual并进行一些修改进行DNA操作，包括DNA制备、消化、连接、扩增、合成、纯化、琼脂糖凝胶电泳等。简而言之，通过连接退火的寡核苷酸(表1)到BsaI消化的pUC19-U6-sgRNA(SEQ ID NO:23-30)载体中构建用于细胞转染测定的靶向sgRNA。

表1.人类基因组靶靶序列。

采用PSI-BLAST程序(Altschul,S.F.et al.Nucleic Acids Res 25,3389-3402(1997))鉴定新的CRISPR-C2c1直系同源物。其编码序列进行人源化(Grote,A.et al.,Nucleic Acids Res33,W526-531(2005))，并且使用GeneDesign程序(Richardson,S.M.etal.,Genome Res 16,550-556(2006))设计用于C2c1基因和sgRNA合成的寡核苷酸。根据文献(Li,G.et al.,Methods Mol Biol 1073,9-17(2013))合成各C2c1基因。使用

将人胚胎肾293T细胞在补充有10％胎牛血清(FBS，Gibco)和1％抗生素-抗真菌剂(Gibco)的Dulbecco's Modified Eagle培养基(DMEM，Gibco)中于37℃，5％CO

将收获的细胞直接用补充有蛋白酶K的Buffer L(Bimake)裂解，并在55℃下孵育3小时并在95℃下灭活10分钟。对每个基因的C2c1靶位点周围的基因组区域进行PCR扩增。将200-400ng PCR产物与ddH

实施例1、新C2c1蛋白鉴定

选择并从头合成来自不同细菌的六种代表性C2c1蛋白，以及之前报道的四种C2c1直系同源物，在人胚胎肾293T细胞中进行基因组编辑(图1、2和SEQ ID NO:1-10)。在这10种C2c1直系同源物中，来自D.inopinatus(DiC2c1)和T.calidus(TcC2c1)的C2c1既没有可预测的前体CRISPR RNA(pre-crRNA)也没有反式激活crRNA(tracrRNA)(图1b)，提示这两种C2c1蛋白可能不适合基因组编辑应用。

为了进行哺乳动物基因组编辑，用单独的C2c1酶和其靶向含有适当PAM的人内源基因座的同源单向导RNA(sgRNA)共转染293T细胞(图1)。T7核酸内切酶(T7EI)测定的结果显示，除了发明人先前已经鉴定的AaC2c1和AkC2c1之外，四种新的C2c1直系同源物(AmC2c1、BhC2c1、Bs3C2c1和LsC2c1)稳健地编辑人类基因组，尽管它们的靶向效率在不同的直系同源物之间和在不同的靶向位点不同(图1b和图3a)。还通过简单地使用多个sgRNA，使用Bs3C2c1实现多重基因组编辑，同时编辑人类基因组中的四个位点(图3b，c)。这些新发现的C2c1直系同源物扩展对基于C2c1的基因组编辑的选择。

实施例2、不同C2c1及双RNA的可互换性

为了研究C2c1系统中双RNA(crRNA和tracRRNA)和蛋白质组分之间的可互换性，首先分析C2c1蛋白和双RNA两者的保守性。除了C2c1直系同源物的保守氨基酸序列外(图4a和图2)，前体crRNA:tracrRNA双链体的DNA序列及其二级结构也表现出高保守性(图1b和图5)。接下来，用分别与来自8个C2c1系统的各sgRNA复合的8种C2c1直系同源物，在293T细胞中进行基因组编辑。如T7EI测定的结果所示，衍生自AaC2c1、AkC2c1、AmC2c1、Bs3C2c1和LsC2c1基因座的sgRNA可以替代原始sgRNA用于哺乳动物基因组编辑，尽管在不同C2c1直系同源物和sgRNA之间的活性有所不同(图4c，d和图6)。这些结果证明不同C2c1和来自不同C2c1基因座的双RNA之间的可互换性。

实施例3、利用天然基因座无CRISPR阵列的C2c1进行基因组编辑

本发明进一步选择两个基因座没有携带CRISPR阵列的C2c1直系同源物DiC2c1和TcC2c1进行后续实验(图7a)。基因座没有携带CRISPR阵列使得它们的crRNA:tracrRNA双链体的序列不可预测。在293T细胞中共转染与靶向不同基因组位点的衍生自其他8种C2c1直系同源物的基因座的sgRNA组合的DiC2c1和TcC2c1以及AaC2c1。T7EI测定结果表明衍生自AaC2c1、AkC2c1、AmC2c1、Bs3C2c1和LsC2c1的sgRNA使TcC2c1能够稳健地编辑人类基因组(图7b、c和图8)。此外，AasgRNA或AksgRNA能够使TcC2c1实现多重基因组编辑(图7d和图9)。上述结果表明在来自不同系统的C2c1和双链RNA之间可互换性使得可能利用天然基因座不具有CRISPR阵列的C2c1直系同源物来编辑哺乳动物基因组。

实施例4、设计用于C2c1介导的基因组编辑的人工sgRNA

不同C2c1系统中C2c1蛋白和双RNA之间的可互换性有利于设计新的人工sgRNA(artsgRNA)支架以促进C2c1介导的基因组编辑。考虑到C2c1直系同源物中DNA序列和二级结构的保守性(图1b和3)，设计并从头合成37种sgRNA支架(SEQ ID NO:39-75)，用于靶向人CCR5基因座(图7e，图10a)。T7EI测定的结果表明22种artsgRNA支架有效地工作(图10b)。为了验证artgRNA的普遍适用性，使用artsgRNA13指导TcC2c1或AaC2c1进行多重基因组编辑(图10a)。T7EI测定结果表明，artsgRNA13同时促进TcC2c1和AaC2c1两者的多重基因组编辑(图7f和图10c)。结果表明通过设计和合成artsgRNA能促进C2c1介导的基因组编辑特别是多重基因组编辑。

表2本发明涉及的序列及信息

序列表

<110> 中国科学院动物研究所

<120> 基于C2c1核酸酶的基因组编辑系统和方法

<130> TC5170

<160> 80

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1129

<212> PRT

<213> Alicyclobacillus acidiphilus

<400> 1

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Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Tyr Lys Thr

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Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn

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Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu

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Glu Glu Lys Ala Lys Ala Glu Ala Arg Lys Ser Thr Asp Arg Thr Ala

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Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg

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Val Tyr Thr Asp Ser Asp Met Ser Ser Val Gln Trp Lys Pro Leu Arg

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Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala

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Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Glu

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Ala Tyr Ala Lys Leu Val Glu Gln Lys Ser Arg Phe Glu Gln Lys Asn

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Phe Val Gly Gln Glu His Leu Val Gln Leu Val Asn Gln Leu Gln Gln

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Asp Met Lys Glu Ala Ser His Gly Leu Glu Ser Lys Glu Gln Thr Ala

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His Tyr Leu Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu

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Lys Trp Glu Lys Leu Asp Pro Asp Ala Pro Phe Asp Leu Tyr Asp Thr

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Asp Leu Phe Ala Lys Leu Ala Glu Pro Lys Tyr Gln Ala Leu Trp Arg

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Ala Ile Arg Phe Gln Lys Leu Leu Thr Val Glu Asp Gly Val Ala Lys

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Asp Leu Leu Pro Arg Asp Pro His Glu Leu Val Ala Leu Tyr Phe Gln

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Asp Tyr Gly Ala Glu Gln His Leu Ala Gly Glu Phe Gly Gly Ala Lys

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Ala Arg Asp Val Tyr Leu Asn Leu Ser Val Arg Val Gln Ser Gln Ser

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Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu

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Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp

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Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu

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Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser

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Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro

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Asn Ser Glu Gly Arg Val Pro Phe Cys Phe Pro Ile Glu Gly Asn Glu

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Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly

610 615 620

Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Arg Gln Arg

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Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val

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Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys

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Leu Ile Glu Gln Pro Met Asp Ala Asn Gln Met Thr Pro Asp Trp Arg

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Glu Ala Phe Glu Asp Glu Leu Gln Lys Leu Lys Ser Leu Tyr Gly Ile

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Cys Gly Asp Arg Glu Trp Thr Glu Ala Val Tyr Glu Ser Val Arg Arg

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Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp Val

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Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Gln Lys Asp Val Val

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Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr Lys

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Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val Ile

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Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His Ile

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Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile Ile

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Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Asp Glu Arg Gly Lys

820 825 830

Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu

835 840 845

Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn

850 855 860

Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Leu

865 870 875 880

Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala

885 890 895

Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys

900 905 910

Arg Arg Val Pro Ala Arg Cys Ala Arg Glu Gln Asn Pro Glu Pro Phe

915 920 925

Pro Trp Trp Leu Asn Lys Phe Val Ala Glu His Lys Leu Asp Gly Cys

930 935 940

Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Phe Phe

945 950 955 960

Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala

965 970 975

Asp Leu Asn Ala Ala Gln Asn Leu Gln Arg Arg Leu Trp Ser Asp Phe

980 985 990

Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly

995 1000 1005

Glu Pro Val Leu Ile Pro Arg Thr Thr Gly Lys Arg Thr Ala Asp Ser

1010 1015 1020

Tyr Gly Asn Lys Val Phe Tyr Thr Lys Thr Gly Val Thr Tyr Tyr Glu

1025 1030 1035 1040

Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln Glu Glu Leu

1045 1050 1055

Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp Glu Ala Arg Glu

1060 1065 1070

Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly Ile Ile Asn Arg Gly

1075 1080 1085

Asp Trp Thr Arg Gln Lys Glu Phe Trp Ser Met Val Asn Gln Arg Ile

1090 1095 1100

Glu Gly Tyr Leu Val Lys Gln Ile Arg Ser Arg Val Arg Leu Gln Glu

1105 1110 1115 1120

Ser Ala Cys Glu Asn Thr Gly Asp Ile

1125

<210> 2

<211> 1147

<212> PRT

<213> Alicyclobacillus kakegawensis

<400> 2

Met Ala Val Lys Ser Ile Lys Val Lys Leu Arg Leu Ser Glu Cys Pro

1 5 10 15

Asp Ile Leu Ala Gly Met Trp Gln Leu His Arg Ala Thr Asn Ala Gly

20 25 30

Val Arg Tyr Tyr Thr Glu Trp Val Ser Leu Met Arg Gln Glu Ile Leu

35 40 45

Tyr Ser Arg Gly Pro Asp Gly Gly Gln Gln Cys Tyr Met Thr Ala Glu

50 55 60

Asp Cys Gln Arg Glu Leu Leu Arg Arg Leu Arg Asn Arg Gln Leu His

65 70 75 80

Asn Gly Arg Gln Asp Gln Pro Gly Thr Asp Ala Asp Leu Leu Ala Ile

85 90 95

Ser Arg Arg Leu Tyr Glu Ile Leu Val Leu Gln Ser Ile Gly Lys Arg

100 105 110

Gly Asp Ala Gln Gln Ile Ala Ser Ser Phe Leu Ser Pro Leu Val Asp

115 120 125

Pro Asn Ser Lys Gly Gly Arg Gly Glu Ala Lys Ser Gly Arg Lys Pro

130 135 140

Ala Trp Gln Lys Met Arg Asp Gln Gly Asp Pro Arg Trp Val Ala Ala

145 150 155 160

Arg Glu Lys Tyr Glu Gln Arg Lys Ala Val Asp Pro Ser Lys Glu Ile

165 170 175

Leu Asn Ser Leu Asp Ala Leu Gly Leu Arg Pro Leu Phe Ala Val Phe

180 185 190

Thr Glu Thr Tyr Arg Ser Gly Val Asp Trp Lys Pro Leu Gly Lys Ser

195 200 205

Gln Gly Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala Leu Glu

210 215 220

Arg Leu Met Ser Trp Glu Ser Trp Asn Arg Arg Val Gly Glu Glu Tyr

225 230 235 240

Ala Arg Leu Phe Gln Gln Lys Met Lys Phe Glu Gln Glu His Phe Ala

245 250 255

Glu Gln Ser His Leu Val Lys Leu Ala Arg Ala Leu Glu Ala Asp Met

260 265 270

Arg Ala Ala Ser Gln Gly Phe Glu Ala Lys Arg Gly Thr Ala His Gln

275 280 285

Ile Thr Arg Arg Ala Leu Arg Gly Ala Asp Arg Val Phe Glu Ile Trp

290 295 300

Lys Ser Ile Pro Glu Glu Ala Leu Phe Ser Gln Tyr Asp Glu Val Ile

305 310 315 320

Arg Gln Val Gln Ala Glu Lys Arg Arg Asp Phe Gly Ser His Asp Leu

325 330 335

Phe Ala Lys Leu Ala Glu Pro Lys Tyr Gln Pro Leu Trp Arg Ala Asp

340 345 350

Glu Thr Phe Leu Thr Arg Tyr Ala Leu Tyr Asn Gly Val Leu Arg Asp

355 360 365

Leu Glu Lys Ala Arg Gln Phe Ala Thr Phe Thr Leu Pro Asp Ala Cys

370 375 380

Val Asn Pro Ile Trp Thr Arg Phe Glu Ser Ser Gln Gly Ser Asn Leu

385 390 395 400

His Lys Tyr Glu Phe Leu Phe Asp His Leu Gly Pro Gly Arg His Ala

405 410 415

Val Arg Phe Gln Arg Leu Leu Val Val Glu Ser Glu Gly Ala Lys Glu

420 425 430

Arg Asp Ser Val Val Val Pro Val Ala Pro Ser Gly Gln Leu Asp Lys

435 440 445

Leu Val Leu Arg Glu Glu Glu Lys Ser Ser Val Ala Leu His Leu His

450 455 460

Asp Thr Ala Arg Pro Asp Gly Phe Met Ala Glu Trp Ala Gly Ala Lys

465 470 475 480

Leu Gln Tyr Glu Arg Ser Thr Leu Ala Arg Lys Ala Arg Arg Asp Lys

485 490 495

Gln Gly Met Arg Ser Trp Arg Arg Gln Pro Ser Met Leu Met Ser Ala

500 505 510

Ala Gln Met Leu Glu Asp Ala Lys Gln Ala Gly Asp Val Tyr Leu Asn

515 520 525

Ile Ser Val Arg Val Lys Ser Pro Ser Glu Val Arg Gly Gln Arg Arg

530 535 540

Pro Pro Tyr Ala Ala Leu Phe Arg Ile Asp Asp Lys Gln Arg Arg Val

545 550 555 560

Thr Val Asn Tyr Asn Lys Leu Ser Ala Tyr Leu Glu Glu His Pro Asp

565 570 575

Lys Gln Ile Pro Gly Ala Pro Gly Leu Leu Ser Gly Leu Arg Val Met

580 585 590

Ser Val Asp Leu Gly Leu Arg Thr Ser Ala Ser Ile Ser Val Phe Arg

595 600 605

Val Ala Lys Lys Glu Glu Val Glu Ala Leu Gly Asp Gly Arg Pro Pro

610 615 620

His Tyr Tyr Pro Ile His Gly Thr Asp Asp Leu Val Ala Val His Glu

625 630 635 640

Arg Ser His Leu Ile Gln Met Pro Gly Glu Thr Glu Thr Lys Gln Leu

645 650 655

Arg Lys Leu Arg Glu Glu Arg Gln Ala Val Leu Arg Pro Leu Phe Ala

660 665 670

Gln Leu Ala Leu Leu Arg Leu Leu Val Arg Cys Gly Ala Ala Asp Glu

675 680 685

Arg Ile Arg Thr Arg Ser Trp Gln Arg Leu Thr Lys Gln Gly Arg Glu

690 695 700

Phe Thr Lys Arg Leu Thr Pro Ser Trp Arg Glu Ala Leu Glu Leu Glu

705 710 715 720

Leu Thr Arg Leu Glu Ala Tyr Cys Gly Arg Val Pro Asp Asp Glu Trp

725 730 735

Ser Arg Ile Val Asp Arg Thr Val Ile Ala Leu Trp Arg Arg Met Gly

740 745 750

Lys Gln Val Arg Asp Trp Arg Lys Gln Val Lys Ser Gly Ala Lys Val

755 760 765

Lys Val Lys Gly Tyr Gln Leu Asp Val Val Gly Gly Asn Ser Leu Ala

770 775 780

Gln Ile Asp Tyr Leu Glu Gln Gln Tyr Lys Phe Leu Arg Arg Trp Ser

785 790 795 800

Phe Phe Ala Arg Ala Ser Gly Leu Val Val Arg Ala Asp Arg Glu Ser

805 810 815

His Phe Ala Val Ala Leu Arg Gln His Ile Glu Asn Ala Lys Arg Asp

820 825 830

Arg Leu Lys Lys Leu Ala Asp Arg Ile Leu Met Glu Ala Leu Gly Tyr

835 840 845

Val Tyr Glu Ala Ser Gly Pro Arg Glu Gly Gln Trp Thr Ala Gln His

850 855 860

Pro Pro Cys Gln Leu Ile Ile Leu Glu Glu Leu Ser Ala Tyr Arg Phe

865 870 875 880

Ser Asp Asp Arg Pro Pro Ser Glu Asn Ser Lys Leu Met Ala Trp Gly

885 890 895

His Arg Gly Ile Leu Glu Glu Leu Val Asn Gln Ala Gln Val His Asp

900 905 910

Val Leu Val Gly Thr Val Tyr Ala Ala Phe Ser Ser Arg Phe Asp Ala

915 920 925

Arg Thr Gly Ala Pro Gly Val Arg Cys Arg Arg Val Pro Ala Arg Phe

930 935 940

Val Gly Ala Thr Val Asp Asp Ser Leu Pro Leu Trp Leu Thr Glu Phe

945 950 955 960

Leu Asp Lys His Arg Leu Asp Lys Asn Leu Leu Arg Pro Asp Asp Val

965 970 975

Ile Pro Thr Gly Glu Gly Glu Phe Leu Val Ser Pro Cys Gly Glu Glu

980 985 990

Ala Ala Arg Val Arg Gln Val His Ala Asp Ile Asn Ala Ala Gln Asn

995 1000 1005

Leu Gln Arg Arg Leu Trp Gln Asn Phe Asp Ile Thr Glu Leu Arg Leu

1010 1015 1020

Arg Cys Asp Val Lys Met Gly Gly Glu Gly Thr Val Leu Val Pro Arg

1025 1030 1035 1040

Val Asn Asn Ala Arg Ala Lys Gln Leu Phe Gly Lys Lys Val Leu Val

1045 1050 1055

Ser Gln Asp Gly Val Thr Phe Phe Glu Arg Ser Gln Thr Gly Gly Lys

1060 1065 1070

Pro His Ser Glu Lys Gln Thr Asp Leu Thr Asp Lys Glu Leu Glu Leu

1075 1080 1085

Ile Ala Glu Ala Asp Glu Ala Arg Ala Lys Ser Val Val Leu Phe Arg

1090 1095 1100

Asp Pro Ser Gly His Ile Gly Lys Gly His Trp Ile Arg Gln Arg Glu

1105 1110 1115 1120

Phe Trp Ser Leu Val Lys Gln Arg Ile Glu Ser His Thr Ala Glu Arg

1125 1130 1135

Ile Arg Val Arg Gly Val Gly Ser Ser Leu Asp

1140 1145

<210> 3

<211> 1146

<212> PRT

<213> Alicyclobacillus macrosporangiidus

<400> 3

Met Asn Val Ala Val Lys Ser Ile Lys Val Lys Leu Met Leu Gly His

1 5 10 15

Leu Pro Glu Ile Arg Glu Gly Leu Trp His Leu His Glu Ala Val Asn

20 25 30

Leu Gly Val Arg Tyr Tyr Thr Glu Trp Leu Ala Leu Leu Arg Gln Gly

35 40 45

Asn Leu Tyr Arg Arg Gly Lys Asp Gly Ala Gln Glu Cys Tyr Met Thr

50 55 60

Ala Glu Gln Cys Arg Gln Glu Leu Leu Val Arg Leu Arg Asp Arg Gln

65 70 75 80

Lys Arg Asn Gly His Thr Gly Asp Pro Gly Thr Asp Glu Glu Leu Leu

85 90 95

Gly Val Ala Arg Arg Leu Tyr Glu Leu Leu Val Pro Gln Ser Val Gly

100 105 110

Lys Lys Gly Gln Ala Gln Met Leu Ala Ser Gly Phe Leu Ser Pro Leu

115 120 125

Ala Asp Pro Lys Ser Glu Gly Gly Lys Gly Thr Ser Lys Ser Gly Arg

130 135 140

Lys Pro Ala Trp Met Gly Met Lys Glu Ala Gly Asp Ser Arg Trp Val

145 150 155 160

Glu Ala Lys Ala Arg Tyr Glu Ala Asn Lys Ala Lys Asp Pro Thr Lys

165 170 175

Gln Val Ile Ala Ser Leu Glu Met Tyr Gly Leu Arg Pro Leu Phe Asp

180 185 190

Val Phe Thr Glu Thr Tyr Lys Thr Ile Arg Trp Met Pro Leu Gly Lys

195 200 205

His Gln Gly Val Arg Ala Trp Asp Arg Asp Met Phe Gln Gln Ser Leu

210 215 220

Glu Arg Leu Met Ser Trp Glu Ser Trp Asn Glu Arg Val Gly Ala Glu

225 230 235 240

Phe Ala Arg Leu Val Asp Arg Arg Asp Arg Phe Arg Glu Lys His Phe

245 250 255

Thr Gly Gln Glu His Leu Val Ala Leu Ala Gln Arg Leu Glu Gln Glu

260 265 270

Met Lys Glu Ala Ser Pro Gly Phe Glu Ser Lys Ser Ser Gln Ala His

275 280 285

Arg Ile Thr Lys Arg Ala Leu Arg Gly Ala Asp Gly Ile Ile Asp Asp

290 295 300

Trp Leu Lys Leu Ser Glu Gly Glu Pro Val Asp Arg Phe Asp Glu Ile

305 310 315 320

Leu Arg Lys Arg Gln Ala Gln Asn Pro Arg Arg Phe Gly Ser His Asp

325 330 335

Leu Phe Leu Lys Leu Ala Glu Pro Val Phe Gln Pro Leu Trp Arg Glu

340 345 350

Asp Pro Ser Phe Leu Ser Arg Trp Ala Ser Tyr Asn Glu Val Leu Asn

355 360 365

Lys Leu Glu Asp Ala Lys Gln Phe Ala Thr Phe Thr Leu Pro Ser Pro

370 375 380

Cys Ser Asn Pro Val Trp Ala Arg Phe Glu Asn Ala Glu Gly Thr Asn

385 390 395 400

Ile Phe Lys Tyr Asp Phe Leu Phe Asp His Phe Gly Lys Gly Arg His

405 410 415

Gly Val Arg Phe Gln Arg Met Ile Val Met Arg Asp Gly Val Pro Thr

420 425 430

Glu Val Glu Gly Ile Val Val Pro Ile Ala Pro Ser Arg Gln Leu Asp

435 440 445

Ala Leu Ala Pro Asn Asp Ala Ala Ser Pro Ile Asp Val Phe Val Gly

450 455 460

Asp Pro Ala Ala Pro Gly Ala Phe Arg Gly Gln Phe Gly Gly Ala Lys

465 470 475 480

Ile Gln Tyr Arg Arg Ser Ala Leu Val Arg Lys Gly Arg Arg Glu Glu

485 490 495

Lys Ala Tyr Leu Cys Gly Phe Arg Leu Pro Ser Gln Arg Arg Thr Gly

500 505 510

Thr Pro Ala Asp Asp Ala Gly Glu Val Phe Leu Asn Leu Ser Leu Arg

515 520 525

Val Glu Ser Gln Ser Glu Gln Ala Gly Arg Arg Asn Pro Pro Tyr Ala

530 535 540

Ala Val Phe His Ile Ser Asp Gln Thr Arg Arg Val Ile Val Arg Tyr

545 550 555 560

Gly Glu Ile Glu Arg Tyr Leu Ala Glu His Pro Asp Thr Gly Ile Pro

565 570 575

Gly Ser Arg Gly Leu Thr Ser Gly Leu Arg Val Met Ser Val Asp Leu

580 585 590

Gly Leu Arg Thr Ser Ala Ala Ile Ser Val Phe Arg Val Ala His Arg

595 600 605

Asp Glu Leu Thr Pro Asp Ala His Gly Arg Gln Pro Phe Phe Phe Pro

610 615 620

Ile His Gly Met Asp His Leu Val Ala Leu His Glu Arg Ser His Leu

625 630 635 640

Ile Arg Leu Pro Gly Glu Thr Glu Ser Lys Lys Val Arg Ser Ile Arg

645 650 655

Glu Gln Arg Leu Asp Arg Leu Asn Arg Leu Arg Ser Gln Met Ala Ser

660 665 670

Leu Arg Leu Leu Val Arg Thr Gly Val Leu Asp Glu Gln Lys Arg Asp

675 680 685

Arg Asn Trp Glu Arg Leu Gln Ser Ser Met Glu Arg Gly Gly Glu Arg

690 695 700

Met Pro Ser Asp Trp Trp Asp Leu Phe Gln Ala Gln Val Arg Tyr Leu

705 710 715 720

Ala Gln His Arg Asp Ala Ser Gly Glu Ala Trp Gly Arg Met Val Gln

725 730 735

Ala Ala Val Arg Thr Leu Trp Arg Gln Leu Ala Lys Gln Val Arg Asp

740 745 750

Trp Arg Lys Glu Val Arg Arg Asn Ala Asp Lys Val Lys Ile Arg Gly

755 760 765

Ile Ala Arg Asp Val Pro Gly Gly His Ser Leu Ala Gln Leu Asp Tyr

770 775 780

Leu Glu Arg Gln Tyr Arg Phe Leu Arg Ser Trp Ser Ala Phe Ser Val

785 790 795 800

Gln Ala Gly Gln Val Val Arg Ala Glu Arg Asp Ser Arg Phe Ala Val

805 810 815

Ala Leu Arg Glu His Ile Asp Asn Gly Lys Lys Asp Arg Leu Lys Lys

820 825 830

Leu Ala Asp Arg Ile Leu Met Glu Ala Leu Gly Tyr Val Tyr Val Thr

835 840 845

Asp Gly Arg Arg Ala Gly Gln Trp Gln Ala Val Tyr Pro Pro Cys Gln

850 855 860

Leu Val Leu Leu Glu Glu Leu Ser Glu Tyr Arg Phe Ser Asn Asp Arg

865 870 875 880

Pro Pro Ser Glu Asn Ser Gln Leu Met Val Trp Ser His Arg Gly Val

885 890 895

Leu Glu Glu Leu Ile His Gln Ala Gln Val His Asp Val Leu Val Gly

900 905 910

Thr Ile Pro Ala Ala Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala

915 920 925

Pro Gly Ile Arg Cys Arg Arg Val Pro Ser Ile Pro Leu Lys Asp Ala

930 935 940

Pro Ser Ile Pro Ile Trp Leu Ser His Tyr Leu Lys Gln Thr Glu Arg

945 950 955 960

Asp Ala Ala Ala Leu Arg Pro Gly Glu Leu Ile Pro Thr Gly Asp Gly

965 970 975

Glu Phe Leu Val Thr Pro Ala Gly Arg Gly Ala Ser Gly Val Arg Val

980 985 990

Val His Ala Asp Ile Asn Ala Ala His Asn Leu Gln Arg Arg Leu Trp

995 1000 1005

Glu Asn Phe Asp Leu Ser Asp Ile Arg Val Arg Cys Asp Arg Arg Glu

1010 1015 1020

Gly Lys Asp Gly Thr Val Val Leu Ile Pro Arg Leu Thr Asn Gln Arg

1025 1030 1035 1040

Val Lys Glu Arg Tyr Ser Gly Val Ile Phe Thr Ser Glu Asp Gly Val

1045 1050 1055

Ser Phe Thr Val Gly Asp Ala Lys Thr Arg Arg Arg Ser Ser Ala Ser

1060 1065 1070

Gln Gly Glu Gly Asp Asp Leu Ser Asp Glu Glu Gln Glu Leu Leu Ala

1075 1080 1085

Glu Ala Asp Asp Ala Arg Glu Arg Ser Val Val Leu Phe Arg Asp Pro

1090 1095 1100

Ser Gly Phe Val Asn Gly Gly Arg Trp Thr Ala Gln Arg Ala Phe Trp

1105 1110 1115 1120

Gly Met Val His Asn Arg Ile Glu Thr Leu Leu Ala Glu Arg Phe Ser

1125 1130 1135

Val Ser Gly Ala Ala Glu Lys Val Arg Gly

1140 1145

<210> 4

<211> 1108

<212> PRT

<213> Bacillus hisashii

<400> 4

Met Ala Thr Arg Ser Phe Ile Leu Lys Ile Glu Pro Asn Glu Glu Val

1 5 10 15

Lys Lys Gly Leu Trp Lys Thr His Glu Val Leu Asn His Gly Ile Ala

20 25 30

Tyr Tyr Met Asn Ile Leu Lys Leu Ile Arg Gln Glu Ala Ile Tyr Glu

35 40 45

His His Glu Gln Asp Pro Lys Asn Pro Lys Lys Val Ser Lys Ala Glu

50 55 60

Ile Gln Ala Glu Leu Trp Asp Phe Val Leu Lys Met Gln Lys Cys Asn

65 70 75 80

Ser Phe Thr His Glu Val Asp Lys Asp Glu Val Phe Asn Ile Leu Arg

85 90 95

Glu Leu Tyr Glu Glu Leu Val Pro Ser Ser Val Glu Lys Lys Gly Glu

100 105 110

Ala Asn Gln Leu Ser Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn

115 120 125

Ser Gln Ser Gly Lys Gly Thr Ala Ser Ser Gly Arg Lys Pro Arg Trp

130 135 140

Tyr Asn Leu Lys Ile Ala Gly Asp Pro Ser Trp Glu Glu Glu Lys Lys

145 150 155 160

Lys Trp Glu Glu Asp Lys Lys Lys Asp Pro Leu Ala Lys Ile Leu Gly

165 170 175

Lys Leu Ala Glu Tyr Gly Leu Ile Pro Leu Phe Ile Pro Tyr Thr Asp

180 185 190

Ser Asn Glu Pro Ile Val Lys Glu Ile Lys Trp Met Glu Lys Ser Arg

195 200 205

Asn Gln Ser Val Arg Arg Leu Asp Lys Asp Met Phe Ile Gln Ala Leu

210 215 220

Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Leu Lys Val Lys Glu Glu

225 230 235 240

Tyr Glu Lys Val Glu Lys Glu Tyr Lys Thr Leu Glu Glu Arg Ile Lys

245 250 255

Glu Asp Ile Gln Ala Leu Lys Ala Leu Glu Gln Tyr Glu Lys Glu Arg

260 265 270

Gln Glu Gln Leu Leu Arg Asp Thr Leu Asn Thr Asn Glu Tyr Arg Leu

275 280 285

Ser Lys Arg Gly Leu Arg Gly Trp Arg Glu Ile Ile Gln Lys Trp Leu

290 295 300

Lys Met Asp Glu Asn Glu Pro Ser Glu Lys Tyr Leu Glu Val Phe Lys

305 310 315 320

Asp Tyr Gln Arg Lys His Pro Arg Glu Ala Gly Asp Tyr Ser Val Tyr

325 330 335

Glu Phe Leu Ser Lys Lys Glu Asn His Phe Ile Trp Arg Asn His Pro

340 345 350

Glu Tyr Pro Tyr Leu Tyr Ala Thr Phe Cys Glu Ile Asp Lys Lys Lys

355 360 365

Lys Asp Ala Lys Gln Gln Ala Thr Phe Thr Leu Ala Asp Pro Ile Asn

370 375 380

His Pro Leu Trp Val Arg Phe Glu Glu Arg Ser Gly Ser Asn Leu Asn

385 390 395 400

Lys Tyr Arg Ile Leu Thr Glu Gln Leu His Thr Glu Lys Leu Lys Lys

405 410 415

Lys Leu Thr Val Gln Leu Asp Arg Leu Ile Tyr Pro Thr Glu Ser Gly

420 425 430

Gly Trp Glu Glu Lys Gly Lys Val Asp Ile Val Leu Leu Pro Ser Arg

435 440 445

Gln Phe Tyr Asn Gln Ile Phe Leu Asp Ile Glu Glu Lys Gly Lys His

450 455 460

Ala Phe Thr Tyr Lys Asp Glu Ser Ile Lys Phe Pro Leu Lys Gly Thr

465 470 475 480

Leu Gly Gly Ala Arg Val Gln Phe Asp Arg Asp His Leu Arg Arg Tyr

485 490 495

Pro His Lys Val Glu Ser Gly Asn Val Gly Arg Ile Tyr Phe Asn Met

500 505 510

Thr Val Asn Ile Glu Pro Thr Glu Ser Pro Val Ser Lys Ser Leu Lys

515 520 525

Ile His Arg Asp Asp Phe Pro Lys Val Val Asn Phe Lys Pro Lys Glu

530 535 540

Leu Thr Glu Trp Ile Lys Asp Ser Lys Gly Lys Lys Leu Lys Ser Gly

545 550 555 560

Ile Glu Ser Leu Glu Ile Gly Leu Arg Val Met Ser Ile Asp Leu Gly

565 570 575

Gln Arg Gln Ala Ala Ala Ala Ser Ile Phe Glu Val Val Asp Gln Lys

580 585 590

Pro Asp Ile Glu Gly Lys Leu Phe Phe Pro Ile Lys Gly Thr Glu Leu

595 600 605

Tyr Ala Val His Arg Ala Ser Phe Asn Ile Lys Leu Pro Gly Glu Thr

610 615 620

Leu Val Lys Ser Arg Glu Val Leu Arg Lys Ala Arg Glu Asp Asn Leu

625 630 635 640

Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn Val Leu His Phe

645 650 655

Gln Gln Phe Glu Asp Ile Thr Glu Arg Glu Lys Arg Val Thr Lys Trp

660 665 670

Ile Ser Arg Gln Glu Asn Ser Asp Val Pro Leu Val Tyr Gln Asp Glu

675 680 685

Leu Ile Gln Ile Arg Glu Leu Met Tyr Lys Pro Tyr Lys Asp Trp Val

690 695 700

Ala Phe Leu Lys Gln Leu His Lys Arg Leu Glu Val Glu Ile Gly Lys

705 710 715 720

Glu Val Lys His Trp Arg Lys Ser Leu Ser Asp Gly Arg Lys Gly Leu

725 730 735

Tyr Gly Ile Ser Leu Lys Asn Ile Asp Glu Ile Asp Arg Thr Arg Lys

740 745 750

Phe Leu Leu Arg Trp Ser Leu Arg Pro Thr Glu Pro Gly Glu Val Arg

755 760 765

Arg Leu Glu Pro Gly Gln Arg Phe Ala Ile Asp Gln Leu Asn His Leu

770 775 780

Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile

785 790 795 800

Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln

805 810 815

Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn

820 825 830

Tyr Asn Pro Tyr Glu Glu Arg Ser Arg Phe Glu Asn Ser Lys Leu Met

835 840 845

Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu

850 855 860

Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg

865 870 875 880

Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Ser Val Val Thr

885 890 895

Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu

900 905 910

Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu

915 920 925

Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg

930 935 940

Lys Cys Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln

945 950 955 960

Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys

965 970 975

Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp

980 985 990

Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu

995 1000 1005

Lys Asp Gly Val Tyr Glu Trp Val Asn Ala Gly Lys Leu Lys Ile Lys

1010 1015 1020

Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser Asp Ile

1025 1030 1035 1040

Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly Glu Lys Leu

1045 1050 1055

Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro Ser Asp Lys Trp

1060 1065 1070

Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu Arg Ile Leu Ile Ser

1075 1080 1085

Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr Ile Glu Asp Asp Ser Ser

1090 1095 1100

Lys Gln Ser Met

1105

<210> 5

<211> 1108

<212> PRT

<213> Bacillus

<400> 5

Met Ala Ile Arg Ser Ile Lys Leu Lys Leu Lys Thr His Thr Gly Pro

1 5 10 15

Glu Ala Gln Asn Leu Arg Lys Gly Ile Trp Arg Thr His Arg Leu Leu

20 25 30

Asn Glu Gly Val Ala Tyr Tyr Met Lys Met Leu Leu Leu Phe Arg Gln

35 40 45

Glu Ser Thr Gly Glu Arg Pro Lys Glu Glu Leu Gln Glu Glu Leu Ile

50 55 60

Cys His Ile Arg Glu Gln Gln Gln Arg Asn Gln Ala Asp Lys Asn Thr

65 70 75 80

Gln Ala Leu Pro Leu Asp Lys Ala Leu Glu Ala Leu Arg Gln Leu Tyr

85 90 95

Glu Leu Leu Val Pro Ser Ser Val Gly Gln Ser Gly Asp Ala Gln Ile

100 105 110

Ile Ser Arg Lys Phe Leu Ser Pro Leu Val Asp Pro Asn Ser Glu Gly

115 120 125

Gly Lys Gly Thr Ser Lys Ala Gly Ala Lys Pro Thr Trp Gln Lys Lys

130 135 140

Lys Glu Ala Asn Asp Pro Thr Trp Glu Gln Asp Tyr Glu Lys Trp Lys

145 150 155 160

Lys Arg Arg Glu Glu Asp Pro Thr Ala Ser Val Ile Thr Thr Leu Glu

165 170 175

Glu Tyr Gly Ile Arg Pro Ile Phe Pro Leu Tyr Thr Asn Thr Val Thr

180 185 190

Asp Ile Ala Trp Leu Pro Leu Gln Ser Asn Gln Phe Val Arg Thr Trp

195 200 205

Asp Arg Asp Met Leu Gln Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu

210 215 220

Ser Trp Asn Lys Arg Val Gln Glu Glu Tyr Ala Lys Leu Lys Glu Lys

225 230 235 240

Met Ala Gln Leu Asn Glu Gln Leu Glu Gly Gly Gln Glu Trp Ile Ser

245 250 255

Leu Leu Glu Gln Tyr Glu Glu Asn Arg Glu Arg Glu Leu Arg Glu Asn

260 265 270

Met Thr Ala Ala Asn Asp Lys Tyr Arg Ile Thr Lys Arg Gln Met Lys

275 280 285

Gly Trp Asn Glu Leu Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala

290 295 300

Ser His Glu Gln Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu

305 310 315 320

Arg Gly Arg Phe Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu

325 330 335

Lys Asn Arg Leu Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe

340 345 350

Val Ala Arg Asn Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser

355 360 365

Ala Thr Met Thr Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg

370 375 380

Phe Asp Ala Arg Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu

385 390 395 400

Ala Asp Lys Pro Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile

405 410 415

Trp Pro Ser Glu Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu

420 425 430

Leu Ala Leu Ser Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn

435 440 445

Asp Lys Gly Lys Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser

450 455 460

Thr Phe Asn Gly His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly

465 470 475 480

Asp Leu Glu Lys Glu Glu Lys Asn Phe Glu Asp Gly Glu Ile Gly Ser

485 490 495

Val Tyr Leu Asn Val Val Ile Asp Phe Glu Pro Leu Gln Glu Val Lys

500 505 510

Asn Gly Arg Val Gln Ala Pro Tyr Gly Gln Val Leu Gln Leu Ile Arg

515 520 525

Arg Pro Asn Glu Phe Pro Lys Val Thr Thr Tyr Lys Ser Glu Gln Leu

530 535 540

Val Glu Trp Ile Lys Ala Ser Pro Gln His Ser Ala Gly Val Glu Ser

545 550 555 560

Leu Ala Ser Gly Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala

565 570 575

Ala Ala Ala Thr Ser Ile Phe Ser Val Glu Glu Ser Ser Asp Lys Asn

580 585 590

Ala Ala Asp Phe Ser Tyr Trp Ile Glu Gly Thr Pro Leu Val Ala Val

595 600 605

His Gln Arg Ser Tyr Met Leu Arg Leu Pro Gly Glu Gln Val Glu Lys

610 615 620

Gln Val Met Glu Lys Arg Asp Glu Arg Phe Gln Leu His Gln Arg Val

625 630 635 640

Lys Phe Gln Ile Arg Val Leu Ala Gln Ile Met Arg Met Ala Asn Lys

645 650 655

Gln Tyr Gly Asp Arg Trp Asp Glu Leu Asp Ser Leu Lys Gln Ala Val

660 665 670

Glu Gln Lys Lys Ser Pro Leu Asp Gln Thr Asp Arg Thr Phe Trp Glu

675 680 685

Gly Ile Val Cys Asp Leu Thr Lys Val Leu Pro Arg Asn Glu Ala Asp

690 695 700

Trp Glu Gln Ala Val Val Gln Ile His Arg Lys Ala Glu Glu Tyr Val

705 710 715 720

Gly Lys Ala Val Gln Ala Trp Arg Lys Arg Phe Ala Ala Asp Glu Arg

725 730 735

Lys Gly Ile Ala Gly Leu Ser Met Trp Asn Ile Glu Glu Leu Glu Gly

740 745 750

Leu Arg Lys Leu Leu Ile Ser Trp Ser Arg Arg Thr Arg Asn Pro Gln

755 760 765

Glu Val Asn Arg Phe Glu Arg Gly His Thr Ser His Gln Arg Leu Leu

770 775 780

Thr His Ile Gln Asn Val Lys Glu Asp Arg Leu Lys Gln Leu Ser His

785 790 795 800

Ala Ile Val Met Thr Ala Leu Gly Tyr Val Tyr Asp Glu Arg Lys Gln

805 810 815

Glu Trp Cys Ala Glu Tyr Pro Ala Cys Gln Val Ile Leu Phe Glu Asn

820 825 830

Leu Ser Gln Tyr Arg Ser Asn Leu Asp Arg Ser Thr Lys Glu Asn Ser

835 840 845

Thr Leu Met Lys Trp Ala His Arg Ser Ile Pro Lys Tyr Val His Met

850 855 860

Gln Ala Glu Pro Tyr Gly Ile Gln Ile Gly Asp Val Arg Ala Glu Tyr

865 870 875 880

Ser Ser Arg Phe Tyr Ala Lys Thr Gly Thr Pro Gly Ile Arg Cys Lys

885 890 895

Lys Val Arg Gly Gln Asp Leu Gln Gly Arg Arg Phe Glu Asn Leu Gln

900 905 910

Lys Arg Leu Val Asn Glu Gln Phe Leu Thr Glu Glu Gln Val Lys Gln

915 920 925

Leu Arg Pro Gly Asp Ile Val Pro Asp Asp Ser Gly Glu Leu Phe Met

930 935 940

Thr Leu Thr Asp Gly Ser Gly Ser Lys Glu Val Val Phe Leu Gln Ala

945 950 955 960

Asp Ile Asn Ala Ala His Asn Leu Gln Lys Arg Phe Trp Gln Arg Tyr

965 970 975

Asn Glu Leu Phe Lys Val Ser Cys Arg Val Ile Val Arg Asp Glu Glu

980 985 990

Glu Tyr Leu Val Pro Lys Thr Lys Ser Val Gln Ala Lys Leu Gly Lys

995 1000 1005

Gly Leu Phe Val Lys Lys Ser Asp Thr Ala Trp Lys Asp Val Tyr Val

1010 1015 1020

Trp Asp Ser Gln Ala Lys Leu Lys Gly Lys Thr Thr Phe Thr Glu Glu

1025 1030 1035 1040

Ser Glu Ser Pro Glu Gln Leu Glu Asp Phe Gln Glu Ile Ile Glu Glu

1045 1050 1055

Ala Glu Glu Ala Lys Gly Thr Tyr Arg Thr Leu Phe Arg Asp Pro Ser

1060 1065 1070

Gly Val Phe Phe Pro Glu Ser Val Trp Tyr Pro Gln Lys Asp Phe Trp

1075 1080 1085

Gly Glu Val Lys Arg Lys Leu Tyr Gly Lys Leu Arg Glu Arg Phe Leu

1090 1095 1100

Thr Lys Ala Arg

1105

<210> 6

<211> 1112

<212> PRT

<213> Bacillus

<400> 6

Met Ala Ile Arg Ser Ile Lys Leu Lys Met Lys Thr Asn Ser Gly Thr

1 5 10 15

Asp Ser Ile Tyr Leu Arg Lys Ala Leu Trp Arg Thr His Gln Leu Ile

20 25 30

Asn Glu Gly Ile Ala Tyr Tyr Met Asn Leu Leu Thr Leu Tyr Arg Gln

35 40 45

Glu Ala Ile Gly Asp Lys Thr Lys Glu Ala Tyr Gln Ala Glu Leu Ile

50 55 60

Asn Ile Ile Arg Asn Gln Gln Arg Asn Asn Gly Ser Ser Glu Glu His

65 70 75 80

Gly Ser Asp Gln Glu Ile Leu Ala Leu Leu Arg Gln Leu Tyr Glu Leu

85 90 95

Ile Ile Pro Ser Ser Ile Gly Glu Ser Gly Asp Ala Asn Gln Leu Gly

100 105 110

Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn Ser Gln Ser Gly Lys

115 120 125

Gly Thr Ser Asn Ala Gly Arg Lys Pro Arg Trp Lys Arg Leu Lys Glu

130 135 140

Glu Gly Asn Pro Asp Trp Glu Leu Glu Lys Lys Lys Asp Glu Glu Arg

145 150 155 160

Lys Ala Lys Asp Pro Thr Val Lys Ile Phe Asp Asn Leu Asn Lys Tyr

165 170 175

Gly Leu Leu Pro Leu Phe Pro Leu Phe Thr Asn Ile Gln Lys Asp Ile

180 185 190

Glu Trp Leu Pro Leu Gly Lys Arg Gln Ser Val Arg Lys Trp Asp Lys

195 200 205

Asp Met Phe Ile Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu Ser Trp

210 215 220

Asn Arg Arg Val Ala Asp Glu Tyr Lys Gln Leu Lys Glu Lys Thr Glu

225 230 235 240

Ser Tyr Tyr Lys Glu His Leu Thr Gly Gly Glu Glu Trp Ile Glu Lys

245 250 255

Ile Arg Lys Phe Glu Lys Glu Arg Asn Met Glu Leu Glu Lys Asn Ala

260 265 270

Phe Ala Pro Asn Asp Gly Tyr Phe Ile Thr Ser Arg Gln Ile Arg Gly

275 280 285

Trp Asp Arg Val Tyr Glu Lys Trp Ser Lys Leu Pro Glu Ser Ala Ser

290 295 300

Pro Glu Glu Leu Trp Lys Val Val Ala Glu Gln Gln Asn Lys Met Ser

305 310 315 320

Glu Gly Phe Gly Asp Pro Lys Val Phe Ser Phe Leu Ala Asn Arg Glu

325 330 335

Asn Arg Asp Ile Trp Arg Gly His Ser Glu Arg Ile Tyr His Ile Ala

340 345 350

Ala Tyr Asn Gly Leu Gln Lys Lys Leu Ser Arg Thr Lys Glu Gln Ala

355 360 365

Thr Phe Thr Leu Pro Asp Ala Ile Glu His Pro Leu Trp Ile Arg Tyr

370 375 380

Glu Ser Pro Gly Gly Thr Asn Leu Asn Leu Phe Lys Leu Glu Glu Lys

385 390 395 400

Gln Lys Lys Asn Tyr Tyr Val Thr Leu Ser Lys Ile Ile Trp Pro Ser

405 410 415

Glu Glu Lys Trp Ile Glu Lys Glu Asn Ile Glu Ile Pro Leu Ala Pro

420 425 430

Ser Ile Gln Phe Asn Arg Gln Ile Lys Leu Lys Gln His Val Lys Gly

435 440 445

Lys Gln Glu Ile Ser Phe Ser Asp Tyr Ser Ser Arg Ile Ser Leu Asp

450 455 460

Gly Val Leu Gly Gly Ser Arg Ile Gln Phe Asn Arg Lys Tyr Ile Lys

465 470 475 480

Asn His Lys Glu Leu Leu Gly Glu Gly Asp Ile Gly Pro Val Phe Phe

485 490 495

Asn Leu Val Val Asp Val Ala Pro Leu Gln Glu Thr Arg Asn Gly Arg

500 505 510

Leu Gln Ser Pro Ile Gly Lys Ala Leu Lys Val Ile Ser Ser Asp Phe

515 520 525

Ser Lys Val Ile Asp Tyr Lys Pro Lys Glu Leu Met Asp Trp Met Asn

530 535 540

Thr Gly Ser Ala Ser Asn Ser Phe Gly Val Ala Ser Leu Leu Glu Gly

545 550 555 560

Met Arg Val Met Ser Ile Asp Met Gly Gln Arg Thr Ser Ala Ser Val

565 570 575

Ser Ile Phe Glu Val Val Lys Glu Leu Pro Lys Asp Gln Glu Gln Lys

580 585 590

Leu Phe Tyr Ser Ile Asn Asp Thr Glu Leu Phe Ala Ile His Lys Arg

595 600 605

Ser Phe Leu Leu Asn Leu Pro Gly Glu Val Val Thr Lys Asn Asn Lys

610 615 620

Gln Gln Arg Gln Glu Arg Arg Lys Lys Arg Gln Phe Val Arg Ser Gln

625 630 635 640

Ile Arg Met Leu Ala Asn Val Leu Arg Leu Glu Thr Lys Lys Thr Pro

645 650 655

Asp Glu Arg Lys Lys Ala Ile His Lys Leu Met Glu Ile Val Gln Ser

660 665 670

Tyr Asp Ser Trp Thr Ala Ser Gln Lys Glu Val Trp Glu Lys Glu Leu

675 680 685

Asn Leu Leu Thr Asn Met Ala Ala Phe Asn Asp Glu Ile Trp Lys Glu

690 695 700

Ser Leu Val Glu Leu His His Arg Ile Glu Pro Tyr Val Gly Gln Ile

705 710 715 720

Val Ser Lys Trp Arg Lys Gly Leu Ser Glu Gly Arg Lys Asn Leu Ala

725 730 735

Gly Ile Ser Met Trp Asn Ile Asp Glu Leu Glu Asp Thr Arg Arg Leu

740 745 750

Leu Ile Ser Trp Ser Lys Arg Ser Arg Thr Pro Gly Glu Ala Asn Arg

755 760 765

Ile Glu Thr Asp Glu Pro Phe Gly Ser Ser Leu Leu Gln His Ile Gln

770 775 780

Asn Val Lys Asp Asp Arg Leu Lys Gln Met Ala Asn Leu Ile Ile Met

785 790 795 800

Thr Ala Leu Gly Phe Lys Tyr Asp Lys Glu Glu Lys Asp Arg Tyr Lys

805 810 815

Arg Trp Lys Glu Thr Tyr Pro Ala Cys Gln Ile Ile Leu Phe Glu Asn

820 825 830

Leu Asn Arg Tyr Leu Phe Asn Leu Asp Arg Ser Arg Arg Glu Asn Ser

835 840 845

Arg Leu Met Lys Trp Ala His Arg Ser Ile Pro Arg Thr Val Ser Met

850 855 860

Gln Gly Glu Met Phe Gly Leu Gln Val Gly Asp Val Arg Ser Glu Tyr

865 870 875 880

Ser Ser Arg Phe His Ala Lys Thr Gly Ala Pro Gly Ile Arg Cys His

885 890 895

Ala Leu Thr Glu Glu Asp Leu Lys Ala Gly Ser Asn Thr Leu Lys Arg

900 905 910

Leu Ile Glu Asp Gly Phe Ile Asn Glu Ser Glu Leu Ala Tyr Leu Lys

915 920 925

Lys Gly Asp Ile Ile Pro Ser Gln Gly Gly Glu Leu Phe Val Thr Leu

930 935 940

Ser Lys Arg Tyr Lys Lys Asp Ser Asp Asn Asn Glu Leu Thr Val Ile

945 950 955 960

His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Lys Arg Phe Trp Gln

965 970 975

Gln Asn Ser Glu Val Tyr Arg Val Pro Cys Gln Leu Ala Arg Met Gly

980 985 990

Glu Asp Lys Leu Tyr Ile Pro Lys Ser Gln Thr Glu Thr Ile Lys Lys

995 1000 1005

Tyr Phe Gly Lys Gly Ser Phe Val Lys Asn Asn Thr Glu Gln Glu Val

1010 1015 1020

Tyr Lys Trp Glu Lys Ser Glu Lys Met Lys Ile Lys Thr Asp Thr Thr

1025 1030 1035 1040

Phe Asp Leu Gln Asp Leu Asp Gly Phe Glu Asp Ile Ser Lys Thr Ile

1045 1050 1055

Glu Leu Ala Gln Glu Gln Gln Lys Lys Tyr Leu Thr Met Phe Arg Asp

1060 1065 1070

Pro Ser Gly Tyr Phe Phe Asn Asn Glu Thr Trp Arg Pro Gln Lys Glu

1075 1080 1085

Tyr Trp Ser Ile Val Asn Asn Ile Ile Lys Ser Cys Leu Lys Lys Lys

1090 1095 1100

Ile Leu Ser Asn Lys Val Glu Leu

1105 1110

<210> 7

<211> 1149

<212> PRT

<213> Desulfovibrio inopinatus

<400> 7

Met Pro Thr Arg Thr Ile Asn Leu Lys Leu Val Leu Gly Lys Asn Pro

1 5 10 15

Glu Asn Ala Thr Leu Arg Arg Ala Leu Phe Ser Thr His Arg Leu Val

20 25 30

Asn Gln Ala Thr Lys Arg Ile Glu Glu Phe Leu Leu Leu Cys Arg Gly

35 40 45

Glu Ala Tyr Arg Thr Val Asp Asn Glu Gly Lys Glu Ala Glu Ile Pro

50 55 60

Arg His Ala Val Gln Glu Glu Ala Leu Ala Phe Ala Lys Ala Ala Gln

65 70 75 80

Arg His Asn Gly Cys Ile Ser Thr Tyr Glu Asp Gln Glu Ile Leu Asp

85 90 95

Val Leu Arg Gln Leu Tyr Glu Arg Leu Val Pro Ser Val Asn Glu Asn

100 105 110

Asn Glu Ala Gly Asp Ala Gln Ala Ala Asn Ala Trp Val Ser Pro Leu

115 120 125

Met Ser Ala Glu Ser Glu Gly Gly Leu Ser Val Tyr Asp Lys Val Leu

130 135 140

Asp Pro Pro Pro Val Trp Met Lys Leu Lys Glu Glu Lys Ala Pro Gly

145 150 155 160

Trp Glu Ala Ala Ser Gln Ile Trp Ile Gln Ser Asp Glu Gly Gln Ser

165 170 175

Leu Leu Asn Lys Pro Gly Ser Pro Pro Arg Trp Ile Arg Lys Leu Arg

180 185 190

Ser Gly Gln Pro Trp Gln Asp Asp Phe Val Ser Asp Gln Lys Lys Lys

195 200 205

Gln Asp Glu Leu Thr Lys Gly Asn Ala Pro Leu Ile Lys Gln Leu Lys

210 215 220

Glu Met Gly Leu Leu Pro Leu Val Asn Pro Phe Phe Arg His Leu Leu

225 230 235 240

Asp Pro Glu Gly Lys Gly Val Ser Pro Trp Asp Arg Leu Ala Val Arg

245 250 255

Ala Ala Val Ala His Phe Ile Ser Trp Glu Ser Trp Asn His Arg Thr

260 265 270

Arg Ala Glu Tyr Asn Ser Leu Lys Leu Arg Arg Asp Glu Phe Glu Ala

275 280 285

Ala Ser Asp Glu Phe Lys Asp Asp Phe Thr Leu Leu Arg Gln Tyr Glu

290 295 300

Ala Lys Arg His Ser Thr Leu Lys Ser Ile Ala Leu Ala Asp Asp Ser

305 310 315 320

Asn Pro Tyr Arg Ile Gly Val Arg Ser Leu Arg Ala Trp Asn Arg Val

325 330 335

Arg Glu Glu Trp Ile Asp Lys Gly Ala Thr Glu Glu Gln Arg Val Thr

340 345 350

Ile Leu Ser Lys Leu Gln Thr Gln Leu Arg Gly Lys Phe Gly Asp Pro

355 360 365

Asp Leu Phe Asn Trp Leu Ala Gln Asp Arg His Val His Leu Trp Ser

370 375 380

Pro Arg Asp Ser Val Thr Pro Leu Val Arg Ile Asn Ala Val Asp Lys

385 390 395 400

Val Leu Arg Arg Arg Lys Pro Tyr Ala Leu Met Thr Phe Ala His Pro

405 410 415

Arg Phe His Pro Arg Trp Ile Leu Tyr Glu Ala Pro Gly Gly Ser Asn

420 425 430

Leu Arg Gln Tyr Ala Leu Asp Cys Thr Glu Asn Ala Leu His Ile Thr

435 440 445

Leu Pro Leu Leu Val Asp Asp Ala His Gly Thr Trp Ile Glu Lys Lys

450 455 460

Ile Arg Val Pro Leu Ala Pro Ser Gly Gln Ile Gln Asp Leu Thr Leu

465 470 475 480

Glu Lys Leu Glu Lys Lys Lys Asn Arg Leu Tyr Tyr Arg Ser Gly Phe

485 490 495

Gln Gln Phe Ala Gly Leu Ala Gly Gly Ala Glu Val Leu Phe His Arg

500 505 510

Pro Tyr Met Glu His Asp Glu Arg Ser Glu Glu Ser Leu Leu Glu Arg

515 520 525

Pro Gly Ala Val Trp Phe Lys Leu Thr Leu Asp Val Ala Thr Gln Ala

530 535 540

Pro Pro Asn Trp Leu Asp Gly Lys Gly Arg Val Arg Thr Pro Pro Glu

545 550 555 560

Val His His Phe Lys Thr Ala Leu Ser Asn Lys Ser Lys His Thr Arg

565 570 575

Thr Leu Gln Pro Gly Leu Arg Val Leu Ser Val Asp Leu Gly Met Arg

580 585 590

Thr Phe Ala Ser Cys Ser Val Phe Glu Leu Ile Glu Gly Lys Pro Glu

595 600 605

Thr Gly Arg Ala Phe Pro Val Ala Asp Glu Arg Ser Met Asp Ser Pro

610 615 620

Asn Lys Leu Trp Ala Lys His Glu Arg Ser Phe Lys Leu Thr Leu Pro

625 630 635 640

Gly Glu Thr Pro Ser Arg Lys Glu Glu Glu Glu Arg Ser Ile Ala Arg

645 650 655

Ala Glu Ile Tyr Ala Leu Lys Arg Asp Ile Gln Arg Leu Lys Ser Leu

660 665 670

Leu Arg Leu Gly Glu Glu Asp Asn Asp Asn Arg Arg Asp Ala Leu Leu

675 680 685

Glu Gln Phe Phe Lys Gly Trp Gly Glu Glu Asp Val Val Pro Gly Gln

690 695 700

Ala Phe Pro Arg Ser Leu Phe Gln Gly Leu Gly Ala Ala Pro Phe Arg

705 710 715 720

Ser Thr Pro Glu Leu Trp Arg Gln His Cys Gln Thr Tyr Tyr Asp Lys

725 730 735

Ala Glu Ala Cys Leu Ala Lys His Ile Ser Asp Trp Arg Lys Arg Thr

740 745 750

Arg Pro Arg Pro Thr Ser Arg Glu Met Trp Tyr Lys Thr Arg Ser Tyr

755 760 765

His Gly Gly Lys Ser Ile Trp Met Leu Glu Tyr Leu Asp Ala Val Arg

770 775 780

Lys Leu Leu Leu Ser Trp Ser Leu Arg Gly Arg Thr Tyr Gly Ala Ile

785 790 795 800

Asn Arg Gln Asp Thr Ala Arg Phe Gly Ser Leu Ala Ser Arg Leu Leu

805 810 815

His His Ile Asn Ser Leu Lys Glu Asp Arg Ile Lys Thr Gly Ala Asp

820 825 830

Ser Ile Val Gln Ala Ala Arg Gly Tyr Ile Pro Leu Pro His Gly Lys

835 840 845

Gly Trp Glu Gln Arg Tyr Glu Pro Cys Gln Leu Ile Leu Phe Glu Asp

850 855 860

Leu Ala Arg Tyr Arg Phe Arg Val Asp Arg Pro Arg Arg Glu Asn Ser

865 870 875 880

Gln Leu Met Gln Trp Asn His Arg Ala Ile Val Ala Glu Thr Thr Met

885 890 895

Gln Ala Glu Leu Tyr Gly Gln Ile Val Glu Asn Thr Ala Ala Gly Phe

900 905 910

Ser Ser Arg Phe His Ala Ala Thr Gly Ala Pro Gly Val Arg Cys Arg

915 920 925

Phe Leu Leu Glu Arg Asp Phe Asp Asn Asp Leu Pro Lys Pro Tyr Leu

930 935 940

Leu Arg Glu Leu Ser Trp Met Leu Gly Asn Thr Lys Val Glu Ser Glu

945 950 955 960

Glu Glu Lys Leu Arg Leu Leu Ser Glu Lys Ile Arg Pro Gly Ser Leu

965 970 975

Val Pro Trp Asp Gly Gly Glu Gln Phe Ala Thr Leu His Pro Lys Arg

980 985 990

Gln Thr Leu Cys Val Ile His Ala Asp Met Asn Ala Ala Gln Asn Leu

995 1000 1005

Gln Arg Arg Phe Phe Gly Arg Cys Gly Glu Ala Phe Arg Leu Val Cys

1010 1015 1020

Gln Pro His Gly Asp Asp Val Leu Arg Leu Ala Ser Thr Pro Gly Ala

1025 1030 1035 1040

Arg Leu Leu Gly Ala Leu Gln Gln Leu Glu Asn Gly Gln Gly Ala Phe

1045 1050 1055

Glu Leu Val Arg Asp Met Gly Ser Thr Ser Gln Met Asn Arg Phe Val

1060 1065 1070

Met Lys Ser Leu Gly Lys Lys Lys Ile Lys Pro Leu Gln Asp Asn Asn

1075 1080 1085

Gly Asp Asp Glu Leu Glu Asp Val Leu Ser Val Leu Pro Glu Glu Asp

1090 1095 1100

Asp Thr Gly Arg Ile Thr Val Phe Arg Asp Ser Ser Gly Ile Phe Phe

1105 1110 1115 1120

Pro Cys Asn Val Trp Ile Pro Ala Lys Gln Phe Trp Pro Ala Val Arg

1125 1130 1135

Ala Met Ile Trp Lys Val Met Ala Ser His Ser Leu Gly

1140 1145

<210> 8

<211> 1090

<212> PRT

<213> Laceyella sediminis

<400> 8

Met Ser Ile Arg Ser Phe Lys Leu Lys Ile Lys Thr Lys Ser Gly Val

1 5 10 15

Asn Ala Glu Glu Leu Arg Arg Gly Leu Trp Arg Thr His Gln Leu Ile

20 25 30

Asn Asp Gly Ile Ala Tyr Tyr Met Asn Trp Leu Val Leu Leu Arg Gln

35 40 45

Glu Asp Leu Phe Ile Arg Asn Glu Glu Thr Asn Glu Ile Glu Lys Arg

50 55 60

Ser Lys Glu Glu Ile Gln Gly Glu Leu Leu Glu Arg Val His Lys Gln

65 70 75 80

Gln Gln Arg Asn Gln Trp Ser Gly Glu Val Asp Asp Gln Thr Leu Leu

85 90 95

Gln Thr Leu Arg His Leu Tyr Glu Glu Ile Val Pro Ser Val Ile Gly

100 105 110

Lys Ser Gly Asn Ala Ser Leu Lys Ala Arg Phe Phe Leu Gly Pro Leu

115 120 125

Val Asp Pro Asn Asn Lys Thr Thr Lys Asp Val Ser Lys Ser Gly Pro

130 135 140

Thr Pro Lys Trp Lys Lys Met Lys Asp Ala Gly Asp Pro Asn Trp Val

145 150 155 160

Gln Glu Tyr Glu Lys Tyr Met Ala Glu Arg Gln Thr Leu Val Arg Leu

165 170 175

Glu Glu Met Gly Leu Ile Pro Leu Phe Pro Met Tyr Thr Asp Glu Val

180 185 190

Gly Asp Ile His Trp Leu Pro Gln Ala Ser Gly Tyr Thr Arg Thr Trp

195 200 205

Asp Arg Asp Met Phe Gln Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu

210 215 220

Ser Trp Asn Arg Arg Val Arg Glu Arg Arg Ala Gln Phe Glu Lys Lys

225 230 235 240

Thr His Asp Phe Ala Ser Arg Phe Ser Glu Ser Asp Val Gln Trp Met

245 250 255

Asn Lys Leu Arg Glu Tyr Glu Ala Gln Gln Glu Lys Ser Leu Glu Glu

260 265 270

Asn Ala Phe Ala Pro Asn Glu Pro Tyr Ala Leu Thr Lys Lys Ala Leu

275 280 285

Arg Gly Trp Glu Arg Val Tyr His Ser Trp Met Arg Leu Asp Ser Ala

290 295 300

Ala Ser Glu Glu Ala Tyr Trp Gln Glu Val Ala Thr Cys Gln Thr Ala

305 310 315 320

Met Arg Gly Glu Phe Gly Asp Pro Ala Ile Tyr Gln Phe Leu Ala Gln

325 330 335

Lys Glu Asn His Asp Ile Trp Arg Gly Tyr Pro Glu Arg Val Ile Asp

340 345 350

Phe Ala Glu Leu Asn His Leu Gln Arg Glu Leu Arg Arg Ala Lys Glu

355 360 365

Asp Ala Thr Phe Thr Leu Pro Asp Ser Val Asp His Pro Leu Trp Val

370 375 380

Arg Tyr Glu Ala Pro Gly Gly Thr Asn Ile His Gly Tyr Asp Leu Val

385 390 395 400

Gln Asp Thr Lys Arg Asn Leu Thr Leu Ile Leu Asp Lys Phe Ile Leu

405 410 415

Pro Asp Glu Asn Gly Ser Trp His Glu Val Lys Lys Val Pro Phe Ser

420 425 430

Leu Ala Lys Ser Lys Gln Phe His Arg Gln Val Trp Leu Gln Glu Glu

435 440 445

Gln Lys Gln Lys Lys Arg Glu Val Val Phe Tyr Asp Tyr Ser Thr Asn

450 455 460

Leu Pro His Leu Gly Thr Leu Ala Gly Ala Lys Leu Gln Trp Asp Arg

465 470 475 480

Asn Phe Leu Asn Lys Arg Thr Gln Gln Gln Ile Glu Glu Thr Gly Glu

485 490 495

Ile Gly Lys Val Phe Phe Asn Ile Ser Val Asp Val Arg Pro Ala Val

500 505 510

Glu Val Lys Asn Gly Arg Leu Gln Asn Gly Leu Gly Lys Ala Leu Thr

515 520 525

Val Leu Thr His Pro Asp Gly Thr Lys Ile Val Thr Gly Trp Lys Ala

530 535 540

Glu Gln Leu Glu Lys Trp Val Gly Glu Ser Gly Arg Val Ser Ser Leu

545 550 555 560

Gly Leu Asp Ser Leu Ser Glu Gly Leu Arg Val Met Ser Ile Asp Leu

565 570 575

Gly Gln Arg Thr Ser Ala Thr Val Ser Val Phe Glu Ile Thr Lys Glu

580 585 590

Ala Pro Asp Asn Pro Tyr Lys Phe Phe Tyr Gln Leu Glu Gly Thr Glu

595 600 605

Leu Phe Ala Val His Gln Arg Ser Phe Leu Leu Ala Leu Pro Gly Glu

610 615 620

Asn Pro Pro Gln Lys Ile Lys Gln Met Arg Glu Ile Arg Trp Lys Glu

625 630 635 640

Arg Asn Arg Ile Lys Gln Gln Val Asp Gln Leu Ser Ala Ile Leu Arg

645 650 655

Leu His Lys Lys Val Asn Glu Asp Glu Arg Ile Gln Ala Ile Asp Lys

660 665 670

Leu Leu Gln Lys Val Ala Ser Trp Gln Leu Asn Glu Glu Ile Ala Thr

675 680 685

Ala Trp Asn Gln Ala Leu Ser Gln Leu Tyr Ser Lys Ala Lys Glu Asn

690 695 700

Asp Leu Gln Trp Asn Gln Ala Ile Lys Asn Ala His His Gln Leu Glu

705 710 715 720

Pro Val Val Gly Lys Gln Ile Ser Leu Trp Arg Lys Asp Leu Ser Thr

725 730 735

Gly Arg Gln Gly Ile Ala Gly Leu Ser Leu Trp Ser Ile Glu Glu Leu

740 745 750

Glu Ala Thr Lys Lys Leu Leu Thr Arg Trp Ser Lys Arg Ser Arg Glu

755 760 765

Pro Gly Val Val Lys Arg Ile Glu Arg Phe Glu Thr Phe Ala Lys Gln

770 775 780

Ile Gln His His Ile Asn Gln Val Lys Glu Asn Arg Leu Lys Gln Leu

785 790 795 800

Ala Asn Leu Ile Val Met Thr Ala Leu Gly Tyr Lys Tyr Asp Gln Glu

805 810 815

Gln Lys Lys Trp Ile Glu Val Tyr Pro Ala Cys Gln Val Val Leu Phe

820 825 830

Glu Asn Leu Arg Ser Tyr Arg Phe Ser Tyr Glu Arg Ser Arg Arg Glu

835 840 845

Asn Lys Lys Leu Met Glu Trp Ser His Arg Ser Ile Pro Lys Leu Val

850 855 860

Gln Met Gln Gly Glu Leu Phe Gly Leu Gln Val Ala Asp Val Tyr Ala

865 870 875 880

Ala Tyr Ser Ser Arg Tyr His Gly Arg Thr Gly Ala Pro Gly Ile Arg

885 890 895

Cys His Ala Leu Thr Glu Ala Asp Leu Arg Asn Glu Thr Asn Ile Ile

900 905 910

His Glu Leu Ile Glu Ala Gly Phe Ile Lys Glu Glu His Arg Pro Tyr

915 920 925

Leu Gln Gln Gly Asp Leu Val Pro Trp Ser Gly Gly Glu Leu Phe Ala

930 935 940

Thr Leu Gln Lys Pro Tyr Asp Asn Pro Arg Ile Leu Thr Leu His Ala

945 950 955 960

Asp Ile Asn Ala Ala Gln Asn Ile Gln Lys Arg Phe Trp His Pro Ser

965 970 975

Met Trp Phe Arg Val Asn Cys Glu Ser Val Met Glu Gly Glu Ile Val

980 985 990

Thr Tyr Val Pro Lys Asn Lys Thr Val His Lys Lys Gln Gly Lys Thr

995 1000 1005

Phe Arg Phe Val Lys Val Glu Gly Ser Asp Val Tyr Glu Trp Ala Lys

1010 1015 1020

Trp Ser Lys Asn Arg Asn Lys Asn Thr Phe Ser Ser Ile Thr Glu Arg

1025 1030 1035 1040

Lys Pro Pro Ser Ser Met Ile Leu Phe Arg Asp Pro Ser Gly Thr Phe

1045 1050 1055

Phe Lys Glu Gln Glu Trp Val Glu Gln Lys Thr Phe Trp Gly Lys Val

1060 1065 1070

Gln Ser Met Ile Gln Ala Tyr Met Lys Lys Thr Ile Val Gln Arg Met

1075 1080 1085

Glu Glu

1090

<210> 9

<211> 1119

<212> PRT

<213> Spirochaetes

<400> 9

Met Ser Phe Thr Ile Ser Tyr Pro Phe Lys Leu Ile Ile Lys Asn Lys

1 5 10 15

Asp Glu Ala Lys Ala Leu Leu Asp Thr His Gln Tyr Met Asn Glu Gly

20 25 30

Val Lys Tyr Tyr Leu Glu Lys Leu Leu Met Phe Arg Gln Glu Lys Ile

35 40 45

Phe Ile Gly Glu Asp Glu Thr Gly Lys Arg Ile Tyr Ile Glu Glu Thr

50 55 60

Glu Tyr Lys Lys Gln Ile Glu Glu Phe Tyr Leu Ile Lys Lys Thr Glu

65 70 75 80

Leu Gly Arg Asn Leu Thr Leu Thr Leu Asp Glu Phe Lys Thr Leu Met

85 90 95

Arg Glu Leu Tyr Ile Cys Leu Val Ser Ser Ser Met Glu Asn Lys Lys

100 105 110

Gly Phe Pro Asn Ala Gln Gln Ala Ser Leu Asn Ile Phe Ser Pro Leu

115 120 125

Phe Asp Ala Glu Ser Lys Gly Tyr Ile Leu Lys Glu Glu Asn Asn Asn

130 135 140

Ile Ser Leu Ile His Lys Asp Tyr Gly Lys Ile Leu Leu Lys Arg Leu

145 150 155 160

Arg Asp Asn Asn Leu Ile Pro Ile Phe Thr Lys Phe Thr Asp Ile Lys

165 170 175

Lys Ile Thr Ala Lys Leu Ser Pro Thr Ala Leu Asp Arg Met Ile Phe

180 185 190

Ala Gln Ala Ile Glu Lys Leu Leu Ser Tyr Glu Ser Trp Cys Lys Leu

195 200 205

Met Ile Lys Glu Arg Phe Asp Lys Glu Val Lys Ile Lys Glu Leu Glu

210 215 220

Asn Lys Cys Glu Asn Lys Gln Glu Arg Asp Lys Ile Phe Glu Ile Leu

225 230 235 240

Glu Lys Tyr Glu Glu Glu Arg Gln Lys Thr Phe Glu Gln Asp Ser Gly

245 250 255

Phe Ala Lys Lys Gly Lys Phe Tyr Ile Thr Gly Arg Met Leu Lys Gly

260 265 270

Phe Asp Glu Ile Lys Glu Lys Trp Leu Lys Glu Lys Asp Arg Ser Glu

275 280 285

Gln Asn Leu Ile Asn Ile Leu Asn Lys Tyr Gln Thr Asp Asn Ser Lys

290 295 300

Leu Val Gly Asp Arg Asn Leu Phe Glu Phe Ile Ile Lys Leu Glu Asn

305 310 315 320

Gln Cys Leu Trp Asn Gly Asp Ile Asp Tyr Leu Lys Ile Lys Arg Asp

325 330 335

Ile Asn Lys Asn Gln Ile Trp Leu Asp Arg Pro Glu Met Pro Arg Phe

340 345 350

Thr Met Pro Asp Phe Lys Lys His Pro Leu Trp Tyr Arg Tyr Glu Asp

355 360 365

Pro Ser Asn Ser Asn Phe Arg Asn Tyr Lys Ile Glu Val Val Lys Asp

370 375 380

Glu Asn Tyr Ile Thr Ile Pro Leu Ile Thr Glu Arg Asn Asn Glu Tyr

385 390 395 400

Phe Glu Glu Asn Tyr Thr Phe Asn Leu Ala Lys Leu Lys Lys Leu Ser

405 410 415

Glu Asn Ile Thr Phe Ile Pro Lys Ser Lys Asn Lys Glu Phe Glu Phe

420 425 430

Ile Asp Ser Asn Asp Glu Glu Glu Asp Lys Lys Asp Gln Lys Lys Ser

435 440 445

Lys Gln Tyr Ile Lys Tyr Cys Asp Thr Ala Lys Asn Thr Ser Tyr Gly

450 455 460

Lys Ser Gly Gly Ile Arg Leu Tyr Phe Asn Arg Asn Glu Leu Glu Asn

465 470 475 480

Tyr Lys Asp Gly Lys Lys Met Asp Ser Tyr Thr Val Phe Thr Leu Ser

485 490 495

Ile Arg Asp Tyr Lys Ser Leu Phe Ala Lys Glu Lys Leu Gln Pro Gln

500 505 510

Ile Phe Asn Thr Val Asp Asn Lys Ile Thr Ser Leu Lys Ile Gln Lys

515 520 525

Lys Phe Gly Asn Glu Glu Gln Thr Asn Phe Leu Ser Tyr Phe Thr Gln

530 535 540

Asn Gln Ile Thr Lys Lys Asp Trp Met Asp Glu Lys Thr Phe Gln Asn

545 550 555 560

Val Lys Glu Leu Asn Glu Gly Ile Arg Val Leu Ser Val Asp Leu Gly

565 570 575

Gln Arg Phe Phe Ala Ala Val Ser Cys Phe Glu Ile Met Ser Glu Ile

580 585 590

Asp Asn Asn Lys Leu Phe Phe Asn Leu Asn Asp Gln Asn His Lys Ile

595 600 605

Ile Arg Ile Asn Asp Lys Asn Tyr Tyr Ala Lys His Ile Tyr Ser Lys

610 615 620

Thr Ile Lys Leu Ser Gly Glu Asp Asp Asp Leu Tyr Lys Glu Arg Lys

625 630 635 640

Ile Asn Lys Asn Tyr Lys Leu Ser Tyr Gln Glu Arg Lys Asn Lys Ile

645 650 655

Gly Ile Phe Thr Arg Gln Ile Asn Lys Leu Asn Gln Leu Leu Lys Ile

660 665 670

Ile Arg Asn Asp Glu Ile Asp Lys Glu Lys Phe Lys Glu Leu Ile Glu

675 680 685

Thr Thr Lys Arg Tyr Val Lys Asn Thr Tyr Asn Asp Gly Ile Ile Asp

690 695 700

Trp Asn Asn Val Asp Asn Lys Ile Leu Ser Tyr Glu Asn Lys Glu Asp

705 710 715 720

Val Ile Asn Leu His Lys Glu Leu Asp Lys Lys Leu Glu Ile Asp Phe

725 730 735

Lys Glu Phe Ile Arg Glu Cys Arg Lys Pro Ile Phe Arg Ser Gly Gly

740 745 750

Leu Ser Met Gln Arg Ile Asp Phe Leu Glu Lys Leu Asn Lys Leu Lys

755 760 765

Arg Lys Trp Val Ala Arg Thr Gln Lys Ser Ala Glu Ser Ile Val Leu

770 775 780

Thr Pro Lys Phe Gly Tyr Lys Leu Lys Glu His Ile Asn Glu Leu Lys

785 790 795 800

Asp Asn Arg Val Lys Gln Gly Val Asn Tyr Ile Leu Met Thr Ala Leu

805 810 815

Gly Tyr Ile Lys Asp Asn Glu Ile Lys Asn Asp Ser Lys Lys Lys Gln

820 825 830

Lys Glu Asp Trp Val Lys Lys Asn Arg Ala Cys Gln Ile Ile Leu Met

835 840 845

Glu Lys Leu Thr Glu Tyr Thr Phe Ala Glu Asp Arg Pro Arg Glu Glu

850 855 860

Asn Ser Lys Leu Arg Met Trp Ser His Arg Gln Ile Phe Asn Phe Leu

865 870 875 880

Gln Gln Lys Ala Ser Leu Trp Gly Ile Leu Val Gly Asp Val Phe Ala

885 890 895

Pro Tyr Thr Ser Lys Cys Leu Ser Asp Asn Asn Ala Pro Gly Ile Arg

900 905 910

Cys His Gln Val Thr Lys Lys Asp Leu Ile Asp Asn Ser Trp Phe Leu

915 920 925

Lys Ile Val Val Lys Asp Asp Ala Phe Cys Asp Leu Ile Glu Ile Asn

930 935 940

Lys Glu Asn Val Lys Asn Lys Ser Ile Lys Ile Asn Asp Ile Leu Pro

945 950 955 960

Leu Arg Gly Gly Glu Leu Phe Ala Ser Ile Lys Asp Gly Lys Leu His

965 970 975

Ile Val Gln Ala Asp Ile Asn Ala Ser Arg Asn Ile Ala Lys Arg Phe

980 985 990

Leu Ser Gln Ile Asn Pro Phe Arg Val Val Leu Lys Lys Asp Lys Asp

995 1000 1005

Glu Thr Phe His Leu Lys Asn Glu Pro Asn Tyr Leu Lys Asn Tyr Tyr

1010 1015 1020

Ser Ile Leu Asn Phe Val Pro Thr Asn Glu Glu Leu Thr Phe Phe Lys

1025 1030 1035 1040

Val Glu Glu Asn Lys Asp Ile Lys Pro Thr Lys Arg Ile Lys Met Asp

1045 1050 1055

Lys His Glu Lys Glu Ser Thr Asp Glu Gly Asp Asp Tyr Ser Lys Asn

1060 1065 1070

Gln Ile Ala Leu Phe Arg Asp Asp Ser Gly Ile Phe Phe Asp Lys Ser

1075 1080 1085

Leu Trp Val Asp Gly Lys Ile Phe Trp Ser Val Val Lys Asn Lys Met

1090 1095 1100

Thr Lys Leu Leu Arg Glu Arg Asn Asn Lys Lys Asn Gly Ser Lys

1105 1110 1115

<210> 10

<211> 1142

<212> PRT

<213> Tuberibacillus calidus

<400> 10

Met Asn Ile His Leu Lys Glu Leu Ile Arg Met Ala Thr Lys Ser Phe

1 5 10 15

Ile Leu Lys Met Lys Thr Lys Asn Asn Pro Gln Leu Arg Leu Ser Leu

20 25 30

Trp Lys Thr His Glu Leu Phe Asn Phe Gly Val Ala Tyr Tyr Met Asp

35 40 45

Leu Leu Ser Leu Phe Arg Gln Lys Asp Leu Tyr Met His Asn Asp Glu

50 55 60

Asp Pro Asp His Pro Val Val Leu Lys Lys Glu Glu Ile Gln Glu Arg

65 70 75 80

Leu Trp Met Lys Val Arg Glu Thr Gln Gln Lys Asn Gly Phe His Gly

85 90 95

Glu Val Ser Lys Asp Glu Val Leu Glu Thr Leu Arg Ala Leu Tyr Glu

100 105 110

Glu Leu Val Pro Ser Ala Val Gly Lys Ser Gly Glu Ala Asn Gln Ile

115 120 125

Ser Asn Lys Tyr Leu Tyr Pro Leu Thr Asp Pro Ala Ser Gln Ser Gly

130 135 140

Lys Gly Thr Ala Asn Ser Gly Arg Lys Pro Arg Trp Lys Lys Leu Lys

145 150 155 160

Glu Ala Gly Asp Pro Ser Trp Lys Asp Ala Tyr Glu Lys Trp Glu Lys

165 170 175

Glu Arg Gln Glu Asp Pro Lys Leu Lys Ile Leu Ala Ala Leu Gln Ser

180 185 190

Phe Gly Leu Ile Pro Leu Phe Arg Pro Phe Thr Glu Asn Asp His Lys

195 200 205

Ala Val Ile Ser Val Lys Trp Met Pro Lys Ser Lys Asn Gln Ser Val

210 215 220

Arg Lys Phe Asp Lys Asp Met Phe Asn Gln Ala Ile Glu Arg Phe Leu

225 230 235 240

Ser Trp Glu Ser Trp Asn Glu Lys Val Ala Glu Asp Tyr Glu Lys Thr

245 250 255

Val Ser Ile Tyr Glu Ser Leu Gln Lys Glu Leu Lys Gly Ile Ser Thr

260 265 270

Lys Ala Phe Glu Ile Met Glu Arg Val Glu Lys Ala Tyr Glu Ala His

275 280 285

Leu Arg Glu Ile Thr Phe Ser Asn Ser Thr Tyr Arg Ile Gly Asn Arg

290 295 300

Ala Ile Arg Gly Trp Thr Glu Ile Val Lys Lys Trp Met Lys Leu Asp

305 310 315 320

Pro Ser Ala Pro Gln Gly Asn Tyr Leu Asp Val Val Lys Asp Tyr Gln

325 330 335

Arg Arg His Pro Arg Glu Ser Gly Asp Phe Lys Leu Phe Glu Leu Leu

340 345 350

Ser Arg Pro Glu Asn Gln Ala Ala Trp Arg Glu Tyr Pro Glu Phe Leu

355 360 365

Pro Leu Tyr Val Lys Tyr Arg His Ala Glu Gln Arg Met Lys Thr Ala

370 375 380

Lys Lys Gln Ala Thr Phe Thr Leu Cys Asp Pro Ile Arg His Pro Leu

385 390 395 400

Trp Val Arg Tyr Glu Glu Arg Ser Gly Thr Asn Leu Asn Lys Tyr Arg

405 410 415

Leu Ile Met Asn Glu Lys Glu Lys Val Val Gln Phe Asp Arg Leu Ile

420 425 430

Cys Leu Asn Ala Asp Gly His Tyr Glu Glu Gln Glu Asp Val Thr Val

435 440 445

Pro Leu Ala Pro Ser Gln Gln Phe Asp Asp Gln Ile Lys Phe Ser Ser

450 455 460

Glu Asp Thr Gly Lys Gly Lys His Asn Phe Ser Tyr Tyr His Lys Gly

465 470 475 480

Ile Asn Tyr Glu Leu Lys Gly Thr Leu Gly Gly Ala Arg Ile Gln Phe

485 490 495

Asp Arg Glu His Leu Leu Arg Arg Gln Gly Val Lys Ala Gly Asn Val

500 505 510

Gly Arg Ile Phe Leu Asn Val Thr Leu Asn Ile Glu Pro Met Gln Pro

515 520 525

Phe Ser Arg Ser Gly Asn Leu Gln Thr Ser Val Gly Lys Ala Leu Lys

530 535 540

Val Tyr Val Asp Gly Tyr Pro Lys Val Val Asn Phe Lys Pro Lys Glu

545 550 555 560

Leu Thr Glu His Ile Lys Glu Ser Glu Lys Asn Thr Leu Thr Leu Gly

565 570 575

Val Glu Ser Leu Pro Thr Gly Leu Arg Val Met Ser Val Asp Leu Gly

580 585 590

Gln Arg Gln Ala Ala Ala Ile Ser Ile Phe Glu Val Val Ser Glu Lys

595 600 605

Pro Asp Asp Asn Lys Leu Phe Tyr Pro Val Lys Asp Thr Asp Leu Phe

610 615 620

Ala Val His Arg Thr Ser Phe Asn Ile Lys Leu Pro Gly Glu Lys Arg

625 630 635 640

Thr Glu Arg Arg Met Leu Glu Gln Gln Lys Arg Asp Gln Ala Ile Arg

645 650 655

Asp Leu Ser Arg Lys Leu Lys Phe Leu Lys Asn Val Leu Asn Met Gln

660 665 670

Lys Leu Glu Lys Thr Asp Glu Arg Glu Lys Arg Val Asn Arg Trp Ile

675 680 685

Lys Asp Arg Glu Arg Glu Glu Glu Asn Pro Val Tyr Val Gln Glu Phe

690 695 700

Glu Met Ile Ser Lys Val Leu Tyr Ser Pro His Ser Val Trp Val Asp

705 710 715 720

Gln Leu Lys Ser Ile His Arg Lys Leu Glu Glu Gln Leu Gly Lys Glu

725 730 735

Ile Ser Lys Trp Arg Gln Ser Ile Ser Gln Gly Arg Gln Gly Val Tyr

740 745 750

Gly Ile Ser Leu Lys Asn Ile Glu Asp Ile Glu Lys Thr Arg Arg Leu

755 760 765

Leu Phe Arg Trp Ser Met Arg Pro Glu Asn Pro Gly Glu Val Lys Gln

770 775 780

Leu Gln Pro Gly Glu Arg Phe Ala Ile Asp Gln Gln Asn His Leu Asn

785 790 795 800

His Leu Lys Asp Asp Arg Ile Lys Lys Leu Ala Asn Gln Ile Val Met

805 810 815

Thr Ala Leu Gly Tyr Arg Tyr Asp Gly Lys Arg Lys Lys Trp Ile Ala

820 825 830

Lys His Pro Ala Cys Gln Leu Val Leu Phe Glu Asp Leu Ser Arg Tyr

835 840 845

Ala Phe Tyr Asp Glu Arg Ser Arg Leu Glu Asn Arg Asn Leu Met Arg

850 855 860

Trp Ser Arg Arg Glu Ile Pro Lys Gln Val Ala Gln Ile Gly Gly Leu

865 870 875 880

Tyr Gly Leu Leu Val Gly Glu Val Gly Ala Gln Tyr Ser Ser Arg Phe

885 890 895

His Ala Lys Ser Gly Ala Pro Gly Ile Arg Cys Arg Val Val Lys Glu

900 905 910

His Glu Leu Tyr Ile Thr Glu Gly Gly Gln Lys Val Arg Asn Gln Lys

915 920 925

Phe Leu Asp Ser Leu Val Glu Asn Asn Ile Ile Glu Pro Asp Asp Ala

930 935 940

Arg Arg Leu Glu Pro Gly Asp Leu Ile Arg Asp Gln Gly Gly Asp Lys

945 950 955 960

Phe Ala Thr Leu Asp Glu Arg Gly Glu Leu Val Ile Thr His Ala Asp

965 970 975

Ile Asn Ala Ala Gln Asn Leu Gln Lys Arg Phe Trp Thr Arg Thr His

980 985 990

Gly Leu Tyr Arg Ile Arg Cys Glu Ser Arg Glu Ile Lys Asp Ala Val

995 1000 1005

Val Leu Val Pro Ser Asp Lys Asp Gln Lys Glu Lys Met Glu Asn Leu

1010 1015 1020

Phe Gly Ile Gly Tyr Leu Gln Pro Phe Lys Gln Glu Asn Asp Val Tyr

1025 1030 1035 1040

Lys Trp Val Lys Gly Glu Lys Ile Lys Gly Lys Lys Thr Ser Ser Gln

1045 1050 1055

Ser Asp Asp Lys Glu Leu Val Ser Glu Ile Leu Gln Glu Ala Ser Val

1060 1065 1070

Met Ala Asp Glu Leu Lys Gly Asn Arg Lys Thr Leu Phe Arg Asp Pro

1075 1080 1085

Ser Gly Tyr Val Phe Pro Lys Asp Arg Trp Tyr Thr Gly Gly Arg Tyr

1090 1095 1100

Phe Gly Thr Leu Glu His Leu Leu Lys Arg Lys Leu Ala Glu Arg Arg

1105 1110 1115 1120

Leu Phe Asp Gly Gly Ser Ser Arg Arg Gly Leu Phe Asn Gly Thr Asp

1125 1130 1135

Ser Asn Thr Asn Val Glu

1140

<210> 11

<211> 3387

<212> DNA

<213> Alicyclobacillus acidiphilus

<400> 11

atggccgtga agagcatgaa ggtgaagctg cgcctggaca acatgcccga gatccgcgcc 60

ggcctgtgga agctgcacac cgaggtgaac gccggcgtgc gctactacac cgagtggctg 120

agcctgctgc gccaggagaa cctgtaccgc cgcagcccca acggcgacgg cgagcaggag 180

tgctacaaga ccgccgagga gtgcaaggcc gagctgctgg agcgcctgcg cgcccgccag 240

gtggagaacg gccactgcgg ccccgccggc agcgacgacg agctgctgca gctggcccgc 300

cagctgtacg agctgctggt gccccaggcc atcggcgcca agggcgacgc ccagcagatc 360

gcccgcaagt tcctgagccc cctggccgac aaggacgccg tgggcggcct gggcatcgcc 420

aaggccggca acaagccccg ctgggtgcgc atgcgcgagg ccggcgagcc cggctgggag 480

gaggagaagg ccaaggccga ggcccgcaag agcaccgacc gcaccgccga cgtgctgcgc 540

gccctggccg acttcggcct gaagcccctg atgcgcgtgt acaccgacag cgacatgagc 600

agcgtgcagt ggaagcccct gcgcaagggc caggccgtgc gcacctggga ccgcgacatg 660

ttccagcagg ccatcgagcg catgatgagc tgggagagct ggaaccagcg cgtgggcgag 720

gcctacgcca agctggtgga gcagaagagc cgcttcgagc agaagaactt cgtgggccag 780

gagcacctgg tgcagctggt gaaccagctg cagcaggaca tgaaggaggc cagccacggc 840

ctggagagca aggagcagac cgcccactac ctgaccggcc gcgccctgcg cggcagcgac 900

aaggtgttcg agaagtggga gaagctggac cccgacgccc ccttcgacct gtacgacacc 960

gagatcaaga acgtgcagcg ccgcaacacc cgccgcttcg gcagccacga cctgttcgcc 1020

aagctggccg agcccaagta ccaggccctg tggcgcgagg acgccagctt cctgacccgc 1080

tacgccgtgt acaacagcat cgtgcgcaag ctgaaccacg ccaagatgtt cgccaccttc 1140

accctgcccg acgccaccgc ccaccccatc tggacccgct tcgacaagct gggcggcaac 1200

ctgcaccagt acaccttcct gttcaacgag ttcggcgagg gccgccacgc catccgcttc 1260

cagaagctgc tgaccgtgga ggacggcgtg gccaaggagg tggacgacgt gaccgtgccc 1320

atcagcatga gcgcccagct ggacgacctg ctgccccgcg acccccacga gctggtggcc 1380

ctgtacttcc aggactacgg cgccgagcag cacctggccg gcgagttcgg cggcgccaag 1440

atccagtacc gccgcgacca gctgaaccac ctgcacgccc gccgcggcgc ccgcgacgtg 1500

tacctgaacc tgagcgtgcg cgtgcagagc cagagcgagg cccgcggcga gcgccgcccc 1560

ccctacgccg ccgtgttccg cctggtgggc gacaaccacc gcgccttcgt gcacttcgac 1620

aagctgagcg actacctggc cgagcacccc gacgacggca agctgggcag cgagggcctg 1680

ctgagcggcc tgcgcgtgat gagcgtggac ctgggcctgc gcaccagcgc cagcatcagc 1740

gtgttccgcg tggcccgcaa ggacgagctg aagcccaaca gcgagggccg cgtgcccttc 1800

tgcttcccca tcgagggcaa cgagaacctg gtggccgtgc acgagcgcag ccagctgctg 1860

aagctgcccg gcgagaccga gagcaaggac ctgcgcgcca tccgcgagga gcgccagcgc 1920

accctgcgcc agctgcgcac ccagctggcc tacctgcgcc tgctggtgcg ctgcggcagc 1980

gaggacgtgg gccgccgcga gcgcagctgg gccaagctga tcgagcagcc catggacgcc 2040

aaccagatga cccccgactg gcgcgaggcc ttcgaggacg agctgcagaa gctgaagagc 2100

ctgtacggca tctgcggcga ccgcgagtgg accgaggccg tgtacgagag cgtgcgccgc 2160

gtgtggcgcc acatgggcaa gcaggtgcgc gactggcgca aggacgtgcg cagcggcgag 2220

cgccccaaga tccgcggcta ccagaaggac gtggtgggcg gcaacagcat cgagcagatc 2280

gagtacctgg agcgccagta caagttcctg aagagctgga gcttcttcgg caaggtgagc 2340

ggccaggtga tccgcgccga gaagggcagc cgcttcgcca tcaccctgcg cgagcacatc 2400

gaccacgcca aggaggaccg cctgaagaag ctggccgacc gcatcatcat ggaggccctg 2460

ggctacgtgt acgccctgga cgacgagcgc ggcaagggca agtgggtggc caagtacccc 2520

ccctgccagc tgatcctgct ggaggagctg agcgagtacc agttcaacaa cgaccgcccc 2580

cccagcgaga acaaccagct gatgcagtgg agccaccgcg gcgtgttcca ggagctgctg 2640

aaccaggccc aggtgcacga cctgctggtg ggcaccatgt acgccgcctt cagcagccgc 2700

ttcgacgccc gcaccggcgc ccccggcatc cgctgccgcc gcgtgcccgc ccgctgcgcc 2760

cgcgagcaga accccgagcc cttcccctgg tggctgaaca agttcgtggc cgagcacaag 2820

ctggacggct gccccctgcg cgccgacgac ctgatcccca ccggcgaggg cgagttcttc 2880

gtgagcccct tcagcgccga ggagggcgac ttccaccaga tccacgccga cctgaacgcc 2940

gcccagaacc tgcagcgccg cctgtggagc gacttcgaca tcagccagat ccgcctgcgc 3000

tgcgactggg gcgaggtgga cggcgagccc gtgctgatcc cccgcaccac cggcaagcgc 3060

accgccgaca gctacggcaa caaggtgttc tacaccaaga ccggcgtgac ctactacgag 3120

cgcgagcgcg gcaagaagcg ccgcaaggtg ttcgcccagg aggagctgag cgaggaggag 3180

gccgagctgc tggtggaggc cgacgaggcc cgcgagaaga gcgtggtgct gatgcgcgac 3240

cccagcggca tcatcaaccg cggcgactgg acccgccaga aggagttctg gagcatggtg 3300

aaccagcgca tcgagggcta cctggtgaag cagatccgca gccgcgtgcg cctgcaggag 3360

agcgcctgcg agaacaccgg cgacatc 3387

<210> 12

<211> 3441

<212> DNA

<213> Alicyclobacillus kakegawensis

<400> 12

atggccgtga agagcatcaa ggtgaagctg cgcctgagcg agtgccccga catcctggcc 60

ggcatgtggc agctgcaccg cgccaccaac gccggcgtgc gctactacac cgagtgggtg 120

agcctgatgc gccaggagat cctgtacagc cgcggccccg acggcggcca gcagtgctac 180

atgaccgccg aggactgcca gcgcgagctg ctgcgccgcc tgcgcaaccg ccagctgcac 240

aacggccgcc aggaccagcc cggcaccgac gccgacctgc tggccatcag ccgccgcctg 300

tacgagatcc tggtgctgca gagcatcggc aagcgcggcg acgcccagca gatcgccagc 360

agcttcctga gccccctggt ggaccccaac agcaagggcg gccgcggcga ggccaagagc 420

ggccgcaagc ccgcctggca gaagatgcgc gaccagggcg acccccgctg ggtggccgcc 480

cgcgagaagt acgagcagcg caaggccgtg gaccccagca aggagatcct gaacagcctg 540

gacgccctgg gcctgcgccc cctgttcgcc gtgttcaccg agacctaccg cagcggcgtg 600

gactggaagc ccctgggcaa gagccagggc gtgcgcacct gggaccgcga catgttccag 660

caggccctgg agcgcctgat gagctgggag agctggaacc gccgcgtggg cgaggagtac 720

gcccgcctgt tccagcagaa gatgaagttc gagcaggagc acttcgccga gcagagccac 780

ctggtgaagc tggcccgcgc cctggaggcc gacatgcgcg ccgccagcca gggcttcgag 840

gccaagcgcg gcaccgccca ccagatcacc cgccgcgccc tgcgcggcgc cgaccgcgtg 900

ttcgagatct ggaagagcat ccccgaggag gccctgttca gccagtacga cgaggtgatc 960

cgccaggtgc aggccgagaa gcgccgcgac ttcggcagcc acgacctgtt cgccaagctg 1020

gccgagccca agtaccagcc cctgtggcgc gccgacgaga ccttcctgac ccgctacgcc 1080

ctgtacaacg gcgtgctgcg cgacctggag aaggcccgcc agttcgccac cttcaccctg 1140

cccgacgcct gcgtgaaccc catctggacc cgcttcgaga gcagccaggg cagcaacctg 1200

cacaagtacg agttcctgtt cgaccacctg ggccccggcc gccacgccgt gcgcttccag 1260

cgcctgctgg tggtggagag cgagggcgcc aaggagcgcg acagcgtggt ggtgcccgtg 1320

gcccccagcg gccagctgga caagctggtg ctgcgcgagg aggagaagag cagcgtggcc 1380

ctgcacctgc acgacaccgc ccgccccgac ggcttcatgg ccgagtgggc cggcgccaag 1440

ctgcagtacg agcgcagcac cctggcccgc aaggcccgcc gcgacaagca gggcatgcgc 1500

agctggcgcc gccagcccag catgctgatg agcgccgccc agatgctgga ggacgccaag 1560

caggccggcg acgtgtacct gaacatcagc gtgcgcgtga agagccccag cgaggtgcgc 1620

ggccagcgcc gcccccccta cgccgccctg ttccgcatcg acgacaagca gcgccgcgtg 1680

accgtgaact acaacaagct gagcgcctac ctggaggagc accccgacaa gcagatcccc 1740

ggcgcccccg gcctgctgag cggcctgcgc gtgatgagcg tggacctggg cctgcgcacc 1800

agcgccagca tcagcgtgtt ccgcgtggcc aagaaggagg aggtggaggc cctgggcgac 1860

ggccgccccc cccactacta ccccatccac ggcaccgacg acctggtggc cgtgcacgag 1920

cgcagccacc tgatccagat gcccggcgag accgagacca agcagctgcg caagctgcgc 1980

gaggagcgcc aggccgtgct gcgccccctg ttcgcccagc tggccctgct gcgcctgctg 2040

gtgcgctgcg gcgccgccga cgagcgcatc cgcacccgca gctggcagcg cctgaccaag 2100

cagggccgcg agttcaccaa gcgcctgacc cccagctggc gcgaggccct ggagctggag 2160

ctgacccgcc tggaggccta ctgcggccgc gtgcccgacg acgagtggag ccgcatcgtg 2220

gaccgcaccg tgatcgccct gtggcgccgc atgggcaagc aggtgcgcga ctggcgcaag 2280

caggtgaaga gcggcgccaa ggtgaaggtg aagggctacc agctggacgt ggtgggcggc 2340

aacagcctgg cccagatcga ctacctggag cagcagtaca agttcctgcg ccgctggagc 2400

ttcttcgccc gcgccagcgg cctggtggtg cgcgccgacc gcgagagcca cttcgccgtg 2460

gccctgcgcc agcacatcga gaacgccaag cgcgaccgcc tgaagaagct ggccgaccgc 2520

atcctgatgg aggccctggg ctacgtgtac gaggccagcg gcccccgcga gggccagtgg 2580

accgcccagc accccccctg ccagctgatc atcctggagg agctgagcgc ctaccgcttc 2640

agcgacgacc gcccccccag cgagaacagc aagctgatgg cctggggcca ccgcggcatc 2700

ctggaggagc tggtgaacca ggcccaggtg cacgacgtgc tggtgggcac cgtgtacgcc 2760

gccttcagca gccgcttcga cgcccgcacc ggcgcccccg gcgtgcgctg ccgccgcgtg 2820

cccgcccgct tcgtgggcgc caccgtggac gacagcctgc ccctgtggct gaccgagttc 2880

ctggacaagc accgcctgga caagaacctg ctgcgccccg acgacgtgat ccccaccggc 2940

gagggcgagt tcctggtgag cccctgcggc gaggaggccg cccgcgtgcg ccaggtgcac 3000

gccgacatca acgccgccca gaacctgcag cgccgcctgt ggcagaactt cgacatcacc 3060

gagctgcgcc tgcgctgcga cgtgaagatg ggcggcgagg gcaccgtgct ggtgccccgc 3120

gtgaacaacg cccgcgccaa gcagctgttc ggcaagaagg tgctggtgag ccaggacggc 3180

gtgaccttct tcgagcgcag ccagaccggc ggcaagcccc acagcgagaa gcagaccgac 3240

ctgaccgaca aggagctgga gctgatcgcc gaggccgacg aggcccgcgc caagagcgtg 3300

gtgctgttcc gcgaccccag cggccacatc ggcaagggcc actggatccg ccagcgcgag 3360

ttctggagcc tggtgaagca gcgcatcgag agccacaccg ccgagcgcat ccgcgtgcgc 3420

ggcgtgggca gcagcctgga c 3441

<210> 13

<211> 3438

<212> DNA

<213> Alicyclobacillus macrosporangiidus

<400> 13

atgaacgtgg ccgtgaagag catcaaggtg aagctgatgc tgggccacct gcccgagatc 60

cgcgagggcc tgtggcacct gcacgaggcc gtgaacctgg gcgtgcgcta ctacaccgag 120

tggctggccc tgctgcgcca gggcaacctg taccgccgcg gcaaggacgg cgcccaggag 180

tgctacatga ccgccgagca gtgccgccag gagctgctgg tgcgcctgcg cgaccgccag 240

aagcgcaacg gccacaccgg cgaccccggc accgacgagg agctgctggg cgtggcccgc 300

cgcctgtacg agctgctggt gccccagagc gtgggcaaga agggccaggc ccagatgctg 360

gccagcggct tcctgagccc cctggccgac cccaagagcg agggcggcaa gggcaccagc 420

aagagcggcc gcaagcccgc ctggatgggc atgaaggagg ccggcgacag ccgctgggtg 480

gaggccaagg cccgctacga ggccaacaag gccaaggacc ccaccaagca ggtgatcgcc 540

agcctggaga tgtacggcct gcgccccctg ttcgacgtgt tcaccgagac ctacaagacc 600

atccgctgga tgcccctggg caagcaccag ggcgtgcgcg cctgggaccg cgacatgttc 660

cagcagagcc tggagcgcct gatgagctgg gagagctgga acgagcgcgt gggcgccgag 720

ttcgcccgcc tggtggaccg ccgcgaccgc ttccgcgaga agcacttcac cggccaggag 780

cacctggtgg ccctggccca gcgcctggag caggagatga aggaggccag ccccggcttc 840

gagagcaaga gcagccaggc ccaccgcatc accaagcgcg ccctgcgcgg cgccgacggc 900

atcatcgacg actggctgaa gctgagcgag ggcgagcccg tggaccgctt cgacgagatc 960

ctgcgcaagc gccaggccca gaacccccgc cgcttcggca gccacgacct gttcctgaag 1020

ctggccgagc ccgtgttcca gcccctgtgg cgcgaggacc ccagcttcct gagccgctgg 1080

gccagctaca acgaggtgct gaacaagctg gaggacgcca agcagttcgc caccttcacc 1140

ctgcccagcc cctgcagcaa ccccgtgtgg gcccgcttcg agaacgccga gggcaccaac 1200

atcttcaagt acgacttcct gttcgaccac ttcggcaagg gccgccacgg cgtgcgcttc 1260

cagcgcatga tcgtgatgcg cgacggcgtg cccaccgagg tggagggcat cgtggtgccc 1320

atcgccccca gccgccagct ggacgccctg gcccccaacg acgccgccag ccccatcgac 1380

gtgttcgtgg gcgaccccgc cgcccccggc gccttccgcg gccagttcgg cggcgccaag 1440

atccagtacc gccgcagcgc cctggtgcgc aagggccgcc gcgaggagaa ggcctacctg 1500

tgcggcttcc gcctgcccag ccagcgccgc accggcaccc ccgccgacga cgccggcgag 1560

gtgttcctga acctgagcct gcgcgtggag agccagagcg agcaggccgg ccgccgcaac 1620

cccccctacg ccgccgtgtt ccacatcagc gaccagaccc gccgcgtgat cgtgcgctac 1680

ggcgagatcg agcgctacct ggccgagcac cccgacaccg gcatccccgg cagccgcggc 1740

ctgaccagcg gcctgcgcgt gatgagcgtg gacctgggcc tgcgcaccag cgccgccatc 1800

agcgtgttcc gcgtggccca ccgcgacgag ctgacccccg acgcccacgg ccgccagccc 1860

ttcttcttcc ccatccacgg catggaccac ctggtggccc tgcacgagcg cagccacctg 1920

atccgcctgc ccggcgagac cgagagcaag aaggtgcgca gcatccgcga gcagcgcctg 1980

gaccgcctga accgcctgcg cagccagatg gccagcctgc gcctgctggt gcgcaccggc 2040

gtgctggacg agcagaagcg cgaccgcaac tgggagcgcc tgcagagcag catggagcgc 2100

ggcggcgagc gcatgcccag cgactggtgg gacctgttcc aggcccaggt gcgctacctg 2160

gcccagcacc gcgacgccag cggcgaggcc tggggccgca tggtgcaggc cgccgtgcgc 2220

accctgtggc gccagctggc caagcaggtg cgcgactggc gcaaggaggt gcgccgcaac 2280

gccgacaagg tgaagatccg cggcatcgcc cgcgacgtgc ccggcggcca cagcctggcc 2340

cagctggact acctggagcg ccagtaccgc ttcctgcgca gctggagcgc cttcagcgtg 2400

caggccggcc aggtggtgcg cgccgagcgc gacagccgct tcgccgtggc cctgcgcgag 2460

cacatcgaca acggcaagaa ggaccgcctg aagaagctgg ccgaccgcat cctgatggag 2520

gccctgggct acgtgtacgt gaccgacggc cgccgcgccg gccagtggca ggccgtgtac 2580

cccccctgcc agctggtgct gctggaggag ctgagcgagt accgcttcag caacgaccgc 2640

ccccccagcg agaacagcca gctgatggtg tggagccacc gcggcgtgct ggaggagctg 2700

atccaccagg cccaggtgca cgacgtgctg gtgggcacca tccccgccgc cttcagcagc 2760

cgcttcgacg cccgcaccgg cgcccccggc atccgctgcc gccgcgtgcc cagcatcccc 2820

ctgaaggacg cccccagcat ccccatctgg ctgagccact acctgaagca gaccgagcgc 2880

gacgccgccg ccctgcgccc cggcgagctg atccccaccg gcgacggcga gttcctggtg 2940

acccccgccg gccgcggcgc cagcggcgtg cgcgtggtgc acgccgacat caacgccgcc 3000

cacaacctgc agcgccgcct gtgggagaac ttcgacctga gcgacatccg cgtgcgctgc 3060

gaccgccgcg agggcaagga cggcaccgtg gtgctgatcc cccgcctgac caaccagcgc 3120

gtgaaggagc gctacagcgg cgtgatcttc accagcgagg acggcgtgag cttcaccgtg 3180

ggcgacgcca agacccgccg ccgcagcagc gccagccagg gcgagggcga cgacctgagc 3240

gacgaggagc aggagctgct ggccgaggcc gacgacgccc gcgagcgcag cgtggtgctg 3300

ttccgcgacc ccagcggctt cgtgaacggc ggccgctgga ccgcccagcg cgccttctgg 3360

ggcatggtgc acaaccgcat cgagaccctg ctggccgagc gcttcagcgt gagcggcgcc 3420

gccgagaagg tgcgcggc 3438

<210> 14

<211> 3324

<212> DNA

<213> Bacillus hisashii

<400> 14

atggccaccc gcagcttcat cctgaagatc gagcccaacg aggaggtgaa gaagggcctg 60

tggaagaccc acgaggtgct gaaccacggc atcgcctact acatgaacat cctgaagctg 120

atccgccagg aggccatcta cgagcaccac gagcaggacc ccaagaaccc caagaaggtg 180

agcaaggccg agatccaggc cgagctgtgg gacttcgtgc tgaagatgca gaagtgcaac 240

agcttcaccc acgaggtgga caaggacgag gtgttcaaca tcctgcgcga gctgtacgag 300

gagctggtgc ccagcagcgt ggagaagaag ggcgaggcca accagctgag caacaagttc 360

ctgtaccccc tggtggaccc caacagccag agcggcaagg gcaccgccag cagcggccgc 420

aagccccgct ggtacaacct gaagatcgcc ggcgacccca gctgggagga ggagaagaag 480

aagtgggagg aggacaagaa gaaggacccc ctggccaaga tcctgggcaa gctggccgag 540

tacggcctga tccccctgtt catcccctac accgacagca acgagcccat cgtgaaggag 600

atcaagtgga tggagaagag ccgcaaccag agcgtgcgcc gcctggacaa ggacatgttc 660

atccaggccc tggagcgctt cctgagctgg gagagctgga acctgaaggt gaaggaggag 720

tacgagaagg tggagaagga gtacaagacc ctggaggagc gcatcaagga ggacatccag 780

gccctgaagg ccctggagca gtacgagaag gagcgccagg agcagctgct gcgcgacacc 840

ctgaacacca acgagtaccg cctgagcaag cgcggcctgc gcggctggcg cgagatcatc 900

cagaagtggc tgaagatgga cgagaacgag cccagcgaga agtacctgga ggtgttcaag 960

gactaccagc gcaagcaccc ccgcgaggcc ggcgactaca gcgtgtacga gttcctgagc 1020

aagaaggaga accacttcat ctggcgcaac caccccgagt acccctacct gtacgccacc 1080

ttctgcgaga tcgacaagaa gaagaaggac gccaagcagc aggccacctt caccctggcc 1140

gaccccatca accaccccct gtgggtgcgc ttcgaggagc gcagcggcag caacctgaac 1200

aagtaccgca tcctgaccga gcagctgcac accgagaagc tgaagaagaa gctgaccgtg 1260

cagctggacc gcctgatcta ccccaccgag agcggcggct gggaggagaa gggcaaggtg 1320

gacatcgtgc tgctgcccag ccgccagttc tacaaccaga tcttcctgga catcgaggag 1380

aagggcaagc acgccttcac ctacaaggac gagagcatca agttccccct gaagggcacc 1440

ctgggcggcg cccgcgtgca gttcgaccgc gaccacctgc gccgctaccc ccacaaggtg 1500

gagagcggca acgtgggccg catctacttc aacatgaccg tgaacatcga gcccaccgag 1560

agccccgtga gcaagagcct gaagatccac cgcgacgact tccccaaggt ggtgaacttc 1620

aagcccaagg agctgaccga gtggatcaag gacagcaagg gcaagaagct gaagagcggc 1680

atcgagagcc tggagatcgg cctgcgcgtg atgagcatcg acctgggcca gcgccaggcc 1740

gccgccgcca gcatcttcga ggtggtggac cagaagcccg acatcgaggg caagctgttc 1800

ttccccatca agggcaccga gctgtacgcc gtgcaccgcg ccagcttcaa catcaagctg 1860

cccggcgaga ccctggtgaa gagccgcgag gtgctgcgca aggcccgcga ggacaacctg 1920

aagctgatga accagaagct gaacttcctg cgcaacgtgc tgcacttcca gcagttcgag 1980

gacatcaccg agcgcgagaa gcgcgtgacc aagtggatca gccgccagga gaacagcgac 2040

gtgcccctgg tgtaccagga cgagctgatc cagatccgcg agctgatgta caagccctac 2100

aaggactggg tggccttcct gaagcagctg cacaagcgcc tggaggtgga gatcggcaag 2160

gaggtgaagc actggcgcaa gagcctgagc gacggccgca agggcctgta cggcatcagc 2220

ctgaagaaca tcgacgagat cgaccgcacc cgcaagttcc tgctgcgctg gagcctgcgc 2280

cccaccgagc ccggcgaggt gcgccgcctg gagcccggcc agcgcttcgc catcgaccag 2340

ctgaaccacc tgaacgccct gaaggaggac cgcctgaaga agatggccaa caccatcatc 2400

atgcacgccc tgggctactg ctacgacgtg cgcaagaaga agtggcaggc caagaacccc 2460

gcctgccaga tcatcctgtt cgaggacctg agcaactaca acccctacga ggagcgcagc 2520

cgcttcgaga acagcaagct gatgaagtgg agccgccgcg agatcccccg ccaggtggcc 2580

ctgcagggcg agatctacgg cctgcaggtg ggcgaggtgg gcgcccagtt cagcagccgc 2640

ttccacgcca agaccggcag ccccggcatc cgctgcagcg tggtgaccaa ggagaagctg 2700

caggacaacc gcttcttcaa gaacctgcag cgcgagggcc gcctgaccct ggacaagatc 2760

gccgtgctga aggagggcga cctgtacccc gacaagggcg gcgagaagtt catcagcctg 2820

agcaaggacc gcaagtgcgt gaccacccac gccgacatca acgccgccca gaacctgcag 2880

aagcgcttct ggacccgcac ccacggcttc tacaaggtgt actgcaaggc ctaccaggtg 2940

gacggccaga ccgtgtacat ccccgagagc aaggaccaga agcagaagat catcgaggag 3000

ttcggcgagg gctacttcat cctgaaggac ggcgtgtacg agtgggtgaa cgccggcaag 3060

ctgaagatca agaagggcag cagcaagcag agcagcagcg agctggtgga cagcgacatc 3120

ctgaaggaca gcttcgacct ggccagcgag ctgaagggcg agaagctgat gctgtaccgc 3180

gaccccagcg gcaacgtgtt ccccagcgac aagtggatgg ccgccggcgt gttcttcggc 3240

aagctggagc gcatcctgat cagcaagctg accaaccagt acagcatcag caccatcgag 3300

gacgacagca gcaagcagag catg 3324

<210> 15

<211> 3324

<212> DNA

<213> Bacillus

<400> 15

atggccatcc gcagcatcaa gctgaagctg aagacccaca ccggccccga ggcccagaac 60

ctgcgcaagg gcatctggcg cacccaccgc ctgctgaacg agggcgtggc ctactacatg 120

aagatgctgc tgctgttccg ccaggagagc accggcgagc gccccaagga ggagctgcag 180

gaggagctga tctgccacat ccgcgagcag cagcagcgca accaggccga caagaacacc 240

caggccctgc ccctggacaa ggccctggag gccctgcgcc agctgtacga gctgctggtg 300

cccagcagcg tgggccagag cggcgacgcc cagatcatca gccgcaagtt cctgagcccc 360

ctggtggacc ccaacagcga gggcggcaag ggcaccagca aggccggcgc caagcccacc 420

tggcagaaga agaaggaggc caacgacccc acctgggagc aggactacga gaagtggaag 480

aagcgccgcg aggaggaccc caccgccagc gtgatcacca ccctggagga gtacggcatc 540

cgccccatct tccccctgta caccaacacc gtgaccgaca tcgcctggct gcccctgcag 600

agcaaccagt tcgtgcgcac ctgggaccgc gacatgctgc agcaggccat cgagcgcctg 660

ctgagctggg agagctggaa caagcgcgtg caggaggagt acgccaagct gaaggagaag 720

atggcccagc tgaacgagca gctggagggc ggccaggagt ggatcagcct gctggagcag 780

tacgaggaga accgcgagcg cgagctgcgc gagaacatga ccgccgccaa cgacaagtac 840

cgcatcacca agcgccagat gaagggctgg aacgagctgt acgagctgtg gagcaccttc 900

cccgccagcg ccagccacga gcagtacaag gaggccctga agcgcgtgca gcagcgcctg 960

cgcggccgct tcggcgacgc ccacttcttc cagtacctga tggaggagaa gaaccgcctg 1020

atctggaagg gcaaccccca gcgcatccac tacttcgtgg cccgcaacga gctgaccaag 1080

cgcctggagg aggccaagca gagcgccacc atgaccctgc ccaacgcccg caagcacccc 1140

ctgtgggtgc gcttcgacgc ccgcggcggc aacctgcagg actactacct gaccgccgag 1200

gccgacaagc cccgcagccg ccgcttcgtg accttcagcc agctgatctg gcccagcgag 1260

agcggctgga tggagaagaa ggacgtggag gtggagctgg ccctgagccg ccagttctac 1320

cagcaggtga agctgctgaa gaacgacaag ggcaagcaga agatcgagtt caaggacaag 1380

ggcagcggca gcaccttcaa cggccacctg ggcggcgcca agctgcagct ggagcgcggc 1440

gacctggaga aggaggagaa gaacttcgag gacggcgaga tcggcagcgt gtacctgaac 1500

gtggtgatcg acttcgagcc cctgcaggag gtgaagaacg gccgcgtgca ggccccctac 1560

ggccaggtgc tgcagctgat ccgccgcccc aacgagttcc ccaaggtgac cacctacaag 1620

agcgagcagc tggtggagtg gatcaaggcc agcccccagc acagcgccgg cgtggagagc 1680

ctggccagcg gcttccgcgt gatgagcatc gacctgggcc tgcgcgccgc cgccgccacc 1740

agcatcttca gcgtggagga gagcagcgac aagaacgccg ccgacttcag ctactggatc 1800

gagggcaccc ccctggtggc cgtgcaccag cgcagctaca tgctgcgcct gcccggcgag 1860

caggtggaga agcaggtgat ggagaagcgc gacgagcgct tccagctgca ccagcgcgtg 1920

aagttccaga tccgcgtgct ggcccagatc atgcgcatgg ccaacaagca gtacggcgac 1980

cgctgggacg agctggacag cctgaagcag gccgtggagc agaagaagag ccccctggac 2040

cagaccgacc gcaccttctg ggagggcatc gtgtgcgacc tgaccaaggt gctgccccgc 2100

aacgaggccg actgggagca ggccgtggtg cagatccacc gcaaggccga ggagtacgtg 2160

ggcaaggccg tgcaggcctg gcgcaagcgc ttcgccgccg acgagcgcaa gggcatcgcc 2220

ggcctgagca tgtggaacat cgaggagctg gagggcctgc gcaagctgct gatcagctgg 2280

agccgccgca cccgcaaccc ccaggaggtg aaccgcttcg agcgcggcca caccagccac 2340

cagcgcctgc tgacccacat ccagaacgtg aaggaggacc gcctgaagca gctgagccac 2400

gccatcgtga tgaccgccct gggctacgtg tacgacgagc gcaagcagga gtggtgcgcc 2460

gagtaccccg cctgccaggt gatcctgttc gagaacctga gccagtaccg cagcaacctg 2520

gaccgcagca ccaaggagaa cagcaccctg atgaagtggg cccaccgcag catccccaag 2580

tacgtgcaca tgcaggccga gccctacggc atccagatcg gcgacgtgcg cgccgagtac 2640

agcagccgct tctacgccaa gaccggcacc cccggcatcc gctgcaagaa ggtgcgcggc 2700

caggacctgc agggccgccg cttcgagaac ctgcagaagc gcctggtgaa cgagcagttc 2760

ctgaccgagg agcaggtgaa gcagctgcgc cccggcgaca tcgtgcccga cgacagcggc 2820

gagctgttca tgaccctgac cgacggcagc ggcagcaagg aggtggtgtt cctgcaggcc 2880

gacatcaacg ccgcccacaa cctgcagaag cgcttctggc agcgctacaa cgagctgttc 2940

aaggtgagct gccgcgtgat cgtgcgcgac gaggaggagt acctggtgcc caagaccaag 3000

agcgtgcagg ccaagctggg caagggcctg ttcgtgaaga agagcgacac cgcctggaag 3060

gacgtgtacg tgtgggacag ccaggccaag ctgaagggca agaccacctt caccgaggag 3120

agcgagagcc ccgagcagct ggaggacttc caggagatca tcgaggaggc cgaggaggcc 3180

aagggcacct accgcaccct gttccgcgac cccagcggcg tgttcttccc cgagagcgtg 3240

tggtaccccc agaaggactt ctggggcgag gtgaagcgca agctgtacgg caagctgcgc 3300

gagcgcttcc tgaccaaggc ccgc 3324

<210> 16

<211> 3336

<212> DNA

<213> Bacillus

<400> 16

atggccatcc gcagcatcaa gctgaagatg aagaccaaca gcggcaccga cagcatctac 60

ctgcgcaagg ccctgtggcg cacccaccag ctgatcaacg agggcatcgc ctactacatg 120

aacctgctga ccctgtaccg ccaggaggcc atcggcgaca agaccaagga ggcctaccag 180

gccgagctga tcaacatcat ccgcaaccag cagcgcaaca acggcagcag cgaggagcac 240

ggcagcgacc aggagatcct ggccctgctg cgccagctgt acgagctgat catccccagc 300

agcatcggcg agagcggcga cgccaaccag ctgggcaaca agttcctgta ccccctggtg 360

gaccccaaca gccagagcgg caagggcacc agcaacgccg gccgcaagcc ccgctggaag 420

cgcctgaagg aggagggcaa ccccgactgg gagctggaga agaagaagga cgaggagcgc 480

aaggccaagg accccaccgt gaagatcttc gacaacctga acaagtacgg cctgctgccc 540

ctgttccccc tgttcaccaa catccagaag gacatcgagt ggctgcccct gggcaagcgc 600

cagagcgtgc gcaagtggga caaggacatg ttcatccagg ccatcgagcg cctgctgagc 660

tgggagagct ggaaccgccg cgtggccgac gagtacaagc agctgaagga gaagaccgag 720

agctactaca aggagcacct gaccggcggc gaggagtgga tcgagaagat ccgcaagttc 780

gagaaggagc gcaacatgga gctggagaag aacgccttcg cccccaacga cggctacttc 840

atcaccagcc gccagatccg cggctgggac cgcgtgtacg agaagtggag caagctgccc 900

gagagcgcca gccccgagga gctgtggaag gtggtggccg agcagcagaa caagatgagc 960

gagggcttcg gcgaccccaa ggtgttcagc ttcctggcca accgcgagaa ccgcgacatc 1020

tggcgcggcc acagcgagcg catctaccac atcgccgcct acaacggcct gcagaagaag 1080

ctgagccgca ccaaggagca ggccaccttc accctgcccg acgccatcga gcaccccctg 1140

tggatccgct acgagagccc cggcggcacc aacctgaacc tgttcaagct ggaggagaag 1200

cagaagaaga actactacgt gaccctgagc aagatcatct ggcccagcga ggagaagtgg 1260

atcgagaagg agaacatcga gatccccctg gcccccagca tccagttcaa ccgccagatc 1320

aagctgaagc agcacgtgaa gggcaagcag gagatcagct tcagcgacta cagcagccgc 1380

atcagcctgg acggcgtgct gggcggcagc cgcatccagt tcaaccgcaa gtacatcaag 1440

aaccacaagg agctgctggg cgagggcgac atcggccccg tgttcttcaa cctggtggtg 1500

gacgtggccc ccctgcagga gacccgcaac ggccgcctgc agagccccat cggcaaggcc 1560

ctgaaggtga tcagcagcga cttcagcaag gtgatcgact acaagcccaa ggagctgatg 1620

gactggatga acaccggcag cgccagcaac agcttcggcg tggccagcct gctggagggc 1680

atgcgcgtga tgagcatcga catgggccag cgcaccagcg ccagcgtgag catcttcgag 1740

gtggtgaagg agctgcccaa ggaccaggag cagaagctgt tctacagcat caacgacacc 1800

gagctgttcg ccatccacaa gcgcagcttc ctgctgaacc tgcccggcga ggtggtgacc 1860

aagaacaaca agcagcagcg ccaggagcgc cgcaagaagc gccagttcgt gcgcagccag 1920

atccgcatgc tggccaacgt gctgcgcctg gagaccaaga agacccccga cgagcgcaag 1980

aaggccatcc acaagctgat ggagatcgtg cagagctacg acagctggac cgccagccag 2040

aaggaggtgt gggagaagga gctgaacctg ctgaccaaca tggccgcctt caacgacgag 2100

atctggaagg agagcctggt ggagctgcac caccgcatcg agccctacgt gggccagatc 2160

gtgagcaagt ggcgcaaggg cctgagcgag ggccgcaaga acctggccgg catcagcatg 2220

tggaacatcg acgagctgga ggacacccgc cgcctgctga tcagctggag caagcgcagc 2280

cgcacccccg gcgaggccaa ccgcatcgag accgacgagc ccttcggcag cagcctgctg 2340

cagcacatcc agaacgtgaa ggacgaccgc ctgaagcaga tggccaacct gatcatcatg 2400

accgccctgg gcttcaagta cgacaaggag gagaaggacc gctacaagcg ctggaaggag 2460

acctaccccg cctgccagat catcctgttc gagaacctga accgctacct gttcaacctg 2520

gaccgcagcc gccgcgagaa cagccgcctg atgaagtggg cccaccgcag catcccccgc 2580

accgtgagca tgcagggcga gatgttcggc ctgcaggtgg gcgacgtgcg cagcgagtac 2640

agcagccgct tccacgccaa gaccggcgcc cccggcatcc gctgccacgc cctgaccgag 2700

gaggacctga aggccggcag caacaccctg aagcgcctga tcgaggacgg cttcatcaac 2760

gagagcgagc tggcctacct gaagaagggc gacatcatcc ccagccaggg cggcgagctg 2820

ttcgtgaccc tgagcaagcg ctacaagaag gacagcgaca acaacgagct gaccgtgatc 2880

cacgccgaca tcaacgccgc ccagaacctg cagaagcgct tctggcagca gaacagcgag 2940

gtgtaccgcg tgccctgcca gctggcccgc atgggcgagg acaagctgta catccccaag 3000

agccagaccg agaccatcaa gaagtacttc ggcaagggca gcttcgtgaa gaacaacacc 3060

gagcaggagg tgtacaagtg ggagaagagc gagaagatga agatcaagac cgacaccacc 3120

ttcgacctgc aggacctgga cggcttcgag gacatcagca agaccatcga gctggcccag 3180

gagcagcaga agaagtacct gaccatgttc cgcgacccca gcggctactt cttcaacaac 3240

gagacctggc gcccccagaa ggagtactgg agcatcgtga acaacatcat caagagctgc 3300

ctgaagaaga agatcctgag caacaaggtg gagctg 3336

<210> 17

<211> 3447

<212> DNA

<213> Desulfovibrio inopinatus

<400> 17

atgcccaccc gcaccatcaa cctgaagctg gtgctgggca agaaccccga gaacgccacc 60

ctgcgccgcg ccctgttcag cacccaccgc ctggtgaacc aggccaccaa gcgcatcgag 120

gagttcctgc tgctgtgccg cggcgaggcc taccgcaccg tggacaacga gggcaaggag 180

gccgagatcc cccgccacgc cgtgcaggag gaggccctgg ccttcgccaa ggccgcccag 240

cgccacaacg gctgcatcag cacctacgag gaccaggaga tcctggacgt gctgcgccag 300

ctgtacgagc gcctggtgcc cagcgtgaac gagaacaacg aggccggcga cgcccaggcc 360

gccaacgcct gggtgagccc cctgatgagc gccgagagcg agggcggcct gagcgtgtac 420

gacaaggtgc tggacccccc ccccgtgtgg atgaagctga aggaggagaa ggcccccggc 480

tgggaggccg ccagccagat ctggatccag agcgacgagg gccagagcct gctgaacaag 540

cccggcagcc ccccccgctg gatccgcaag ctgcgcagcg gccagccctg gcaggacgac 600

ttcgtgagcg accagaagaa gaagcaggac gagctgacca agggcaacgc ccccctgatc 660

aagcagctga aggagatggg cctgctgccc ctggtgaacc ccttcttccg ccacctgctg 720

gaccccgagg gcaagggcgt gagcccctgg gaccgcctgg ccgtgcgcgc cgccgtggcc 780

cacttcatca gctgggagag ctggaaccac cgcacccgcg ccgagtacaa cagcctgaag 840

ctgcgccgcg acgagttcga ggccgccagc gacgagttca aggacgactt caccctgctg 900

cgccagtacg aggccaagcg ccacagcacc ctgaagagca tcgccctggc cgacgacagc 960

aacccctacc gcatcggcgt gcgcagcctg cgcgcctgga accgcgtgcg cgaggagtgg 1020

atcgacaagg gcgccaccga ggagcagcgc gtgaccatcc tgagcaagct gcagacccag 1080

ctgcgcggca agttcggcga ccccgacctg ttcaactggc tggcccagga ccgccacgtg 1140

cacctgtgga gcccccgcga cagcgtgacc cccctggtgc gcatcaacgc cgtggacaag 1200

gtgctgcgcc gccgcaagcc ctacgccctg atgaccttcg cccacccccg cttccacccc 1260

cgctggatcc tgtacgaggc ccccggcggc agcaacctgc gccagtacgc cctggactgc 1320

accgagaacg ccctgcacat caccctgccc ctgctggtgg acgacgccca cggcacctgg 1380

atcgagaaga agatccgcgt gcccctggcc cccagcggcc agatccagga cctgaccctg 1440

gagaagctgg agaagaagaa gaaccgcctg tactaccgca gcggcttcca gcagttcgcc 1500

ggcctggccg gcggcgccga ggtgctgttc caccgcccct acatggagca cgacgagcgc 1560

agcgaggaga gcctgctgga gcgccccggc gccgtgtggt tcaagctgac cctggacgtg 1620

gccacccagg ccccccccaa ctggctggac ggcaagggcc gcgtgcgcac cccccccgag 1680

gtgcaccact tcaagaccgc cctgagcaac aagagcaagc acacccgcac cctgcagccc 1740

ggcctgcgcg tgctgagcgt ggacctgggc atgcgcacct tcgccagctg cagcgtgttc 1800

gagctgatcg agggcaagcc cgagaccggc cgcgccttcc ccgtggccga cgagcgcagc 1860

atggacagcc ccaacaagct gtgggccaag cacgagcgca gcttcaagct gaccctgccc 1920

ggcgagaccc ccagccgcaa ggaggaggag gagcgcagca tcgcccgcgc cgagatctac 1980

gccctgaagc gcgacatcca gcgcctgaag agcctgctgc gcctgggcga ggaggacaac 2040

gacaaccgcc gcgacgccct gctggagcag ttcttcaagg gctggggcga ggaggacgtg 2100

gtgcccggcc aggccttccc ccgcagcctg ttccagggcc tgggcgccgc ccccttccgc 2160

agcacccccg agctgtggcg ccagcactgc cagacctact acgacaaggc cgaggcctgc 2220

ctggccaagc acatcagcga ctggcgcaag cgcacccgcc cccgccccac cagccgcgag 2280

atgtggtaca agacccgcag ctaccacggc ggcaagagca tctggatgct ggagtacctg 2340

gacgccgtgc gcaagctgct gctgagctgg agcctgcgcg gccgcaccta cggcgccatc 2400

aaccgccagg acaccgcccg cttcggcagc ctggccagcc gcctgctgca ccacatcaac 2460

agcctgaagg aggaccgcat caagaccggc gccgacagca tcgtgcaggc cgcccgcggc 2520

tacatccccc tgccccacgg caagggctgg gagcagcgct acgagccctg ccagctgatc 2580

ctgttcgagg acctggcccg ctaccgcttc cgcgtggacc gcccccgccg cgagaacagc 2640

cagctgatgc agtggaacca ccgcgccatc gtggccgaga ccaccatgca ggccgagctg 2700

tacggccaga tcgtggagaa caccgccgcc ggcttcagca gccgcttcca cgccgccacc 2760

ggcgcccccg gcgtgcgctg ccgcttcctg ctggagcgcg acttcgacaa cgacctgccc 2820

aagccctacc tgctgcgcga gctgagctgg atgctgggca acaccaaggt ggagagcgag 2880

gaggagaagc tgcgcctgct gagcgagaag atccgccccg gcagcctggt gccctgggac 2940

ggcggcgagc agttcgccac cctgcacccc aagcgccaga ccctgtgcgt gatccacgcc 3000

gacatgaacg ccgcccagaa cctgcagcgc cgcttcttcg gccgctgcgg cgaggccttc 3060

cgcctggtgt gccagcccca cggcgacgac gtgctgcgcc tggccagcac ccccggcgcc 3120

cgcctgctgg gcgccctgca gcagctggag aacggccagg gcgccttcga gctggtgcgc 3180

gacatgggca gcaccagcca gatgaaccgc ttcgtgatga agagcctggg caagaagaag 3240

atcaagcccc tgcaggacaa caacggcgac gacgagctgg aggacgtgct gagcgtgctg 3300

cccgaggagg acgacaccgg ccgcatcacc gtgttccgcg acagcagcgg catcttcttc 3360

ccctgcaacg tgtggatccc cgccaagcag ttctggcccg ccgtgcgcgc catgatctgg 3420

aaggtgatgg ccagccacag cctgggc 3447

<210> 18

<211> 3270

<212> DNA

<213> Laceyella sediminis

<400> 18

atgagcatcc gcagcttcaa gctgaagatc aagaccaaga gcggcgtgaa cgccgaggag 60

ctgcgccgcg gcctgtggcg cacccaccag ctgatcaacg acggcatcgc ctactacatg 120

aactggctgg tgctgctgcg ccaggaggac ctgttcatcc gcaacgagga gaccaacgag 180

atcgagaagc gcagcaagga ggagatccag ggcgagctgc tggagcgcgt gcacaagcag 240

cagcagcgca accagtggag cggcgaggtg gacgaccaga ccctgctgca gaccctgcgc 300

cacctgtacg aggagatcgt gcccagcgtg atcggcaaga gcggcaacgc cagcctgaag 360

gcccgcttct tcctgggccc cctggtggac cccaacaaca agaccaccaa ggacgtgagc 420

aagagcggcc ccacccccaa gtggaagaag atgaaggacg ccggcgaccc caactgggtg 480

caggagtacg agaagtacat ggccgagcgc cagaccctgg tgcgcctgga ggagatgggc 540

ctgatccccc tgttccccat gtacaccgac gaggtgggcg acatccactg gctgccccag 600

gccagcggct acacccgcac ctgggaccgc gacatgttcc agcaggccat cgagcgcctg 660

ctgagctggg agagctggaa ccgccgcgtg cgcgagcgcc gcgcccagtt cgagaagaag 720

acccacgact tcgccagccg cttcagcgag agcgacgtgc agtggatgaa caagctgcgc 780

gagtacgagg cccagcagga gaagagcctg gaggagaacg ccttcgcccc caacgagccc 840

tacgccctga ccaagaaggc cctgcgcggc tgggagcgcg tgtaccacag ctggatgcgc 900

ctggacagcg ccgccagcga ggaggcctac tggcaggagg tggccacctg ccagaccgcc 960

atgcgcggcg agttcggcga ccccgccatc taccagttcc tggcccagaa ggagaaccac 1020

gacatctggc gcggctaccc cgagcgcgtg atcgacttcg ccgagctgaa ccacctgcag 1080

cgcgagctgc gccgcgccaa ggaggacgcc accttcaccc tgcccgacag cgtggaccac 1140

cccctgtggg tgcgctacga ggcccccggc ggcaccaaca tccacggcta cgacctggtg 1200

caggacacca agcgcaacct gaccctgatc ctggacaagt tcatcctgcc cgacgagaac 1260

ggcagctggc acgaggtgaa gaaggtgccc ttcagcctgg ccaagagcaa gcagttccac 1320

cgccaggtgt ggctgcagga ggagcagaag cagaagaagc gcgaggtggt gttctacgac 1380

tacagcacca acctgcccca cctgggcacc ctggccggcg ccaagctgca gtgggaccgc 1440

aacttcctga acaagcgcac ccagcagcag atcgaggaga ccggcgagat cggcaaggtg 1500

ttcttcaaca tcagcgtgga cgtgcgcccc gccgtggagg tgaagaacgg ccgcctgcag 1560

aacggcctgg gcaaggccct gaccgtgctg acccaccccg acggcaccaa gatcgtgacc 1620

ggctggaagg ccgagcagct ggagaagtgg gtgggcgaga gcggccgcgt gagcagcctg 1680

ggcctggaca gcctgagcga gggcctgcgc gtgatgagca tcgacctggg ccagcgcacc 1740

agcgccaccg tgagcgtgtt cgagatcacc aaggaggccc ccgacaaccc ctacaagttc 1800

ttctaccagc tggagggcac cgagctgttc gccgtgcacc agcgcagctt cctgctggcc 1860

ctgcccggcg agaacccccc ccagaagatc aagcagatgc gcgagatccg ctggaaggag 1920

cgcaaccgca tcaagcagca ggtggaccag ctgagcgcca tcctgcgcct gcacaagaag 1980

gtgaacgagg acgagcgcat ccaggccatc gacaagctgc tgcagaaggt ggccagctgg 2040

cagctgaacg aggagatcgc caccgcctgg aaccaggccc tgagccagct gtacagcaag 2100

gccaaggaga acgacctgca gtggaaccag gccatcaaga acgcccacca ccagctggag 2160

cccgtggtgg gcaagcagat cagcctgtgg cgcaaggacc tgagcaccgg ccgccagggc 2220

atcgccggcc tgagcctgtg gagcatcgag gagctggagg ccaccaagaa gctgctgacc 2280

cgctggagca agcgcagccg cgagcccggc gtggtgaagc gcatcgagcg cttcgagacc 2340

ttcgccaagc agatccagca ccacatcaac caggtgaagg agaaccgcct gaagcagctg 2400

gccaacctga tcgtgatgac cgccctgggc tacaagtacg accaggagca gaagaagtgg 2460

atcgaggtgt accccgcctg ccaggtggtg ctgttcgaga acctgcgcag ctaccgcttc 2520

agctacgagc gcagccgccg cgagaacaag aagctgatgg agtggagcca ccgcagcatc 2580

cccaagctgg tgcagatgca gggcgagctg ttcggcctgc aggtggccga cgtgtacgcc 2640

gcctacagca gccgctacca cggccgcacc ggcgcccccg gcatccgctg ccacgccctg 2700

accgaggccg acctgcgcaa cgagaccaac atcatccacg agctgatcga ggccggcttc 2760

atcaaggagg agcaccgccc ctacctgcag cagggcgacc tggtgccctg gagcggcggc 2820

gagctgttcg ccaccctgca gaagccctac gacaaccccc gcatcctgac cctgcacgcc 2880

gacatcaacg ccgcccagaa catccagaag cgcttctggc accccagcat gtggttccgc 2940

gtgaactgcg agagcgtgat ggagggcgag atcgtgacct acgtgcccaa gaacaagacc 3000

gtgcacaaga agcagggcaa gaccttccgc ttcgtgaagg tggagggcag cgacgtgtac 3060

gagtgggcca agtggagcaa gaaccgcaac aagaacacct tcagcagcat caccgagcgc 3120

aagcccccca gcagcatgat cctgttccgc gaccccagcg gcaccttctt caaggagcag 3180

gagtgggtgg agcagaagac cttctggggc aaggtgcaga gcatgatcca ggcctacatg 3240

aagaagacca tcgtgcagcg catggaggag 3270

<210> 19

<211> 3357

<212> DNA

<213> Spirochaetes

<400> 19

atgagcttca ccatcagcta ccccttcaag ctgatcatca agaacaagga cgaggccaag 60

gccctgctgg acacccacca gtacatgaac gagggcgtga agtactacct ggagaagctg 120

ctgatgttcc gccaggagaa gatcttcatc ggcgaggacg agaccggcaa gcgcatctac 180

atcgaggaga ccgagtacaa gaagcagatc gaggagttct acctgatcaa gaagaccgag 240

ctgggccgca acctgaccct gaccctggac gagttcaaga ccctgatgcg cgagctgtac 300

atctgcctgg tgagcagcag catggagaac aagaagggct tccccaacgc ccagcaggcc 360

agcctgaaca tcttcagccc cctgttcgac gccgagagca agggctacat cctgaaggag 420

gagaacaaca acatcagcct gatccacaag gactacggca agatcctgct gaagcgcctg 480

cgcgacaaca acctgatccc catcttcacc aagttcaccg acatcaagaa gatcaccgcc 540

aagctgagcc ccaccgccct ggaccgcatg atcttcgccc aggccatcga gaagctgctg 600

agctacgaga gctggtgcaa gctgatgatc aaggagcgct tcgacaagga ggtgaagatc 660

aaggagctgg agaacaagtg cgagaacaag caggagcgcg acaagatctt cgagatcctg 720

gagaagtacg aggaggagcg ccagaagacc ttcgagcagg acagcggctt cgccaagaag 780

ggcaagttct acatcaccgg ccgcatgctg aagggcttcg acgagatcaa ggagaagtgg 840

ctgaaggaga aggaccgcag cgagcagaac ctgatcaaca tcctgaacaa gtaccagacc 900

gacaacagca agctggtggg cgaccgcaac ctgttcgagt tcatcatcaa gctggagaac 960

cagtgcctgt ggaacggcga catcgactac ctgaagatca agcgcgacat caacaagaac 1020

cagatctggc tggaccgccc cgagatgccc cgcttcacca tgcccgactt caagaagcac 1080

cccctgtggt accgctacga ggaccccagc aacagcaact tccgcaacta caagatcgag 1140

gtggtgaagg acgagaacta catcaccatc cccctgatca ccgagcgcaa caacgagtac 1200

ttcgaggaga actacacctt caacctggcc aagctgaaga agctgagcga gaacatcacc 1260

ttcatcccca agagcaagaa caaggagttc gagttcatcg acagcaacga cgaggaggag 1320

gacaagaagg accagaagaa gagcaagcag tacatcaagt actgcgacac cgccaagaac 1380

accagctacg gcaagagcgg cggcatccgc ctgtacttca accgcaacga gctggagaac 1440

tacaaggacg gcaagaagat ggacagctac accgtgttca ccctgagcat ccgcgactac 1500

aagagcctgt tcgccaagga gaagctgcag ccccagatct tcaacaccgt ggacaacaag 1560

atcaccagcc tgaagatcca gaagaagttc ggcaacgagg agcagaccaa cttcctgagc 1620

tacttcaccc agaaccagat caccaagaag gactggatgg acgagaagac cttccagaac 1680

gtgaaggagc tgaacgaggg catccgcgtg ctgagcgtgg acctgggcca gcgcttcttc 1740

gccgccgtga gctgcttcga gatcatgagc gagatcgaca acaacaagct gttcttcaac 1800

ctgaacgacc agaaccacaa gatcatccgc atcaacgaca agaactacta cgccaagcac 1860

atctacagca agaccatcaa gctgagcggc gaggacgacg acctgtacaa ggagcgcaag 1920

atcaacaaga actacaagct gagctaccag gagcgcaaga acaagatcgg catcttcacc 1980

cgccagatca acaagctgaa ccagctgctg aagatcatcc gcaacgacga gatcgacaag 2040

gagaagttca aggagctgat cgagaccacc aagcgctacg tgaagaacac ctacaacgac 2100

ggcatcatcg actggaacaa cgtggacaac aagatcctga gctacgagaa caaggaggac 2160

gtgatcaacc tgcacaagga gctggacaag aagctggaga tcgacttcaa ggagttcatc 2220

cgcgagtgcc gcaagcccat cttccgcagc ggcggcctga gcatgcagcg catcgacttc 2280

ctggagaagc tgaacaagct gaagcgcaag tgggtggccc gcacccagaa gagcgccgag 2340

agcatcgtgc tgacccccaa gttcggctac aagctgaagg agcacatcaa cgagctgaag 2400

gacaaccgcg tgaagcaggg cgtgaactac atcctgatga ccgccctggg ctacatcaag 2460

gacaacgaga tcaagaacga cagcaagaag aagcagaagg aggactgggt gaagaagaac 2520

cgcgcctgcc agatcatcct gatggagaag ctgaccgagt acaccttcgc cgaggaccgc 2580

ccccgcgagg agaacagcaa gctgcgcatg tggagccacc gccagatctt caacttcctg 2640

cagcagaagg ccagcctgtg gggcatcctg gtgggcgacg tgttcgcccc ctacaccagc 2700

aagtgcctga gcgacaacaa cgcccccggc atccgctgcc accaggtgac caagaaggac 2760

ctgatcgaca acagctggtt cctgaagatc gtggtgaagg acgacgcctt ctgcgacctg 2820

atcgagatca acaaggagaa cgtgaagaac aagagcatca agatcaacga catcctgccc 2880

ctgcgcggcg gcgagctgtt cgccagcatc aaggacggca agctgcacat cgtgcaggcc 2940

gacatcaacg ccagccgcaa catcgccaag cgcttcctga gccagatcaa ccccttccgc 3000

gtggtgctga agaaggacaa ggacgagacc ttccacctga agaacgagcc caactacctg 3060

aagaactact acagcatcct gaacttcgtg cccaccaacg aggagctgac cttcttcaag 3120

gtggaggaga acaaggacat caagcccacc aagcgcatca agatggacaa gcacgagaag 3180

gagagcaccg acgagggcga cgactacagc aagaaccaga tcgccctgtt ccgcgacgac 3240

agcggcatct tcttcgacaa gagcctgtgg gtggacggca agatcttctg gagcgtggtg 3300

aagaacaaga tgaccaagct gctgcgcgag cgcaacaaca agaagaacgg cagcaag 3357

<210> 20

<211> 3426

<212> DNA

<213> Tuberibacillus calidus

<400> 20

atgaacatcc acctgaagga gctgatccgc atggccacca agagcttcat cctgaagatg 60

aagaccaaga acaaccccca gctgcgcctg agcctgtgga agacccacga gctgttcaac 120

ttcggcgtgg cctactacat ggacctgctg agcctgttcc gccagaagga cctgtacatg 180

cacaacgacg aggaccccga ccaccccgtg gtgctgaaga aggaggagat ccaggagcgc 240

ctgtggatga aggtgcgcga gacccagcag aagaacggct tccacggcga ggtgagcaag 300

gacgaggtgc tggagaccct gcgcgccctg tacgaggagc tggtgcccag cgccgtgggc 360

aagagcggcg aggccaacca gatcagcaac aagtacctgt accccctgac cgaccccgcc 420

agccagagcg gcaagggcac cgccaacagc ggccgcaagc cccgctggaa gaagctgaag 480

gaggccggcg accccagctg gaaggacgcc tacgagaagt gggagaagga gcgccaggag 540

gaccccaagc tgaagatcct ggccgccctg cagagcttcg gcctgatccc cctgttccgc 600

cccttcaccg agaacgacca caaggccgtg atcagcgtga agtggatgcc caagagcaag 660

aaccagagcg tgcgcaagtt cgacaaggac atgttcaacc aggccatcga gcgcttcctg 720

agctgggaga gctggaacga gaaggtggcc gaggactacg agaagaccgt gagcatctac 780

gagagcctgc agaaggagct gaagggcatc agcaccaagg ccttcgagat catggagcgc 840

gtggagaagg cctacgaggc ccacctgcgc gagatcacct tcagcaacag cacctaccgc 900

atcggcaacc gcgccatccg cggctggacc gagatcgtga agaagtggat gaagctggac 960

cccagcgccc cccagggcaa ctacctggac gtggtgaagg actaccagcg ccgccacccc 1020

cgcgagagcg gcgacttcaa gctgttcgag ctgctgagcc gccccgagaa ccaggccgcc 1080

tggcgcgagt accccgagtt cctgcccctg tacgtgaagt accgccacgc cgagcagcgc 1140

atgaagaccg ccaagaagca ggccaccttc accctgtgcg accccatccg ccaccccctg 1200

tgggtgcgct acgaggagcg cagcggcacc aacctgaaca agtaccgcct gatcatgaac 1260

gagaaggaga aggtggtgca gttcgaccgc ctgatctgcc tgaacgccga cggccactac 1320

gaggagcagg aggacgtgac cgtgcccctg gcccccagcc agcagttcga cgaccagatc 1380

aagttcagca gcgaggacac cggcaagggc aagcacaact tcagctacta ccacaagggc 1440

atcaactacg agctgaaggg caccctgggc ggcgcccgca tccagttcga ccgcgagcac 1500

ctgctgcgcc gccagggcgt gaaggccggc aacgtgggcc gcatcttcct gaacgtgacc 1560

ctgaacatcg agcccatgca gcccttcagc cgcagcggca acctgcagac cagcgtgggc 1620

aaggccctga aggtgtacgt ggacggctac cccaaggtgg tgaacttcaa gcccaaggag 1680

ctgaccgagc acatcaagga gagcgagaag aacaccctga ccctgggcgt ggagagcctg 1740

cccaccggcc tgcgcgtgat gagcgtggac ctgggccagc gccaggccgc cgccatcagc 1800

atcttcgagg tggtgagcga gaagcccgac gacaacaagc tgttctaccc cgtgaaggac 1860

accgacctgt tcgccgtgca ccgcaccagc ttcaacatca agctgcccgg cgagaagcgc 1920

accgagcgcc gcatgctgga gcagcagaag cgcgaccagg ccatccgcga cctgagccgc 1980

aagctgaagt tcctgaagaa cgtgctgaac atgcagaagc tggagaagac cgacgagcgc 2040

gagaagcgcg tgaaccgctg gatcaaggac cgcgagcgcg aggaggagaa ccccgtgtac 2100

gtgcaggagt tcgagatgat cagcaaggtg ctgtacagcc cccacagcgt gtgggtggac 2160

cagctgaaga gcatccaccg caagctggag gagcagctgg gcaaggagat cagcaagtgg 2220

cgccagagca tcagccaggg ccgccagggc gtgtacggca tcagcctgaa gaacatcgag 2280

gacatcgaga agacccgccg cctgctgttc cgctggagca tgcgccccga gaaccccggc 2340

gaggtgaagc agctgcagcc cggcgagcgc ttcgccatcg accagcagaa ccacctgaac 2400

cacctgaagg acgaccgcat caagaagctg gccaaccaga tcgtgatgac cgccctgggc 2460

taccgctacg acggcaagcg caagaagtgg atcgccaagc accccgcctg ccagctggtg 2520

ctgttcgagg acctgagccg ctacgccttc tacgacgagc gcagccgcct ggagaaccgc 2580

aacctgatgc gctggagccg ccgcgagatc cccaagcagg tggcccagat cggcggcctg 2640

tacggcctgc tggtgggcga ggtgggcgcc cagtacagca gccgcttcca cgccaagagc 2700

ggcgcccccg gcatccgctg ccgcgtggtg aaggagcacg agctgtacat caccgagggc 2760

ggccagaagg tgcgcaacca gaagttcctg gacagcctgg tggagaacaa catcatcgag 2820

cccgacgacg cccgccgcct ggagcccggc gacctgatcc gcgaccaggg cggcgacaag 2880

ttcgccaccc tggacgagcg cggcgagctg gtgatcaccc acgccgacat caacgccgcc 2940

cagaacctgc agaagcgctt ctggacccgc acccacggcc tgtaccgcat ccgctgcgag 3000

agccgcgaga tcaaggacgc cgtggtgctg gtgcccagcg acaaggacca gaaggagaag 3060

atggagaacc tgttcggcat cggctacctg cagcccttca agcaggagaa cgacgtgtac 3120

aagtgggtga agggcgagaa gatcaagggc aagaagacca gcagccagag cgacgacaag 3180

gagctggtga gcgagatcct gcaggaggcg agcgtgatgg ccgacgagct gaagggcaac 3240

cgcaagaccc tgttccgcga ccccagcggc tacgtgttcc ccaaggaccg ctggtacacc 3300

ggcggccgct acttcggcac cctggagcac ctgctgaagc gcaagctggc cgagcgccgc 3360

ctgttcgacg gcggcagcag ccgccgcggc ctgttcaacg gcaccgacag caacaccaac 3420

gtggag 3426

<210> 21

<211> 2870

<212> DNA

<213> Artificial Sequence

<220>