一种核酸检测试剂盒、检测方法及应用

本申请是申请日为2020年05月19日申请号为202010426283.1、发明创造名称为“一种核酸检测试剂盒、检测方法及应用”的专利申请的分案申请。

技术领域

本发明属于生物技术领域，特别涉及一种包括Cpf1检测体系的核酸检测试剂盒、检测方法。本发明还涉及所述核酸检测试剂盒的应用，特别是在严重急性呼吸综合征相关冠状病毒和中东呼吸综合征冠状病毒的核酸的快速检测中的应用。

背景技术

冠状病毒属于套式病毒目Nidovirales，冠状病毒科Coronaviridae，冠状病毒属Coronavirus[6]。成熟的冠状病毒表面有囊膜，囊膜表面覆有纤突，纤突末端呈球形，故整个纤突呈花瓣状或梨状，纤突之间有较宽的间隙。电镜下，囊膜纤突规则地排列成王冠状，病毒颗粒因此而得名。目前尚无针对高度传染性的冠状病毒的有效疫苗和治疗药物，及时诊断和隔离是控制该病暴发流行的有效方法。

快速、高效的核酸快速检测是临床上病毒性疾病防控的有效方法，针对病毒性疾病现有的检测方法多以核酸检测为主，WHO发布的严重急性呼吸系统综合症(Severe AcuteRespiratory Syndrome，缩写为SARS)和中东呼吸综合征冠状病毒(Middle Eastrespiratory syndrome coronavirus，MERS-CoV)的实验室确诊程序指导文件中推荐使用实时反转录聚合酶链式反应(real time reverse transcription PCR，rRT-PCR)方法对病毒进行检测确诊。rRT-PCR检测病毒核酸主要受制于：昂贵的PCR实验设备、能进行熟练稳定操作的工作人员人数、检测时间较长等。免疫学检测方法也是病毒检测的重要手段，如双抗体夹心ELISA、胶体金免疫层析试纸等方法。免疫学检查容易受到病程的影响，早期病人体内抗体较少，需要等待一定的时间才可以产生相应的抗体，容易出现漏诊；患病后的康复病人体内可能携带该类抗体，容易导致免疫诊断出现假阳性。为了有效的控制病毒性传染疾病的传播，需要建立一种简便、快速、特异的新型检测方法。

CRISPR-Cas(Clustered regularly interspaced short palindromic repeats，CRISPRs)是细菌中的一种适应性免疫系统，Cas蛋白通过RNA引导的核酸酶靶向降解外来核酸[1,2]。其中，CRISPR-Cpf1(Cpf1)属于Cas酶第二家族，用来引导RNA指导双链DNA裂解单个RuvC催化结构域。Cpf1酶识别富含胸腺嘧啶(Thymine，T)核苷酸的间隔区相邻基序(PAM)[3]，催化它们自身的指导CRISPR RNA(crRNA)成熟[4]，并特异性识别和切割互补配对的双链DNA(dsDNA)[3]。当CRISPR/Cpf1蛋白以序列特异性方式识别切割靶双链DNA时，能诱导强大的非特异性单链DNA(ssDNA)反式切割活性[5]。Cpf1是具有核酸内切酶活性，核酸内切酶的配体离子通常为镁离子。现有以Cpf1为基础的核酸检测，信号强度仍旧不高，从而导致在实际检测中的检测时间过长，低浓度样品检测效率低等缺陷。因此还需要进一步增强检测信号的强度以满足实际检测的需要。此外，在检测核酸时的灵敏度、特异性和稳定性也迫切需要提高。

发明内容

本发明所要解决的技术问题是为了克服现有技术中检测核酸时成本过高、检测试剂较长、检测效率低、特异性和灵敏性不够高等缺陷，提供了一种核酸检测试剂盒、一种突变的Cpf1蛋白、一种crRNA、检测方法及应用。利用本发明所述的核酸检测试剂盒、突变的Cpf1蛋白或crRNA检测核酸(例如检测SARS相关冠状病毒或MERS冠状病毒的核酸)时，可以显著提高检测时的信号强度从而减少检测时间、提高检测效率，且灵敏度高、特异性高、准确性高、检测可视化、成本低、无需复杂大型实验设备、操作简便。

Cpf1是具有核酸内切酶活性，而在本领域中核酸内切酶的配体离子通常为镁离子。而本发明人首次发现锰离子能够促进Cpf1对核酸探针(例如单链DNA探针)的切割能力，进而提高Cpf1检测的信号强度，从而可用于增强Cpf1介导的体外检测的信号以减少检测时间、提高检测效率。此外，本发明还通过大量实验，意外发现当将某些Cpf1蛋白进行特定突变后，也能够显著提高检测核酸时的信号强度。本发明人还首次采用Cpf1荧光法检测SARS相关冠状病毒和MERS冠状病毒，经过大量实验摸索设计出能够用于检测这些病毒的crRNA，将其用于检测病毒时灵敏度和特异性非常高，且准确性也很高。

为了解决上述技术问题，本发明第一方面提供了一种核酸检测试剂盒，其包括Cpf1检测体系，所述Cpf1检测体系包括：向导RNA、Cpf1蛋白、核酸探针和含锰离子的溶液。

较佳地，所述含锰离子的溶液为硫酸锰、氯化锰或醋酸锰溶液。

较佳地，所述核酸探针为单链DNA探针，所述单链DNA探针优选包括荧光标记，更优选在其5’端和3’端分别具有荧光基团和荧光淬灭基团，进一步更优选为在其5’端具有荧光基团和3’端具有荧光淬灭基团。

其中，所述荧光基团可以为本领域常规，例如为6-FAM、TET、CY3、CY5或ROX。所述荧光淬灭基团可以为本领域常规，例如为BHQ1、BHQ2或BHQ3。所述单链DNA探针的序列按照本领域常规方法进行制备，例如可以为TTTATTT。

较佳地，所述Cpf1蛋白选自AsCpf1(Acidaminococcus sp.BV3L6)、BbCpf1(Beauveria bassiana KA00251)、BoCpf1(Bacteroidetes oral)、FnCpf1(Francisellanovicida U112)、HkCpf1(Helcococcus kunzii)、Lb4Cpf1(Lachnospiraceae bacteriumMC2017)、Lb5Cpf1(Lachnospiraceae bacterium NC2008)、LbCpf1(Lachnospiraceaebacterium ND2006)、Oscpf1(Oribacterium sp.)和TsCpf1(Thiomicrospira sp.XS5)中的一种或多种。

较佳地，所述Cpf1蛋白为经过密码子优化的Cpf1蛋白，其核苷酸序列优选如SEQID NO.14-23中的一个或多个所示。

较佳地，所述Cpf1蛋白为突变的Cpf1蛋白，所述突变的Cpf1蛋白优选其氨基酸序列与天然Cpf1蛋白具有98％优选99％以上的序列同源性。更佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.55所示的K180、E184、N607和K613中的一个或多个位点突变为R后的序列，或将如SEQ ID NO.54所示的T148、T152、G532和K538中的一个或多个位点突变为R后的序列。例如可以是将如SEQ ID NO.55所示的E184和N607突变为R后的序列，可以是将如SEQ ID NO.55所示的E184、N607和K613突变为R后的序列，可以是将如SEQ ID NO.55所示的K180、E184、N607和K613突变为R后的序列，可以是将如SEQ ID NO.54所示的T152和G532突变为R后的序列，可以是将如SEQ ID NO.54所示的T152、G532和K538突变为R后的序列，可以是将如SEQ ID NO.54所示的T148、T152、G532和K538突变为R后的序列。进一步更佳地，所述突变的Cpf1蛋白的核苷酸序列如SEQ ID NO.30-41所示。

较佳地，所述向导RNA为引导Cpf1蛋白特异性结合所述核酸的RNA，优选为crRNA；所述crRNA优选为检测病毒核酸的crRNA；更优选为检测SARS相关冠状病毒或MERS-CoV病毒的crRNA；所述SARS相关冠状病毒优选为冠状病毒SARS-CoV(严重急性呼吸综合征冠状病毒)或SARS-CoV-2(严重急性呼吸综合征冠状病毒2型)。更佳地，所述crRNA为检测SARS-CoV病毒orf1ab基因的crRNA、检测MERS-CoV病毒orf1a基因的crRNA、检测MERS-CoV病毒upE基因的crRNA、检测SARS-CoV-2病毒E基因的crRNA、检测SARS-CoV-2病毒S基因的crRNA、检测SARS-CoV-2病毒M基因的crRNA和/或检测SARS-CoV-2病毒N基因的crRNA，所述orf1ab基因的核苷酸序列如SEQ ID NO.11所示，所述orf1a基因的核苷酸序列如SEQ ID NO.12所示，所述upE基因的核苷酸序列如SEQ ID NO.13所示，所述E基因的核苷酸序列如SEQ ID NO.46所示，所述SARS-CoV-2-S基因的核苷酸序列如SEQ ID NO.79所示；所述SARS-CoV-2-M基因的核苷酸序列如SEQ ID NO.80所示；所述SARS-CoV-2-N基因的核苷酸序列如SEQ ID NO.81所示。进一步更佳地，所述crRNA的核苷酸序列优选如SEQ ID NO.1-10、SEQ ID NO.47、SEQ IDNO.48和SEQ ID NO.53、SEQ ID NO.56-72中的一个或多个所示。

较佳地，所述含锰离子的溶液中锰离子的浓度为5-600mM，例如6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500mM等。所列点值仅为示例，并不是限制于此。并且应当理解的是，在这浓度范围内的任意点值均可以自由组合，从而任意组合所行成的浓度范围均应该在本发明保护的范围内。例如5-50、5-100、5-200、5-300、5-400、5-500、100-200、100-300、200-300等等这些均应该在本发明的保护范围内。在实验中发明人发现锰离子的工作浓度在0.5-60mM都有效果，试剂盒中一般是混合的10倍的储液，故储液浓度可以是5-600mM。在本发明某一较佳实施例中，所用的含锰离子的溶液中锰离子的浓度为100mM。

较佳地，所述核酸探针的浓度为1-100pmol/μL；优选25pmol/μL。

较佳地，所述Cpf1蛋白的浓度为20-1000ng/μL；优选200ng/μL。

较佳地，所述向导RNA的浓度为0.1-50μM；优选1μM。

较佳地，所述核酸检测试剂盒还包括RNA酶抑制剂和缓冲液；所述RNA酶抑制剂的浓度优选为10-200U/μL，优选40U/μL。

较佳地，所述缓冲液优选包括NaCl、Tris和BSA，所述缓冲液的pH优选7.9。在本发明某一较佳实施例中，所用缓冲液为10×缓冲液，其组成为：1000mM NaCl，500mM Tris，1000ug/ml BSA，pH 7.9。

为了解决上述技术问题，本发明第二方面提供了一种突变的Cpf1蛋白，其序列包括将SEQ ID NO.55所示的K180、E184、N607和K613中的一个或多个位点突变为R后的序列；或者，其序列包括将SEQ ID NO.54所示的T148、T152、G532和K538中的一个或多个位点突变为R后的序列。

较佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.55所示的E184和N607突变为R后的序列。

较佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.55所示的E184、N607和K613突变为R后的序列。

较佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.55所示的K180、E184、N607和K613突变为R后的序列。

较佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.54所示的T152和G532突变为R后的序列。

较佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.54所示的T152、G532和K538突变为R后的序列。

较佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.54所示的或T148、T152、G532和K538突变为R后的序列。

更佳地，所述突变的Cpf1蛋白的核苷酸序列如SEQ ID NO.30-41所示。

为了解决上述技术问题，本发明第三方面提供了一种核酸检测试剂盒，其包括Cpf1检测体系，所述Cpf1检测体系包括：向导RNA、核酸探针和如本发明第二方面所述的突变的Cpf1蛋白。

较佳地，所述向导RNA为引导Cpf1蛋白特异性结合所述核酸的RNA，优选为crRNA；所述crRNA优选为检测病毒核酸的crRNA；更优选为检测SARS相关冠状病毒或MERS-CoV病毒的crRNA；所述SARS相关冠状病毒优选为冠状病毒SARS-CoV或SARS-CoV-2；所述crRNA优选为检测SARS-CoV病毒orf1ab基因的crRNA、检测MERS-CoV病毒orf1a基因的crRNA、检测MERS-CoV病毒upE基因的crRNA、检测SARS-CoV-2病毒E基因的crRNA、检测SARS-CoV-2病毒S基因的crRNA、检测SARS-CoV-2病毒M基因的crRNA和/或检测SARS-CoV-2病毒N基因的crRNA，所述orf1ab基因的核苷酸序列如SEQ ID NO.11所示，所述orf1a基因的核苷酸序列如SEQ ID NO.12所示，所述upE基因的核苷酸序列如SEQ ID NO.13所示，所述E基因的核苷酸序列如SEQ ID NO.46所示，所述SARS-CoV-2-S基因的核苷酸序列如SEQ ID NO.79所示；所述SARS-CoV-2-M基因的核苷酸序列如SEQ ID NO.80所示；所述SARS-CoV-2-N基因的核苷酸序列如SEQ ID NO.81所示。更佳地，所述crRNA的核苷酸序列如SEQ ID NO.1-10、SEQ IDNO.47、SEQ ID NO.48和SEQ ID NO.53、SEQ ID NO.56-72中的一个或多个所示。

较佳地，所述核酸探针为单链DNA探针，所述单链DNA探针优选包括荧光标记，更优选为在其两端分别具有荧光基团和荧光淬灭基团，进一步更优选为在5’端具有荧光基团和3’端具有荧光淬灭基团。所述荧光基团可为本领域常规，例如为6-FAM、TET、CY3、CY5或ROX。所述荧光淬灭基团可为本领域常规，例如为BHQ1、BHQ2或BHQ3。所述单链DNA探针可按本领域常规方法进行制备，其序列例如可以为TTTATTT。

较佳地，所述向导RNA的浓度为0.1-50μM；优选1μM。

较佳地，所述突变的Cpf1蛋白的浓度为20-1000ng/μL；优选200ng/μL。

较佳地，所述核酸探针的浓度为1-100pmol/μL；优选25pmol/μL。

较佳地，所述核酸检测试剂盒还包括含金属离子的溶液，例如含镁离子的溶液、含锰离子的溶液等；所述溶液中金属离子的浓度优选为5-600mM，例如6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500mM等。所列点值仅为示例，并不是限制于此。并且应当理解的是，在这浓度范围内的任意点值均可以自由组合，从而任意组合所行成的浓度范围均应该在本发明保护的范围内。例如5-50、5-100、5-200、5-300、5-400、5-500、100-200、100-300、200-300等等这些均应该在本发明的保护范围内。在实验中发明人发现金属离子的工作浓度在0.5-60mM都有效果，试剂盒中一般是混合的10倍的储液，故储液浓度可以是5-600mM。在本发明某一较佳实施例中，所用的含金属离子的溶液中金属离子的浓度为100mM。

较佳地，所述核酸检测试剂盒还包括RNA酶抑制剂和缓冲液；所述RNA酶抑制剂的浓度优选为10-200U/μL，优选40U/μL。

较佳地，所述缓冲液优选包括NaCl、Tris和BSA，所述缓冲液的pH优选7.9。在本发明某一较佳实施例中，所用缓冲液为10×缓冲液，其组成为：1000mM NaCl，500mM Tris，1000ug/ml BSA，pH 7.9。

为了解决上述技术问题，本发明第四方面提供了一种检测冠状病毒核酸的crRNA，所述crRNA为检测SARS-CoV病毒orf1ab基因的crRNA、检测MERS-CoV病毒orf1a基因的crRNA、检测MERS-CoV病毒upE基因的crRNA、检测SARS-CoV-2病毒E基因的crRNA、检测SARS-CoV-2病毒S基因的crRNA、检测SARS-CoV-2病毒M基因的crRNA和/或检测SARS-CoV-2病毒N基因的crRNA，所述orf1ab基因的核苷酸序列如SEQ ID NO.11所示，所述orf1a基因的核苷酸序列如SEQ ID NO.12所示，所述upE基因的核苷酸序列如SEQ ID NO.13所示，所述E基因的核苷酸序列如SEQ ID NO.46所示，所述SARS-CoV-2-S基因的核苷酸序列如SEQ ID NO.79所示；所述SARS-CoV-2-M基因的核苷酸序列如SEQ ID NO.80所示；所述SARS-CoV-2-N基因的核苷酸序列如SEQ ID NO.81所示。

较佳地，所述crRNA为检测SARS-CoV病毒orf1ab基因的crRNA的核苷酸序列如SEQID NO.1-3中的一个或多个所示。

较佳地，所述检测MERS-CoV病毒orf1a基因的crRNA的核苷酸序列如SEQ ID NO.4-7中的一个或多个所示。

较佳地，所述检测MERS-CoV病毒upE基因的crRNA的核苷酸序列如SEQ ID NO.8-10中的一个或多个所示。

较佳地，所述检测SARS-CoV-2病毒E基因的crRNA的核苷酸序列如SEQ ID NO.47、SEQ ID NO.48和SEQ ID NO.53中的一个或多个所示。

较佳地，所述检测SARS-CoV-2病毒S基因的crRNA的核苷酸序列如SEQ ID NO.56-63中的一个或多个所示。

较佳地，所述检测SARS-CoV-2病毒M基因的crRNA的核苷酸序列如SEQ ID NO.64-68中的一个或多个所示。

较佳地，所述检测SARS-CoV-2病毒N基因的crRNA的核苷酸序列如SEQ ID NO.69-72中的一个或多个所示。

在本发明某一较佳实施例中，所述crRNA的制备方法包括：针对SARS病毒核酸orf1ab基因，其序列为SEQ ID NO.11，寻找包含cpf1识别序列(PAM)TTTN的靶向序列，设计44bp长度的crRNA。针对MERS病毒核酸upE基因，其序列为SEQ ID NO.12，寻找包含cpf1识别序列(PAM)TTTN的靶向序列，设计44bp长度的crRNA。针对MERS病毒核酸Orf1基因，其序列为SEQ ID NO.13，寻找包含cpf1识别序列(PAM)TTTN的靶向序列，设计44bp长度的crRNA。针对SARS-CoV-2病毒E基因，其序列为SEQ ID NO.46，寻找包含cpf1识别序列(PAM)TTTN的靶向序列，设计44bp长度的crRNA。针对SARS-CoV-2病毒S基因，其序列为SEQ ID NO.79，寻找包含cpf1识别序列(PAM)TTTN的靶向序列，设计44bp长度的crRNA。针对SARS-CoV-2病毒M基因，其序列为SEQ ID NO.80，寻找包含cpf1识别序列(PAM)TTTN的靶向序列，设计44bp长度的crRNA。针对SARS-CoV-2病毒N基因，其序列为SEQ ID NO.81，寻找包含cpf1识别序列(PAM)TTTN的靶向序列，设计44bp长度的crRNA。设计完成后，制备crRNA。crRNA的制备，可构建到载体pUC57-T7-crRNA，通过体外转录获得目的crRNA；或可公司合成RNA序列。

为了解决上述技术问题，本发明第五方面提供了一种核酸检测试剂盒，其包括Cpf1检测体系，所述Cpf1检测体系包括：如本发明第四方面所述的crRNA、Cpf1蛋白和核酸探针。

较佳地，所述Cpf1蛋白选自AsCpf1(Acidaminococcus sp.BV3L6)、BbCpf1(Beauveria bassiana KA00251)、BoCpf1(Bacteroidetes oral)、FnCpf1(Francisellanovicida U112)、HkCpf1(Helcococcus kunzii)、Lb4Cpf1(Lachnospiraceae bacteriumMC2017)、Lb5Cpf1(Lachnospiraceae bacterium NC2008)、LbCpf1(Lachnospiraceaebacterium ND2006)、Oscpf1(Oribacterium sp.)和TsCpf1(Thiomicrospira sp.XS5)、或与其氨基酸序列具有98％优选99％以上序列同源性的一种或多种。

较佳地，所述Cpf1蛋白为经过密码子优化的Cpf1蛋白，其核苷酸序列优选如SEQID NO.14-23中的一个或多个所示。

较佳地，所述Cpf1蛋白为突变的Cpf1蛋白，所述突变的Cpf1蛋白优选其氨基酸序列与天然Cpf1蛋白具有98％优选99％以上的序列同源性。更佳地，所述突变的Cpf1蛋白的序列包括将如SEQ ID NO.55所示的K180、E184、N607和K613中的一个或多个位点突变为R后的序列，或将如SEQ ID NO.54所示的T148、T152、G532和K538中的一个或多个位点突变为R后的序列。例如可以是将如SEQ ID NO.55所示的E184和N607突变为R后的序列，可以是将如SEQ ID NO.55所示的E184、N607和K613突变为R后的序列，可以是将如SEQ ID NO.55所示的K180、E184、N607和K613突变为R后的序列，可以是将如SEQ ID NO.54所示的T152和G532突变为R后的序列，可以是将如SEQ ID NO.54所示的T152、G532和K538突变为R后的序列，可以是将如SEQ ID NO.54所示的T148、T152、G532和K538突变为R后的序列。进一步更佳地，所述突变的Cpf1蛋白的核苷酸序列如SEQ ID NO.30-41所示。

较佳地，所述核酸探针为单链DNA探针，所述单链DNA探针优选包括荧光标记，更优选为在其两端分别具有荧光基团和荧光淬灭基团，进一步更优选为在5’端具有荧光基团和3’端具有荧光淬灭基团。所述荧光基团可以为本领域常规，例如为6-FAM、TET、CY3、CY5或ROX。所述荧光淬灭基团可以为本领域常规，例如为BHQ1、BHQ2或BHQ3。所述单链DNA探针可按本领域常规方法制备，其序列例如可以为TTTATTT。

较佳地，所述crRNA的浓度为0.1-50μM；优选1μM。

较佳地，所述Cpf1蛋白的浓度为20-1000ng/μL；优选200ng/μL。

较佳地，所述核酸探针的浓度为1-100pmol/μL；优选25pmol/μL。

较佳地，所述核酸检测试剂盒还包括含金属离子的溶液，例如含镁离子的溶液、含锰离子的溶液等；所述溶液中金属离子的浓度优选为5-600mM，例如6、7、8、9、10、20、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450或500mM等。所列点值仅为示例，并不是限制于此。并且应当理解的是，在这浓度范围内的任意点值均可以自由组合，从而任意组合所行成的浓度范围均应该在本发明保护的范围内。例如5-50、5-100、5-200、5-300、5-400、5-500、100-200、100-300、200-300等等这些均应该在本发明的保护范围内。在实验中发明人发现金属离子的工作浓度在0.5-60mM都有效果，试剂盒中一般是混合的10倍的储液，故储液浓度可以是5-600mM。在本发明某一较佳实施例中，所用的含金属离子的溶液中金属离子的浓度为100mM。

较佳地，所述核酸检测试剂盒还包括RNA酶抑制剂和缓冲液；所述RNA酶抑制剂的浓度优选为10-200U/μL，优选40U/μL。

较佳地，所述缓冲液优选包括NaCl、Tris和BSA，所述缓冲液的pH优选7.9。在本发明某一较佳实施例中，所用缓冲液为10×缓冲液，其组成为：1000mM NaCl，500mM Tris，1000ug/ml BSA，pH 7.9。

为了解决上述技术问题，本发明第六方面提供了一种核酸的检测方法，利用如本发明第一、三、五方面所述的核酸检测试剂盒中所述的Cpf1检测体系检测所述核酸。

较佳地，利用核酸快速释放试剂释放待测样品中的核酸。

较佳地，所述核酸通过扩增得到，优选利用RT-RPA扩增待测样品中的核酸。其中，所述扩增时的温度优选为25±5min，和/或，所述扩增的时间优选为39±5℃，和/或，所述扩增时的引物优选如SEQ ID NO.24-29、SEQ ID NO.44-45、SEQ ID NO.73-78所示。

较佳地，所述Cpf1检测体系的反应温度为37±5℃。

较佳地，所述Cpf1检测体系的反应时间25±5min。

较佳地，所述方法还包括读取结果的步骤，优选利用酶标仪或荧光灯下肉眼进行读取。

在本发明某一较佳实施例中，所述检测方法包括以下步骤：

步骤a：利用核酸快速释放试剂对样品病毒灭活和释放待测样品中核酸；

步骤b：利用等温扩增引物扩增待测样品中核酸：将步骤a获得产物，利用SARS病毒核酸orf1ab基因片段的特异性引物SEQ ID NO.24和SEQ ID NO.25，或者MERS病毒核酸upE基因片段的特异性引物SEQ ID NO.26和SEQ ID NO.27，或者MERS病毒核酸upE基因片段的特异性引物，SEQ ID NO.28和SEQ ID NO.29，或者SARS-CoV-2病毒E基因片段的特异性引物，SEQ ID NO.44和SEQ ID NO.45加入RT-RPA等温扩增体系，或者SARS-CoV-2病毒S基因片段的特异性引物，SEQ ID NO.73和SEQ ID NO.74加入RT-RPA等温扩增体系，或者SARS-CoV-2病毒M基因片段的特异性引物，SEQ ID NO.75和SEQ ID NO.76加入RT-RPA等温扩增体系，或者SARS-CoV-2病毒N基因片段的特异性引物，SEQ ID NO.77和SEQ ID NO.78加入RT-RPA等温扩增体系，在37℃反应30min扩增获得特异性产物；

步骤c：利用Cpf1检测体系识别切割SARS病毒核酸orf1ab或者MERS病毒核酸upE基因片段或者MERS病毒核酸Orf1基因片段或者SARS-CoV-2病毒E基因片段或者SARS-CoV-2病毒S基因片段或者SARS-CoV-2病毒M基因片段或者SARS-CoV-2病毒N基因片段时：将步骤b中产物加入Cpf1检测体系，于37℃反应25min；

步骤d：利用荧光酶标仪或荧光灯肉眼直接检测判读待检测样品中是否存在病毒核酸。

为了解决上述技术问题，本发明第七方面提供了如本发明第一、三、五方面所述的核酸检测试剂盒、如本发明第二方面所述的突变的Cpf1蛋白或如本发明第四方面所述的crRNA在检测核酸优选病毒核酸，或制备检测核酸优选病毒核酸的试剂中的应用。

较佳地，所述病毒为SARS相关冠状病毒或MERS-CoV病毒；所述SARS相关冠状病毒优选为SARS-CoV或SARS-CoV-2。

本发明提供的用于SARS和MERS病毒核酸快速检测的核酸检测试剂盒既可利用酶标仪荧光检测也可以利用荧光肉眼检测。本发明中核酸探针(例如ssDNA FQ reporter)，当使用酶标仪荧光检测时，设定检测激发光为485nm-520nm；当使用肉眼直接检测时，选用可产生485nm波长光源的发光器进行检测。

本发明中，通常可以对临床样品进行病毒灭活处理，释放待检样品中核酸。在等温条件下，对病毒RNA进行反转录(RT)获得DNA，并进行重组酶聚合酶扩增(RPA)[9]。在使用荧光检测时，当Cpf1检测体系中存在病毒核酸，会由在特异性crRNA介导下特异性激活Cpf1蛋白的核酸内切酶活性。激活后的Cpf1蛋白会切割由荧光基团和淬灭基团标记的ssDNAFQreporter，从而释放激活荧光基团，使用酶标仪可以检测到荧光读数或肉眼可见绿色反应。相对应的，待检测样品中不存在病毒核酸时，不会有荧光读数或肉眼不可见绿色反应。

本发明中，所述的核酸探针一般也可称为荧光-淬灭单链DNA(ssDNA)报告系统，其一般含有荧光基团和淬灭基团，在完整状态荧光被淬灭基团淬灭，在反应过程中被切割之后，从而发出荧光。在本发明某一较佳实施例中，所述核酸探针为5’端-6-FAM/TTTATTT/BHQ1-3’端。

在本发明某一较佳实施例中，所述Cpf1蛋白的制备方法可以包括：针对多种不同来源的cpf1蛋白核酸序列进行原核密码子优化获得序列SEQ ID NO.14-23，构建到pET28a表达载体，进行低温诱导可溶蛋白表达，通过亲和纯化和分子筛纯化获得目的蛋白。

为了解决上述技术问题，本发明还提供了一种检测受试者中是否含有某种病毒(例如被某种病毒感染)的方法，其包括使用如本发明第一、三、五方面所述的核酸检测试剂盒、如本发明第二方面所述的突变的Cpf1蛋白或如本发明第四方面所述的crRNA进行检测。检测的目的是判断受试者中是否含有该病毒的核酸。

术语解释

术语crRNA是指CRISPR RNA，是短的引导Cas12a(Cpf1)到结合到靶标DNA序列的RNA。

术语CRISPR是指成簇的、规律间隔的短回文重复序列(clustered regularlyinterspaced short palindromic repeats)，该序列是许多原核生物的免疫系统。

术语Cas蛋白是指CRISPR-associated蛋白，它是CRISPR系统中的相关蛋白。

术语Cpf1(又称Cas12a)是指crRNA依赖的内切酶，它是CRISPR系统分类中V型(type V)的酶。

术语PAM是指前间区序列邻近基序(protospacer-adjacent motif)，是Cas12a切割所必须，FnCas12a的PAM为TTN序列，LbCas12a的PAM为TTTN序列。

术语RT-RPA(reverse transcription-recombinase polymeraseamplification)为本领域常规含义，通常是指反转录等温扩增，又称逆转录重组酶聚合酶扩增。

本发明中，所提供的核苷酸序列，一般默认是包含了终止子，这对于本领域技术人员来说都是可被理解的。

在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。

本发明所用试剂和原料均市售可得。

本发明的积极进步效果在于：

利用本发明所述的核酸检测试剂盒、突变的Cpf1蛋白或crRNA检测核酸(例如检测SARS相关冠状病毒或MERS冠状病毒的核酸)时，可以显著提高检测时的信号强度从而减少检测时间、提高检测效率，且灵敏度高、特异性高、准确性高、检测可视化(可在荧光灯下肉眼直接可视检测)、成本低、无需复杂大型实验设备、操作简便。这些优势使得本发明的试剂盒和检测方法更加适用于基层实验和临床一线对病毒例如SARS相关冠状病毒和MERS冠状病毒的基层快速检测和鉴定诊断。在本发明某一较佳实施例中，检测病毒时的信号强度较现有技术提高了高达3-5倍不等。在本发明某一较佳实施例中，检测病毒的DNA样品时的检测灵敏度为1E8个拷贝病毒，检测病毒的RNA样品时的检测灵敏度为5个拷贝病毒。

附图说明

图1为增强型Cpf1快速检测病毒核酸方法示意图。

图2为不同金属离子对于不同Cpf1信号的影响。

图3为锰离子对于不同Cpf1蛋白荧光检测效果的影响。

图4为不同锰盐对于不同Cpf1蛋白荧光检测效果的影响。

图5为不同crRNA检测SARS和MERS病毒不同基因荧光检测效果。

图6为增强型Cpf1荧光检测SARS和MERS病毒DNA灵敏度。

图7为增强型Cpf1荧光检测SARS和MERS病毒DNA灵敏度。

图8为荧光检测Cpf1快速检测SARS和MERS病毒DNA特异性。

图9为荧光检测Cpf1快速检测SARS和MERS病毒RNA特异性。

图10为模拟检测临床核酸样品荧光检测结果。

图11为锰离子对于不同Cpf1突变蛋白荧光检测效果的影响。

图12为不同crRNA检测SARS-CoV-2病毒基因的荧光检测效果。

图13为模拟检测临床核酸样品荧光检测结果。

图14为针对SARS-CoV-2的S基因、M基因和N基因的检测结果。

图15为cpf1的不同突变体通过酶标仪检测SARS-CoV2的E基因的结果图。

图16为cpf1的不同突变体通过酶标仪检测SARS-CoV2的S基因的结果图。

图17为cpf1的不同突变体通过酶标仪检测SARS-CoV2的M基因的结果图。

图18为cpf1的不同突变体通过酶标仪检测SARS-CoV2的N基因的结果图。

具体实施方式

下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。

本发明中，RPA扩增试剂盒

本发明的总体技术示意图如图1所示，包括以下3大部分：待检测核酸样品制备、Cpf1检测成分设计制备和体系构建和荧光检测。

实施例1：灵敏度增强型Cpf1检测方法

1.不同金属离子对于Cpf1检测的荧光强度的影响

本实施例中的不同物种的Cpf1基因经过密码子优化后，序列分别如SEQ IDNO.14-23(密码子优化后的核苷酸序列分别对应AsCpf1、BbCpf1、BoCpf1、FnCpf1(氨基酸序列如SEQ ID NO.55所示)、HkCpf1、Lb4Cpf1、Lb5Cpf1、LbCpf1(氨基酸序列如SEQ ID NO.54所示)、OsCpf1、TsCpf1)所示，将其克隆到pet28a质粒中(由南京金斯瑞公司完成该步骤)，在大肠杆菌中表达，经过纯化后用于检测实验。

增强型Cpf1检测采用20μL体系如表1所示，但不限于此，包含对相应成分的比例调整，金属离子包括CaCl

表1病毒cpf1检测体系

其中，10×缓冲液的组成为：1000mM NaCl，500mM Tris，1000ug/ml BSA，pH 7.9。ssDNA FQ reporter为6FAM/TTTATTT/3BHQ1。

依次检测不同的金属离子针对MERS病毒核酸的crRNA3(具体信息如下表2-1中的SEQ ID NO.6所示)检测效率。在Cpf1检测体系中，各加入1μL 100mM金属离子母液，其它各组分维持一致，混合均匀，37℃反应30min，对上述反应产物进行后续结果检测判定。

利用荧光检测判定Cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，读取检测30min荧光数值为反应值。其反应检测结果如图2。结果表明，本发明检测体系中，针对AsCpf1、FnCpf1和LbCpf1，锰离子均能提高信号强度，其荧光强度较其他金属离子显著提高，较镁离子、铁离子提高3-5倍不等(图2中显示了AsCpf1、BbCpf1、BoCpf1的结果图，其他物种的Cpf1所得结果类似)。

2.锰离子对于不同种属的Cpf1检测的荧光强度的影响

依次检测锰离子和镁离子对于不同种属的Cpf1的检测的荧光强度的影响。在Cpf1的检测体系中，各加入1μL上述不同种属的Cpf1，包括AsCpf1、BbCpf1、BoCpf1、FnCpf1、HkCpf1、Lb4Cpf1、Lb5Cpf1、LbCpf1、OsCpf1、TsCpf1，其它各组分维持一致(同第1部分)，混合均匀，37℃反应30min，对上述反应产物进行后续荧光强度检测判定，用锰离子的荧光信号除以镁离子的荧光信号，其结果如图3所示。结果表明，本发明的不同种属的Cpf1检测体系中，锰离子均能提高荧光信号强度，锰离子相对于镁离子的应该强度增加比例为3.2-8倍不等。

3.锰离子对于不同种属的Cpf1突变蛋白检测的荧光强度的影响

依次检测锰离子和镁离子对于不同种属的Cpf1(LbCpf1和FnCpf1)的突变蛋白检测的荧光强度的影响。在Cpf1的检测体系中，各加入1μL不同种属的Cpf1突变蛋白，包括LbCpf1(氨基酸序列如SEQ ID NO.54所示)、LbCpf1-T148R(SEQ ID NO.36)、LbCpf1-T152R(SEQ ID NO.37)、LbCpf1-G532R(SEQ ID NO.38)、LbCpf1-K536R(SEQ ID NO.42)、LbCpf1-K538R(SEQ ID NO.39)、LbCpf1-T152R/G532R(SEQ ID NO.40)、LbCpf1-T152R/G532R/K538R(SEQ ID NO.41)、FnCpf1(氨基酸序列如SEQ ID NO.55所示)、FnCpf1-K180R(SEQ IDNO.30)、FnCpf1-E184R(SEQ ID NO.31)、FnCpf1-N607R(SEQ ID NO.32)、FnCpf1-K611R(SEQID NO.43)、FnCpf1-K613R(SEQ ID NO.33)、FnCpf1-E184R/N607R(SEQ ID NO.34)、FnCpf1-E184R/N607R/K613R(SEQ ID NO.35)，其它各组分维持一致(同第1部分)，混合均匀，37℃反应30min，对上述反应产物进行后续荧光强度检测，检测结果如图11所示。结果表明，本发明的不同种属的Cpf1突变检测体系中，部分突变能够增强信号强度，但是LbCpf1-K536R和FnCpf1-K611R不能增加信号强度。锰离子能提高具有活性的突变蛋白的荧光信号强度，锰离子相对于镁离子的应该强度增加比例为3-5倍不等。此外，相对于未突变的Cpf1蛋白而言，Cpf1的突变蛋白也相应提高了荧光信号强度，其中突变体FnCpf1-E184R、FnCpf1-N607R、FnCpf1-K613R、FnCpf1-E184R/N607R、FnCpf1-E184R/N607R/K613R和LbCpf1-T152R、LbCpf1-G532R、LbCpf1-K538R、LbCpf1-T152R/G532R、LbCpf1-T152R/G532R/K538R相对于其未突变的蛋白而言，荧光信号强度提高了1/2-1倍不等。

4.不同形式的锰盐对于信号的增强作用

依次检测MnSO

实施例2：SARS和MERS病毒基因片段快速灵敏检测

2.1核酸制备

本实施例中，SARS-CoV病毒orf1ab基因参考NCBI上的AY278487基因，MERS病毒upE基因片段参考NCBI上的JX869059基因，MERS病毒ORF1a基因片段参考NCBI上的JX869059基因，SARS-CoV-2病毒E基因片段、S基因片段、M基因片段和N基因片段参考NCBI上的NC045512基因组，均由南京金斯瑞公司合成，分别命名为SARS-ORF1ab、MERS-upE、MERS-ORF1a、SARS-CoV-2-E、SARS-CoV-2-S、SARS-CoV-2-M、SARS-CoV-2-N对应序列分别为SEQ ID NO.11-13、SEQ ID No.46、SEQ ID No.79-81。

通过体外转录制备SARS-CoV病毒orf1ab基因，MERS病毒upE基因片段、ORF1a、SARS-CoV-2病毒E基因、SARS-CoV-2病毒S基因、SARS-CoV-2病毒M基因、SARS-CoV-2病毒N基因对应的RNA核酸样品。具体操作如下：使用MEGAshortscript T7转录试剂盒(ThermoFisher Scientific)，分别以上述SARS-ORF1ab、MERS-upE和ORF1a、SARS-CoV-2-E、SARS-CoV-2-S、SARS-CoV-2-M、SARS-CoV-2-N作为模板转录对应的基因RNA样品pcRNA-SARS-orf1ab、pcRNA-MERS-upE和pcRNA-MERS-ORF1a、pcRNA-SARS-CoV-2-E、pcRNA-SARS-CoV-2-S、pcRNA-SARS-CoV-2-M、pcRNA-SARS-CoV-2-N。转录后的RNA用MEGAclear试剂盒(ThermoFisher Scientific)纯化，并通过乙醇沉淀法回收。检测转录后所得单链RNA(ssRNA)的质量和浓度，并保存在-80℃直至使用。

2.2针对特异性crRNA的设计制备

针对特异性crRNA制备按照如下方案进行，针对SARS-ORF1ab、MERS-upE基因、MERS-ORF1a基因、SARS-CoV-2-E基因、SARS-CoV-2-S基因、SARS-CoV-2-M基因、SARS-CoV-2-N基因，寻找包含cpf1识别序列(PAM，Protospacer Adjacent Motif)TTTN的靶向序列，设计44bp长度的crRNA。分别命名为针对SARS-ORF1ab基因的SARS-orf1ab-crRNA1到SARS-orf1a-crRNA3、针对MERS-upE基因的MERS-upE-crRNA1到MERS-upE-crRNA4、针对MERS-ORF1a基因的MERS-ORF1a-crRNA1到MERS-ORF1a-crRNA3、针对SARS-CoV2-E基因的Cov2-E-crRNA1到CoV2-E-crRNA7、针对SARS-CoV2-S基因的Cov2-S-cr3到CoV2-S-cr10、针对SARS-CoV2-M基因的Cov2-M-cr1到CoV2-M-cr5、针对SARS-CoV2-N基因的Cov2-N-cr1到CoV2-N-cr4。设计完成后，制备crRNA。crRNA的制备，可交与南京金斯瑞公司合成oligo，构建到载体pUC57-T7-crRNA，通过体外转录获得目的crRNA，或可交由南京金斯瑞公司直接合成crRNA序列对应的RNA。

本发明提供的SARS-ORF1ab、MERS-upE基因、MERS-ORF1a基因和SARS-CoV-2-E基因、SARS-CoV-2-S基因、SARS-CoV-2-M基因、SARS-CoV-2-N基因的crRNA包括SEQ ID NO.1到SEQ ID NO.10、SEQ ID NO.47到SEQ ID NO.53、SEQ ID NO.56到SEQ ID NO.72，具体信息如表2-1和表2-2所示：

表2-1针对SARS和MERS病毒核酸特异性crRNA

表2-2针对SARS和MERS病毒核酸特异性crRNA

2.3反转录等温扩增反应

本实施例中，利用反转录等温扩增(RT-RPA)对上述2.1中所制备的单链RNA(ssRNA)病毒核酸进行预扩增，以便进行cpf1检测反应。按照等温扩增反应要求，设计并合成RT-RPA扩增引物RT-RPA-F(正向引物)和RT-RPA-R(反向引物)(表3)。参考RT-RPA等温扩增操作步骤，扩增获得待检测样品。具体操作如下：等温扩增在50μL反应体系进行。将xμLRNA样品，(18.5-x)μL ddH

表3针对冠状病毒CoV核酸特异性RT-RPA引物序列

2.4本实施例中，增强型cpf1检测采用20μL体系如表4所示，但不限于此，包含对相应成分的比例调整：

表4病毒cpf1检测体系

2.5全波长酶标仪荧光检测

酶标仪荧光检测中，Cpf1对目的基因检测体系依次加入2μL 10×缓冲液、1μLRnase Inhibitors、1μL Cpf1、1μL ssDNA FQ reporter、5μL上述2.3中所得RT-RPA产物样品、1μL crRNA和9μL H

首先，依次检测针对SARS-CoV病毒orf1ab基因、MERS病毒upE基因、MERS病毒ORF1a基因、SARS-CoV-2病毒E基因、SARS-CoV-2病毒S基因、SARS-CoV-2病毒M基因、SARS-CoV-2病毒N基因的crRNA检测效率。在Cpf1检测体系中，各加入1μL crRNA，其它各组分维持一致，混合均匀，37℃反应30min，对上述反应产物进行后续结果检测判定。

利用荧光检测判定Cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，读取检测30min荧光数值为反应值。针对SARS-CoV病毒和MERS病毒的不同crRNA，其反应检测结果如图5。结果表明，本发明检测体系中，针对不同基因片段的crRNA均能高效特异的检测对应的基因片段。针对SARS-CoV-2的E基因的检测结果如图12所示，其中，CoV2-E-Cr4和CoV2-E-Cr5无法检出，CoV2-E-Cr3和CoV2-E-Cr6检测效率较低，CoV2-E-Cr1、CoV2-E-Cr2和CoV2-E-Cr7检测效率较高。后续实验将CoV2-E-Cr1、CoV2-E-Cr2和CoV2-E-Cr7混合在一起制备SARS-CoV2-Crmix用于后续检测SARS-CoV-2-E基因。针对SARS-CoV-2的S基因、M基因和N基因的检测结果如图14所示，针对不同基因片段的crRNA均能高效特异的检测对应的基因片段。其中，CoV2-S-Cr3、CoV2-S-Cr5和CoV2-S-Cr6对SARS-CoV-2-S基因的检测效率较高，后续实验将这三个crRNA混合，制备成CoV2-S-Crmix用于后续监测SARS-CoV-2-S基因；其中CoV2-M-Cr1、CoV2-M-Cr2和CoV2-M-Cr4对SARS-CoV-2-M基因的检测效率较高，后续实验将这三个crRNA混合，制备成CoV2-M-Crmix用于后续监测SARS-CoV-2-M基因；其中CoV2-N-Cr1、CoV2-N-Cr2和CoV2-N-Cr6对SARS-CoV-2-N的检测效率较高，后续实验将这三个crRNA混合，制备成CoV2-N-Crmix用于后续监测SARS-CoV-2-N基因。

鉴于本实施例中针对SARS-ORF1ab和MERS-upE基因的crRNA有一致的检测效果，将针对SARS-ORF1ab基因的SARS-O-crRNA1到SARS-O-crRNA3等比例混合获得SARS-O-Crmix；将针对MERS-upE基因的MERS-E-crRNA1到MERS-E-crRNA4等比例混合获得MERS-E-Crmix；将针对MERS-Orf基因的MERS-O-crRNA1到MERS-O-crRNA3等比例混合获得MERS-O-Crmix。后续检测中，SARS-O-Crmix检测SARS-ORF1ab基因，MERS-E-Crmix检测MERS-upE基因，MERS-O-Crmix检测MERS-Orf基因。

实施例3：基于cpf1荧光法高灵敏度检测病毒核酸

本实施例中，为测定cpf1荧光法对检测灵敏度的影响，进行以下检测。

(1)首先，根据实施例2中2.1所述的SARS-ORF1ab、MERS-upE和MERS-Orf1基因片段对应的DNA样品，计算检测片段分子量，并进行梯度稀释，获得每微升含有5E9、1E9、5E8、1E8、5E7、1E7拷贝数(copy/μL)的测试样品。

利用荧光法，对上述稀释样品进行cpf1特异性反应，参照实施例2中检测步骤进行检测和荧光结果判读。操作简述如下：1μL梯度稀释样品，加入到20μL cpf1荧光核酸检测体系(其中所添加的crRNA即为上述检测SARS-ORF1ab基因的SARS-O-Crmix、检测MERS-upE基因的MERS-E-Crmix或检测MERS-Orf基因的MERS-O-Crmix)，混合均匀，37℃反应30min进行荧光结果判读。

本实施例中，应用增强型cpf1荧光法检测SARS和MERS病毒，分别采用酶标仪检测(结果见图6)，可实现对1E8个拷贝病毒的高灵敏度检出，灵敏度高。

(2)其次，根据实施例2中2.1所述的SARS-ORF1ab、MERS-upE和MERS-Orf1基因片段对应的RNA样品，计算检测片段分子量，并进行梯度稀释，获得每微升含有1000、100、10、5拷贝数(copy/μL)的测试样品。

利用荧光法，对上述稀释样品进行cpf1特异性反应，参照实施例2中检测步骤进行检测和荧光结果判读。操作简述如下：1μL梯度稀释样品，加入到50μL RT-RPA等温扩增反应体系进行扩增。取10μL等温扩增产物，加入到20μL cpf1荧光核酸检测体系(其中所添加的crRNA即为上述检测SARS-ORF1ab基因的SARS-O-Crmix、检测MERS-upE基因的MERS-E-Crmix或检测MERS-Orf基因的MERS-O-Crmix)，混合均匀，37℃反应25min进行荧光结果判读。

本实施例中，应用cpf1荧光法检测SARS和MERS病毒RNA，分别采用酶标仪检测(结果见图7)，可实现对5个拷贝病毒的高灵敏度检出，灵敏度高。

实施例4：基于cpf1荧光法高特异性检测病毒核酸

本实施例中，为检测cpf1荧光法能否进行高特异反应，能否有效区分SARS-CoV病毒和MERS病毒，进行以下检测。

(1)首先，针对病毒DNA的特异性进行测试。

根据本发明中针对SARS-CoV病毒和MERS病毒核酸检测区段SARS-ORF1ab和MERS-upE，参照实施例2中的方法，制备MERS病毒对应基因序列DNA样品：SARS-ORF1ab，MERS-upE和MERS-Orf1。

参照实施例2，将上述DNA样品混合，在增强型Cpf1检测体系中，依次加入2μLBuffer、1μLRnase Inhibitors、1μL Cpf1、1μL ssDNA FQ reporter、1μL crRNA(其中所添加的crRNA即为上述检测SARS-ORF1ab基因的SARS-O-Crmix、检测MERS-upE基因的MERS-E-Crmix或检测MERS-Orf基因的MERS-O-Crmix)和1μL检测样品。各组分混合均匀，在37℃反应30min。本检测体系中，Rnase Inhibitors浓度为40U/μL，Cpf1浓度为200ng/μL，ssDNA DBreporter浓度为25pM/μL，crRNA浓度为1nM/μL。

本实施例中，利用荧光检测判定Cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，取检测30min时荧光数值为反应值。检测结果如图8a(图8中的T1、T2、T3、T4分别为配制样品，其中，T1：已知是没有SARS或者MERS-upE基因的DNA，T2：已知是含有SARS基因的DNA；T3：已知是含有MERS-upE基因的DNA；T4：已知是含有SARS和MERS-upE基因的DNA。图8中的MERS-Crmix是指上述MERS-E-Crmix，SARS-Crmix是指上述SARS-O-Crmix)。同时，将上述cpf1检测反应30min后产物，置于在485nm激光灯下，可由肉眼直接进行结果判读。当crRNA特异性识别靶核酸片段后，反应产物颜色由无色变为荧光绿；对应的，如果待检测无对应靶核酸，反应产物颜色仍为无色。在cpf1检测反应30min后，在荧光下进行肉眼判读，并拍照记录。如图8b所示，针对不同基因片段的crRNA均能高效特异的检测对应的基因片段，特异性高且检测结果准确。

(2)其次，针对病毒RNA的特异性进行测试。

根据本发明中针对SARS-CoV病毒和MERS病毒核酸检测区段SARS-ORF1ab，MERS-upE和MERS-Orf1，参照实施例2中方法，制备MERS病毒对应基因序列RNA样品：SARS-ORF1ab，MERS-upE和MERS-Orf1。

参照实施例2，通过RT-RPA反应，分别扩增制备SARS-CoV病毒和MERS病毒检测样品。将RNA样品混合，在增强型Cpf1检测体系中，依次加入2μL Buffer、1μLRnaseInhibitors、1μL Cpf1、1μL ssDNA FQ reporter、1μL crRNA和10μL检测样品。各组分混合均匀，在37℃反应30min。本检测体系中，Rnase Inhibitors浓度为40U/μL，Cpf1浓度为200ng/μL，ssDNA DB reporter浓度为25pM/μL，crRNA浓度为1nM/μL。

本实施例中，利用荧光检测判定Cpf1检测体系检测活性。使用全波长酶标仪用于测量检测反应的荧光，其中激发波长为485nm，发射波长为520nm，取检测30min时荧光数值为反应值。检测结果如图9a(图9中的T5、T6、T7、T8分别为配制样品，其中，T5：已知是没有SARS或者MERS-upE基因的RNA，T6：已知是含有SARS基因的RNA；T7：已知是含有MERS-upE基因的RNA；T8：已知是含有SARS和MERS-upE基因的RNA。图9中的MERS-Crmix是指MERS-E-Crmix，SARS-Crmix是指上述SARS-O-Crmix)。同时，将上述cpf1检测反应30min后产物，置于在485nm激光灯下，可由肉眼直接进行结果判读。当crRNA特异性识别靶核酸片段后，反应产物颜色由无色变为荧光绿；对应的，如果待检测无对应靶核酸，反应产物颜色仍为无色。在cpf1检测反应30min后，在荧光下进行肉眼判读，并拍照记录。如图9b所示，针对不同基因片段的crRNA均能高效特异的检测对应的基因片段，特异性高且检测结果准确。

实施例5：模拟cpf1荧光法快速检测临床病毒核酸

本实施例模拟临床组织样品的核酸进行快速检测，本检测不涉及任何临床SARS和MERS病毒样品，且所有操作合格按照有关法律法规和相关规定进行。

本实施例中，首先，将实施例2中SARS-CoV病毒核酸对应RNA样品(SARS-orf1ab)和MERS病毒核酸对应RNA样品(MERS-orf1ab和MERS-upE)，点涂到咽拭子，模拟临床样品进行检测。

(1)首先，对病毒灭活和病毒核酸释放。本实施例使用购自诺唯赞公司的快速核酸释放剂获得预处理的核酸。步骤如下：将咽拭子加入50μL PBS，模拟溶解病毒到液体。取3μl待检测样品加入20μl核酸裂解液，加入RNA酶抑制剂，常温静置3分钟，加入20μL中和液，混匀后进行下一步检测。在Cpf1检测中，每个待检测样品取5μL进行RT-RPA预扩增，步骤与实施例1中相同，获得Cpf1检测样品。

在Cpf1检测体系中，依次加入2μL Buffer、1μL Rnase Inhibitors、1μL Cpf1、1μLssDNA FQ reporter、10μL RPA sample、1μL crRNA。各组分混合均匀，在37℃反应25min。

本实施例中，可通过荧灯裸眼观察或者酶标仪直接判定cpf1检测结果。如图10所示，本发明中cpf1荧光法可通过酶标仪检测可有效对模拟临床阴阳性样品判定。同样的，在荧光灯下裸眼可有效对模拟临床阴阳性样品判定(图10，图中的CS1-CS6分别为配制样品，其中，CS1、CS2和CS6为已知是SARS-CoV病毒阳性的样品，其余均为已知是SARS阴性和MERS阴性的样品。图中的MERS-Crmix是MERS-E-Crmix，SARS-Crmix是指上述SARS-O-Crmix)。可见本发明的检测方法快速、可视、成本低、操作简便的同时，所得结果准确性还很高。

(2)类似的，模拟临床制备CS7到CS12的样品，其中CS8、CS10和CS11已知为SARS-CoV-2阳性的样品，CS9已知为MERS-CoV阳性的样品，其余均为已知是SARS-CoV-2阴性和MERS-CoV阴性的样品。通过与上述(1)中类似的方法进行检测，然后通过荧灯裸眼观察或者酶标仪直接判定cpf1检测结果。如图13所示(图中MERS-Crmix是MERS-E-Crmix)，本发明中cpf1荧光法可通过酶标仪检测可有效对模拟临床阴阳性样品判定。可见本发明的检测方法准确性很高。

此外，综合上述实施例的结果进一步可知，将反应体系中Cpf1的配体离子替换为锰离子时，在检测不同的病毒(SARS-CoV、MERS-CoV、SARS-CoV-2等)核酸时都能够显著提高检测的信号强度，可见该锰离子体系具有普适性。

实施例6：模拟cpf1荧光法快速检测临床SARS-CoV-2病毒核酸的不同基因

本实施例模拟临床组织样品的核酸进行快速检测，本检测不涉及任何临床SARS-CoV-2病毒样品，且所有操作合格按照有关法律法规和相关规定进行。

本实施例中，首先，将SARS-CoV-2病毒核酸对应RNA样品(CoV2-E-基因，CoV2-S-基因,CoV2-M-基因,CoV2-N-基因)，点涂到咽拭子，模拟临床样品进行检测。

(1)首先，对病毒灭活和病毒核酸释放。本实施例使用购自诺唯赞公司的快速核酸释放剂获得预处理的核酸。步骤如下：将咽拭子加入50μL PBS，模拟溶解病毒到液体。取3μl待检测样品加入20μl核酸裂解液，加入RNA酶抑制剂，常温静置3分钟，加入20μL中和液，混匀后进行下一步检测。在Cpf1检测中，每个待检测样品取5μL进行RT-RPA预扩增，步骤与实施例1中相同，获得Cpf1检测样品。

在Cpf1检测体系中，依次加入2μL Buffer、1μL Rnase Inhibitors、1μL Cpf1或Cpf1突变蛋白、1μL ssDNA FQ reporter、10μL RPA sample、1μL crRNA、加入1μL Mn

本实施例中，可通过酶标仪直接判定cpf1及其突变的检测结果。如图15、16、17、18所示，本发明中cpf1的不同突变体可以通过酶标仪检测SARS-CoV2的S基因、M基因、N基因和E基因。在加入Mg

1.R,B.,et al.,CRISPR provides acquired resistance against viruses inprokaryotes.Science(New York,N.Y.),2007.315(5819):p.1709-12.

2.LA,M.and S.EJ,CRISPR interference limits horizontal gene transferin staphylococci by targeting DNA.Science(New York,N.Y.),2008.322(5909):p.1843-5.

3.B,Z.,et al.,Cpf1 is a single RNA-guided endonuclease of a class2CRISPR-Cas system.Cell,2015.163(3):p.759-71.

4.I,F.,et al.,The CRISPR-associated DNA-cleaving enzyme Cpf1 alsoprocesses precursor CRISPR RNA.Nature,2016.532(7600):p.517-21.

5.JS,C.,et al.,CRISPR-Cpf1 target binding unleashes indiscriminatesingle-stranded DNase activity.Science(New York,N.Y.),2018.360(6387):p.436-439.

6.González,JM.et al.A comparative sequence analysis to revise thecurrent taxonomy of the family Coronaviridae.,Gomez-Puertas P,Cavanagh D,Gorbalenya AE,Enjuanes L.Arch Virol.2003；148(11):p.2207-35.

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SEQUENCE LISTING

<110> 上海科技大学

<120> 一种核酸检测试剂盒、检测方法及应用

<130> P20015854CD

<150> 202010251367.6

<151> 2020-04-01

<160> 81

<170> PatentIn version 3.5

<210> 1

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> SARS-O-Cr1

<400> 1

uaauuucuac uaaguguaga uuacagguua gcuaacgagu gugc 44

<210> 2

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> SARS-O-Cr2

<400> 2

uaauuucuac uaaguguaga uucaagcugu uacagccaau guaa 44

<210> 3

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> SARS-O-Cr3

<400> 3

uaauuucuac uaaguguaga uaacugaugg uaauaagaua gcug 44

<210> 4

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> MC-E-crRNA1

<400> 4

uaauuucuac uaaguguaga ugacauaugg aaaacgaacu augu 44

<210> 5

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> MC-E-crRNA2

<400> 5

uaauuucuac uaaguguaga uuccaagaac gaauaggguu guuc 44

<210> 6

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> MC-E-crRNA3

<400> 6

uaauuucuac uaaguguaga ucuaugaaca acccuauucg uucu 44

<210> 7

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> MC-E-crRNA4

<400> 7

uaauuucuac uaaguguaga ucauaugucc aaagagagac uaau 44

<210> 8

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> MC-O-Cr1

<400> 8

uaauuucuac uaaguguaga uacuuaugca aacauagucu acga 44

<210> 9

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> MC-O-Cr2

<400> 9

uaauuucuac uaaguguaga ugucagcgcu gauugcaguu gcaa 44

<210> 10

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> MC-O-Cr3

<400> 10

uaauuucuac uaaguguaga ucaacugcaa ucagcgcuga cgaa 44

<210> 11

<211> 444

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-Orf1ab

<400> 11

taatacgact cactataggt ctagaaataa ttttgtttaa ctttaagaag gagatatacc 60

atgctaacat gcttaggata atggcctctc ttgttcttgc tcgcaaacat aacacttgct 120

gtaacttatc acaccgtttc tacaggttag ctaacgagtg tgcgcaagta ttaagtgaga 180

tggtcatgtg tggcggctca ctatatgtta aaccaggtgg aacatcatcc ggtgatgcta 240

caactgctta tgctaatagt gtctttaaca tttgtcaagc tgttacagcc aatgtaaatg 300

cacttctttc aactgatggt aataagatag ctgacaagta tgtccgcaat ctacaacaca 360

ggctctatga gtgtctctat agaaataggg atgttgatca tgaattcgtg gatgagtttt 420

acgcttacct gtttttttga attc 444

<210> 12

<211> 345

<212> DNA

<213> Artificial Sequence

<220>

<223> MERS-upE

<400> 12

taatacgact cactataggt ctagaaataa ttttgtttaa ctttaagaag gagatatacc 60

atgctgtttt cgtgcctgca acgcgcgatt cagttcctct tcacataatc gccccgagct 120

cgcttatcgt ttaagcagct ctgcgctact atgggtcccg tgtagaggct aatccattag 180

tctctctttg gacatatgga aaacgaacta tgttaccctt tgtccaagaa cgaatagggt 240

tgttcatagt aaactttttc atttttaccg tagtatgtgc tataacactc ttggtgtgta 300

tggctttcct tacggctact agattatgtg tgtttttttg aattc 345

<210> 13

<211> 449

<212> DNA

<213> Artificial Sequence

<220>

<223> MERS-Orf1a

<400> 13

taatacgact cactataggg agatctagaa ataattttgt ttaactttaa gaaggagata 60

taccatgact attcccacac agttgttccc actcttattt gtgactatgg ccttcgttat 120

gttgttggtt aaacacaaac acaccttttt gacacttttc ttgttgcctg tggctatttg 180

tttgacttat gcaaacatag tctacgagcc cactactccc atttcgtcag cgctgattgc 240

agttgcaaat tggcttgccc ccactaatgc ttatatgcgc actacacata ctgatattgg 300

tgtctacatt agtatgtcac ttgtattagt cattgtagtg aagagattgt acaacccatc 360

actttctaac tttgcgttag cattgtgcag tggtgtaatg tggttgtaca cttatagcat 420

tggagaagcc tcaagctttt tttgaattc 449

<210> 14

<211> 3924

<212> DNA

<213> Artificial Sequence

<220>

<223> AsCpf1

<400> 14

atgactcagt tcgaggggtt caccaatttg taccaggtct ctaagacact tcgctttgag 60

cttatccctc agggcaagac gctgaagcat atccaggagc aaggatttat cgaggaggac 120

aaggcacgta atgaccacta taaagagctg aaaccgatca ttgatcgcat ttacaagacc 180

tatgcagatc agtgtctgca gcttgtgcaa ttagattggg aaaatctgag cgccgcaatc 240

gactcctatc gtaaagagaa gactgaagaa acacgtaatg cattaattga agaacaggcc 300

acgtatcgca atgctatcca cgattatttt attgggcgca ccgacaattt aactgatgcc 360

atcaataagc gtcacgcaga gatctacaag ggacttttta aagccgaatt atttaatggc 420

aaggttttaa aacagcttgg tactgtaaca acaacagagc acgagaatgc ccttctgcgc 480

tcgttcgata agtttaccac gtacttctcg ggcttttacg aaaaccgcaa aaacgtgttc 540

tcggcggagg acatctctac agctatccca caccgtattg tacaagataa cttccccaaa 600

tttaaggaga attgtcacat tttcacgcgt ttgattactg cggtaccgtc tttacgtgag 660

catttcgaaa acgttaagaa ggcaattggc atttttgtca gcacgtctat tgaagaagtg 720

ttctcgtttc ccttctacaa tcagctgttg actcaaacgc agatcgactt atataaccag 780

ctgttgggtg gcatctcacg cgaggcaggc acagaaaaaa ttaaaggcct gaacgaggtc 840

ttgaatttgg ctatccagaa gaacgatgag accgcgcaca tcattgcttc cttacctcat 900

cgctttattc cgttgtttaa acaaattctt agtgatcgta atactttatc cttcatctta 960

gaggaattta aatccgacga ggaggttatt cagtccttct gcaaatataa aactctgtta 1020

cgcaacgaga acgtgcttga aaccgcggag gctttattca acgagctgaa ctcgattgac 1080

ttgacacata ttttcatctc acacaagaaa ctggaaacga tttcgtcagc cctttgcgac 1140

cactgggata ctctgcgtaa cgcattatat gaacgtcgca tttccgaatt gacaggaaaa 1200

atcactaaga gcgcaaagga gaaggtacag cgttcgctga aacatgaaga catcaacctt 1260

caggagatta tctctgcggc gggcaaagaa cttagtgaag catttaagca aaaaaccagt 1320

gagatcctta gccatgcgca cgctgctctt gaccaaccac ttccgacaac gctgaaaaaa 1380

caggaggaaa aggagattct gaagagccag ctggattctt tacttggact ttatcatttg 1440

ttggattggt ttgccgtgga cgaaagtaac gaagtagatc ccgaattttc ggcgcgcctt 1500

accggcatca agttggaaat ggaaccctca ctgtcgttct acaataaagc ccgcaactac 1560

gccactaaaa agccctattc ggtagaaaag tttaagttga actttcagat gccaactctt 1620

gcttcaggat gggatgtgaa caaagaaaag aataacggag ctattttgtt cgttaagaac 1680

ggtctgtatt atcttggaat catgccgaaa cagaagggac gttacaaagc gctttcgttc 1740

gaaccaacag aaaagacctc cgaggggttc gataagatgt attacgacta ttttccggac 1800

gcagcaaaaa tgatcccgaa gtgctcaacg cagctgaaag ctgtgactgc gcattttcaa 1860

actcacacga cccccatttt gttatccaat aatttcatcg aacctttgga aattacgaag 1920

gagatctatg acttaaacaa tcctgaaaaa gagccgaaaa aatttcaaac cgcgtacgcc 1980

aagaaaaccg gggaccaaaa agggtaccgc gaagctcttt gcaaatggat cgacttcact 2040

cgtgattttc tttctaagta tacgaaaact acgtcgattg acttatcgag tttacgccca 2100

tcctctcaat acaaggatct gggggaatat tatgccgaac tgaatcctct tttatatcac 2160

atcagcttcc aacgcatcgc cgaaaaagag attatggacg cggtcgagac gggaaagctt 2220

tacttatttc aaatctataa taaagatttc gccaaaggtc atcacgggaa acccaatctg 2280

cacacccttt attggacagg tctgttctct cccgaaaact tagcgaaaac atccattaag 2340

cttaacgggc aggcagagtt gttctatcgt cccaaatccc gtatgaagcg catggcacac 2400

cgtcttggag aaaaaatgct taacaaaaag cttaaagacc aaaaaactcc gatccctgat 2460

accctttacc aagagttata tgattatgtc aatcatcgct tatcacatga cttgtccgac 2520

gaggcacgcg cacttttacc aaacgtcatc acgaaggaag tcagtcacga aatcattaag 2580

gatcgtcgtt tcacatccga taaatttttt tttcacgtac caattacttt gaactatcag 2640

gctgccaata gtccgagcaa gttcaatcaa cgtgtaaacg cgtacttaaa ggagcacccc 2700

gaaaccccca ttatcgggat cgaccgcggc gaacgcaatc ttatctatat cacggttatc 2760

gattctactg gaaaaatttt agagcagcgt tccctgaaca ccattcaaca gtttgattat 2820

cagaagaaac ttgacaatcg cgaaaaagaa cgtgttgcgg cgcgtcaagc atggagcgtt 2880

gttggtacaa tcaaagacct taaacaaggt tacttatcgc aggtcatcca cgagatcgtt 2940

gacctgatga tccactacca agctgtcgtc gtacttgaaa atcttaattt cggcttcaag 3000

tccaaacgta caggaattgc ggaaaaggct gtataccaac aattcgaaaa aatgcttatc 3060

gataagctta actgccttgt gttgaaagat taccctgcgg aaaaagtcgg aggtgtatta 3120

aacccatatc aattgaccga tcaattcact tcatttgcca aaatgggcac gcagtcaggg 3180

tttttgttct atgtaccagc tccatatacc tctaagattg atccgttaac gggctttgtt 3240

gatcctttcg tctggaagac catcaaaaac catgaaagcc gcaaacactt ccttgagggc 3300

tttgactttc ttcactacga tgtaaagacc ggagatttca tcttacactt caaaatgaac 3360

cgcaatctgt ctttccaacg tggtttacct ggatttatgc ctgcgtggga catcgtattc 3420

gagaaaaatg aaactcagtt tgatgcaaaa ggcacgccgt ttatcgccgg aaagcgcatt 3480

gtaccagtta tcgagaacca ccgtttcaca gggcgttacc gcgacctgta ccccgcgaac 3540

gaattgatcg cgctgttaga agaaaaggga atcgttttcc gtgacgggtc caacatctta 3600

cccaagttac ttgaaaacga cgacagtcac gcaatcgaca caatggttgc tttaatccgc 3660

tccgttctgc aaatgcgtaa cagcaacgct gcgactggtg aggactacat taatagcccg 3720

gtccgcgacc ttaacggcgt atgtttcgac tcccgtttcc agaatcctga gtggcccatg 3780

gacgcagacg caaatggggc ctaccatatt gcgctgaaag gtcaattact tctgaatcac 3840

ttgaaggaat ctaaggacct taaactgcag aatgggatct caaaccaaga ctggttagcg 3900

tacattcagg aattacgtaa ctag 3924

<210> 15

<211> 3729

<212> DNA

<213> Artificial Sequence

<220>

<223> BbCpf1

<400> 15

atgaaaaagt ttaccaatct gtatccggtt caaaaaacac ttcgttttga acttatcccg 60

caaggcaata cctctaaaca tctttgcaaa attattcagg aggacgagca gatcgcagag 120

gactcgcaag aagtgaaaaa attattagac cgttatcata aagagttcat cgcaattgct 180

ttaagcagct tccctacgtc tcctttagcg aaagaaatta tcccgaaact gaaagagttt 240

gctcaaatcc gtgctacagg ggatgcaaag cagatttcaa caattcaaga tgaacttcgt 300

gagctggtgg ttaaaggttt caagggggag ggggagcaag aacgtcgtta caaaatcttg 360

atcggagcaa aggggaatcc aaacgccgat gaactgttta acactgagtt gattaacttt 420

cttaaagatc cggctgagca agccctggta aaaaagtttc agaaacatac tggttatttc 480

ttgggattta acgaaaatcg caagaacatg tactcggcga aggctcagag tacggctatt 540

gcgtatcgct taattcatga gaacttgcca cgctttcttg acaatattac aacttacgaa 600

aaagtcaaga catacctgaa agaggagatc cctcagttgg agaaggaact ggtacgcgct 660

ggggcgagcc ttgtaagtca tgtcgacagt gtcttcacaa tcgacttttt tttagaggtg 720

tttacgcagt ccggcattga ccagtacaac gctcttattg gaaaaattgt caaccaggaa 780

caaggggagg tgaaaggatt aaatgagcgc atcaatttat acaatcaaca acataagcaa 840

gaggcgaagc tgccactttt taaacctttg tacaaacaga ttttaagcga tcgcgagcaa 900

ttgtcttggc ttgccgaagc atataattct gacaaagatt tattggactc gatccagaaa 960

tattatcaac tgttaatcga caaccaaatc tttgaacgta ttccgcgcct gatgcacacg 1020

ctggaaaagg caccgttaga taagatttgg atcacttacg atacgcaact gacaagtatc 1080

tcaaacacac tttacggctc ttggcgcgtc atcggtgagg ctctgggccg taacgctaag 1140

agtgagaagg agcgtaaatc atcgcaaaaa aaggcgttaa attactccct ggagtccatt 1200

aatcaggcca tcgccaagat gcccagcgat gaggagcttc cacctatcca gaaatacttt 1260

atcgcacttg ggagtaaccc gtccaaaaaa gacgctagct ccggtacgtt agtagataaa 1320

gtccgttcgt catataaagc ctgtcaagat attcttacaa atcctgatca tacgggcaag 1380

aaacttatcc aagacaaaaa gcaagttgat ctgttaaagc aattactgga tgacttattg 1440

atcttacagc gtttcatcaa acctttgctg tatagcaata acgagaatga gactcacaaa 1500

gatgaagttt tctatacaga attaacggat atcatggatt tgcttaatcc tattgtagga 1560

ctgtataaca aagtacgcaa ttatttgact caaaagccat attcgactga aaagtttaag 1620

atcaatttca aatcatcgtc cctgttggcg ggttgggacc gcaacaagga gaaagacaac 1680

ttaggggtaa tcttgaaacg cgaagataaa tactaccttg ctattatgga caaggcacac 1740

aacgccacat ttaaaaacaa gtcacttcct actcaaggcg aatgttacga gaaaatggaa 1800

tataagcttc tgccgggagc caataaaatg ctgccgaagg tatatatcac ctcgaaaaaa 1860

ggtatcgaat ccttccatcc atctgaagaa ttacaaaaaa agtacaaatt gggaacgcac 1920

aagaaaggcg cttcgttcaa cctgagcgac atgcgtgcat tgatcgatta cttcaaagaa 1980

tccttggaga agcacgagga gcattcacaa tttggattcc acttttcgga cacgagtacc 2040

tacgaagaca tttctggatt ttaccgtgaa gttgagcagc aggcttataa aattacgttc 2100

cgtaaagtat cggtcgaata catcgatcaa ttggttaacg aaggcaagct ttatctgttc 2160

cagatttata acaaagattt ttctccctat tctaaaggca cacctaattt acacactttg 2220

tactggaaga tgttgtttga tcccgcgaac ttacaggata ttgtttataa gttgaatggt 2280

gaggcggaag tattttttcg caaaaagtcg ctgcagtacg accgtcctac acatccaaaa 2340

ggccaaccta tcaataagaa aagtttgctg aatgaggggg aaacatccct tttcgactat 2400

gaccttatca aggaccgccg ctttactgta gacaaatttc agtttcatgt ccctattacg 2460

atgaatttca aggccacgca aggtacaaag gtcaaccaaa tggtccagga ggaggtcaag 2520

aaatccaaag gattccactt gatcggcatc gatcgcggag aacgcaacct tttatacatc 2580

gtcgtgatca atgaacgcgg ggagatcatt gagcagtgtt cattaaacaa gattgtgaat 2640

acataccagg aaaaggaaca caccgttgat tataaagctc tgttagagaa acgctcacag 2700

tctcgtcttg aggagcgcaa gtcgtggcaa actatcgaaa atatcaagga gttaaagggc 2760

ggatacttat cacaagttgt gcacaaaatc gctcagttga tgatcaaata taacgcaatt 2820

gccgttctgg aggacttaaa cttcggtttc attcgtactc gcaagaagtt tgaattttcg 2880

gtgtatcagg agttcgagaa gaaactgatc gataaattgg gttacgtggt agacaagaaa 2940

gccccgattc agcaggaggg tggtttactt caagcttatc agttaaccgc cccattcaag 3000

tcattccgtg agatggggaa acagaacggt tttttgtttt acgtacccgc gtggaacaca 3060

agcgcaatcg atccgcgtac cggatttgtc aatttactgg acacacgcta tgaatcgatc 3120

gcaaaaacca aagagcttat taagaaattg aaagacattc gttacaatag ccaaaaggac 3180

tggttcgaaa tcgatcttga ctataatgcc tttgggaacc gtgcgaaggg ttctcgctcg 3240

aaatggcgct tatgtagcta cggcgaacgt attgagcaca ctcgtaaaca ggatagtaat 3300

ggtcaagagg agagcgattc gatggtagta ttgacggaag catttaaaga tgtctttacg 3360

aagtatcaga tcgattaccg cgagaatctg aaggaacagt tacttctgca gtcagataaa 3420

gctttttttg tggatttcct gagcctgtta cgtttgacgc tgcaactgcg taacagttta 3480

tccaactccc ttattgacta tattctttcg ccggtagccg acgagaatgg ggagtttttt 3540

gactcgcgta aagcccttag taatgagcca caggatgctg acgccaacgg tgcataccac 3600

attgctttaa agggtttgtg ggtattggat aaaatccgta aaacggagaa ggttacgcca 3660

gctaagttgg cgctttccaa ccaggaatgg ttgtcgtttg cccaagagaa gccctttttc 3720

aatgagtga 3729

<210> 16

<211> 3789

<212> DNA

<213> Artificial Sequence

<220>

<223> BoCpf1

<400> 16

atgcgcaagt tcaacgagtt cgtcggattg taccctattt ctaaaacctt acgttttgag 60

cttaagccga ttggtaagac gttggagcat attcagcgta ataaactttt ggagcatgac 120

gcggtacgcg ctgatgatta cgtaaaggtg aagaaaatta ttgacaaata tcacaaatgt 180

ttgattgacg aagcactgtc tggcttcacc tttgacacgg aagcagacgg tcgctcgaac 240

aatagtcttt ctgagtacta cctgtattac aatctgaaaa aacgcaacga acaggaacaa 300

aaaacgttta agacaattca aaacaatttg cgtaagcaga ttgtcaacaa acttactcaa 360

tcggagaagt acaaacgcat tgacaaaaaa gagttaatta cgactgactt accggacttt 420

ctgacaaacg agagcgagaa ggagcttgta gaaaaattta agaatttcac gacgtatttt 480

acagagttcc ataagaatcg taaaaatatg tactccaaag aagaaaaatc gaccgcaatt 540

gcatttcgct tgattaatga gaatttgccc aaattcgtag ataacattgc ggcgttcgag 600

aaagtcgtgt cttccccgct ggccgaaaaa atcaacgctc tttacgagga ctttaaggaa 660

tacctgaacg tagaggaaat cagtcgtgtg tttcgcttag actactatga tgaattgtta 720

actcaaaaac agattgatct ttacaatgct atcgtaggcg ggcgtacgga agaagacaat 780

aaaattcaga ttaaaggtct taaccaatac attaacgagt acaatcagca acagactgac 840

cgctccaatc gtttacctaa attgaagcct ctgtataagc aaattctgag cgatcgcgaa 900

agcgtaagct ggttgcctcc taaatttgac tctgacaaga atttgctgat caaaatcaag 960

gaatgctatg atgcactgtc tgagaaggag aaagttttcg acaagttaga gtctattctt 1020

aagagtttaa gcacttatga tttgagcaag atttacatta gtaatgacag tcaattgagt 1080

tacatctccc aaaagatgtt tggccgctgg gatatcattt ccaaggcaat ccgcgaggat 1140

tgtgcgaagc gtaaccctca gaaatctcgt gagtcactgg agaaatttgc agagcgcatt 1200

gataaaaaac ttaaaactat tgatagtatt tccatcgggg atgtagacga atgcttggcc 1260

cagttgggtg aaacttacgt aaaacgtgtc gaggattact ttgttgcgat gggagaatca 1320

gagatcgacg atgaacagac tgacacaacg tccttcaaaa agaacattga aggcgcctac 1380

gagagcgtta aggaacttct taacaatgca gacaatatca ccgacaacaa tcttatgcag 1440

gataaaggta atgtcgaaaa gatcaagaca ctgttggatg ctatcaagga tcttcaacgc 1500

tttattaagc cgttattagg gaaaggtgac gaagcggaca aagatggggt cttctatggt 1560

gaattcacga gcttgtggac caaattagac caagtgacac cgctttataa catggttcgc 1620

aactatttga ccagtaagcc ctatagtaca aaaaagatca agctgaattt cgagaattcc 1680

acccttatgg acgggtggga tttgaacaaa gagcctgata ataccactgt gattttttgt 1740

aaggacggtt tgtactattt gggtatcatg ggaaaaaagt ataatcgcgt ctttgtggat 1800

cgtgaagatt tgccgcacga tggagagtgt tatgataaaa tggaatataa actgcttcca 1860

ggagctaata agatgctgcc aaaagtattc ttctcggaaa ccgggattca acgttttctg 1920

ccttccgagg aacttttagg caagtacgag cgtggaacgc ataagaaggg tgccggcttt 1980

gaccttggag actgtcgcgc tttaatcgat tttttcaaga agtcgatcga acgccacgat 2040

gactggaaaa aattcgattt caagttttcg gacacttcta cctatcagga tatctccgag 2100

ttctaccgtg aggttgaaca acaaggctat aaaatgtcat ttcgcaaggt ctcagtggat 2160

tatatcaaat ctctggtaga ggaaggcaag ttgtatttat ttcaaatcta taataaagat 2220

ttttcggctc attccaaggg cacacccaat atgcacacat tgtactggaa gatgttgttt 2280

gacgaagaga acttaaaaga cgttgtttat aagcttaacg gagaggctga agtcttcttt 2340

cgtaagtcaa gtatcacagt ccaatcaccc acccatcccg caaattcccc aatcaagaat 2400

aagaataaag acaaccaaaa aaaagagagt aagtttgaat atgatttgat caaagatcgt 2460

cgttacaccg tcgataaatt tttgtttcac gttcctatca ccatgaactt caagagtgtg 2520

ggtggttcga atattaatca gttagttaag cgtcacattc gctcggcgac tgacttacac 2580

atcatcggca tcgatcgcgg tgagcgccat ctgctgtacc tgaccgtgat cgattcacgc 2640

ggcaatatta aagaacaatt ttctcttaac gagatcgtca atgaatacaa cggaaacacc 2700

taccgcaccg attaccacga actgcttgac acgcgcgaag gggagcgtac cgaagcacgt 2760

cgtaactggc aaacgattca gaatatccgc gaacttaagg aaggatattt gagccaggtg 2820

atccataaga tttccgaatt ggcaattaaa tacaacgcgg taattgtgct ggaagacctt 2880

aactttggct tcatgcgcag tcgtcagaag gtggaaaaac aggtatacca aaaatttgag 2940

aaaatgttga tcgacaagct taattatttg gtagacaaaa aaaagcctgt ggccgaaacg 3000

gggggattat tgcgcgccta tcaactgacg ggagaatttg agtcattcaa gactcttggc 3060

aagcaatctg gaatcttgtt ctacgtacca gcatggaata ctagcaagat tgaccctgtt 3120

acaggcttcg taaatctttt cgatacacat tacgagaata ttgagaaagc gaaggtgttc 3180

ttcgataagt ttaagtcgat tcgctacaat tcggacaaag attggtttga gttcgtcgtc 3240

gatgactaca ctcgtttttc tcccaaggct gagggaactc gtcgtgattg gacgatttgt 3300

actcagggta aacgtatcca gatttgtcgc aaccatcagc gcaacaatga atgggaaggg 3360

caagaaatcg accttacgaa agccttcaag gaacacttcg aggcgtatgg tgtagacatt 3420

tctaaggatt tacgtgagca aattaacaca cagaataaga aagaattttt tgaagaatta 3480

ttgcgtttac ttcgcttgac gctgcagatg cgcaattcaa tgccgtcctc ggatattgat 3540

taccttatct ctccagttgc taatgacact ggatgtttct ttgactctcg caaacaggcg 3600

gaattgaagg aaaatgccgt tttgccgatg aacgcggatg ctaatggagc gtacaacatc 3660

gctcgtaaag gacttttggc gattcgcaaa atgaaacagg aggagaacga ctccgcgaaa 3720

attagcttag ccattagtaa taaggagtgg ttgaagttcg cacagactaa accgtatctg 3780

gaagattga 3789

<210> 17

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1

<400> 17

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttcaaa 540

gggttccacg agaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgaa cggctgggat aaaaataagg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 18

<211> 3933

<212> DNA

<213> Artificial Sequence

<220>

<223> HkCpf1

<400> 18

atgttcgaaa agcttagtaa catcgtaagt atttcgaaga cgatccgctt caaattgatt 60

ccagtaggta aaaccctgga gaatattgaa aagcttggca aactggaaaa ggactttgag 120

cgctcggact tttacccaat cctgaagaat atctcggatg actattaccg ccaatacatc 180

aaagaaaaat tgtccgattt aaacctggat tggcagaagc tttacgatgc tcatgagttg 240

ctggactcca gtaagaaaga gtcgcagaaa aaccttgaga tgatccaagc tcagtatcgc 300

aaagtccttt tcaacatttt atcgggcgag cttgataaga gcggagaaaa gaatagcaag 360

gacctgatta agaataataa agccttatac ggaaagttgt tcaaaaaaca atttattctg 420

gaggtcctgc ctgactttgt gaataataac gattcctatt ctgaagaaga tcttgaggga 480

cttaatcttt atagcaaatt tacaactcgc ttgaaaaact tctgggaaac acgtaaaaat 540

gtcttcactg ataaagacat tgtcacggcg attcccttcc gtgcggtgaa tgagaacttt 600

ggattttact acgataacat taaaattttt aacaaaaata ttgagtatct ggagaacaaa 660

atcccaaatt tggaaaatga gttgaaggag gccgatatcc ttgacgacaa tcgctctgtg 720

aaagattact ttacaccaaa cggattcaat tacgtcatca cccaagacgg tattgacgtc 780

taccaagcaa ttcgtggcgg gttcaccaaa gagaatggcg agaaggtcca gggtatcaac 840

gagattttga atcttacaca gcaacagctt cgccgcaagc cggaaactaa aaacgttaag 900

cttggagttt tgacgaagtt gcgtaaacaa atcctggagt atagcgagag cacaagtttt 960

ttgattgatc agattgaaga tgataatgat ttagtagacc gtattaataa gttcaacgta 1020

tcgttctttg aaagcacaga agttagcccc tcattattcg aacagatcga acgcttgtat 1080

aatgccctga agtctattaa aaaagaggaa gtatatattg acgctcgtaa tacacagaaa 1140

ttctcccaga tgcttttcgg tcaatgggac gttatccgcc gcggttacac ggtcaagatt 1200

actgaagggt ctaaggaaga aaagaagaag tacaaagagt atctggagtt agatgaaaca 1260

tctaaggcta agcgttatct taatattcgt gaaattgaag aattagtcaa cttagtagag 1320

gggtttgagg aggtagacgt cttctcagtc ttgctggaga agtttaaaat gaataatatt 1380

gagcgctcag agtttgaggc gccaatttat ggaagcccta ttaaattaga ggcgattaag 1440

gagtacttgg agaaacattt ggaggagtat cacaaatgga agctgttgct tattggaaat 1500

gacgaccttg ataccgacga gaccttctac cctctgctta atgaggtcat ttctgattac 1560

tatatcatcc cactttataa tcttacgcgc aactatttga cccgcaagca cagtgacaag 1620

gacaagatca aagtgaactt tgactttcct accctggctg acgggtggag cgaatcgaag 1680

atctctgaca accgctccat tatcttgcgt aaggggggct attattactt ggggatctta 1740

atcgataata agttattaat taataaaaaa aataagtcaa aaaagattta cgagatcctg 1800

atttacaatc agatccctga attctctaaa tcaattccaa attacccctt tacaaagaaa 1860

gttaaggaac acttcaaaaa caatgttagc gacttccagt taattgatgg ctatgttagc 1920

ccgcttatta ttactaagga aatctatgat attaaaaagg aaaagaagta caaaaaggac 1980

ttctataaag acaacaatac caacaagaac tacttgtata caatttacaa atggatcgaa 2040

ttctgtaagc agtttcttta taagtacaag ggacccaata aggaatccta caaggaaatg 2100

tatgattttt ccacgttaaa ggacacgtcg ttatacgtta acttgaatga cttctacgcc 2160

gatgttaact cttgtgctta tcgcgtcctg ttcaataaga tcgacgagaa tactattgac 2220

aacgcggttg aagatgggaa gctgctgtta tttcaaattt acaataaaga ctttagccct 2280

gaatcgaaag gcaaaaagaa tttgcacaca ctgtactggc ttagcatgtt cagcgaagag 2340

aaccttcgca cgcgtaaatt gaagctgaat ggccaggcag agatctttta ccgcaaaaag 2400

ctggagaaga aaccgattat ccataaggaa ggcagtatcc tgcttaacaa gatcgataaa 2460

gaagggaaca caatccccga aaacatttac catgagtgct accgttattt aaataaaaaa 2520

attggtcgcg aggatttgtc agatgaggcg attgcgcttt tcaataaaga cgtcttgaaa 2580

tataaggaag ctcgcttcga tattattaag gaccgccgtt actccgagtc tcaatttttc 2640

tttcatgtgc cgatcacgtt caactgggat atcaagacca acaaaaacgt taaccaaatc 2700

gtgcagggga tgattaaaga tggggaaatt aagcacatta tcgggattga ccgtggcgag 2760

cgtcatttgc tgtactattc cgtcatcgac ttagaaggta atatcgttga acaagggtct 2820

cttaatacct tggagcagaa tcgctttgat aactcgaccg taaaggtaga ttaccaaaac 2880

aaacttcgca cgcgtgaaga agatcgtgac cgtgctcgca aaaattggac gaatattaac 2940

aaaatcaaag aactgaaaga tggttacctg tcccatgtgg tccacaagtt aagtcgcttg 3000

atcattaaat atgaagcaat tgtaatcatg gaaaacttga accaaggttt taagcgcgga 3060

cgttttaagg tagagcgtca agtttaccaa aagttcgagt tggcacttat gaacaagttg 3120

tccgccttgt cgttcaaaga gaagtacgat gaacgcaaaa acttggagcc atcaggaatc 3180

cttaatccta tccaagcatg ttacccagta gatgcctatc aagagttgca aggtcagaac 3240

gggatcgtct tttacttgcc agccgcttat acctccgtta ttgacccggt gactggattt 3300

acaaatttat ttcgcctgaa aagtatcaac agctcgaaat atgaggaatt tattaaaaag 3360

tttaaaaaca tttactttga caatgaagaa gaggacttca aattcatttt taactacaag 3420

gatttcgcaa aggctaatct tgtgatttta aacaatatta agagcaaaga ctggaagatt 3480

agcacgcgcg gtgagcgcat tagctacaat tccaagaaaa aggagtattt ctatgttcaa 3540

cctactgagt tcttaatcaa taaacttaaa gaattaaata tcgattacga aaacattgac 3600

atcatcccat taattgataa tctggaggaa aaggccaaac gtaagattct taaggcgctt 3660

tttgatacct tcaagtactc cgttcaactt cgtaattatg actttgagaa cgattacatc 3720

atctctccga cagcagacga caatggcaat tattacaact caaatgaaat cgacatcgac 3780

aaaacgaatc tgcctaataa cggggacgct aacggtgctt ttaatatcgc tcgcaaaggg 3840

ttgctgctga aggaccgtat cgtcaactct aatgaatcta aggttgacct taaaatcaag 3900

aacgaagact ggatcaattt tatcattagc tga 3933

<210> 19

<211> 3675

<212> DNA

<213> Artificial Sequence

<220>

<223> Lb4Cpf1

<400> 19

atgggtttgt atgatggctt cgttaatcgt tatagtgtca gtaaaaccct gcgtttcgaa 60

cttattccac aagggcgcac tcgtgaatac atcgaaacca atggaatcct gtcagatgac 120

gaggagcgcg ccaaagacta caagacaatc aagcgtctga ttgacgagta ccacaaagac 180

tatatttccc gttgccttaa aaatgtaaac atctcttgtt tggaggagta ctatcacctg 240

tataactcct ctaaccgtga taagcgtcac gaggagctgg atgcattatc cgaccagatg 300

cgcggcgaga ttgcatcttt tttaactggg aacgacgagt ataaggaaca gaaaagtcgc 360

gacatcatta ttaatgaacg tattattaac tttgcgtcta cggacgaaga gttggcggca 420

gtcaagcgct tccgcaagtt cacaagttat tttactgggt ttttcactaa ccgcgagaac 480

atgtactcgg ccgagaaaaa atctacggcc atcgcacatc gtatcattga cgtgaatctt 540

ccaaagtacg tggataacat taaggcattt aacacggcta tcgaagcggg agtctttgat 600

atcgctgaat ttgaatcaaa tttcaaggcc attaccgatg agcacgaagt aagcgattta 660

cttgacatca ccaaatactc tcgctttatc cgtaatgagg acattatcat ttacaatact 720

cttctggggg gtattagcat gaaagacgaa aaaattcagg ggctgaatga gttaattaat 780

cttcataacc agaagcatcc tggtaagaaa gtgcctttac ttaaggtcct ttacaagcag 840

attttggggg attcacagac tcacagtttt gtcgacgatc agttcgagga cgaccaacag 900

gtcatcaacg ccgtgaaggc ggttacggac accttttcag agacgctgtt aggctctttg 960

aaaattatca ttaataacat tggtcattac gaccttgacc gtatttacat taaagcggga 1020

caggatatca ccacgctgag taaacgtgca ttaaatgatt ggcacattat caccgagtgc 1080

ttggagtcag aatacgatga taaattccct aagaacaaga aaagtgatac ttacgaggaa 1140

atgcgtaacc gctacgttaa gtcctttaag tcgtttagta ttgggcgcct taacagtctg 1200

gtaaccacat atacagaaca agcgtgcttc cttgagaact acttgggctc gtttgggggc 1260

gatactgata agaactgttt gacagatttt accaactcac ttatggaagt agaacatctg 1320

cttaattcag agtatcctgt gacaaatcgt cttatcacgg attacgaatc cgtgcgcatt 1380

cttaagcgcc tgttagattc agaaatggaa gttattcact tcttaaagcc gttgttaggg 1440

aatggaaacg aatcagataa agatcttgtt ttttacgggg aatttgaggc agaatatgag 1500

aaattgctgc ctgtcatcaa agtctataac cgcgtgcgta actatttaac tcgcaagccg 1560

ttttcaacgg agaagatcaa attaaacttt aactccccaa ctcttctgtg cggatggtct 1620

cagagtaaag agaaagaata catgggcgtg attttgcgta aagacggaca gtattacctt 1680

ggtatcatga ctccgagcaa taaaaagatt ttcagcgaag cgcccaagcc tgacgaagat 1740

tgttacgaaa aaatggtctt acgctacatt ccccacccct accagatgct gcctaaggtg 1800

ttcttctcca agtcgaacat tgcctttttt aacccttcgg atgaaatctt gcgcattaaa 1860

aagcaggagt catttaagaa agggaagagt tttaatcgcg atgactgcca caaattcatt 1920

gacttctata aagactcaat caaccgccac gaggagtggc gcaaatttaa cttcaaattt 1980

tctgatacag actcttatga agatatctcg cgtttttaca aagaggtaga gaaccaggct 2040

ttttccatga gctttactaa gatccccacc gtatatattg attcattagt ggacgaggga 2100

aagctttacc tgttcaaact gcacaacaag gattttagcg aacattctaa gggaaagccg 2160

aacttacata cggtttactg gaacgcttta ttctcagaat acaacttaca gaacacggtc 2220

tatcaattga acgggtctgc ggagatcttc ttccgcaaag cctcgattcc agaaaatgag 2280

cgcgtaattc acaagaaaaa tgttcctatt actcgcaaag tagcagaact taatggaaag 2340

aaggaagttt cggtctttcc ttatgatatc atcaaaaacc gccgctacac agtagacaag 2400

tttcaatttc acgtgccact gaaaatgaat ttcaaggccg atgaaaagaa gcgtattaac 2460

gacgacgtaa tcgaagccat ccgtagtaac aaggggattc acgtcatcgg catcgatcgt 2520

ggtgaacgca atcttctgta cttaagctta attaacgagg aagggcgtat catcgaacaa 2580

cgttcgttga atattatcga tagtggagag gggcacactc agaactaccg cgatctgtta 2640

gactcacgtg aaaaagatcg cgaaaaagct cgtgagaact ggcaggaaat ccaagaaatc 2700

aaagatttga agactggcta tttgtcccag gcgatccata caatcacgaa atggatgaag 2760

gagtacaatg caatcattgt tcttgaagat cttaatgacc gctttacaaa tggccgtaag 2820

aaagttgaaa agcaagtgta tcagaagttc gagaaaatgt taattgataa gcttaattat 2880

tacgtcgata aggacgagga gtttgaccgt atgggtggaa ctcaccgcgc ccttcaattg 2940

acggagaagt tcgagtcatt tcagaaactt ggccgccaga cggggtttat tttctatgtg 3000

cctgcatgga atacctcgaa gttagaccca acaactgggt ttgtagactt gctttatccg 3060

aaatataaat ctgtagatgc gaccaaggat tttatcaaga agtttgactt tatccgcttt 3120

aatagcgaga agaactactt tgagtttggc cttcattatt ccaactttac tgagcgcgct 3180

atcggttgtc gcgatgaatg gattttatgc tcttacggta accgcattgt taatttccgc 3240

aacgccgcaa agaataacag ttgggactac aaagaaatcg acattacgaa acagttactg 3300

gacctgtttg aaaaaaacgg aattgacgta aagcaagaaa atttgatcga cagcatctgt 3360

gaaatgaaag ataaaccttt tttcaagagc cttatcgcga acatcaaact tatcttacag 3420

atccgcaact ccgcgtcggg gactgatatc gactatatga tctctcccgc tatgaatgac 3480

cgcggggagt tctttgacac tcgtaaaggt ttgcagcaat tacctcttga cgccgatgcg 3540

aatggtgcat acaacatcgc gaagaaggga ttatggatcg ttgatcaaat ccgtaatacg 3600

actggcaaca atgtgaagat ggccatgtcc aaccgcgaat ggatgcactt tgcacaggaa 3660

agtcgtctgg cttga 3675

<210> 20

<211> 3621

<212> DNA

<213> Artificial Sequence

<220>

<223> Lb5Cpf1

<400> 20

atggagaatt attatgattc attgacgcgc caatatccgg ttactaagac cattcgccaa 60

gaacttaaac ccgtggggaa gactttagag aacattaaga atgcggaaat catcgaggcc 120

gacaagcaaa aaaaggaagc atacgtcaaa gtgaaagagc ttatggatga atttcataaa 180

agcatcattg agaaatcatt ggtgggtatc aagctggacg ggttgtcgga atttgaaaag 240

ctgtataaga ttaagacaaa gaccgacgag gacaagaatc gtattagcga gctgttctat 300

tacatgcgca aacagatcgc agacgcgctt aaaaacagcc gtgattacgg gtacgtagat 360

aataaagatc ttatcgaaaa gatcttacca gaacgcgtta aggatgaaaa ctccctgaat 420

gccttgtcct gttttaaggg atttacgact tatttcacgg attattataa aaaccgcaaa 480

aacatctata gtgatgaaga gaaacattcc acggtggggt atcgctgcat caacgagaac 540

ttattgattt tcatgtctaa tattgaggta tatcaaatct ataagaaggc gaacatcaag 600

aatgacaatt acgatgaaga gaccctggat aagaccttca tgatcgagtc gttcaacgag 660

tgcttaacgc agtctggagt tgaggcctat aattcggtag tcgcctctat caagacggcg 720

acaaatctgt atattcaaaa aaataacaag gaggaaaact tcgttcgcgt gcctaagatg 780

aaggtgttgt tcaaacaaat tctgtctgac cgtacgtcat tattcgacgg tctgattatc 840

gagtctgatg acgagttact ggacaaactt tgtagttttt ctgcggaggt agataagttc 900

ttgccgatca atattgatcg ctacatcaag accctgatgg acagcaacaa tggcacgggc 960

atttatgtaa agaacgatag cagtctgacg accttaagca actatttgac agactcgtgg 1020

tcgtcaattc gtaatgcatt taacgagaac tacgacgcta agtataccgg taaagtcaac 1080

gataagtacg aggaaaagcg tgagaaagca tataaatcca atgactcctt tgaacttaat 1140

tacatccaaa acttattagg aattaatgtg attgacaagt atattgagcg tatcaacttt 1200

gacatcaagg aaatctgtga ggcttataaa gagatgacca aaaattgctt cgaggatcat 1260

gataagacaa aaaaattgca gaagaatatt aaagctgtag cttccattaa gagttatctt 1320

gacagtctga agaacattga gcgtgatatc aagctgttaa acgggacagg attggagtcg 1380

cgcaatgaat ttttttatgg tgaacagagt acggtgttag aagaaatcac gaaagtagat 1440

gagttatata atattacccg caattacctt acgaaaaagc ccttttctac cgaaaaaatg 1500

aagttaaatt tcaacaatcc ccaactttta ggaggttggg acgttaacaa ggagcgtgat 1560

tgttacggtg tgattctgat taaagacaat aactattact tgggtatcat ggataaatcg 1620

gcgaataaga gttttcttaa tattaaggaa agcaaaaacg aaaatgcgta caagaaggtt 1680

aactgcaagc tgcttcctgg acccaacaag atgtttccaa aggtgttctt cgctaagtcg 1740

aacattgact actacgatcc gactcacgaa atcaagaagc tttacgacaa gggaactttt 1800

aaaaagggga attctttcaa tttagaagac tgccataagt tgattgattt ctataaggag 1860

tcgattaaaa agaatgacga ctggaaaaac tttaacttta atttcagtga taccaaggac 1920

tatgaggata tttcgggatt ctttcgtgag gttgaagcgc aaaattataa gattacatat 1980

accaacgtta gctgcgattt cattgagtca cttgttgacg agggtaagct ttatttattt 2040

caaatctaca acaaggattt ttcagagtac gctaccggca atttgaactt acatactctg 2100

tacttaaaaa tgcttttcga cgaacgcaac ttaaaagact tatgcatcaa gatgaacggc 2160

gaggccgagg tgttctaccg ccccgcgtca attttggatg aagacaaagt ggtgcacaaa 2220

gcaaatcaga aaattacgaa caagaacaca aactcgaaaa agaaagaaag tattttcagc 2280

tatgacattg taaaagacaa acgctacact gtcgataaat ttttcattca tttgcccatt 2340

acgttgaact ataaggagca aaatgtatcg cgtttcaacg attacattcg tgaaatttta 2400

aaaaagtcta agaacattcg tgtgattggc attgatcgcg gagagcgtaa ccttctgtat 2460

gttgtggtct gtgactctga tgggtcaatt ttgtatcagc gttccattaa tgagatcgta 2520

agcggttccc acaaaacaga ttaccacaag cttttggaca acaaggaaaa agaacgtttg 2580

tccagccgtc gtgattggaa gactatcgaa aatattaaag atctgaaggc gggatatatg 2640

tcgcaagttg ttaatgaaat ttataatttg attttgaagt ataatgctat cgttgtctta 2700

gaagacctta acattggttt taagaatggc cgcaagaagg tggaaaaaca agtgtaccag 2760

aattttgaaa aagccctgat cgacaaactg aactacctgt gtatcgacaa gacgcgcgaa 2820

caattgtcac catcatcacc aggaggagtc ttaaatgcct atcaactgac tgccaagttc 2880

gagagttttg agaagattgg aaaacaaaca ggatgcatct tctacgtgcc agcctacctg 2940

acttcccaga tcgacccaac gacagggttt gtaaacttgt tctatcagaa agatacttcc 3000

aagcaaggcc ttcagctttt cttccgtaag tttaaaaaga tcaacttcga taaggtggcc 3060

agcaattttg aatttgtttt cgattacaat gactttacta ataaagctga gggtactaag 3120

acaaattgga ccatctcaac tcagggtacg cgtatcgcca aataccgctc agatgacgca 3180

aatggaaagt ggatctcgcg tactgtgcat ccgaccgaca ttatcaaaga agctctgaat 3240

cgtgagaaga ttaactataa tgatggccat gacttgattg atgagattgt aagtattgag 3300

aagagcgctg tgctgaagga gatttactac ggatttaagc tgaccctgca attacgtaat 3360

tcgactttgg ctaatgagga ggagcaggaa gattacatca tctccccagt aaaaaacagt 3420

tcgggaaact acttcgattc ccgcatcaca tcgaaggagt taccttgcga tgctgacgcc 3480

aatggcgctt acaacatcgc acgcaaaggt ttatgggctt tagaacaaat tcgcaacagt 3540

gagaacgtat caaaagtaaa attagcgatc tcgaacaagg agtggtttga gtacacgcaa 3600

aacaatatcc caagtttgtg a 3621

<210> 21

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1

<400> 21

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt cacgactgcc tttacgggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgggtggat gggataagga caaagagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 22

<211> 3669

<212> DNA

<213> Artificial Sequence

<220>

<223> OsCpf1

<400> 22

atggagaccg aaattttaaa gtacgatttt tttgaacgcg aaggtaagta tatgtattat 60

gatggcctga caaaacagta cgctttaagt aagacaatcc gtaacgagct ggtgccgatt 120

ggtaaaactc ttgacaacat caagaaaaac cgtattttag aagcggatat caaacgcaaa 180

tctgattatg agcacgttaa aaagttgatg gatatgtatc ataaaaaaat cattaacgag 240

gcgcttgaca actttaaatt gtctgttctg gaagacgcgg cggacattta cttcaacaaa 300

cagaacgatg aacgtgatat tgatgctttc ttgaaaattc aggacaaact gcgcaaggaa 360

atcgtcgaac agttgaaggg ccatacggac tactccaaag tcggaaataa ggacttcttg 420

ggtctgctta aggccgctag tacagaagag gatcgtatct tgattgagag cttcgacaac 480

ttttacacat atttcacatc gtacaataag gttcgttcaa acctgtactc tgccgaagac 540

aagtcgtcga cagtagcgta tcgtctgatc aatgaaaatt taccaaagtt tttcgataat 600

atcaaagcct atcgcacggt tcgtaacgct ggggtaatct ccggtgacat gtccattgtc 660

gagcaagatg agctgtttga ggtagacacg tttaaccata ctctgacaca atatgggatc 720

gacacgtata accatatgat tggccagttg aacagtgcaa ttaatcttta caatcaaaag 780

atgcatggcg ctggttcttt taagaagtta cccaaaatga aggagttata caagcagctg 840

cttactgagc gtgaggagga attcatcgaa gaatataccg atgacgaagt gctgatcacg 900

agtgttcata attacgtttc atacttgatc gattacctga attccgacaa ggttgagtct 960

ttttttgata ctttacgtaa gagcgacggt aaagaggtat ttatcaaaaa cgatgtaagt 1020

aaaactacga tgtctaacat tttatttgac aattggagca ccattgatga tttgattaac 1080

catgaatatg attcagctcc agaaaacgtt aagaaaacga aagatgacaa atatttcgag 1140

aaacgtcaaa aggatttaaa aaagaacaag tcgtactctt tgtcgaagat tgctgctctt 1200

tgtcgcgata caacgattct ggagaagtat atccgtcgtt tggtagacga cattgaaaag 1260

atttacacaa gtaacaatgt cttcagcgac attgttttat ccaaacatga tcgcagtaag 1320

aaattgagca agaatacgaa tgccgtacaa gccattaaga atatgcttga ttctatcaaa 1380

gactttgagc atgatgtcat gcttattaac ggttcaggac aggagatcaa gaaaaatctt 1440

aacgtttact cagaacagga agcattggcc ggcatcttac gccaggtcga ccacatctat 1500

aatcttacac gcaactactt gacaaaaaag cccttctcca cagaaaaaat caaactgaat 1560

tttaatcgcc cgacgttcct ggatgggtgg gacaaaaata aggaggaagc aaacctgggc 1620

atcttgttaa tcaaagataa tcgctactac ctggggatca tgaatacaag tagtaacaag 1680

gcttttgtaa accccccaaa agccattagc aatgatattt ataaaaaggt cgattacaaa 1740

cttttgcctg gccccaacaa gatgttgccc aaggtgtttt ttgcgacgaa gaacattgca 1800

tactatgccc ccagcgagga gttgctgtcg aaatatcgta aagggacgca caagaaagga 1860

gatagtttca gcatcgacga ttgtcgtaac ctgattgatt tttttaaatc gtcaattaac 1920

aaaaacactg attggtcgac gtttggattc aatttctctg atactaacag ttataacgat 1980

atctcggact tttatcgtga agtcgagaaa cagggctaca aattatcgtt cacggatatt 2040

gatgcttgtt acattaaaga cttggtcgat aataatgaat tgtatctgtt tcagatttac 2100

aacaaggact tttcccctta tagtaaaggt aaattaaatc tgcatacact gtacttcaaa 2160

atgttatttg atcaacgcaa ccttgacaac gtagtctata agctgaatgg tgaggcggaa 2220

gtattttacc gccctgcatc catcgagtca gacgagcaaa ttattcacaa gtcaggacaa 2280

aacattaaga ataagaatca gaaacgctca aattgcaaga agacgagcac tttcgactat 2340

gatattgtga aagatcgccg ttattgcaaa gataagttta tgcttcattt gcccatcact 2400

gttaatttcg gtacaaatga gagcggaaaa tttaatgaat tggttaacaa cgcgattcgc 2460

gctgataagg acgttaatgt gattgggatc gaccgtggtg aacgtaattt actttatgta 2520

gttgtcgttg acccctgcgg taagattatc gaacagatct cgcttaacac aattgtggat 2580

aaagagtatg acatcgagac ggattaccac caattattgg acgaaaaaga ggggtcccgt 2640

gataaggccc gcaaggattg gaatacgatt gagaacatca aagaattgaa ggagggctat 2700

ctgtcgcaag ttgtaaatat cattgccaag ttggtactga aatatgacgc catcatttgt 2760

ttagaagact taaactttgg atttaaacgc ggacgccaaa aagtagaaaa gcaggtgtac 2820

cagaaattcg aaaagatgct gatcgataag atgaattatt tagtgttgga taaatcgcgc 2880

aaacaggaat cccctcaaaa accaggcggc gcgttaaatg ccctgcagtt gactagtgcc 2940

tttaagtcgt ttaaggagtt gggtaaacag acaggtatca tctactatgt accagcgtat 3000

ttgacgagta aaatcgatcc caccaccgga ttcgcaaatc ttttttacat caagtatgaa 3060

tccgtcgata aagcccgtga cttcttcagc aaatttgatt ttatccgtta taaccaaatg 3120

gataactatt tcgaatttgg attcgactac aaatcgttca cagaacgcgc gtcagggtgt 3180

aagtccaaat ggattgcttg cacgaacggc gaacgtatcg tgaaatatcg caattctgat 3240

aaaaataact ctttcgatga taagacagtc atcttgaccg atgaatatcg ctcgctgttc 3300

gacaaatact tacaaaacta tattgatgag gacgatctta aggaccaaat cttgcagatt 3360

gattctgcag acttttacaa gaatcttatt aaactgttcc agttgaccct tcaaatgcgc 3420

aatagcagct ccgacggaaa gcgtgattat attatttccc ccgtgaagaa ttatcgcgag 3480

gagttctttt gcagtgagtt cagtgatgac accttccctc gtgacgccga cgcaaatgga 3540

gcatataaca tcgctcgcaa agggctgtgg gtcatcaagc agatccgtga gacaaaaagc 3600

ggcacgaaaa tcaacttggc gatgagcaat agcgagtggt tggaatacgc ccaatgcaat 3660

ttgctttga 3669

<210> 23

<211> 3897

<212> DNA

<213> Artificial Sequence

<220>

<223> TsCpf1

<400> 23

atgaccaaga ctttcgatag cgagttcttc aacctgtaca gcttgcaaaa aacggtccgt 60

tttgagttaa agccagtcgg tgaaactgca tcctttgtcg aggatttcaa aaatgaagga 120

ttaaagcgcg tagtgagcga ggacgaacgt cgcgcggtcg attaccagaa ggtgaaggaa 180

atcatcgatg attaccatcg cgatttcatc gaagaatccc ttaactattt tcctgagcaa 240

gtgagtaagg acgcccttga gcaggcgttt catttatatc agaagttgaa agccgctaag 300

gtagaagagc gcgagaaagc gttgaaggaa tgggaagccc tgcaaaaaaa attgcgtgaa 360

aaagtagtga agtgtttctc cgacagcaat aaagcgcgtt tctctcgtat cgataagaaa 420

gagctgatca aggaagatct tatcaactgg ttagtcgccc aaaatcgtga agacgacatt 480

cctactgtcg agacgttcaa taattttacg acgtacttca caggttttca tgaaaatcgt 540

aagaacattt attcaaaaga cgaccatgct accgctatca gtttccgctt gatccatgag 600

aacctgccaa agtttttcga caacgtcatt tcttttaaca agttaaaaga aggttttccc 660

gagcttaagt tcgataaggt gaaagaagat ttggaagtag attatgactt gaaacacgct 720

tttgaaattg agtactttgt taattttgtt acccaggccg gtatcgacca atacaattac 780

ttgttaggcg gaaagacgtt agaagatgga acaaagaaac aaggaatgaa tgagcaaatt 840

aacttattca agcagcaaca gacgcgcgac aaggcacgtc agattcccaa attgattcca 900

ctttttaaac aaatcctgtc tgagcgtaca gagtcccaga gtttcattcc aaaacagttt 960

gaatccgacc aagagctgtt tgattcgttg caaaaattac ataacaactg tcaggataag 1020

ttcacggttt tgcaacaagc catcttaggc ttagcagaag cggatcttaa aaaggtattc 1080

atcaaaacgt cagatttgaa cgcgctgagt aacaccattt tcggtaacta ttcggtattc 1140

tctgatgcgc tgaacctgta caaagaaagt cttaaaacta aaaaagccca agaggccttt 1200

gaaaaacttc ccgcacatag catccacgat ttaatccaat accttgaaca gtttaacagc 1260

tctctggacg cagagaaaca acaatcgacc gatacggtat tgaactattt cattaagact 1320

gacgaattgt atagccgctt tatcaagagc acatctgagg cctttacgca agtgcagcct 1380

ctttttgaat tggaggctct gtcgtcaaag cgccgcccac cagaatcaga agacgaagga 1440

gcgaagggtc aggaaggatt cgagcagatt aagcgtatta aagcttacct tgacaccctg 1500

atggaggccg tacacttcgc taagccgctt tacttggtca aaggccgtaa gatgatcgaa 1560

ggcttggaca aagaccaatc tttttacgaa gcattcgaga tggcatatca ggaattagaa 1620

agtcttatta ttcctattta caataaggct cgttcatatc ttagccgcaa accgtttaag 1680

gcggataaat tcaaaattaa ctttgacaac aacactctgt tatctggctg ggacgctaat 1740

aaagaaactg ctaatgcgtc gatcctgttt aaaaaagatg gactttatta cttagggatt 1800

atgcctaaag ggaaaacgtt tctttttgat tatttcgttt cgagtgaaga ctcggaaaaa 1860

ctgaaacagc gccgccagaa aacggcagag gaagctttgg cacaagacgg ggagtcatat 1920

ttcgaaaaga ttcgctataa actgctgccc ggcgcaagca aaatgttgcc gaaggtattt 1980

ttttccaaca aaaatattgg gttctataac ccgtcagacg atatcttgcg tattcgcaat 2040

acagcgtcac acactaagaa tggcactccg cagaaagggc atagcaaggt ggaatttaac 2100

ttgaatgact gtcacaagat gatcgacttt tttaagtctt cgattcaaaa gcacccagag 2160

tgggggtcct ttggatttac tttctccgat actagcgact tcgaagatat gagtgcgttt 2220

tatcgtgaag tggagaacca gggatacgtt atttcattcg acaagatcaa ggaaacctac 2280

attcagagcc aagtcgaaca ggggaacctt tatctgtttc aaatctacaa caaggatttt 2340

agtccctact ctaagggcaa acccaactta cacaccttat actggaaggc tctttttgag 2400

gaagcaaacc ttaataatgt agttgcgaaa ttaaatggcg aggctgaaat ttttttccgc 2460

cgtcatagta tcaaagcctc tgacaaggtc gtacacccgg ccaaccaagc aatcgacaac 2520

aaaaatcccc atacagagaa aacgcaaagt acgtttgaat atgatcttgt gaaggacaag 2580

cgctatactc aagataagtt cttcttccac gtcccaatca gtctgaattt taaagcgcag 2640

ggtgtttcga aatttaacga taaagttaac gggttcctta aagggaaccc ggatgtaaat 2700

atcattggga tcgaccgtgg ggaacgtcat ttgttgtact ttacggtagt aaatcagaaa 2760

ggtgagattt tggtgcaaga gtcacttaat acactgatga gcgataaggg tcacgtgaac 2820

gattatcagc agaagttaga taagaaggag caggaacgtg acgccgcccg caaatcttgg 2880

acaactgtcg agaacattaa ggaactgaaa gaaggttatc tgtctcatgt ggttcacaag 2940

ttagcgcatc tgatcattaa atacaatgcg atcgtgtgtc tggaagactt aaatttcggt 3000

tttaagcgtg gacgctttaa agtcgaaaaa caggtctatc aaaagttcga aaaggcgctg 3060

atcgataagc tgaactattt agttttcaag gagaaggagt taggtgaggt cgggcactac 3120

ttaacagctt accaactgac agctccattc gagtccttta aaaagttagg caagcagagc 3180

ggaatccttt tttatgttcc cgctgactat acatcgaaga tcgacccgac cacgggcttt 3240

gttaacttcc ttgatctgcg ctatcaaagc gttgaaaagg caaagcaact tctttcagat 3300

tttaatgcca ttcgtttcaa ttctgtccaa aactactttg agttcgaaat cgactacaaa 3360

aaattgacac caaagcgtaa agttgggacc cagtcaaagt gggtgatctg cacgtatgga 3420

gatgtccgtt atcagaatcg tcgcaatcaa aaggggcact gggaaactga ggaggtaaat 3480

gtgactgaga aattaaaggc tttgttcgcg agcgactcta aaaccactac agtcattgat 3540

tacgcgaatg atgacaactt aatcgacgtt atcttagagc aggacaaagc atcgtttttt 3600

aaggaattat tgtggctgct gaagctgacg atgacgttac gtcatagcaa gatcaagtct 3660

gaagatgact ttatcttgag ccctgtcaag aacgagcagg gcgaatttta cgacagtcgc 3720

aaagcaggtg aagtctggcc gaaggatgcc gacgctaacg gtgcgtatca cattgcgctt 3780

aagggactgt ggaacctgca acagattaat cagtgggaaa aaggtaagac attgaatctg 3840

gcaattaaaa accaggattg gttttccttt atccaagaga agccatacca agagtga 3897

<210> 24

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-Orf1ab-RPA-F

<400> 24

ctaacatgct taggataatg gcctctcttg ttc 33

<210> 25

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-Orf1ab -RPA-R

<400> 25

gtaagcgtaa aactcatcca cgaattcatg atc 33

<210> 26

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> MERS-upE-RPA-F

<400> 26

ctcgcttatc gtttaagcag ctctgcgcta ctatg 35

<210> 27

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> MERS-upE-RPA-F

<400> 27

ctcgcttatc gtttaagcag ctctgcgcta ctatg 35

<210> 28

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> MERS-Orf1-RPA-F

<400> 28

ctattcccac acagttgttc ccactcttat 30

<210> 29

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> MERS-Orf1-RPA-R

<400> 29

cttgaggctt ctccaatgct ataagtgtac 30

<210> 30

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1-K180R

<400> 30

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttccgt 540

gggttccacg agaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgaa cggctgggat aaaaataagg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 31

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1-E184R

<400> 31

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttcaaa 540

gggttccacc gtaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgaa cggctgggat aaaaataagg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 32

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1-N607R

<400> 32

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttcaaa 540

gggttccacg agaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgcg tggctgggat aaaaataagg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 33

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1-K613R

<400> 33

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttcaaa 540

gggttccacg agaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgaa cggctgggat aaaaatcgtg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 34

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1-E184R-N607R

<400> 34

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttcaaa 540

gggttccacc gtaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgcg tggctgggat aaaaataagg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 35

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1-E184R-N607R-K613R

<400> 35

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttcaaa 540

gggttccacc gtaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgcg tggctgggat aaaaatcgtg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 36

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1-T148R

<400> 36

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt ccgtactgcc tttacgggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgggtggat gggataagga caaagagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 37

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1-T152R

<400> 37

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt cacgactgcc tttcgtggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgggtggat gggataagga caaagagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 38

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1-G532R

<400> 38

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt cacgactgcc tttacgggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgcgtggat gggataagga caaagagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 39

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1-K538R

<400> 39

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt cacgactgcc tttacgggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgggtggat gggataagga ccgtgagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 40

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1-T152R-G532R

<400> 40

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt cacgactgcc tttcgtggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgcgtggat gggataagga caaagagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 41

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1-T152R-G532R-K538R

<400> 41

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt cacgactgcc tttcgtggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgcgtggat gggataagga ccgtgagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 42

<211> 3687

<212> DNA

<213> Artificial Sequence

<220>

<223> LbCpf1-K536R

<400> 42

atgtctaagc tggaaaaatt caccaactgc tacagtttaa gcaagacgtt acgcttcaag 60

gcgatcccgg ttgggaaaac acaagagaat attgacaaca aacgcctttt agttgaagat 120

gagaagcgtg cagaggacta taaaggagtt aagaagcttt tggaccgtta ctacttatcg 180

tttattaatg atgtgctgca ttccatcaaa ctgaagaatc tgaataatta cattagtctt 240

ttccgtaaga aaacacgtac tgagaaggaa aacaaggagc tggaaaatct tgagatcaat 300

ttgcgcaagg agattgctaa ggcattcaaa ggtaacgagg gatataagtc gttgtttaaa 360

aaagacatta ttgaaacgat ccttccggaa ttccttgacg ataaagacga gattgcactg 420

gtgaactcct tcaacgggtt cacgactgcc tttacgggtt tttttgacaa ccgcgagaat 480

atgttcagtg aagaagccaa atctacctct atcgctttcc gctgcattaa tgaaaacctg 540

actcgttata tctctaacat ggacatcttc gaaaaggtag acgcgatctt tgataaacac 600

gaagtgcagg agatcaagga gaaaattctt aatagcgact atgatgttga agatttcttt 660

gagggggaat tctttaactt tgtactgacg caagaaggga tcgacgtcta taacgctatt 720

atcggaggat tcgtaaccga gagtggagag aaaattaagg gtcttaatga gtacattaat 780

ttgtacaatc aaaaaacgaa acagaaattg cccaagttta aacctctgta taaacaagtg 840

ctgagtgatc gcgaaagttt gtcattctat ggagaggggt atactagcga cgaggaggtt 900

cttgaggtgt ttcgcaacac cttaaacaag aactctgaga ttttcagctc aatcaaaaaa 960

cttgagaaat tatttaaaaa tttcgacgaa tactcgtcag ccggcatttt cgtgaaaaat 1020

ggtccggcca tttccacaat ctctaaggac atctttggag aatggaacgt tatccgtgac 1080

aaatggaacg ctgaatatga tgacatccat ttgaaaaaga aggctgtcgt tacagaaaaa 1140

tatgaagatg atcgccgcaa gagcttcaaa aagatcggtt cgttctcgct tgagcagttg 1200

caagaatacg cagatgccga tctgtcagta gtagagaaat taaaggaaat cattattcaa 1260

aaagtggacg agatttacaa agtttacgga tcctctgaga agctttttga tgcagatttt 1320

gtgttggaga aatcgttaaa aaagaatgat gccgtcgtgg caattatgaa agacttatta 1380

gatagcgtga agtccttcga gaattatatt aaggcgttct ttggagaggg taaggagaca 1440

aaccgcgacg aatcgtttta cggcgatttc gtgttggcct acgacatctt gctgaaagtt 1500

gatcacattt acgacgccat ccgtaactat gtaactcaga aaccctacag taaggacaag 1560

tttaagttat actttcagaa tccgcagttc atgggtggat gggatcgcga caaagagaca 1620

gactaccgtg ctactatctt acgctacggg tcgaagtatt accttgcaat tatggataag 1680

aagtacgcaa aatgcttgca gaagatcgat aaagacgacg tcaacggaaa ttatgaaaag 1740

atcaattata aattattacc cgggcctaat aaaatgttgc ccaaagtatt tttctctaag 1800

aaatggatgg cctactacaa tccatcagaa gacattcaga agatctataa aaacggcaca 1860

ttcaaaaaag gcgatatgtt caacttgaac gattgccaca aactgattga tttctttaag 1920

gatagcatta gccgttaccc gaagtggtcc aacgcttatg actttaactt cagcgaaaca 1980

gagaagtata aagacattgc agggttctac cgtgaggtag aagaacaagg atataaagtt 2040

tctttcgaaa gtgcaagtaa gaaggaagta gataaattgg ttgaggaggg caaactgtat 2100

atgtttcaga tttacaataa ggatttctct gacaagtccc acggcacacc caatttgcat 2160

actatgtact tcaaacttct gtttgacgag aataaccatg ggcagatccg tcttagtggg 2220

ggagctgagt tatttatgcg ccgtgcgtct ttgaaaaagg aggagttagt tgttcacccg 2280

gcaaattcac ccattgccaa caaaaatcca gataacccca agaagacaac cactctttca 2340

tacgatgtgt acaaagataa gcgtttctct gaagatcaat acgaactgca tattccgatc 2400

gcaatcaaca agtgcccaaa gaacatcttt aaaatcaata cggaagttcg cgttcttctg 2460

aaacatgatg ataacccgta tgttattgga attgatcgtg gggagcgtaa cttgttgtat 2520

attgtcgtag tagatggaaa aggtaacatc gtcgaacagt actcgctgaa tgagattatt 2580

aacaacttca acgggattcg catcaagact gattaccact cacttcttga caaaaaggag 2640

aaggagcgct ttgaagcccg tcaaaattgg acatcaatcg aaaatattaa ggagctgaaa 2700

gccggataca tctctcaagt ggtccataaa atttgtgaac tggtcgagaa atatgatgcc 2760

gtgatcgcgc ttgaggacct gaatagtgga ttcaaaaact cgcgtgtgaa agttgagaaa 2820

caggtgtatc agaaatttga gaaaatgtta attgacaaat tgaactacat ggtagataaa 2880

aagagtaacc catgtgcgac gggcggtgca ttgaagggat accagatcac caataagttt 2940

gagtctttca aaagtatgtc tacgcagaat ggttttattt tctacatccc ggcttggctg 3000

acatctaaaa ttgacccttc gacgggtttt gtcaatctgc tgaaaacaaa gtatacaagt 3060

attgcggatt caaaaaaatt catctcatct ttcgatcgca tcatgtacgt cccagaagaa 3120

gacttatttg agtttgcact ggattataag aatttttccc gtactgacgc agactatatc 3180

aagaaatgga aattatattc atacgggaac cgcattcgta tcttccgtaa tcctaaaaag 3240

aacaacgttt tcgactggga agaagtatgc ttaaccagtg cttacaaaga attgttcaac 3300

aaatacggca ttaactacca gcagggcgac attcgcgcat tattatgcga acagtcagat 3360

aaagcgttct actccagctt tatggcgtta atgtcgctta tgttacagat gcgcaacagc 3420

attacaggcc gcaccgatgt cgattttctg atttcgccgg tgaaaaacag cgatggcatt 3480

ttttacgatt ctcgcaatta cgaggcgcag gaaaatgcaa ttcttccgaa gaacgcagac 3540

gcaaacggcg cttacaacat cgcacgtaaa gttctttggg cgatcggaca atttaaaaaa 3600

gccgaggatg aaaaattgga caaggttaaa attgctatct cgaataaaga gtggcttgag 3660

tacgcgcaaa cttctgttaa gcactga 3687

<210> 43

<211> 3903

<212> DNA

<213> Artificial Sequence

<220>

<223> FnCpf1-K611R

<400> 43

atgtctatct accaggagtt tgtaaacaag tattctctta gtaaaacgtt gcgttttgaa 60

cttattccac agggtaaaac cctggagaat attaaggctc gtggcttaat tcttgatgac 120

gaaaagcgcg caaaggatta caaaaaggca aaacaaatta ttgataagta ccatcagttc 180

ttcattgaag agattttatc cagcgtttgt atctccgagg acctgttaca gaactacagc 240

gatgtatatt tcaaattgaa aaaatccgat gatgacaatc tgcaaaagga ttttaaatcg 300

gccaaggaca ctatcaagaa acaaatttct gagtacatca aagatagcga gaaattcaag 360

aatttattta accaaaatct gatcgacgcg aagaaggggc aggagtcaga cttgattttg 420

tggttgaaac agtcgaaaga taatggaatc gaactgttca aagccaactc tgacattaca 480

gatattgatg aagctctgga aattattaag agctttaagg ggtggaccac ctatttcaaa 540

gggttccacg agaaccgtaa aaacgtgtac tcttcgaatg atattcctac tagcattatt 600

tatcgtattg tagacgacaa ccttcctaaa ttcttggaga ataaagctaa atatgaatcg 660

ctgaaggata aggcgcccga agcaatcaat tacgaacaaa tcaaaaaaga tctggctgaa 720

gaactgacct ttgacattga ctataaaact tctgaagtaa accagcgcgt tttcagtctt 780

gacgaggttt ttgaaatcgc taattttaat aattatttaa atcaatccgg gattacaaaa 840

tttaacacga ttattggtgg caagttcgta aatggagaga acactaagcg taagggaatt 900

aacgagtaca ttaatttata ctcgcagcag attaacgaca aaacgctgaa aaaatataaa 960

atgagcgttt tatttaagca aattctgagc gataccgagt caaagtcctt cgtaattgac 1020

aagcttgagg atgactctga cgttgtcacg acgatgcagt cattctatga acagattgcg 1080

gcttttaaaa ccgtggaaga aaaatcaatc aaagagacct tgtcattatt atttgacgat 1140

ctgaaagctc aaaagttgga cttaagtaag atctacttta agaatgataa gagtctgacg 1200

gatttatccc aacaagtctt tgacgactac tctgtaattg gcacagccgt cttggaatac 1260

atcacccaac aaatcgctcc taaaaattta gacaatccga gtaaaaagga acaagagctg 1320

attgcaaaaa agaccgagaa agcgaaatat ttatcgttag aaactattaa attagcgctg 1380

gaagaattta ataaacaccg tgacattgac aagcaatgtc gtttcgaaga gattttagct 1440

aactttgcgg ccatcccgat gatctttgac gagattgcac aaaataaaga caatcttgcg 1500

cagatctcga ttaagtatca gaaccaaggt aagaaagacc tgttgcaggc ttccgcagag 1560

gatgacgtta aagcgatcaa agatctgtta gaccaaacca acaatttgct tcacaagtta 1620

aagatttttc acatttcaca gtcagaggat aaagcaaaca ttcttgataa agacgaacac 1680

ttttatttag ttttcgagga atgttatttt gagctggcga acattgttcc cctttataat 1740

aagatccgta attatatcac tcagaaacct tattctgacg agaaattcaa gttgaatttt 1800

gaaaactcca ctcttgcgaa cggctgggat cgcaataagg aacccgataa tactgccatt 1860

ctgtttatta aagacgacaa atattactta ggagttatga ataagaaaaa caataagatc 1920

tttgatgaca aagctattaa ggagaacaaa ggcgagggct acaaaaaaat tgtatacaaa 1980

cttctgcctg gagctaacaa gatgttgcca aaagtatttt tcagtgcaaa atcgattaag 2040

ttctacaacc catcggagga tattttacgt attcgcaacc attccaccca tacgaaaaat 2100

gggtcacccc aaaagggtta cgaaaagttt gagtttaata ttgaagattg ccgtaagttt 2160

attgattttt ataaacaatc tatctctaaa caccccgaat ggaaggattt cggattccgt 2220

ttctctgaca cacaacgcta caattcgatc gatgaattct atcgtgaggt tgagaaccaa 2280

ggatataagt tgacatttga aaacatttcc gaatcataca tcgattcggt cgtgaaccag 2340

ggtaagttat atttatttca gatctacaac aaggattttt ccgcctatag caaaggccgt 2400

ccgaacttac atacgttgta ctggaaggcc ctgtttgatg agcgcaatct gcaagatgtc 2460

gtatataagt taaatggcga agctgaactg ttttaccgca agcagtcgat cccgaagaag 2520

atcacacatc ctgctaagga ggcaattgct aacaagaata aagacaatcc aaagaaagaa 2580

agtgtcttcg agtatgactt aattaaagat aaacgtttta ctgaggacaa gtttttcttt 2640

cattgcccaa tcacaatcaa tttcaaatca agtggagcaa acaagtttaa cgacgaaatt 2700

aacttactgc tgaaggaaaa ggcgaatgac gtgcacattc tgtccattga tcgcggagaa 2760

cgtcacctgg cgtactatac gttggtcgac gggaagggaa acatcattaa acaggacacg 2820

tttaatatta tcggcaatga tcgcatgaag acaaattatc acgacaagct ggcggcaatt 2880

gagaaggatc gtgactcagc tcgcaaagac tggaagaaga ttaacaacat caaggagatg 2940

aaagagggtt atcttagcca agtcgtccat gagattgcaa aattggttat tgaatacaat 3000

gcgattgttg tcttcgaaga tcttaatttt gggttcaaac gtggtcgctt caaagtcgag 3060

aagcaggttt accaaaagct ggaaaagatg cttattgaga aattgaatta cttggttttc 3120

aaggacaatg aattcgataa gacaggcggt gtacttcgtg cgtatcaact tacggcaccc 3180

tttgaaacct tcaaaaaaat gggcaaacaa actgggatca tctactacgt gccggcgggg 3240

ttcaccagca aaatttgtcc tgtaactggg ttcgtgaacc agttgtaccc caaatacgaa 3300

tcagtcagta agtcccagga gtttttttct aagttcgata agatctgtta taatcttgac 3360

aaaggctact ttgagttttc tttcgattat aagaactttg gcgataaagc agccaaaggg 3420

aagtggacta ttgcgtcgtt cggctcacgt cttatcaact ttcgcaacag cgataagaac 3480

cataactggg atacacgtga agtttatcca acgaaggagc ttgagaagtt attaaaagat 3540

tacagtattg aatatgggca cggggagtgc attaaagcag ctatctgtgg tgagtcggac 3600

aagaaattct tcgcaaaact gacgtctgtt cttaatacca tccttcaaat gcgtaattcc 3660

aagacgggta cggagctgga ttacctgatc agtcccgttg ccgatgttaa tggtaatttt 3720

ttcgactcgc gtcaggcccc gaagaacatg cctcaggacg ccgatgccaa cggtgcgtac 3780

catattggcc ttaagggctt aatgctgtta ggacgtatta agaacaacca ggagggtaaa 3840

aaactgaatc tggtcattaa aaacgaagaa tatttcgaat ttgtacagaa ccgcaataac 3900

tga 3903

<210> 44

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> CoV2-E- RPA-F

<400> 44

tgtactcatt cgtttcggaa gagacaggta cg 32

<210> 45

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Cov2-E- RPA-R

<400> 45

tagaccagaa gatcaggaac tctagaagaa ttcag 35

<210> 46

<211> 308

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV-2-E

<400> 46

taatacgact cactataggg agatctagaa ataattttgt ttaactttaa gaaggagata 60

taccatgatg tactcattcg tttcggaaga gacaggtacg ttaatagtta atagcgtact 120

tctttttctt gctttcgtgg tattcttgct agttacacta gccatcctta ctgcgcttcg 180

attgtgtgcg tactgctgca atattgttaa cgtgagtctt gtaaaacctt ctttttacgt 240

ttactctcgt gttaaaaatc tgaattcttc tagagttcct gatcttctgg tctaattttt 300

ttgaattc 308

<210> 47

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-E-Cr1

<400> 47

uaauuucuac uaaguguaga uacaagacuc acguuaacaa uauu 44

<210> 48

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-E-Cr2

<400> 48

uaauuucuac uaaguguaga uacacgagag uaaacguaaa aaga 44

<210> 49

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-E-Cr3

<400> 49

uaauuucuac uaaguguaga uaacacgaga guaaacguaa aaag 44

<210> 50

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-E-Cr4

<400> 50

uaauuucuac uaaguguaga uuacguuuac ucucguguua aaaa 44

<210> 51

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-E-Cr5

<400> 51

uaauuucuac uaaguguaga uuaacacgag aguaaacgua aaaa 44

<210> 52

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-E-Cr6

<400> 52

uaauuucuac uaaguguaga uacguuuacu cucguguuaa aaau 44

<210> 53

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-E-Cr7

<400> 53

uaauuucuac uaaguguaga ucguuuacuc ucguguuaaa aauc 44

<210> 54

<211> 1228

<212> PRT

<213> Lachnospiraceae bacterium ND2006

<400> 54

Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu

65 70 75 80

Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn

145 150 155 160

Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile

165 170 175

Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys

180 185 190

Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys

195 200 205

Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe

210 215 220

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

225 230 235 240

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

245 250 255

Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys

260 265 270

Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser

275 280 285

Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys

450 455 460

Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr

465 470 475 480

Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile

485 490 495

Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr

500 505 510

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

515 520 525

Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala

530 535 540

Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys

545 550 555 560

Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly

565 570 575

Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met

580 585 590

Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro

595 600 605

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

610 615 620

Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys

625 630 635 640

Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn

645 650 655

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

660 665 670

Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys

675 680 685

Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile

690 695 700

Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His

705 710 715 720

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

725 730 735

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

740 745 750

Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys

755 760 765

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

770 775 780

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

785 790 795 800

Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val

805 810 815

Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp

820 825 830

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

835 840 845

Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn

850 855 860

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

865 870 875 880

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

885 890 895

Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys

900 905 910

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

915 920 925

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

930 935 940

Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys

945 950 955 960

Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile

965 970 975

Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe

980 985 990

Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr

995 1000 1005

Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp

1010 1015 1020

Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro

1025 1030 1035

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

1040 1045 1050

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

1055 1060 1065

Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val

1070 1075 1080

Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu

1085 1090 1095

Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala

1100 1105 1110

Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met

1115 1120 1125

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

1130 1135 1140

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

1145 1150 1155

Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala

1160 1165 1170

Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala

1175 1180 1185

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

1190 1195 1200

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

1205 1210 1215

Leu Glu Tyr Ala Gln Thr Ser Val Lys His

1220 1225

<210> 55

<211> 1300

<212> PRT

<213> Francisella novicida U112

<400> 55

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

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

20 25 30

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

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

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

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

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

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

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

370 375 380

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

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

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

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

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

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

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

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035

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

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

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

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

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

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

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

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

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

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 56

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr3

<400> 56

uaauuucuac uaaguguaga uacaagacuc acguuaacaa uauu 44

<210> 57

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr4

<400> 57

uaauuucuac uaaguguaga uacacgagag uaaacguaaa aaga 44

<210> 58

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr5

<400> 58

uaauuucuac uaaguguaga uaacacgaga guaaacguaa aaag 44

<210> 59

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr6

<400> 59

uaauuucuac uaaguguaga uuacguuuac ucucguguua aaaa 44

<210> 60

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr7

<400> 60

uaauuucuac uaaguguaga uuaacacgag aguaaacgua aaaa 44

<210> 61

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr8

<400> 61

uaauuucuac uaaguguaga uacguuuacu cucguguuaa aaau 44

<210> 62

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr9

<400> 62

uaauuucuac uaaguguaga ucguuuacuc ucguguuaaa aauc 44

<210> 63

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-S-cr10

<400> 63

uaauuucuac uaaguguaga uacuccuggu gauucuucuu cagg 44

<210> 64

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-M-cr1

<400> 64

uaauuucuac uaaguguaga uuagaagcgg ucuggucaga auag 44

<210> 65

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-M-cr2

<400> 65

uaauuucuac uaaguguaga uggcaggucc uugaugucac agcg 44

<210> 66

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-M-cr3

<400> 66

uaauuucuac uaaguguaga uuuuaggcag guccuugaug ucac 44

<210> 67

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-M-cr4

<400> 67

uaauuucuac uaaguguaga uuaauaagaa agcguucgug augu 44

<210> 68

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-M-cr5

<400> 68

uaauuucuac uaaguguaga uuuauuacaa auugggagcu ucgc 44

<210> 69

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-N-cr1

<400> 69

uaauuucuac uaaguguaga uuugaacugu ugcgacuacg ugau 44

<210> 70

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-N-cr2

<400> 70

uaauuucuac uaaguguaga ucugcugcuu gacagauuga acca 44

<210> 71

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-N-cr3

<400> 71

uaauuucuac uaaguguaga ucucucaagc ugguucaauc uguc 44

<210> 72

<211> 44

<212> RNA

<213> Artificial Sequence

<220>

<223> CoV2-N-cr4

<400> 72

uaauuucuac uaaguguaga ugccuuguug uuguuggccu uuac 44

<210> 73

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> CoV2-S- RPA-F

<400> 73

ggaaagtgag ttcagagttt attctagtgc ga 32

<210> 74

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> CoV2-S- RPA-R

<400> 74

cacagtctac agcatctgta atggttccat 30

<210> 75

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> CoV2-M- RPA-F

<400> 75

ctacttcatt gcttctttca gactgtttgc 30

<210> 76

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> CoV2-M- RPA-R

<400> 76

ttatagttgc caatcctgta gcgactgtat 30

<210> 77

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> CoV2-N- RPA-F

<400> 77

atcgtgctac aacttcctca aggaacaaca 30

<210> 78

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> CoV2-N- RPA-R

<400> 78

gcaatttgcg gccaatgttt gtaatcagtt 30

<210> 79

<211> 416

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV2-S-基因

<400> 79

ggaaagtgag ttcagagttt attctagtgc gaataattgc acttttgaat atgtctctca 60

gccttttctt atggaccttg aaggaaaaca gggtaatttc aaaaatctta gggaatttgt 120

gtttaagaat attgatggtt attttaaaat atattctaag cacacgccta ttaatttagt 180

gcgtgatctc cctcagggtt tttcggcttt agaaccattg gtagatttgc caataggtat 240

taacatcact aggtttcaaa ctttacttgc tttacataga agttatttga ctcctggtga 300

ttcttcttca ggttggacag ctggtgctgc agcttattat gtgggttatc ttcaacctag 360

gacttttcta ttaaaatata atgaaaatgg aaccattaca gatgctgtag actgtg 416

<210> 80

<211> 333

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV2-M-基因

<400> 80

ctacttcatt gcttctttca gactgtttgc gcgtacgcgt tccatgtggt cattcaatcc 60

agaaactaac attcttctca acgtgccact ccatggcact attctgacca gaccgcttct 120

agaaagtgaa ctcgtaatcg gagctgtgat ccttcgtgga catcttcgta ttgctggaca 180

ccatctagga cgctgtgaca tcaaggacct gcctaaagaa atcactgttg ctacatcacg 240

aacgctttct tattacaaat tgggagcttc gcagcgtgta gcaggtgact caggttttgc 300

tgcatacagt cgctacagga ttggcaacta taa 333

<210> 81

<211> 446

<212> DNA

<213> Artificial Sequence

<220>

<223> SARS-CoV2-N-基因

<400> 81

atcgtgctac aacttcctca aggaacaaca ttgccaaaag gcttctacgc agaagggagc 60

agaggcggca gtcaagcctc ttctcgttcc tcatcacgta gtcgcaacag ttcaagaaat 120

tcaactccag gcagcagtag gggaacttct cctgctagaa tggctggcaa tggcggtgat 180

gctgctcttg ctttgctgct gcttgacaga ttgaaccagc ttgagagcaa aatgtctggt 240

aaaggccaac aacaacaagg ccaaactgtc actaagaaat ctgctgctga ggcttctaag 300

aagcctcggc aaaaacgtac tgccactaaa gcatacaatg taacacaagc tttcggcaga 360

cgtggtccag aacaaaccca aggaaatttt ggggaccagg aactaatcag acaaggaact 420

gattacaaac attggccgca aattgc 446