CN115380111A - 用于碱基多样化的组合物、系统和方法 - Google Patents

用于碱基多样化的组合物、系统和方法 Download PDF

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CN115380111A
CN115380111A CN202180021501.6A CN202180021501A CN115380111A CN 115380111 A CN115380111 A CN 115380111A CN 202180021501 A CN202180021501 A CN 202180021501A CN 115380111 A CN115380111 A CN 115380111A
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deaminase
nucleic acid
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crispr
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Y·金
A·哈默尔
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Pairing Plant Service Co ltd
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Abstract

本申请描述了修饰或编辑靶核酸的方法，例如将胞嘧啶编辑为胸腺嘧啶和将腺嘌呤编辑为鸟嘌呤的方法和/或将胞嘧啶编辑为胸腺嘧啶、腺嘌呤或鸟嘌呤的方法。本申请还描述了用于修饰或编辑靶核酸的组合物和系统。本申请所述的方法、组合物和系统可用于产生等位基因多样性。

Description

用于碱基多样化的组合物、系统和方法
关于序列表电子提交的声明
根据37C.F.R.§1.821提交的ASCII文本格式的序列表，标题为1499-9WO_ST25，大小为609,139字节，生成于2021年1月29日，并通过EFS-Web提交，提供代替纸质副本。该序列表在此通过引用并入本说明书的公开内容。
技术领域
本发明涉及修饰或编辑靶核酸的方法，例如将胞嘧啶编辑为胸腺嘧啶和将腺嘌呤编辑为鸟嘌呤的方法和/或将胞嘧啶编辑为胸腺嘧啶、腺嘌呤或鸟嘌呤的方法。本发明进一步涉及用于修饰或编辑靶核酸的组合物和系统。
背景技术
虽然CRISPR-Cas9和相关技术提供了一种在基因座内产生靶向突变的方法，但它们产生的产物类型是非常确定的。当前的CRISPR技术不擅长以半随机方式产生等位基因多样性。等位基因多样性的产生对于发现新的表型和性状是有价值的。因此，能够从单一工具产生多种结果的新方法将是有利的。
发明内容
本发明的第一方面涉及修饰靶核酸的方法，该方法包括：使靶核酸接触：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(指导RNA)、胞嘧啶脱氨酶和腺嘌呤脱氨酶，其中CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶形成复合物或包含在复合物中，从而修饰靶核酸。该方法可以进一步包括使用修饰的靶核酸确定期望的或优选的表型。
本发明的另一方面涉及碱基编辑组合物或系统，其包含：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)、胞嘧啶脱氨酶和腺嘌呤脱氨酶，其中CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶形成复合物或包含在复合物中。
本发明的另一方面涉及修饰靶核酸的方法，该方法包括：使靶核酸接触：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)和胞嘧啶脱氨酶，其中该方法将靶核酸的胞嘧啶(C)修饰为腺嘌呤(A)、鸟嘌呤(G)或胸腺嘧啶(T)，从而修饰靶核酸。该方法可以进一步包括使用修饰的靶核酸确定期望的或优选的表型。
本发明的另一方面涉及碱基编辑组合物或系统，其包含：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)和胞嘧啶脱氨酶，其中所述组合物或系统不含糖基化酶抑制剂(例如，尿嘧啶糖基化酶抑制剂(UGI))。
本发明进一步提供了包含本发明的核酸构建体的表达盒和/或载体，以及包含本发明的多肽、融合蛋白和/或核酸构建体的细胞。此外，本发明提供了包含本发明的核酸构建体和包含其的表达盒、载体和/或细胞的试剂盒。
应当注意，关于一个实施方案描述的本发明的方面可以并入不同的实施方案中，尽管没有相对于其的具体描述。也就是说，所有实施方案和/或任何实施方案的特征可以以任何方式和/或组合进行组合。申请人保留更改任何最初提交的权利要求和/或相应地提交任何新的权利要求的权利，包括能够修改任何最初提交的权利要求以依赖和/或合并任何其他权利要求的任何特征的权利要求，尽管最初没有以这种方式提出要求。本发明的这些和其他目的和/或方面在下面阐述的说明书中详细解释。本领域普通技术人员通过阅读附图和随后的优选实施方案的详细描述将理解本发明的其他特征、优点和细节，这样的描述仅是对本发明的说明。
附图说明
图1是显示根据本发明一些实施方案使用MS2/MCP系统的C-和A-碱基编辑结果的图。
图2是显示根据本发明一些实施方案使用具有Cas9的SunTag系统的C-和A-碱基编辑结果的图。
图3提供了显示根据本发明一些实施方案使用TREE系统的C-和A-碱基编辑结果的图。
图4是显示根据本发明一些实施方案使用与MCP融合的各种脱氨酶结构域的反式募集而由Cas9(D10A)介导的碱基多样化的图。
图5是显示根据本发明的一些实施方案的碱基多样化可以在不存在UGI的情况下产生显著量的插入缺失突变，而无论脱氨酶结构域如何的图。
图6提供了显示根据本发明一些实施方案对靶碱基进行C编辑以及CRT0044876降低插入缺失突变率的图。
图7-26是显示根据本发明一些实施方案的关于各个间隔区序列的碱基编辑百分比的图。
图27是显示根据本发明一些实施方案由具有或不具有Gam的胞嘧啶脱氨酶产生的插入缺失(indels)百分比的图。
具体实施方式
现在将在下文中参考附图和实施例来描述本发明，其中示出了本发明的实施方案。该描述并非旨在成为可以实施本发明的所有不同方式或可以添加到本发明的所有特征的详细目录。例如，关于一个实施方案说明的特征可以并入其他实施方案中，并且关于特定实施方案说明的特征可以从该实施方案中删除。因此，本发明设想在本发明的一些实施方案中，可以排除或省略本文阐述的任何特征或特征组合。此外，根据本公开内容，本文提出的各种实施方案的许多变化和添加对于本领域技术人员来说将是显而易见的，它们不脱离本发明。因此，以下描述旨在示例说明本发明的一些特定实施方案，而不是详尽地指定其所有排列、组合和变化。
除非另有定义，本文使用的所有技术和科学术语与本发明所属领域的普通技术人员通常理解的含义相同。在本文的本发明的描述中使用的术语仅用于描述特定实施方案的目的，并不旨在限制本发明。
本文引用的所有出版物、专利申请、专利和其他参考文献通过引用整体并入，以用于与在其中呈现参考文献的句子和/或段落相关的教导。
除非上下文另有说明，否则本文所述的本发明的各种特征特别旨在以任何组合形式使用。此外，本发明还设想在本发明的一些实施方案中，可以排除或省略本文阐述的任何特征或特征的组合。为了示例说明，如果说明书声明组合物包含组分A、B和C，则特别意在A、B或C中的任何一个或其组合可以单独或以任何组合被省略和排除。
如在本发明的描述和所附权利要求中使用的，单数形式“一个”、“一种”和“该”旨在也包括复数形式，除非上下文另有明确说明。
同样如本文所用，“和/或”是指并涵盖一个或多个相关列出的项目的任何和所有可能的组合，以及当解释为备选方案(“或”)时缺少组合。
如本文所用的术语“约”在提及诸如量或浓度等的可测量值时，意在包括所述值的±10％、±5％、±1％、±0.5％或甚至±0.1％的变化以及所述值。例如，“约X”，其中X是可测量的值，意味着包括X以及X的±10％、±5％、±1％、±0.5％或甚至±0.1％的变化。此处提供的可测量值的范围可以包括其中的任何其他范围和/或单个值。
如本文所用，诸如“在X和Y之间”和“在约X和Y之间”的短语应当被解释为包括X和Y。如在本文中使用的那样，诸如“在约X和Y之间”的短语是指“在约X和约Y之间”和诸如“从约X到Y”之类的短语是指“从约X到约Y”。
除非在本文中另有说明，否则本文中数值范围的列举仅旨在用作单独引用落入该范围内的每个单独值的速记方法，并且每个单独的值被并入说明书中，就好像它在本文中单独列举一样。例如，如果公开了10到15的范围，则还公开了11、12、13和14。
如本文所用，术语“包括”、“包含”和“含有”指定了所述特征、整数、步骤、操作、元素和/或组件的存在，但不排除一种或多种其他特征、整数、步骤、操作、元素、组件和/或它们的组的存在或添加。
如本文所用，过渡短语“基本上由......组成”是指权利要求的范围应解释为包括权利要求中列举的具体材料或步骤，以及那些不实质影响所要求保护的发明的基本和新颖特征的材料或步骤。因此，当在本发明的权利要求中使用时，术语“基本上由......组成”并不旨在解释为等同于“包括”。
如本文所用，术语“增加”、“增加了”、“增强”、“增强了”、“改善”和“改善了”(及其语法变体)描述了至少约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％、150％、200％、300％、400％、500％或更多的提升，例如与另一个可测量的特性或数量(例如，对照值)相比。
如本文所用，术语“减少”、“减少的”、“减少了”、“减少量”、“减弱”和“降低”(及其语法变体)描述例如至少约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％或100％的减少，例如与另一个可测量的特性或数量(例如，对照值)相比。在一些实施方案中，减少可导致没有或基本上没有(即，微不足道的量，例如，小于约10％或甚至5％)可检测的活性或量。
“异源”或“重组”核苷酸序列是与引入其的宿主细胞不天然相关的核苷酸序列，包括天然存在的核苷酸序列的非天然存在的多个拷贝。
“天然”或“野生型”核酸、核苷酸序列、多肽或氨基酸序列是指天然存在的或内源性核酸、核苷酸序列、多肽或氨基酸序列。因此，例如，“野生型mRNA”是在参考生物体中天然存在或对其是内源的mRNA。“同源”核酸序列是与引入其的宿主细胞天然相关的核苷酸序列。
如本文所用，术语“核酸”、“核酸分子”、“核苷酸序列”和“多核苷酸”是指线性或分支、单链或双链、或其杂合体的RNA或DNA。该术语还包括RNA/DNA杂合体。当合成产生dsRNA时，不太常见的碱基，如肌苷、5-甲基胞嘧啶、6-甲基腺嘌呤、次黄嘌呤等也可用于反义、dsRNA和核酶配对。例如，含有尿苷和胞苷的C-5丙炔类似物的多核苷酸已显示以高亲和力结合RNA，并且是基因表达的有效反义抑制剂。也可以进行其他修饰，例如对磷酸二酯骨架或RNA的核糖糖基团中的2′-羟基的修饰。
如本文所用，术语“核苷酸序列”是指核苷酸的杂聚物或这些核苷酸从核酸分子的5′到3′端的序列，并且包括DNA或RNA分子，包括cDNA、DNA片段或部分、基因组DNA、合成(例如化学合成)DNA、质粒DNA、mRNA和反义RNA，其中任何一种都可以是单链或双链的。术语“核苷酸序列”、“核酸”、“核酸分子”、“核酸构建体”、“重组核酸”、“寡核苷酸”和“多核苷酸”在本文中也可互换使用以指核苷酸的杂聚物。本文提供的核酸分子和/或核苷酸序列在本文中以5′到3′方向从左到右呈现，并使用用于表示核苷酸字符的标准代码表示，如美国序列规则37CFR§§1.821-1.825和世界知识产权组织(WIPO)标准ST.25。如本文所用，“5′区域”可以指多核苷酸中最接近多核苷酸的5′末端的区域。因此，例如，多核苷酸的5′区域中的元件可以位于从位于多核苷酸的5′末端的第一个核苷酸到位于多核苷酸中途的核苷酸的任何位置。如本文所用，“3′区域”可以指多核苷酸中最接近多核苷酸的3′末端的区域。因此，例如，多核苷酸的3′区域中的元件可以位于从位于多核苷酸的3′端的第一个核苷酸到位于多核苷酸中途的核苷酸的任何位置。
如本文所用，术语“基因”是指能够用于产生mRNA、反义RNA、miRNA、抗微RNA反义寡脱氧核糖核苷酸(AMO)等的核酸分子。基因可以或可以不能用于产生功能性蛋白质或基因产物。基因可以包括编码区和非编码区(例如，内含子、调控元件、启动子、增强子、终止序列和/或5′和3′非翻译区)。基因可以是“分离的”，其意指基本上或实质上不含通常发现的与其天然状态的核酸相关联的组分的核酸。此类组分包括其他细胞材料、来自重组生产的培养基和/或用于化学合成核酸的各种化学品。
术语“突变”是指点突变(例如，错义，或无义，或导致移码的单个碱基对的插入或缺失)、插入、缺失和/或截短。当突变是氨基酸序列中的一个残基被另一个残基取代，或者是序列中一个或多个残基的缺失或插入时，突变通常通过识别原始残基、然后是该残基在序列中的位置以及通过新取代残基的身份来描述。
如本文所用，术语“互补”或“互补性”是指多核苷酸在允许的盐和温度条件下通过碱基配对的天然结合。例如，序列“A-G-T”(5′至3′)与互补序列“T-C-A”(3′至5′)结合。两个单链分子之间的互补性可以是“部分的”，其中只有一些核苷酸结合，或者当单链分子之间存在完全互补性时，它可以是完全的。核酸链之间的互补程度对核酸链之间杂交的效率和强度有显著影响。
如本文所用，“互补”可以表示与比较核苷酸序列100％互补性，或者它可以表示小于100％的互补性(例如，“基本上的互补性”，例如约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％等的互补性)。
核苷酸序列或多肽序列的“部分”或“片段”将理解为表示长度减少的核苷酸或多肽序列(例如，相对于参考核苷酸或多肽序列，分别减少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个残基(例如，核苷酸或肽))，并且包括以下、基本上由以下组成和/或由以下组成：与参考核苷酸或多肽序列分别相同或几乎相同(例如，70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相同)的连续残基的核苷酸或多肽序列。根据本发明的这种核酸片段或部分可以酌情包含在其作为构件的更大多核苷酸中。例如，本发明的指导核酸的重复序列可以包含野生型CRISPR-Cas重复序列的部分(例如，野生型V型CRISPR Cas重复，例如来自CRISPR Cas系统的重复，包括但不限于Cas12a(Cpf1)、Cas12b、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12g、Cas12h、Cas12i、C2c1、C2c4、C2c5、C2c8、C2c9、C2c10、Cas14a、Cas14b和/或Cas14c等)。
具有同源性的不同核酸或蛋白质在本文中称为“同源物”。术语同源物包括来自相同和其他物种的同源序列以及来自相同和其他物种的直系同源序列。“同源性”是指两个或更多个核酸和/或氨基酸序列之间在位置同一性百分比(即序列相似性或同一性)方面的相似性水平。同源性也指不同核酸或蛋白质之间具有相似功能特性的概念。因此，本发明的组合物和方法进一步包含本发明的核苷酸序列和多肽序列的同源物。如本文所用，“直系同源”是指不同物种中在物种形成过程中由共同祖先基因产生的同源核苷酸序列和/或氨基酸序列。本发明的核苷酸序列的同源物对本发明的所述核苷酸序列具有基本的序列同一性(例如，至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或100％)。
如本文所用，“序列同一性”是指两个最佳比对的多核苷酸或多肽序列在整个组分(例如核苷酸或氨基酸)的比对窗口中不变的程度。“同一性”可以很容易地通过已知方法计算，包括但不限于：Computational Molecular Biology(Lesk，A.M.编)Oxford UniversityPress，New York(1988)；Biocomputing：Informatics and Genome Projects(Smith，D.W.编)Academic Press，New York(1993)；Computer Analysis of Sequence Data，Part I(Griffin，A.M.和Griffin，H.G.编)Humana Press，New Jersey(1994)；Sequence Analysisin Molecular Biology(von Heinje，G.编)Academic Press(1987)；和Sequence AnaysisPrimer(Gribskov，M.和Devereux，J.编)Stockton Press，New York(1991)。
如本文所用，术语“序列同一性百分比”或“同一性百分比”是指当两个序列最佳比对时，与测试(“主题”)多核苷酸分子(或其互补链)相比，参考(“查询”)多核苷酸分子(或其互补链)的线性多核苷酸序列中相同核苷酸的百分比。在一些实施方案中，“同一性百分比”可以指与参考多肽相比，氨基酸序列中相同氨基酸的百分比。
如本文所用，在两个核酸分子、核苷酸序列或蛋白质序列的上下文中的短语“基本上相同”或“基本上同一性”是指当比较和比对最大对应时，具有至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或100％核苷酸或氨基酸残基同一性的两条或更多条序列或子序列，如使用以下序列比较算法之一或通过目视检查所测量的。在本发明的一些实施方案中，基本上同一性存在于本发明核苷酸序列的连续核苷酸区域上，其为约10个核苷酸至约20个核苷酸、约10个核苷酸至约25个核苷酸、约10个核苷酸至约30个核苷酸，约15个核苷酸至约25个核苷酸、约30个核苷酸至约40个核苷酸、约50个核苷酸至约60个核苷酸、约70个核苷酸至约80个核苷酸、约90个核苷酸至约100个核苷酸或更多核苷酸的长度，以及其中的任何范围，直到序列的全长。在一些实施方案中，核苷酸序列可以在至少约20个核苷酸(例如，约20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40个核苷酸)上基本上相同。在一些实施方案中，基本上相同的核苷酸或蛋白质序列执行与其基本上相同的核苷酸(或编码的蛋白质序列)基本上相同的功能。
对于序列比较，通常一个序列充当与测试序列进行比较的参考序列。当使用序列比较算法时，将测试序列和参考序列输入计算机，必要时指定子序列坐标，并指定序列算法程序参数。然后基于指定的程序参数，序列比较算法计算一个或多个测试序列相对于参考序列的百分比序列同一性。
用于比对比较窗口的序列的最佳比对是本领域技术人员熟知的，并且可以通过诸如Smith和Waterman的局部同源性算法、Needleman和Wunsch的同源性比对算法、Pearson和Lipman的相似性搜索方法等工具，以及可选地通过这些算法的计算机化实现来进行，所述计算机化实现例如GAP、BESTFIT、FASTA和TFASTA，这些作为
Figure BDA0003846855640000091
Wisconsin
Figure BDA0003846855640000092
(Accelrys Inc.，San Diego，CA)的一部分提供。测试序列和参考序列的比对片段的“同一性分数”是两个比对序列共享的相同组分的数量除以参考序列片段中的组分总数(例如整个参考序列或参考序列的较小定义部分)。序列同一性百分比表示为同一性分数乘以100。一个或多个多核苷酸序列的比较可以是与全长多核苷酸序列或其一部分、或与更长的多核苷酸序列比较。对于本发明的目的，“百分比同一性”也可以使用用于翻译的核苷酸序列的BLASTX 2.0版和用于多核苷酸序列的BLASTN 2.0版来确定。
当两条核苷酸序列在严格条件下相互杂交时，也可以认为两条核苷酸序列基本上互补。在一些代表性实施方案中，被认为基本上互补的两个核苷酸序列在高度严格的条件下彼此杂交。
核酸杂交实验如Southem和Northern杂交中的“严格杂交条件”和“严格杂交洗涤条件”是序列依赖性的，并且在不同的环境参数下是不同的。在Tijssen LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization with NucleicAcid Probes第I部分第2章″Overview of principles of hybridization and thestrategy of nucleic acid probe assays″Elsevier，New York(1993)有对核酸杂交的详细指导。通常，高度严格的杂交和洗涤条件选择为比特定序列在确定的离子强度和pH值下的热解链点(Tm)低约5℃。
Tm是50％的靶序列与完美匹配的探针杂交的温度(在规定的离子强度和pH下)。选择非常严格的条件以等于针对特定探针的Tm。在Southern或Northern印迹中滤纸上，具有超过100个互补残基的互补核苷酸序列杂交的严格杂交条件的示例是50％甲酰胺与1mg肝素在42℃下进行杂交过夜。高度严格洗涤条件的示例是0.15M NaCl在72℃洗涤约15分钟。严格洗涤条件的示例是0.2x SSC在65℃洗涤15分钟(参见Sambrook，下文，对SSC缓冲液的描述)。通常，高严格性洗涤之前是低严格性洗涤以去除背景探针信号。对于例如超过100个核苷酸的双链体，中等严格性洗涤的示例是1x SSC在45℃下15分钟。对于例如超过100个核苷酸的双链体的低严格性洗涤示例是4-6x SSC在40℃下15分钟。对于短探针(例如，约10至50个核苷酸)，严格条件通常涉及在pH 7.0至8.3下小于约1.0M Na离子的盐浓度，通常为约0.01至1.0M Na离子浓度(或其他盐)，以及温度通常至少约为30℃。添加去稳定剂如甲酰胺也可以达到严格的条件。一般而言，在特定杂交测定中观察到的信噪比是不相关探针的2倍(或更高)表明检测到特异性杂交。如果它们编码的蛋白质基本上相同，则在严格条件下不相互杂交的核苷酸序列仍然基本上相同。例如，当使用遗传密码允许的最大密码子简并性创建核苷酸序列的副本时，可能会发生这种情况。
本发明的多核苷酸和/或重组核酸构建体可以针对表达进行密码子优化。在一些实施方案中，本发明的多核苷酸、核酸构建体、表达盒和/或载体(例如，其包含/编码核酸结合多肽(例如，DNA结合结构域，例如来自多核苷酸引导的核酸内切酶、锌指核酸酶、转录激活因子样效应核酸酶(TALEN)、Argonaute蛋白和/或CRISPR-Cas效应蛋白的序列特异性DNA结合结构域)、指导核酸、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶)可以针对在生物体(例如动物、植物、真菌、古细菌或细菌)中表达而进行密码子优化。在一些实施方案中，本发明的密码子优化的核酸构建体、多核苷酸、表达盒和/或载体与未经密码子优化的参考核酸构建体、多核苷酸、表达盒和/或载体具有约70％至约99.9％(例如，70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％或100％)同一性或更高。
在本文所述的任何实施方案中，本发明的多核苷酸或核酸构建体可以与多种启动子和/或其他调节元件可操作地关联用于在生物体或其细胞(例如植物和/或植物细胞)中表达。因此，在一些实施方案中，本发明的多核苷酸或核酸构建体可以进一步包含与一种或多种核苷酸序列可操作地连接的一种或多种启动子、内含子、增强子和/或终止子。在一些实施方案中，启动子可以与内含子(例如，Ubi1启动子和内含子)可操作地关联。在一些实施方案中，与内含子关联的启动子可以称为“启动子区域”(例如，Ubil启动子和内含子)。
如本文所用的关于多核苷酸的“可操作地连接”或“可操作地关联”是指所示元件在功能上彼此相关，并且通常也是物理相关的。因此，如本文所用，术语“可操作地连接”或“可操作地关联”是指单个核酸分子上功能关联的核苷酸序列。因此，与第二核苷酸序列可操作地连接的第一核苷酸序列是指当第一核苷酸序列与第二核苷酸序列处于功能关系时的情况。例如，如果启动子影响所述核苷酸序列的转录或表达，则启动子与核苷酸序列可操作地相关。本领域技术人员将理解，控制序列(例如，启动子)不需要与其可操作地关联的核苷酸序列连续，只要控制序列的功能是指导其表达即可。因此，例如，插入的未翻译但转录的核酸序列可以存在于启动子和核苷酸序列之间，并且启动子仍然可以被认为“可操作地连接”到核苷酸序列。
如本文所用，关于多肽的术语“连接的”或“融合的”是指一种多肽与另一种多肽的附接。多肽可以直接地(例如，通过肽键)或通过接头(例如，肽接头)连接或融合至另一个多肽(在N-末端或C-末端)。
关于多肽的术语“接头”是本领域公认的并且是指化学基团或连接两个分子或部分的分子，例如融合蛋白的两个结构域，例如CRISPR-Cas效应蛋白和肽标签和/或目标多肽。接头可以由单个连接分子(例如，单个氨基酸)组成或可以包含多于一个的连接分子。在一些实施方案中，接头可以是有机分子、基团、聚合物或化学部分，例如二价有机部分。在一些实施方案中，接头可以是氨基酸或者它可以是肽。在一些实施方案中，接头是肽。
在一些实施方案中，可用于本发明的肽接头长度可为约2至约100个或更多个氨基酸，例如约2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100个或更多个氨基酸的长度(例如，约2至约40、约2至约50、约2至约60、约4至约40、约4至约50、约4至约60、约5至约40、约5至约50、约5至约60、约9至约40、约9至约50、约9至约60、约10至约40、约10至约50、约10至约60、或约2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25个氨基酸至约26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100或更多个氨基酸的长度(例如，约105、110、115、120、130、140、150或更多个氨基酸的长度))。在一些实施方案中，肽接头可以是GS接头。
如本文所用，关于多核苷酸的术语“连接的”或“融合的”是指一个多核苷酸与另一个多核苷酸的附接。在一些实施方案中，两个或更多个多核苷酸分子可以通过接头连接，接头可以是有机分子、基团、聚合物或化学部分，例如二价有机部分。多核苷酸可以通过共价或非共价键或结合(包括例如Watson-Crick碱基配对)或通过一个或多个连接核苷酸而连接或融合至另一个多核苷酸(在5′端或3′端)。在一些实施方案中，可以将某种结构的多核苷酸基序插入另一个多核苷酸序列内(例如，指导RNA中发夹结构的延伸)。在一些实施方案中，连接核苷酸可以是天然存在的核苷酸。在一些实施方案中，连接核苷酸可以是非天然存在的核苷酸。
“启动子”是控制或调节与启动子可操作关联的核苷酸序列(例如，编码序列)转录的核苷酸序列。由启动子控制或调节的编码序列可以编码多肽和/或功能性RNA。通常，“启动子”是指包含用于RNA聚合酶II的结合位点并指导转录起始的核苷酸序列。通常，启动子位于相对于相应编码序列的编码区起点的5′或上游。启动子可以包含作为基因表达的调节因子的其他元件；例如，启动子区域。这些包括TATA盒共有序列，并且通常是CAAT盒共有序列(Breathnach和Chambon，(1981)Annu.Rev.Biochem.50：349)。在植物中，CAAT盒可以被AGGA盒替代(Messing等人，(1983)Genetic Engineering of Plants，T.Kosuge，C.Meredith和A.Hollaender(编)，Plenum Press，pp.211-227)。在一些实施方案中，启动子区域可以包含至少一个内含子(例如，SEQ ID NO：1或SEQ ID NO：2)。
可用于本发明的启动子可以包括例如组成型、诱导型、时间调节型、发育调节型、化学调节型、组织优选和/或组织特异性启动子，用于制备重组核酸分子，例如“合成核酸酸构建体”或“蛋白质-RNA复合物”。这些不同类型的启动子是本领域已知的。
启动子的选择可能因表达的时间和空间要求而异，并且也可能因要转化的宿主细胞而异。许多不同生物体的启动子在本领域中是众所周知的。基于本领域现有的广泛知识，可以为目标特定宿主生物体选择合适的启动子。因此，例如，对模式生物体中高度组成型表达基因上游的启动子了解很多，并且可以在其他系统中适当地访问和实施此类知识。
在一些实施方案中，在植物中起作用的启动子可以与本发明的构建体一起使用。可用于在植物中驱动表达的启动子的非限制性实例包括RubisCo小亚基基因1的启动子(PrbcS1)、肌动蛋白基因的启动子(Pactin)、硝酸还原酶基因的启动子(Pnr)和重复碳酸酐酶基因1的启动子(Pdcal)(参见Walker等人Plant Cell Rep.23：727-735(2005)；Li等人Gene403：132-142(2007)；Li等人Mol Biol.Rep.37：1143-1154(2010))。PrbcS1和Pactin是组成型启动子，Pnr和Pdca1是诱导型启动子。Pnr由硝酸盐诱导并由铵抑制(Li等人Gene403：132-142(2007))，Pdca1由盐诱导(Li等人Mol Biol.Rep.37：1143-1154(2010))。
可用于植物的组成型启动子的实例包括但不限于：cestrum病毒启动子(cmp)(美国专利号7,166,770)、水稻肌动蛋白1启动子(Wang等人(1992)Mol.Cell.Biol.12：3399-3406；以及美国专利号5,641,876)、CaMV35S启动子(Odell等人(1985)Nature 313：810-812)、CaMV 19S启动子(Lawton等人(1987)Plant Mol.Biol.9：315-324)、nos启动子(Ebert等人(1987)Proc.Natl.Acad.Sci USA 84：5745-5749)、Adh启动子(Walker等人(1987)Proc.Natl.Acad.Sci.USA 84：6624-6629)、蔗糖合酶启动子(Yang&Russell(1990)Proc.Natl.Acad.Sci.USA 87：4144-4148)和泛素启动子。源自泛素的组成型启动子在许多细胞类型中积累。泛素启动子已经从几种植物物种中克隆出来用于转基因植物，例如向日葵(Binet等人，1991.Plant Science 79：87-94)、玉蜀黍(Christensen等人，1989.PlantMolec.Biol.12：619-632)和拟南芥(Norris等人1993.Plant Molec.Biol.21：895-906)。玉蜀黍泛素启动子(UbiP)已在转基因单子叶植物系统中开发，其序列和构建用于单子叶植物转化的载体在欧洲专利公开EP0342926中公开。泛素启动子适用于在转基因植物，尤其是单子叶植物中表达本发明的核苷酸序列。此外，McElroy等人(Mol.Gen.Genet.231：150-160(1991))描述的启动子表达盒可以容易地被修饰以表达本发明的核苷酸序列并且特别适用于单子叶植物宿主。
在一些实施方案中，组织特异性/组织优选启动子可用于在植物细胞中表达异源多核苷酸。组织特异性或优选表达模式包括但不限于绿色组织特异性或优选的、根特异性或优选的、茎特异性或优选的、花特异性或优选的或者花粉特异性或优选的。适合在绿色组织中表达的启动子包括许多调节参与光合作用的基因的启动子，其中许多已从单子叶植物和双子叶植物两者中克隆。在一个实施方案中，可用于本发明的启动子是来自磷酸烯醇羧化酶基因的玉蜀黍PEPC启动子(Hudspeth&Grula，Plant Molec.Biol.12：579-589(1989))。组织特异性启动子的非限制性实例包括与编码种子贮藏蛋白(例如β-伴大豆球蛋白、十字花科蛋白、napin和菜豆蛋白)、玉米醇溶蛋白或油体蛋白(例如油质蛋白)或参与脂肪酸的蛋白质(包括酰基载体蛋白、硬脂酰-ACP去饱和酶和脂肪酸去饱和酶(fad 2-1))的基因相关联的那些，以及在胚发育过程中表达的其他核酸(例如Bce4，参见例如，Kridl等人(1991)Seed Sci.Res.1：209-219；以及EP专利号255378)。用于在植物，特别是玉蜀黍中表达本发明的核苷酸序列的组织特异性或组织优先的启动子包括但不限于指导在根、髓、叶或花粉中表达的那些。此类启动子公开于例如WO 93/07278中，其公开的启动子通过引用并入本文。可用于本发明的组织特异性或组织优选启动子的其他非限制性实例是美国专利6,040,504中公开的棉花rubisco启动子；美国专利5,604,121中公开的水稻蔗糖合酶启动子；deFramond描述的根特异性启动子(FEBS 290：103-106(1991)；Ciba-Geigy的欧洲专利EP0452269)；在美国专利5,625,136(Ciba-Geigy)中描述的茎特异性启动子，其驱动玉蜀黍trpA基因的表达；WO 01/73087中公开的certrum黄卷叶病毒启动子；和花粉特异性或优选启动子，包括但不限于来自水稻的ProOsLPS10和ProOsLPS11(Nguyen等人PlantBiotechnol.Reports 9(5)：297-306(2015))，来自玉蜀黍的ZmSTK2_USP((Wang等人Genome60(6)：485-495(2017))、来自番茄的LAT52和LAT59(Twell等人Development 109(3)：705-713(1990))、Zm13(美国专利号10,421,972)、来自拟南芥的PLA2-δ启动子(美国专利号7,141,424)和/或来自玉蜀黍的ZmC5启动子(国际PCT公开号WO1999/042587)。
植物组织特异性/组织优选启动子的其他实例包括但不限于根毛特异性顺式元件(RHE)(KIM等人The Plant Cell 18：2958-2970(2006))、根-特异性启动子RCc3(Jeong等人Plant Physiol.153：185-197(2010))和RB7(美国专利号5459252)，凝集素启动子(Lindstrom等人(1990)Der.Genet.11：160-167；和Vodkin(1983)Prog.Clin.Biol.Res.138：87-98)，玉米醇脱氢酶1启动子(Dennis等人(1984)NucleicAcids Res.12：3983-4000)，S-腺苷-L-甲硫氨酸合成酶(SAMS)(Vander Mijnsbrugge等人(1996)Plant and Cell Physiology，37(8)：1108-1115)，玉米捕光复合体启动子(Bansal等人(1992)Proc.Natl.Acad.Sci.USA 89：3654-3658)、玉米热休克蛋白启动子(O′Dell等人(1985)EMBO J.5：451-458；和Rochester等人(1986)EMBO J.5：451-458)、豌豆小亚基RuBP羧化酶启动子(Cashmore，″Nuclear genes encoding the small subunit ofribulose-l，5-bisphosphate carboxylase″pp.29-39于：Genetic Engineering ofPlants(Hollaender编，Plenum Press 1983；和Poulsen等人(1986)Mol.Gen.Genet.205：193-200)、Ti质粒甘露碱合酶启动子(Langridge等人(1989)Proc.Natl.Acad.Sci.USA 86：3219-3223)、Ti质粒胭脂碱合酶启动子(Langridge等人(1989)，同上)、矮牵牛查尔酮异构酶启动子(van Tunen等人(1988)EMBO J.7：1257-1263)、大豆富含甘氨酸蛋白1启动子(Keller等人(1989)Genes Dev.3：1639-1646)、截短的CaMV 35S启动子(O′Dell等人(1985)Nature 313：810-812)、马铃薯块茎储藏蛋白启动子(Wenzler等人(1989)PlantMol.Biol.13：347-354)、根细胞启动子(Yamamoto等人(1990)Nucleic Acids Res.18：7449)，玉蜀黍玉米醇溶蛋白启动子(Kriz等人(1987)Mol.Gen.Genet.207：90-98；Langridge等人(1983)Cell 34：1015-1022；Reina等人(1990)Nucleic Acids Res.18：6425；Reina等人(1990)Nucleic Acids Res.18：7449；和Wandelt等人(1989)NucleicAcids Res.17：2354)、球蛋白-1启动子(Belanger等人(1991)Genetics 129：863-872)、α-微管蛋白cab启动子(Sullivan等人(1989)Mol.Gen.Genet.215：431-440)、PEPCase启动子(Hudspeth&Grula(1989)Plant Mol.Biol.12：579-589)、R基因复合物相关启动子(Chandler等人(1989)Plant Cell 1：1175-1183)和查耳酮合酶启动子(Franken等人(1991)EMBO J.10：2605-2612)。
对种子特异性表达有用的是豌豆球蛋白启动子(Czako等人(1992)Mol.Gen.Genet.235：33-40；以及美国专利号5,625,136中公开的种子特异性启动子。在成熟叶中表达的有用启动子是在衰老开始时被转换的那些，例如来自拟南芥的SAG启动子(Gan等人(1995)Science 270：1986-1988)。
此外，可以使用在叶绿体中起作用的启动子。此类启动子的非限制性实例包括噬菌体T3基因95′UTR和美国专利号7,579,516中公开的其他启动子。可用于本发明的其他启动子包括但不限于S-E9小亚基RuBP羧化酶启动子和Kunitz胰蛋白酶抑制剂基因启动子(Kti3)。
可用于本发明的其他调节元件包括但不限于内含子、增强子、终止序列和/或5′和3′非翻译区。
可用于本发明的内含子可以是在植物中鉴定和分离的内含子，然后插入到表达盒中以用于植物的转化。如本领域技术人员将理解的，内含子可包含自我切除所需的序列，并以框内的方式并入核酸构建体/表达盒中。内含子可用作间隔物以分隔一个核酸构建体中的多个蛋白质编码序列，或者内含子可用于一个蛋白质编码序列内以例如稳定mRNA。如果它们在蛋白质编码序列内使用，它们将被插入“框内”并包含切除位点。内含子也可以与启动子相关联以改善或修饰表达。例如，可用于本发明的启动子/内含子组合包括但不限于玉蜀黍Ubi1启动子和内含子的组合。
可用于本发明的内含子的非限制性例子包括来自以下的内含子：ADHI基因(例如，Adh1-S内含子1、2和6)、泛素基因(Ubi1)、RuBisCO小亚基(rbcS)基因、RuBisCO大亚基(rbcL)基因、肌动蛋白基因(例如，肌动蛋白-1内含子)、丙酮酸脱氢酶激酶基因(pdk)、硝酸还原酶基因(nr)、重复碳酸酐酶基因1(Tdca1)、psbA基因、atpA基因或其任意组合。
如本文所用，“编辑系统”是指现在已知或以后开发的任何位点特异性(例如，序列特异性)核酸编辑系统，该系统可以以靶标特异性方式在核酸中引入修饰(例如，突变)。例如，编辑系统可以包括但不限于CRISPR-Cas编辑系统、大范围核酸酶编辑系统、锌指核酸酶(ZFN)编辑系统、转录激活因子样效应核酸酶(TALEN)编辑系统，碱基编辑系统和/或启动编辑系统，其各自可包含一种或多种多肽和/或一种或多种多核苷酸，当它们在细胞中一起存在和/或表达时可以以序列特异性方式修饰(例如，突变)靶核酸。在一些实施方案中，编辑系统(例如，位点和/或序列特异性编辑系统)包含一种或多种编码多肽(包括核酸结合多肽(例如DNA结合结构域)和/或核酸酶)的多核苷酸和/或一种或多种多肽(包括核酸结合多肽(例如DNA结合结构域)和/或核酸酶)。在一些实施方案中，编辑系统由一种或多种多核苷酸编码。
在一些实施方案中，编辑系统包含一种或多种序列特异性核酸结合多肽(例如DNA结合结构域)，其可以来自例如多核苷酸引导的核酸内切酶、CRISPR-Cas效应蛋白(例如，CRISPR-Cas核酸内切酶)、锌指核酸酶、转录激活因子样效应核酸酶(TALEN)和/或Argonaute蛋白。在一些实施方案中，编辑系统包含一种或多种切割多肽(例如核酸酶)，例如但不限于核酸内切酶(例如Fok1)、多核苷酸引导的核酸内切酶、CRISPR-Cas效应蛋白(例如，CRISPR-Cas核酸内切酶)、锌指核酸酶和/或转录激活因子样效应核酸酶(TALEN)。
如本文所用，“核酸结合多肽”是指以位点和/或序列特异性方式结合和/或能够结合核酸的多肽。在一些实施方案中，核酸结合多肽包含DNA结合结构域。在一些实施方案中，核酸结合多肽可以是序列特异性核酸结合多肽，例如但不限于来自以下的序列特异性结合多肽和/或结构域：例如来自多核苷酸引导的核酸内切酶、CRISPR-Cas效应蛋白(例如，CRISPR-Cas核酸内切酶)、锌指核酸酶、转录激活因子样效应核酸酶(TALEN)和/或Argonaute蛋白。在一些实施方案中，核酸结合多肽包含切割多肽(例如，核酸酶多肽和/或结构域)，例如但不限于，核酸内切酶(例如，Fok1)、多核苷酸引导的核酸内切酶、CRISPR-Cas核酸内切酶、锌指核酸酶和/或转录激活因子样效应核酸酶(TALEN)。在一些实施方案中，核酸结合多肽与一种或多种核酸分子相关联和/或能够与其相关联(例如，形成复合物)(例如，与本文所述的指导核酸形成复合物)，所述一种或多种核酸分子可以将核酸结合多肽引导或指导至与所述一种或多种核酸分子(或其部分或区域)互补的特定靶核苷酸序列(例如基因组的基因座)，从而引起核酸结合多肽在特定靶位点与核苷酸序列结合。在一些实施方案中，核酸结合多肽是如本文所述的CRISPR-Cas效应蛋白。在一些实施方案中，为简单起见，特指CRISPR-Cas效应蛋白，但可使用本文所述的核酸结合多肽。
在一些实施方案中，编辑系统包含核糖核蛋白，例如组装的核糖核蛋白复合物(例如，包含复合物形式的CRISPR-Cas效应蛋白和指导核酸的核糖核蛋白)。编辑系统的复合物可以是共价和/或非共价结合的复合物。如本文所用，编辑系统可以在引入植物细胞中时组装成复合物(例如，在引入植物细胞中之前组装成复合物)和/或可以在引入植物细胞中之后和/或期间组装成复合物(例如，共价和/或非共价结合的复合物)。示例性核糖核蛋白及其使用方法包括但不限于Malnoy等人，(2016)Front.Plant Sci.7：1904；Subburaj等人，(2016)Plant Cell Rep.35：1535；Woo等人，(2015)Nat.Biotechnol.33：1162；Liang等人，(2017)Nat.Commun.8：14261；Svitashev等人，Nat.Commun.7，13274(2016)；Zhang等人，(2016)Nat.Commun.7：12617；Kim等人，(2017)Nat.Commun.8：14406。
如本文所用的“编辑的细胞”、“编辑的植物”、“编辑的植物部分”、“编辑的根”、“编辑的愈伤组织”和/或类似物分别是指包含修饰的核酸的细胞、植物、植物部分、根、愈伤组织和/或类似物，其中使用如本文所述的编辑系统修饰靶核酸以提供修饰的核酸。因此，“编辑的细胞”、“编辑的植物”、“编辑的植物部分”、“编辑的根”、“编辑的愈伤组织”和/或类似物包含与其未修饰或天然序列和/或结构相比已被修饰(即，修饰的核酸)和/或发生变化的核酸。
如本文所用，术语“转基因”或“转基因的”是指至少一种这样的核酸序列：其取自一种生物体的基因组，或合成产生，然后被引入目标宿主细胞(例如植物细胞)或生物体或组织中，随后通过“稳定”转化或转染方法整合到宿主基因组中。相反，术语“瞬时”转化或转染或引入是指引入分子工具的方式，包括至少一种核酸(DNA、RNA、单链或双链或其混合物)和/或至少一种氨基酸序列，任选地包含合适的化学或生物试剂，以实现转移到细胞的至少一个目标区室中，包括但不限于细胞质、细胞器，包括细胞核、线粒体、液泡、叶绿体或膜中，导致引入的至少一种分子的转录和/或翻译和/或缔合和/或活性，而没有实现稳定的整合或掺入并因此遗传引入到细胞的基因组中的相应的至少一种分子。术语“无转基因”是指在目标宿主细胞或组织或生物体的基因组中不存在或发现转基因的情况。
在一些实施方案中，本发明的多核苷酸和/或核酸构建体可以是“表达盒”或可以包含在表达盒内。如本文所用，“表达盒”意指重组核酸分子，其包含例如本发明的核酸构建体(例如，编码CRISPR-Cas效应蛋白的多核苷酸、编码CRISPR-Cas融合蛋白的多核苷酸、编码胞嘧啶脱氨酶的多核苷酸、编码腺嘌呤脱氨酶的多核苷酸、编码脱氨酶融合蛋白的多核苷酸、编码肽标签的多核苷酸、编码亲和多肽的多核苷酸和/或包含指导核酸的多核苷酸)，其中核酸构建体与至少一个控制序列(例如，启动子)可操作地关联。因此，本发明的一些实施方案提供了设计用于表达例如本发明的核酸构建体的表达盒。当表达盒包含多于一个多核苷酸时，可以将多核苷酸可操作地连接至驱动所有多核苷酸表达的单个启动子，或者可以将多核苷酸可操作地连接至一个或多个单独的启动子(例如，三个多核苷酸可以由一个、两个或三个任意组合的启动子驱动)。因此，例如，包含在表达盒中的编码CRISPR-Cas效应蛋白的多核苷酸、编码胞嘧啶脱氨酶的多核苷酸和包含指导核酸的多核苷酸可以各自与单独的启动子(例如，任何组合的两个或三个启动子)可操作地关联，这些启动子可以彼此相同或不同。作为另一个实例，包含在表达盒中的编码CRISPR-Cas效应蛋白的多核苷酸、编码胞嘧啶脱氨酶的多核苷酸、编码腺嘌呤脱氨酶的多核苷酸和包含指导核酸的多核苷酸可以各自与单个启动子可操作地关联或者一种或多种多核苷酸可以与任何组合中的单独启动子(例如，可以相同或不同的任何组合中的两个、三个或四个启动子)可操作地关联。
在一些实施方案中，可以优化包含本发明的多核苷酸/核酸构建体的表达盒以在生物体(例如，动物、植物、细菌等)中表达。
包含本发明的核酸构建体的表达盒可以是嵌合的，意味着它的至少一种组分相对于它的至少一种其他组分是异源的(例如，来自宿主生物体的启动子可操作地连接至待在宿主生物体中表达的目标多核苷酸，其中目标多核苷酸来自与宿主不同的生物体或通常不与该启动子关联)。表达盒也可以是天然存在的但已经以可用于异源表达的重组形式获得的表达盒。
表达盒可以任选地包括在所选宿主细胞中起作用的转录和/或翻译终止区(即终止区)和/或增强子区。多种转录终止子和增强子是本领域已知的并且可用于表达盒。转录终止子负责转录的终止和正确的mRNA多聚腺苷酸化。终止区和/或增强子区可以对于转录起始区是天然的，可以对于编码CRISPR-Cas效应蛋白的基因或编码脱氨酶的基因是天然的，可以对于宿主细胞是天然的，或可以对于另一个来源是天然的(例如，对于启动子，对于编码CRISPR-Cas效应蛋白的基因或编码脱氨酶的基因，对于宿主细胞，或其任何组合而言是外来的或异源的)。
本发明的表达盒还可包括编码可选择标志物的多核苷酸，其可用于选择转化的宿主细胞。如本文所用，“可选择标志物”是指多核苷酸序列，当其表达时赋予表达该标志物的宿主细胞不同的表型并因此允许将此类转化的细胞与不具有该标志物的那些细胞区分开来。这样的多核苷酸序列可以编码可选择的或可筛选的标志物，这取决于标志物是否赋予可以通过化学手段选择的性状，例如通过使用选择剂(例如抗生素等)选择，或者取决于标志物是否只是一种可以通过观察或测试来识别的特征，例如通过筛选(例如荧光)识别。合适的可选择标志物的许多例子是本领域已知的并且可以用于本文所述的表达盒中。
本文所述的表达盒、核酸分子/构建体和多核苷酸序列可以与载体结合使用。术语“载体”是指用于将一种(或多种)核酸转移、递送或引入细胞中的组合物。载体包括核酸构建体，其包含待转移、递送或引入的核苷酸序列。用于宿主生物体转化的载体在本领域是众所周知的。一般类型的载体的非限制性实例包括双链或单链线性或环状形式的病毒载体、质粒载体、噬菌体载体、噬菌粒载体、粘粒载体(cosmid vectors)、黏粒载体(fosmidvectors)、噬菌体、人工染色体、小环或农杆菌双元载体，其可以是或者可以不是自我传播或移动的。在一些实施方案中，病毒载体可以包括但不限于逆转录病毒、慢病毒、腺病毒、腺相关病毒或单纯疱疹病毒载体。本文定义的载体可以通过整合到细胞基因组中或存在于染色体外(例如，具有复制起点的自主复制质粒)来转化原核或真核宿主。此外，包括穿梭载体，其意指能够在两种不同的宿主生物体中自然或通过设计复制的DNA媒介物，所述宿主生物体可以选自放线菌和相关物种、细菌和真核生物(例如高等植物、哺乳动物、酵母或真菌细胞)。在一些实施方案中，载体中的核酸在用于宿主细胞中转录的合适启动子或其他调控元件的控制下并且与其可操作地连接。载体可以是在多个宿主中起作用的双功能表达载体。在基因组DNA的情况下，这可以包含其自身的启动子和/或其他调控元件，而在cDNA的情况下，这可以在用于在宿主细胞中表达的合适启动子和/或其他调控元件的控制下。因此，本发明的核酸构建体和/或包含其的表达盒可以包含在如本文所述和本领域已知的载体中。
如本文所用，“接触”、“在接触”、“接触的”及其语法变体是指将所需反应的组分一起放在适合进行所需反应(例如，转化、转录控制、基因组编辑、切口和/或切割)的条件下。因此，例如，靶核酸可以与本发明的核酸构建体，例如核酸结合多肽(例如，DNA结合结构域，例如序列特异性DNA结合蛋白(例如，多核苷酸引导的核酸内切酶、CRISPR-Cas效应蛋白(例如，CRISPR-Cas核酸内切酶)、锌指核酸酶、转录激活因子样效应核酸酶(TALEN)和/或Argonaute蛋白)、指导核酸和胞嘧啶脱氨酶和/或腺嘌呤脱氨酶，在一定的条件下接触，从而表达核酸结合多肽，并且核酸结合多肽与指导核酸形成复合物，复合物与靶核酸杂交，和任选地胞嘧啶脱氨酶和/或腺嘌呤脱氨酶被募集到核酸结合多肽(并因此募集到靶核酸)或胞嘧啶脱氨酶和/或腺嘌呤脱氨酶融合至核酸结合多肽，从而修饰靶核酸。在一些实施方案中，CRISPR-Cas效应蛋白、指导核酸和脱氨酶接触靶核酸从而修饰核酸。在一些实施方案中，CRISPR-Cas效应蛋白、指导核酸和/或脱氨酶可以是复合物的形式(例如，核糖核蛋白，例如组装的核糖核蛋白复合物)并且复合物接触靶核酸。在一些实施方案中，复合物或其组分(例如，指导核酸)与靶核酸杂交，从而修饰靶核酸(例如，通过CRISPR-Cas效应蛋白和/或脱氨酶的作用)。在一些实施方案中，胞嘧啶脱氨酶和/或腺嘌呤脱氨酶和核酸结合多肽定位于靶核酸，任选地通过共价和/或非共价相互作用。
如本文所用，关于靶核酸的“进行修饰”或“修饰”包括编辑(例如，突变)、共价修饰、交换/取代核酸/核苷酸碱基、缺失、切割和/或切口靶核酸核酸从而提供修饰的核酸和/或改变靶核酸的转录控制从而提供修饰的核酸。在一些实施方案中，修饰可以包括任何大小的插入和/或缺失和/或任何类型的单碱基改变(SNP)。在一些实施方案中，修饰包括SNP。在一些实施方案中，修饰包括交换和/或取代一个或多个(例如，1、2、3、4、5或更多个)核苷酸。在一些实施方案中，插入或缺失可为约1个碱基至约30,000个碱基的长度(例如，约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10,000、10,500、11,000、11,500、12,000、12,500、13,000、13,500、14,000、14,500、15,000、15,500、16,000、16,500、17,000、17,500、18,000、18,500、19,000、19,500、20,000、20,500、21,000、21,500、22,000、22,500、23,000、23,500、24,000、24,500、25,000、25,500、26,000、26,500、27,000、27,500、28,000、28,500、29,000、29,500、30,000个碱基或更多的长度，或其中的任何数值或范围)。因此，在一些实施方案中，插入或缺失可为约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300至约310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990、1000个碱基的长度，或其中的任何范围或数值；约50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300个碱基至约310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000个碱基或更多的长度，或其中的任何范围或数值；约500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000个碱基至约2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500或10,000个碱基或更多的长度，或其中的任何范围或数值；或约400、410、420、430、440、450、460、470、480、490、500、510、520、530、540、550、560、570、580、590、600、610、620、630、640、650、660、670、680、690或700个碱基至约710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2500、3000、3500、4000、4500或5000个碱基或更多的长度，或其中的任何范围或数值。在一些实施方案中，插入或缺失可以是约1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500或10,000个碱基至约10,500、11,000、11,500、12,000、12,500、13,000、13,500、14,000、14,500、15,000、15,500、16,000、16,500、17,000、17,500、18,000、18,500、19,000、19,500、20,000、20,500、21,000、21,500、22,000、22,500、23,000、23,500、24,000、24,500、25,000、25,500、26,000、26,500、27,000、27,500、28,000、28,500、29,000、29,500或30,000个碱基或更多的长度，或其中的任何范围或数值。
如本文所用，“募集”、“进行募集”或“募集作用”是指使用蛋白质-蛋白质相互作用、核酸-蛋白质相互作用(例如，RNA-蛋白质相互作用)和/或化学相互作用将一个或多个多肽或多核苷酸吸引至另一多肽或多核苷酸(例如，至基因组中的特定位置)。蛋白质-蛋白质相互作用可以包括但不限于肽标签(表位、多聚化表位)和相应的亲和多肽、RNA募集基序和相应的亲和多肽，和/或化学相互作用。可用于募集目的的多肽和多核苷酸的示例化学相互作用可包括但不限于雷帕霉素诱导的FRB-FKBP二聚化；生物素-链霉亲和素相互作用；SNAP标签(Hussain等人Curr Pharm Des.19(30)：5437-42(2013))；Halo标签(Los等人ACSChem Biol.3(6)：373-82(2008))；CLIP标签(Gautier等人Chemistry&Biology 15：128-136(2008))；由化合物诱导的DmrA-DmrC异二聚体(Tak等人Nat Methods 14(12)：1163-1166(2017))；双功能配体方法(将两种蛋白质结合化学物质融合在一起)(VoB等人Curr OpinChemical Biology 28：194-201(2015))(例如二氢叶酸还原酶(DHFR)(Kopyteck等人CellCehm Biol 7(5)：313-321(2000))。
在目标多核苷酸的上下文中，“进行引入”、“引入”、“引入的”(及其语法变体)是指以使核苷酸序列进入细胞内部的方式对宿主生物体或所述生物体的细胞(例如，宿主细胞；例如，植物细胞)呈现目标核苷酸序列(例如，多核苷酸、核酸构建体和/或指导核酸)。因此，例如，可以将编码CRISPR-Cas效应蛋白、指导核酸和胞嘧啶脱氨酶和/或腺嘌呤脱氨酶的本发明的核酸构建体引入生物体的细胞中，从而用CRISPR-Cas效应蛋白、指导核酸和胞嘧啶脱氨酶和/或腺嘌呤脱氨酶转化细胞。在一些实施方案中，可以将包含核酸结合多肽(例如，CRISPR-Cas效应蛋白)的多肽和/或指导核酸引入生物体的细胞中，任选地其中核酸结合多肽和指导核酸可以包含在复合物中(例如，核糖核蛋白)。在一些实施方案中，生物体是真核生物(例如，哺乳动物，如人)。
如本文所用，术语“转化”是指将异源核酸引入细胞。细胞的转化可以是稳定的或短暂的。因此，在一些实施方案中，宿主细胞或宿主生物体可以用本发明的多核苷酸/核酸分子稳定转化。在一些实施方案中，宿主细胞或宿主生物体可以用本发明的核酸构建体瞬时转化。
在多核苷酸的上下文中，“瞬时转化”是指多核苷酸被引入细胞中并且不整合到细胞的基因组中。
在将多核苷酸引入细胞中的情形中，“稳定地引入”或“稳定地引入的”意指引入的多核苷酸被稳定地掺入细胞的基因组中，因此细胞被多核苷酸稳定地转化。
如本文所用，“稳定转化”或“稳定转化的”是指将核酸分子引入细胞中并整合到细胞的基因组中。因此，整合的核酸分子能够被其后代，更具体地，被多个连续世代的后代遗传。如本文所用，“基因组”包括核基因组和质体基因组，因此包括将核酸整合到例如叶绿体或线粒体基因组中。如本文所用，稳定转化还可以指在染色体外维持的转基因，例如，作为微染色体或质粒。
瞬时转化可以通过例如酶联免疫吸附测定法(ELISA)或Western印迹来检测，其可以检测由引入生物体的一种或多种转基因编码的肽或多肽的存在。细胞的稳定转化可以通过例如细胞的基因组DNA与核酸序列的Southern印迹杂交测定来检测，所述核酸序列与引入生物体(例如植物)中的转基因的核苷酸序列特异性杂交。细胞的稳定转化可以通过例如细胞的RNA与核酸序列的Northern印迹杂交测定来检测，所述核酸序列与引入宿主生物中的转基因的核苷酸序列特异性杂交。细胞的稳定转化也可以通过例如聚合酶链式反应(PCR)或本领域熟知的其他扩增反应来检测，使用与转基因的靶序列杂交的特异性引物序列，导致转基因序列的扩增，可以根据标准方法检测。转化也可以通过本领域已知的直接测序和/或杂交方案检测。
因此，在一些实施方案中，本发明的核苷酸序列、多核苷酸、核酸构建体和/或表达盒可以瞬时表达和/或它们可以稳定地掺入宿主生物的基因组中。因此，在一些实施方案中，可以将本发明的核酸构建体与指导核酸一起瞬时引入细胞中，因此，在细胞中没有DNA得到维持。
可以通过本领域技术人员已知的任何方法将本发明的核酸构建体引入细胞中。在一些实施方案中，转化方法包括通过细菌介导的核酸递送(例如，通过农杆菌)、病毒介导的核酸递送、碳化硅和/或核酸晶须介导的核酸递送、脂质体介导的核酸递送、显微注射、微粒轰击、磷酸钙介导的转化、环糊精介导的转化、电穿孔、纳米粒子介导的转化、超声处理、渗入、PEG介导的核酸摄取，以及任何其他电、化学、物理(机械)和/或导致将核酸引入植物细胞中的生物学机制，包括其任何组合的转化。在本发明的一些实施方案中，细胞的转化包括核转化。在一些实施方案中，细胞的转化包括质体转化(例如，叶绿体转化)。在一些实施方案中，可以通过常规育种技术将本发明的重组核酸构建体引入细胞中。
用于转化真核生物和原核生物两者的程序在本领域中是众所周知的并且是常规的，并且在整个文献中都有描述(参见，例如，Jiang等人2013.Nat.Biotechnol.31：233-239；Ran等人Nature Protocols 8：2281-2308(2013))。本领域已知的多种植物转化方法的一般指导包括Miki等人(“Procedures for Introducing Foreign DNA into Plants”inMethods in Plant Molecular Biology and Biotechnology，Glick，B.R.和Thompson，J.E.编.(CRC Press，Inc.，Boca Raton，1993)，第67-88页)和Rakowoczy-Trojanowska(Cell.Mol.Biol.Lett.7：849-858(2002))。
多核苷酸和/或多肽可以以本领域公知的许多方式被引入宿主生物体或其细胞(任选地植物、植物部分和/或植物细胞)中。本发明的方法不依赖于将一种或多种核苷酸序列引入生物体中的特定方法，仅依赖于它们能够进入生物体的至少一个细胞的内部。在要引入多于一个核苷酸序列的情况下，它们可以组装为单个核酸构建体的部分，或作为单独的核酸构建体，并且可以位于相同或不同的核酸构建体上。多核苷酸和/或多肽可以在单个转化事件中或在单独的转化事件中被引入目标细胞中，或者备选地，多核苷酸和/或多肽可以被掺入植物中，例如，作为育种方案的部分。在一些实施方案中，细胞是真核细胞(例如，哺乳动物，例如人类细胞)。
根据一些实施方案，提供了一种碱基编辑组合物或系统，其包含：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)、胞嘧啶脱氨酶和腺嘌呤脱氨酶，其中CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶形成复合物或包含在复合物中。在一些实施方案中，复合物还包含指导核酸。在一些实施方案中，CRISPR-Cas效应蛋白是V型CRISPR-Cas效应蛋白。在一些实施方案中，本发明提供了一种核酸构建体，其包含：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)、胞嘧啶脱氨酶和腺嘌呤脱氨酶，各自如本文所述。核酸构建体可进一步包含糖基化酶抑制剂(例如，尿嘧啶糖基化酶抑制剂(UGI))。
指导核酸可包含如本文所述的RNA募集基序(例如，一个或多个MS2发夹)。在一些实施方案中，CRISPR-Cas效应蛋白与指导核酸(例如，指导RNA)相互作用、结合和/或复合。
可以将CRISPR-Cas效应蛋白融合至糖基化酶抑制剂、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。在一些实施方案中，将CRISPR-Cas效应蛋白在单一融合物中融合至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶或者单独地融合至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶之一或两者。在一些实施方案中，将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶。在一些实施方案中，将CRISPR-Cas效应蛋白融合至腺嘌呤脱氨酶。在一些实施方案中，将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶。在一些实施方案中，将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶不融合至Cas9和/或任选地胞嘧啶脱氨酶和/或腺嘌呤脱氨酶可以通过非共价相互作用被募集至靶位点。在一些实施方案中，将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶融合或募集至V型CRISPR-Cas结构域(例如，Cpf1)。在一些实施方案中，将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶募集至V型CRISPR-Cas结构域(例如，Cpf1)。
在一些实施方案中，将胞嘧啶脱氨酶和腺嘌呤脱氨酶融合在一起。在一些实施方案中，胞嘧啶脱氨酶和/或腺嘌呤脱氨酶包含MS2加帽蛋白(MCP)或其部分。可以将MCP或其部分在单一融合物中融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶两者或者单独地融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶之一或两者。例如，在一些实施方案中，可以将胞嘧啶脱氨酶单独地融合至MCP或其部分，和/或在一些实施方案中，可以将腺嘌呤脱氨酶单独地融合至MCP或其部分。MCP或其部分可以结合或能够结合如本文所述的RNA募集基序，例如MS2发夹。
在一些实施方案中，将糖基化酶抑制剂融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。在一些实施方案中，将糖基化酶抑制剂融合至CRISPR-Cas效应蛋白。在一些实施方案中，将糖基化酶抑制剂在单一融合物中融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶或单独地融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶之一或两者。例如，在一些实施方案中，可以将胞嘧啶脱氨酶单独地融合至糖基化酶抑制剂，和/或在一些实施方案中，可以将腺嘌呤脱氨酶单独地融合至糖基化酶抑制剂。
在一些实施方案中，CRISPR-Cas效应蛋白包含一种或多种(例如，1、2、4、6、8、10或更多种)如本文所述的肽标签。在一些实施方案中，肽标签可以是SunTag和/或肽标签可以包含一个或多个(例如，1、2、3、4或更多个)GCN4表位。
在一些实施方案中，腺嘌呤脱氨酶和/或胞嘧啶脱氨酶包含如本文所述的亲和多肽(例如，scFv)，并且亲和多肽可以能够结合肽标签(例如，融合至CRISPR-Cas效应蛋白的肽标签)。在一些实施方案中，将亲和多肽在单一融合物中融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶两者，或者将亲和多肽单独地融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶之一或两者。当将亲和多肽单独地融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶两者时，融合至胞嘧啶脱氨酶的亲和多肽可以与融合至腺嘌呤脱氨酶的亲和多肽相同或不同。
在一些实施方案中，腺嘌呤脱氨酶和/或胞嘧啶脱氨酶包含一种或多种(例如，1、2、4、6、8、10或更多种)肽标签。在一些实施方案中，肽标签可以是SunTag和/或肽标签可以包含一个或多个(例如，1、2、3、4或更多个)GCN4表位。在一些实施方案中，将肽标签在单一融合物中融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶两者，或者将肽标签单独地融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶之一或两者。当将肽标签单独地融合至胞嘧啶脱氨酶和腺嘌呤脱氨酶两者时，融合至胞嘧啶脱氨酶的肽标签可与融合至腺嘌呤脱氨酶的肽标签相同或不同。
在一些实施方案中，CRISPR-Cas效应蛋白包含如本文所述的亲和多肽(例如，scFv)，并且亲和多肽可以能够结合肽标签(例如，融合至腺嘌呤脱氨酶和/或胞嘧啶脱氨酶的肽标签脱氨酶)。
在一些实施方案中，腺嘌呤脱氨酶和/或胞嘧啶脱氨酶包含DNA结合多肽。在一些实施方案中，本发明的融合蛋白包含CRISPR-Cas效应蛋白、DNA结合多肽和腺嘌呤脱氨酶和/或胞嘧啶脱氨酶。在一些实施方案中，将DNA结合多肽不融合或连接至不同的多肽。在一些实施方案中，DNA结合多肽在细胞中表达，任选地在存在于细胞中和/或引入细胞中的本发明的核酸构建体中表达。如本文所用，“DNA结合多肽”是指能够结合或有能力结合(非特异性地和/或特异性地(例如，以位点和/或序列特异性方式))DNA的蛋白质或多肽或其结构域。在一些实施方案中，将腺嘌呤脱氨酶和/或胞嘧啶脱氨酶融合(例如，连接)至任选地非特异性地结合DNA的DNA结合多肽，并且任选地将CRISPR-Cas效应蛋白融合至脱氨酶和/或DNA结合多肽。在一些实施方案中，DNA结合多肽结合至少一条DNA链，任选地结合双链DNA的一条或两条链。在一些实施方案中，DNA结合多肽结合双链DNA断裂的一端或两端。在一些实施方案中，DNA结合多肽结合双链断裂、捕获双链断裂和/或不结合任何蛋白质。在一些实施方案中，DNA结合多肽与SEQ ID NO：100或SEQ ID NO：113具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性，任选地其中DNA结合多肽包含SEQID NO：100或SEQ ID NO：113的序列。在一些实施方案中，DNA结合多肽包含SEQ ID NO：100或SEQ ID NO：113的至少约50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的连续氨基酸。在一些实施方案中，DNA结合多肽减少或最小化在靶核酸的修饰期间(例如，在碱基编辑期间)的不期望插入缺失的形成，提高修饰靶核酸的效率(例如，提高碱基编辑效率)，增加或提高碱基多样化活性，和/或提高修饰靶核酸的准确性。
根据一些实施方案，提供了一种碱基编辑组合物或系统，其包含：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)和胞嘧啶脱氨酶，其中组合物或系统缺乏糖基化酶抑制剂(例如，尿嘧啶糖基化酶抑制剂(UGI)，例如尿嘧啶-N-糖基化酶(UNG)抑制剂)。在一些实施方案中，碱基编辑组合物或系统包含：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)和胞嘧啶脱氨酶，其中CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和任选的指导核酸形成复合物或包含在复合物中，任选地其中复合物缺乏糖基化酶抑制剂(例如，UGI，例如UNG抑制剂)。在一些实施方案中，本发明提供一种核酸构建体，其包含：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)和胞嘧啶脱氨酶，任选地其中核酸构建体缺乏糖基化酶抑制剂(例如，UGI，例如UNG抑制剂)。在一些实施方案中，组合物、系统和/或核酸构建体包含糖基化酶结构域。指导核酸可以与靶核酸具有不完全的互补性，例如小于100％的互补性(例如，小于99％、98％、97％、96％、95％、94％、93％、92％、91％、90％等)。胞嘧啶脱氨酶可以是rAPOBECl、APOBEC3A、APOBEC3B、hAID和pmCDA1中的一种或多种。CRISPR-Cas效应蛋白可以包含V型CRISPR-Cas效应蛋白和/或II型CRISPR-Cas效应蛋白，例如Cas9，任选地与靶核酸具有减弱的相互作用的Cas9。在一些实施方案中，CRISPR-Cas效应蛋白可以包含(例如，融合至)任选地经密码子优化的外源聚合酶。在一些实施方案中，CRISPR-Cas效应蛋白包含如本文所述的肽标签(例如，SunTag)，并且胞嘧啶脱氨酶包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将胞嘧啶脱氨酶和亲和多肽融合在一起。在一些实施方案中，胞嘧啶脱氨酶包含如本文所述的肽标签(例如，SunTag)，并且CRISPR-Cas效应蛋白包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将CRISPR-Cas效应蛋白和亲和多肽融合在一起。在一些实施方案中，胞嘧啶脱氨酶包含MCP或其部分，任选其中将MCP或其部分融合至胞嘧啶脱氨酶氨基酸序列的N末端。在一些实施方案中，胞嘧啶脱氨酶包含(例如，融合至)Cas9、Cas12、Cas13或Cas14结构域。在一些实施方案中，胞嘧啶脱氨酶包含Cas9结构域，任选地其中将胞嘧啶脱氨酶融合至Cas9结构域。在一些实施方案中，胞嘧啶脱氨酶包含失活的LbCpf1(dLbCpf1)，任选地其中将胞嘧啶脱氨酶融合至dLbCpf1。在一些实施方案中，胞嘧啶脱氨酶是密码子优化的，任选地用于单子叶植物表达和/或双子叶植物表达。
在一些实施方案中，CRISPR-Cas效应蛋白可以包含Cas12a(Cpf1)效应蛋白或其多肽或结构域，例如LbCpf1[毛螺菌科(Lachnospiraceae)细菌]、AsCpfl[氨基酸球菌属(Acidaminococcus)物种]、BpCpf1[Butyrivibrio proteoclasticus]、CMtCpf1[Candidatus Methanoplasma termitum]、EeCpf1[挑剔真杆菌(Eubacterium eligens)]、FnCpf1(新凶手弗朗西丝氏菌(Francisella novicida)U112)、Lb2Cpf1[毛螺菌科细菌]、＞Lb3Cpf1[毛螺菌科细菌]、LiCpf1[稻田氏钩端螺旋体(Leptospira inadai)]、MbCpf1[牛眼莫拉氏菌(Moraxella bovoculi)237]、PbCpf1[Parcubacteria细菌GWC2011_GWC2_44_17]、PcCpf1[狗口腔卟啉单胞菌(Porphyromonas crevioricanis)]、PdCpf1[解糖胨普雷沃氏菌(Prevotella disiens)]、PeCpf1[Peregrinibacteria细菌GW2011_GWA_33_10]、PmCpfl[Porphyromonas macacae]和/或SsCpf1[史密斯氏菌属(Smithella)物种SC_K08D17](例如，SEQ ID NO：3-22)。在一些实施方案中，Cas12a效应蛋白结构域可以是毛螺菌科细菌ND2006 Cas12a(LbCas12a)(LbCpfl)(例如，SEQ ID NO：3、9-11)、氨基酸球菌属物种Cpf1(AsCas12a)(AsCpf1)(例如，SEQ ID NO：4)和/或enAsCas12a(例如，SEQ ID NO：20-22)。
在一些实施方案中，本发明的核酸构建体(例如，编码CRISPR-Cas效应蛋白的多核苷酸、编码CRISPR-Cas融合蛋白的多核苷酸、编码脱氨酶的多核苷酸、编码脱氨酶融合蛋白的多核苷酸、编码肽标签的多核苷酸、编码亲和多肽的多核苷酸、RNA募集基序、募集指导核酸和/或指导核酸和/或包含它们的表达盒和/或载体)可以可操作地连接至至少一个调控序列，任选地，其中至少一个调控序列可以经优化密码子以在植物中表达。在一些实施方案中，至少一个调控序列可以是例如启动子、操纵子、终止子或增强子。在一些实施方案中，至少一个调控序列可以是启动子。在一些实施方案中，调控序列可以是内含子。在一些实施方案中，至少一个调控序列可以是例如与内含子或包含内含子的启动子区域可操作地结合的启动子。在一些实施方案中，至少一个调控序列可以是例如泛素启动子及其相关内含子(例如，蒺藜苜蓿(Medicago truncatula)和/或玉蜀黍(Zea mays)及其相关内含子)。在一些实施方案中，至少一个调控序列可以是终止子核苷酸序列和/或增强子核苷酸序列。
在一些实施方案中，本发明的核酸构建体可以与启动子区域可操作地关联，其中启动子区域包含内含子，任选地其中启动子区域可以是泛素启动子和内含子(例如，苜蓿属(Medicago)或玉蜀黍泛素启动子和内含子，例如，SEQ ID NO：1或SEQ ID NO：2)。在一些实施方案中，与包含内含子的启动子区可操作地关联的本发明的核酸构建体可以经优化密码子以在植物中表达。
在一些实施方案中，本发明的核酸构建体可以编码一种或多种目标多肽，任选地其中一种或多种目标多肽可以经优化密码子以在植物中表达。
可用于本发明的目标多肽可以包括但不限于具有以下活性的多肽或蛋白质结构域：脱氨酶活性、切口酶活性、重组酶活性、转座酶活性、甲基化酶活性、糖基化酶(DNA糖基化酶)活性、糖基化酶抑制剂活性(例如，尿嘧啶-DNA糖基化酶抑制剂(UGI))、去甲基化酶活性、转录激活活性、转录抑制活性、转录释放因子活性、组蛋白修饰活性、核酸酶活性、单链RNA切割活性、双链RNA切割活性、限制性核酸内切酶活性(例如，Fok1)、核酸结合活性、甲基转移酶活性、DNA修复活性、DNA损伤活性、歧化酶活性、烷基化活性、脱嘌呤活性、氧化活性、嘧啶二聚体形成活性、整合酶活性、转座酶活性、聚合酶活性、连接酶活性、解旋酶活性和/或光裂合酶活性。在一些实施方案中，目标多肽是Fok1核酸酶或尿嘧啶-DNA糖基化酶抑制剂。在一些实施方案中，目标多肽是这样的多肽：其在碱基编辑期间减少或最小化不希望的插入缺失的形成，增加靶核酸的修饰(例如，在碱基编辑期间)，提高修饰靶核酸的效率(例如，增加碱基编辑的效率)，增加或改善碱基多样化活性，和/或增加修饰靶核酸的准确性。当在核酸(多核苷酸、表达盒和/或载体)中编码时，所编码的多肽或蛋白质结构域可以经优化密码子以在生物体中表达。在一些实施方案中，可以将目标多肽连接至CRISPR-Cas效应蛋白以提供包含CRISPR-Cas效应蛋白和目标多肽的CRISPR-Cas融合蛋白。在一些实施方案中，也可以将包含连接至肽标签的CRISPR-Cas效应蛋白的CRISPR-Cas融合蛋白连接至目标多肽(例如，可以将CRISPR-Cas效应蛋白例如连接至肽标签(或亲和多肽)和例如目标多肽(例如UGI)两者)。在一些实施方案中，目标多肽可以是尿嘧啶糖基化酶抑制剂(例如，尿嘧啶-DNA糖基化酶抑制剂(UGI))。在一些实施方案中，可以将目标多肽连接至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶以提供包含胞嘧啶脱氨酶和/或腺嘌呤脱氨酶和目标多肽的脱氨酶融合蛋白。在一些实施方案中，目标多肽可以在细胞(例如植物细胞)中表达并且可以不融合至另一个多肽。
在一些实施方案中，本发明的编码CRISPR-Cas效应蛋白和胞嘧啶脱氨酶和/或腺嘌呤脱氨酶并包含指导核酸的核酸构建体可以进一步编码目标多肽，任选地其中目标多肽可以经优化密码子以在生物体(例如植物或真核生物)中表达。
如本文所用，“CRISPR-Cas效应蛋白”是切割、切开或切口核酸、结合核酸(例如，靶核酸和/或指导核酸)和/或鉴定、识别或结合本文定义的指导核酸的蛋白质或多肽或其结构域。在一些实施方案中，CRISPR-Cas效应蛋白可以是酶(例如，核酸酶、核酸内切酶、切口酶等)或其部分和/或可以作为酶起作用。在一些实施方案中，CRISPR-Cas效应蛋白是指CRISPR-Cas核酸酶多肽或其结构域，其包含核酸酶活性或其中核酸酶活性已被降低或消除，和/或包含切口酶活性或其中切口酶已被降低或消除，和/或包含单链DNA切割活性(ssDNA酶活性)或其中ssDNA酶活性已被降低或消除，和/或包含自加工RNA酶活性或其中自加工RNA酶活性已被降低或消除。CRISPR-Cas效应蛋白可以结合靶核酸。CRISPR-Cas效应蛋白可以是I、II、III、IV、V或VI型CRISPR-Cas效应蛋白。在一些实施方案中，CRISPR-Cas效应蛋白可以来自I型CRISPR-Cas系统、II型CRISPR-Cas系统、III型CRISPR-Cas系统、IV型CRISPR-Cas系统、V型CRISPR-Cas系统或VI型CRISPR-Cas系统。在一些实施方案中，本发明的CRISPR-Cas效应蛋白可以来自II型CRISPR-Cas系统或V型CRISPR-Cas系统。在一些实施方案中，CRISPR-Cas效应蛋白可以是II型CRISPR-Cas效应蛋白，例如Cas9效应蛋白。在一些实施方案中，CRISPR-Cas效应蛋白可以是V型CRISPR-Cas效应蛋白，例如Cas12效应蛋白。
在一些实施方案中，CRISPR-Cas效应蛋白可以是或包括但不限于Cas9、C2c1、C2c3、Cas12a(也称为Cpf1)、Cas12b、Cas12c、Cas12d、Cas12e、Cas13a、Cas13b、Cas13c、Cas13d、Casl、CaslB、Cas2、Cas3、Cas3′、Cas3”、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csyl、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf、Csf2、Csf3、Csf4(dinG)和/或Csf5核酸酶，任选地其中CRISPR-Cas效应蛋白可以是Cas9、Cas12a(Cpf1)、Cas12b、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12g、Cas12h、Cas12i、C2c4、C2c5、C2c8、C2c9、C2c10、Cas14a、Cas14b和/或Cas14c效应蛋白。
在一些实施方案中，用于本发明的CRISPR-Cas效应蛋白可以在其核酸酶活性位点(例如，RuvC、HNH，例如Cas12a核酸酶结构域的RuvC位点；例如，Cas9核酸酶结构域的RuvC位点和/或HNH位点)中包含突变。在其核酸酶活性位点具有突变并因此不再包含核酸酶活性的CRISPR-Cas效应蛋白通常被称为“dead”，例如dCas9。在一些实施方案中，与没有突变的相同CRISPR-Cas效应蛋白(例如切口酶，例如Cas9切口酶，Cas12a切口酶)相比，在其核酸酶活性位点具有突变的CRISPR-Cas效应蛋白结构域或多肽可能具有受损的活性或降低的活性。
可用于本发明的CRISPR Cas9效应蛋白或CRISPR Cas9效应结构域可以是任何已知的或后来鉴定的Cas9核酸酶。在一些实施方案中，CRISPR Cas9多肽可以是来自例如链球菌属(Streptococcus)物种(例如，酿脓链球菌(S.pyogenes)、嗜热链球菌(S.thermophilus)、乳杆菌属(Lactobacillus)物种、双歧杆菌属(Bifidobacterium)物种、坎德勒菌属(Kandleria)物种、明串珠菌属(Leuconostoc)物种、酒球菌属(Oenococcus)物种、片球菌属(Pediococcus)物种、魏斯氏菌属(Weissella)物种和/或欧陆森氏菌属(Olseniella)物种的Cas9多肽。在一些实施方案中，CRISPR-Cas效应蛋白可以是Cas9多肽或其结构域，并且任选地可以具有SEQ ID NO：23-37中任一项的核苷酸序列和/或SEQ IDNO：38-39中任一项的氨基酸序列。
在一些实施方案中，CRISPR-Cas效应蛋白可以是源自酿脓链球菌的Cas9多肽并且识别PAM序列基序NGG、NAG、NGA(Mali等人，Science 2013；339(6121)：823-826)。在一些实施方案中，CRISPR-Cas效应蛋白可以是源自嗜热链球菌的Cas9多肽并且识别PAM序列基序NGGNG和/或NNAGAAW(W＝A或T)(参见例如Horvath等人，Science，2010；327(5962)：167-170和Deveau等人，J Bacteriol 2008；190(4)：1390-1400)。在一些实施方案中，CRISPR-Cas效应蛋白可以是源自变形链球菌(Streptococcus mutans)的Cas9多肽并且识别PAM序列基序NGG和/或NAAR(R＝A或G)(参见例如Deveau等人，J BACTERIOL 2008；190(4)：1390-1400)。在一些实施方案中，CRISPR-Cas效应蛋白可以是源自金色链球菌(Streptococcus aureus)的Cas9多肽并识别PAM序列基序NNGRR(R＝A或G)。在一些实施方案中，CRISPR-Cas效应蛋白可以是源自金黄色葡萄球菌(S.aureus)的Cas9蛋白，其识别PAM序列基序N GRRT(R＝A或G)。在一些实施方案中，CRISPR-Cas效应蛋白可以是源自金黄色葡萄球菌的Cas9多肽，其识别PAM序列基序NGRRV(R＝A或G)。在一些实施方案中，CRISPR-Cas效应蛋白可以是源自脑膜炎奈瑟菌(Neisseria meningitidis)的Cas9多肽并且识别PAM序列基序NGATT或N GCTT(R＝A或G，V＝A、G或C)(参见，例如，Hou等人，PNAS 2013，1-6)。在上述实施方案中，N可以是任何核苷酸残基，例如A、G、C或T中的任何一个。在一些实施方案中，CRISPR-Cas效应蛋白可以是源自沙氏纤毛菌(Leptotrichia shahii)的Cas13a蛋白，其识别原型间隔区侧翼序列(PFS)(或RNA PAM(rPAM))单个3′A、U或C的序列基序，所述序列基序可能位于靶核酸内。
可用于本发明实施方案的V型CRISPR-Cas效应蛋白可以是任何V型CRISPR-Cas核酸酶。可用于本发明作为效应蛋白的V型CRISPR-Cas核酸酶可包括但不限于Cas12a(Cpf1)、Cas12b、Cas12c(C2c3)、Cas12d(CasY)、Cas12e(CasX)、Cas12g、Cas12h、Cas12i、C2c1、C2c4、C2c5、C2c8、C2c9、C2c10、Cas14a、Cas14b和/或Cas14c核酸酶。在一些实施方案中，可用于本发明实施方案的V型CRISPR-Cas核酸酶多肽或结构域可以是Cas12a多肽或结构域。在一些实施方案中，可用于本发明实施方案的V型CRISPR-Cas效应蛋白或结构域可以是切口酶，任选地，Cas12a切口酶。在一些实施方案中，CRISPR-Cas效应蛋白可以是Cas12a多肽或其结构域并且任选地可以具有SEQ ID NO：3-19中任一项的氨基酸序列和/或SEQ ID NO：20-22中任一项的核苷酸序列。
在一些实施方案中，CRISPR-Cas效应蛋白可以源自Cas12a，其是V型成簇规则间隔短回文重复序列(CRISPR)-Cas核酸酶。Cas12a在几个方面与更知名的II型CRISPR Cas9核酸酶不同。例如，Cas9识别富含G的原型间隔区邻近基序(PAM)，它位于其指导RNA(gRNA、sgRNA、crRNA、crDNA、CRISPR阵列)结合位点(原型间隔区、靶核酸、靶DNA)的3′(3′-NGG)，而Cas12a识别位于靶标核酸的5′的富含T的PAM(5′-TTN，5′-TTTN)。事实上，Cas9和Cas12a结合其指导RNA的方向与它们的N和C末端的关系几乎相反。此外，Cas12a酶使用单个指导RNA(gRNA、CRISPR阵列、crRNA)而不是在天然Cas9系统中发现的双指导RNA(sgRNA(例如crRNA和tracrRNA))，并且Cas12a处理其gRNA。此外，Cas12a核酸酶活性产生交错的DNA双链断裂，而不是由Cas9核酸酶活性产生的平末端，Cas12a依靠单个RuvC结构域来切割两条DNA链，而Cas9利用HNH结构域和RuvC结构域用于切割。
可用于本发明的CRISPR Cas12a效应蛋白/结构域可以是任何已知的或以后鉴定的Cas12a多肽(以前称为Cpf1)(参见例如美国专利号9,790,490，通过引用将其公开的Cpf1(Cas12a)序列并入本文)。术语“Cas12a”、“Cas12a多肽”或“Cas12a结构域”是指包含Cas12a多肽或其片段的RNA指导的核酸酶，其包含Cas12a的指导核酸结合结构域和/或Cas12a的活性、非活性、或部分活性的DNA切割结构域。在一些实施方案中，可用于本发明的Cas12a可包含核酸酶活性位点(例如，Cas12a结构域的RuvC位点)中的突变。在其核酸酶活性位点具有突变并因此不再包含核酸酶活性的Cas12a结构域或Cas12a多肽通常称为deadCas12a(例如，dCas12a)。在一些实施方案中，在其核酸酶活性位点中具有突变的Cas12a结构域或Cas12a多肽可能具有受损的活性，例如，可能具有切口酶活性。
在一些实施方案中，可以优化CRISPR-Cas效应蛋白以在生物体中表达，例如在动物(例如，哺乳动物，如人)、植物、真菌、古细菌或细菌中表达。在一些实施方案中，可以优化CRISPR-Cas效应蛋白(例如，Cas12a多肽/结构域或Cas9多肽/结构域)以在植物中表达。
可用于碱基编辑的任何脱氨酶结构域/多肽可用于本发明。如本文所用，“胞嘧啶脱氨酶”和“胞苷脱氨酶”是指催化或能够催化胞嘧啶脱氨的多肽或其结构域，因为该多肽或结构域催化或能够催化从胞嘧啶碱基除去胺基团。因此，胞嘧啶脱氨酶可导致胞嘧啶转化为胸苷(通过尿嘧啶中间体)，导致C至T的转换或基因组互补链中的G至A的转换。因此，在一些实施方案中，由本发明的多核苷酸编码的胞嘧啶脱氨酶产生靶核酸的有义(例如“+”；模板)链的C→T转换或靶核酸的反义(例如，“-”，互补)链的G→A转换。在一些实施方案中，由本发明的多核苷酸编码的胞嘧啶脱氨酶在基因组的互补链中产生C至T、G或A的转换。
可用于本发明的胞嘧啶脱氨酶可以是来自任何生物体的任何已知或后来鉴定的胞嘧啶脱氨酶(参见例如美国专利号10,167,457和Thuronyi等人Nat.Biotechnol.37：1070-1079(2019)，其中每一个通过引用将其公开的胞嘧啶脱氨酶并入本文)。胞嘧啶脱氨酶可以催化胞苷或脱氧胞苷分别水解脱氨为尿苷或脱氧尿苷。因此，在一些实施方案中，可用于本发明的脱氨酶或脱氨酶结构域可以是胞苷脱氨酶结构域，催化胞嘧啶至尿嘧啶的水解脱氨。在一些实施方案中，胞嘧啶脱氨酶可以是天然存在的胞嘧啶脱氨酶的变体，包括但不限于灵长类动物(例如人、猴、黑猩猩、大猩猩)、狗、牛、大鼠或小鼠。因此，在一些实施方案中，可用于本发明的胞嘧啶脱氨酶可以与野生型胞嘧啶脱氨酶具有约70％至约100％的同一性(例如，与天然存在的胞嘧啶脱氨酶约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同，以及其中的任何范围或数值)。
在一些实施方案中，可用于本发明的胞嘧啶脱氨酶可以是载脂蛋白BmRNA-编辑复合物(APOBEC)家族脱氨酶。在一些实施方案中，胞嘧啶脱氨酶可以是APOBEC1脱氨酶、APOBEC2脱氨酶、APOBEC3A脱氨酶、APOBEC3B脱氨酶、APOBEC3C脱氨酶、APOBEC3D脱氨酶、APOBEC3F脱氨酶、APOBEC3G脱氨酶、APOBEC3H脱氨酶、APOBEC4脱氨酶、人活化诱导脱氨酶(hAID)、rAPOBECl、FERNY和/或CDA1，任选地pmCDA1、atCDA1(例如，At2g19570)，以及它们的进化版本。进化的脱氨酶公开在例如，美国专利号10,113,163，Gaudelli等人Nature 551(7681)：464-471(2017))和Thuronyi等人(Nature Biotechnology 37：1070-1079(2019))中，它们中的每一个都通过引用将它们公开的脱氨酶和进化的脱氨酶并入本文。在一些实施方案中，胞嘧啶脱氨酶可以是具有SEQ ID NO：40的氨基酸序列的APOBEC1脱氨酶。在一些实施方案中，胞嘧啶脱氨酶可以是具有SEQ ID NO：41的氨基酸序列的APOBEC3A脱氨酶。在一些实施方案中，胞嘧啶脱氨酶可以是CDA1脱氨酶，任选地是具有SEQ ID NO：42的氨基酸序列的CDA1。在一些实施方案中，胞嘧啶脱氨酶可以是FERNY脱氨酶，任选地是具有SEQ IDNO：43的氨基酸序列的FERNY。在一些实施方案中，胞嘧啶脱氨酶可以是rAPOBEC1脱氨酶，任选地是具有SEQ ID NO：44的氨基酸序列的rAPOBEC1脱氨酶。在一些实施方案中，胞嘧啶脱氨酶可以是hAID脱氨酶，任选地是具有SEQ ID NO：45或SEQ ID NO：46的氨基酸序列的hAID。在一些实施方案中，可用于本发明的胞嘧啶脱氨酶可以与天然存在的胞嘧啶脱氨酶(例如，“进化的脱氨酶”)的氨基酸序列(参见，例如，SEQ ID NO：47、SEQ ID NO：48、SEQ IDNO：49)约70％至约100％相同(例如，70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或100％相同)。在一些实施方案中，可用于本发明的胞嘧啶脱氨酶可以与SEQ ID NO：40-49中任一项的氨基酸序列约70％至约99.5％相同(例如，约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.5％相同)(例如，与SEQ ID NO：40-49中任一项的氨基酸序列至少80％、至少85％、至少90％、至少92％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同)。在一些实施方案中，编码胞嘧啶脱氨酶的多核苷酸可以经密码子优化以在植物中表达，并且密码子优化的多核苷酸可以与参考多核苷酸约70％至99.5％相同。
如本文所用，“腺嘌呤脱氨酶”和“腺苷脱氨酶”是指催化或能够催化腺嘌呤或腺苷的水解脱氨(例如，从腺嘌呤去除胺基团)的多肽或其结构域。在一些实施方案中，腺嘌呤脱氨酶可以催化腺苷或脱氧腺苷分别水解脱氨为肌苷或脱氧肌苷。在一些实施方案中，腺苷脱氨酶可以催化DNA中腺嘌呤或腺苷的水解脱氨。在一些实施方案中，由本发明的核酸构建体编码的腺嘌呤脱氨酶可以在靶核酸的有义(例如“+”；模板)链中产生A→G转换或在靶核酸的反义(例如，“-”，互补)链中产生T→C转换。可用于本发明的腺嘌呤脱氨酶可以是来自任何生物体的任何已知的或后来鉴定的腺嘌呤脱氨酶(参见例如美国专利号10,113,163，其公开的腺嘌呤脱氨酶通过引用并入本文)。
在一些实施方案中，腺苷脱氨酶可以是天然存在的腺嘌呤脱氨酶的变体。因此，在一些实施方案中，腺苷脱氨酶可以与野生型腺嘌呤脱氨酶约70％至100％相同(例如，与天然存在的腺嘌呤脱氨酶约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同，以及其中的任何范围或数值)。在一些实施方案中，所述脱氨酶或脱氨酶在自然界中不存在并且可以称为工程化的、突变的或进化的腺苷脱氨酶。因此，例如，工程化的、突变的或进化的腺嘌呤脱氨酶多肽或腺嘌呤脱氨酶结构域可以与天然存在的腺嘌呤脱氨酶多肽/结构域约70％至99.9％相同(例如，与天然存在的腺嘌呤脱氨酶多肽或腺嘌呤脱氨酶结构域约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同，以及其中的任何范围或数值)。在一些实施方案中，腺苷脱氨酶可以来自细菌(例如，大肠杆菌、金黄色葡萄球菌、流感嗜血杆菌(Haemophilusinfluenzae)、新月柄杆菌(Caulobacter crescentus)等)。在一些实施方案中，编码腺嘌呤脱氨酶多肽/结构域的多核苷酸可以经密码子优化以在植物中表达。
在一些实施方案中，腺嘌呤脱氨酶结构域可以是野生型tRNA特异性腺苷脱氨酶结构域，例如tRNA特异性腺苷脱氨酶(TadA)和/或突变/进化的腺苷脱氨酶结构域，例如突变/进化的tRNA特异性腺苷脱氨酶结构域(TadA*)。在一些实施方案中，TadA结构域可以来自大肠杆菌。在一些实施方案中，TadA可以被修饰，例如，相对于全长TadA，截短、缺失一个或多个N末端和/或C末端氨基酸(例如，相对于全长TadA，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、6、17、18、19或20个N末端和/或C末端氨基酸残基可以缺失)。在一些实施方案中，TadA多肽或TadA结构域不包含N末端甲硫氨酸。在一些实施方案中，野生型大肠杆菌TadA包含SEQID NO：50的氨基酸序列。在一些实施方案中，突变的/进化的大肠杆菌TadA*包含SEQ IDNO：51-54(例如，SEQ ID NO：51、52、53或54)的氨基酸序列。在一些实施方案中，编码TadA/TadA*的多核苷酸可以经密码子优化以在植物中表达。在一些实施方案中，腺嘌呤脱氨酶可包含SEQ ID NO：55-60中任一项的氨基酸序列的全部或部分。在一些实施方案中，腺嘌呤脱氨酶可包含SEQ ID NO：50-60中任一项的氨基酸序列的全部或部分。
在一些实施方案中，本发明的核酸构建体可以进一步编码糖基化酶抑制剂(例如，尿嘧啶糖基化酶抑制剂(UGI)，例如尿嘧啶-DNA糖基化酶抑制剂)。因此，在一些实施方案中，编码CRISPR-Cas效应蛋白和胞嘧啶脱氨酶和/或腺嘌呤脱氨酶的核酸构建体可以进一步编码糖基化酶抑制剂，任选地其中糖基化酶抑制剂可以经密码子优化以在植物中表达。在一些实施方案中，本发明提供了包含CRISPR-Cas效应多肽和UGI的融合蛋白和/或一种或多种编码其的多核苷酸，任选地其中一种或多种多核苷酸可以经密码子优化以在植物中表达。在一些实施方案中，本发明提供了包含CRISPR-Cas效应多肽、脱氨酶结构域(例如，腺嘌呤脱氨酶结构域和/或胞嘧啶脱氨酶结构域)和UGI的融合蛋白和/或编码其的一种或多种多核苷酸，任选地其中一种或多种多核苷酸可以经密码子优化以在植物中表达。在一些实施方案中，本发明提供了融合蛋白，其中可以将CRISPR-Cas效应多肽、脱氨酶结构域和/或UGI融合至如本文所述的肽标签和亲和多肽的任何组合，从而可以募集脱氨酶结构域和/或UGI至CRISPR-Cas效应多肽和至靶核酸。在一些实施方案中，可以将指导核酸连接至募集RNA基序，并且可以将脱氨酶结构域和/或UGI中的一个或多个融合至能够与募集RNA基序相互作用的亲和多肽，从而募集脱氨酶域和UGI至靶核酸。
可用于本发明的“尿嘧啶糖基化酶抑制剂”或“UGI”可以是能够抑制尿嘧啶-DNA糖基化酶碱基-切除修复酶的任何蛋白质或多肽或其结构域。在一些实施方案中，UGI包括野生型UGI或其片段。在一些实施方案中，可用于本发明的UGI可以与天然存在的UGI的氨基酸序列约70％至约100％相同(例如，70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或100％相同以及其中的任何范围或数值)。在一些实施方案中，UGI可包含SEQ ID NO：61的氨基酸序列或与SEQ ID NO：61的氨基酸序列具有约70％至约99.5％同一性的多肽(例如，与SEQ ID NO：61的氨基酸序列至少80％、至少85％、至少90％、至少92％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.5％相同)。例如，在一些实施方案中，UGI可以包含与SEQ ID NO：61的氨基酸序列的连续核苷酸的部分(例如，10、15、20、25、30、35、40、45、50、55、60、65、70、75、80个连续核苷酸；例如，约10、15、20、25、30、35、40、45至约50、55、60、65、70、75、80个连续核苷酸)100％相同的SEQ ID NO：61的氨基酸序列的片段。在一些实施方案中，UGI可以是与已知UGI具有约70％至约99.5％同一性(例如，70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％同一性，以及其中的任何范围或数值)的已知UGI(例如SEQID NO：61)的变体。在一些实施方案中，编码UGI的多核苷酸可以经密码子优化以在植物(例如植物)中表达，并且密码子优化的多核苷酸可以与参考多核苷酸约70％至约99.5％相同。
包含CRISPR-Cas效应蛋白或其融合蛋白的本发明的核酸构建体可以与设计用于与所编码的CRISPR-Cas效应蛋白或其结构域发挥功能的指导核酸(例如，指导RNA(gRNA)、CRISPR阵列、CRISPR RNA、crRNA)组合使用，以修饰靶核酸。可用于本发明的指导核酸可以包含至少一个间隔区序列和至少一个重复序列。指导核酸能够与由本发明的核酸构建体所编码和表达的CRISPR-Cas核酸酶结构域形成复合物，并且间隔区序列能够与靶核酸杂交，从而将复合物引导至靶核酸，其中靶核酸可以被脱氨酶(例如胞嘧啶脱氨酶和/或腺嘌呤脱氨酶，任选地存在于复合物中和/或募集到复合物)修饰(例如，切割或编辑)和/或调节(例如，调节转录)。
例如，可以将编码连接至胞嘧啶脱氨酶结构域的Cas9结构域(例如融合蛋白)的核酸构建体与Cas9指导核酸组合使用以修饰靶核酸，其中融合蛋白的胞嘧啶脱氨酶结构域使靶核酸中的胞嘧啶碱基脱氨基，从而编辑靶核酸。在另一个实施例中，可以将编码连接至腺嘌呤脱氨酶结构域的Cas9结构域(例如，融合蛋白)的核酸构建体与Cas9指导核酸组合使用以修饰靶核酸，其中融合蛋白的腺嘌呤脱氨酶结构域使靶核酸中的腺苷碱基脱氨基，从而编辑靶核酸。在一些实施方案中，将CRISPR-Cas效应蛋白(例如，Cas9)不融合至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
同样，可以将编码Cas12a结构域(或其他选择的CRISPR-Cas核酸酶，例如C2c1、C2c3、Cas12b、Cas12c、Cas12d、Cas12e、Cas13a、Cas13b、Cas13c、Cas13d、Cas1、Cas1B、Cas2、Cas3、Cas3’、Cas3”、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csnl和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4(dinG)和/或Csf5)的核酸构建体连接至胞嘧啶脱氨酶结构域或腺嘌呤脱氨酶结构域(例如融合蛋白)，并可与Cas12a指导核酸(或用于其他选择的CRISPR-Cas核酸酶的指导核酸)组合使用以修饰靶核酸，其中融合蛋白的胞嘧啶脱氨酶结构域或腺嘌呤脱氨酶结构域分别将靶核酸中的胞嘧啶碱基或腺苷碱基脱氨基，从而编辑靶核酸酸。
如本文所用，“指导核酸”、“指导RNA”、“gRNA”、“CRISPR RNA/DNA”、“crRNA”或“crDNA”是指核酸，其包含至少一个间隔区序列(该间隔区序列与靶DNA互补(并杂交)(例如，原型间隔区))，和至少一个重复序列(例如，V型Cas12a CRISPR-Cas系统的重复序列，或其片段或部分；II型Cas9 CRISPR-Cas的重复序列或其片段；V型C2c1 CRISPR Cas系统的重复序列或其片段；CRISPR-Cas系统的重复序列，例如C2c3、Cas12a(也称为Cpf1)、Cas12b、Cas12c、Cas12d、Cas12e、Cas13a、Cas13b、Cas13c、Cas13d、Cas1、Cas1B、Cas2、Cas3、Cas3’、Cas3”、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9(也称为Csn1和Csx12)、Cas10、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmrl、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4(dinG)和/或Csf5，或其片段)，其中可以将重复序列连接至间隔区序列的5’端和/或3’端。在一些实施方案中，指导核酸包含DNA。在一些实施方案中，指导核酸包含RNA。本发明的gRNA的设计可以基于I型、II型、III型、IV型、V型或VI型CRISPR-Cas系统。
在一些实施方案中，Cas12a gRNA可包含从5’到3’的重复序列(全长或其部分(“柄”)；例如，假结样结构)和间隔区序列。
在一些实施方案中，指导核酸可以包含多于一个重复序列-间隔区序列(例如，2、3、4、5、6、7、8、9、10或更多个重复-间隔区序列)(例如，重复-间隔区-重复，例如，重复-间隔区-重复-间隔区-重复-间隔区-重复-间隔区-重复-间隔区等)。本发明的指导核酸是合成的、人造的并且在自然界中不存在。gRNA可以很长，可以用作适配体(如在MS2募集策略中)或悬挂在间隔区外的其他RNA结构。
如本文所用，“重复序列”是指例如野生型CRISPR Cas基因座(例如，Cas9基因座、Cas12a基因座、C2c1基因座等)的任何重复序列或与本发明的核酸构建体所编码的CRISPR-Cas效应蛋白一起起作用的合成crRNA的重复序列。可用于本发明的重复序列可以是任何已知或以后鉴定的CRISPR-Cas基因座的重复序列(例如，I型、II型、III型、IV型、V型或VI型)，或者它可以是设计用于在I、II、III、IV、V或VI型CRISPR-Cas系统中起作用的合成的重复。重复序列可以包含发夹结构和/或茎环结构。在一些实施方案中，重复序列可在其5’端形成假结样结构(即，“柄”)。因此，在一些实施方案中，重复序列可以与来自野生型I型CRISPR-Cas基因座、II型CRISPR-Cas基因座、III型CRISPR-Cas基因座、IV型CRISPR-Cas基因座、V型CRISPR-Cas基因座和/或VI型CRISPR-Cas基因座的重复序列相同或基本相同。来自野生型CRISPR-Cas基因座的重复序列可以通过已建立的算法来确定，例如使用通过CRISPRdb提供的CRISPRfinder(参见Grissa等人Nucleic Acids Res.35(Web Server issue)：W52-7)。在一些实施方案中，将重复序列或其部分在其3’端连接至间隔区序列的5’端，从而形成重复-间隔区序列(例如，指导核酸、指导RNA/DNA、crRNA、crDNA)。
在一些实施方案中，取决于特定的重复以及包含重复的指导核酸是加工的还是未加工的，重复序列包含至少10个核苷酸、基本上由至少10个核苷酸组成或由至少10个核苷酸组成(例如，约10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50至100个或更多个核苷酸，或其中的任何范围或数值；例如，约)。在一些实施方案中，重复序列包含、基本上由或由以下组成：约10至约20、约10至约30、约10至约45、约10至约50、约15至约30、约15至约40、约15至约45、约15至约50、约20至约30、约20至约40、约20至约50、约30至约40、约40至约80、约50至约100或更多个的核苷酸。
连接至间隔区序列的5’端的重复序列可以包含重复序列的部分(例如，野生型重复序列的5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或更多个连续核苷酸)。在一些实施方案中，连接至间隔区序列的5’端的重复序列的部分的长度可以是约5至约10个连续核苷酸(例如，约5、6、7、8、9、10个核苷酸)并且与野生型CRISPR Cas重复核苷酸序列的相同区域(例如，5’端)具有至少90％的序列同一性(例如，至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多)。在一些实施方案中，重复序列的部分可以在其5’端包含假结样结构(例如，“柄”)。
如本文所用，“间隔区序列”是与靶核酸(例如，靶DNA)互补的核苷酸序列(例如，原型间隔区)。间隔区序列可以与靶核酸完全互补或基本上互补(例如，至少约70％互补(例如，约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、或更多))。因此，在一些实施方案中，与靶核酸相比，间隔区序列可以具有1、2、3、4或5个错配，这些错配可以是连续的或不连续的。在一些实施方案中，间隔区序列可以与靶核酸具有70％的互补性。在其他实施方案中，间隔区核苷酸序列可以与靶核酸具有80％的互补性。在其他实施方案中，间隔区核苷酸序列可以与靶核酸(原型间隔区)具有85％、90％、95％、96％、97％、98％、99％或99.5％的互补性等。在一些实施方案中，间隔区序列与靶核酸100％互补。间隔区序列可具有约15个核苷酸至约30个核苷酸(例如，15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸，或其中的任何范围或数值)的长度。因此，在一些实施方案中，间隔区序列可在长度为至少约15个核苷酸至约30个核苷酸的靶核酸(例如，原型间隔区)的区域上具有完全互补性或基本互补性。在一些实施方案中，间隔区的长度为约20个核苷酸。在一些实施方案中，间隔区的长度为约21、22或23个核苷酸。
在一些实施方案中，指导核酸的间隔区序列的5’区可以与靶DNA相同，而间隔区的3’区可以与靶DNA基本上互补(例如，V型CRISPR-Cas)，或指导核酸的间隔区序列的3’区可以与靶DNA相同，而间隔区的5’区可以与靶DNA基本上互补(例如II型CRISPR-Cas)，并且因此，间隔区序列与靶DNA的总体互补性可以低于100％。因此，例如，在V型CRISPR-Cas系统的指导中，例如，20个核苷酸的间隔区序列5’区(即种子区)中的前1、2、3、4、5、6、7、8、9、10个核苷酸可以与靶DNA 100％互补，而间隔区序列的3’区的剩余核苷酸与靶DNA基本上互补(例如，至少约70％互补)。在一些实施方案中，间隔区序列的5’端的前1至8个核苷酸(例如，前1、2、3、4、5、6、7、8个核苷酸，以及其中的任何范围)可以与靶DNA 100％互补，而间隔区序列的3’区中的剩余核苷酸与靶DNA基本上互补(例如，至少约50％互补(例如，50％、55％、60％、65％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多))。
作为另一个实例，在II型CRISPR-Cas系统的指导中，例如，20个核苷酸的间隔区序列的3’区(即种子区)中的前1、2、3、4、5、6、7、8、9、10个核苷酸可以与靶DNA 100％互补，而间隔区序列的5’区的剩余核苷酸与靶DNA基本上互补(例如，至少约70％互补)。在一些实施方案中，间隔区序列的3’端的前1至10个核苷酸(例如，前1、2、3、4、5、6、7、8、9、10个核苷酸，以及其中的任何范围)可以与靶DNA 100％互补，而间隔区序列的5’区中的剩余核苷酸与靶标DNA基本上互补(例如，至少约50％互补(例如，至少约50％、55％、60％、65％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多，或其中任何范围或值))。募集指导RNA还包含一个或多个如本文所述的募集基序，其可以连接至指导的5’端或3’端，或者它可以插入募集指导核酸中(例如，在发夹环内)。
在一些实施方案中，间隔区的种子区可以是约8至约10个核苷酸的长度，约5至约6个核苷酸的长度，或约6个核苷酸的长度。
如本文所用，“靶核酸”、“靶DNA”、“靶核苷酸序列”、“靶区域”或“基因组中的靶区域”是指生物体基因组中与本发明的指导核酸中的间隔区序列完全互补(100％互补)或基本上互补(例如，至少70％互补(例如，70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多))的区域。可用于CRISPR-Cas系统的靶区域可以位于生物体基因组(例如，植物基因组或真核(例如，人类)基因组)中的PAM序列的紧邻3’(例如，V型CRISPR-Cas系统)或紧邻5’(例如，II型CRISPR-Cas系统)的位置。靶区域可以选自位于紧邻PAM序列的至少15个连续核苷酸(例如，16、17、18、19、20、21、22、23、24、25、26、27、28、29、30个核苷酸等)的任何区域。
“原型间隔区序列”是指靶双链DNA，特别是与CRISPR重复-间隔区序列的间隔区序列(例如，引导核酸、CRISPR阵列、crRNA)完全或基本上互补(和杂交)的靶DNA部分(例如，或基因组中的靶区)。
在V型CRISPR-Cas(例如，Cas12a)系统和II型CRISPR-Cas(Cas9)系统的情况下，原型间隔区序列侧接(例如，紧邻)有原型间隔区邻近基序(PAM)。对于IV型CRISPR-Cas系统，PAM位于非靶标链的5’端和靶标链的3’端(例如，见下文)。
Figure BDA0003846855640000461
对于II型CRISPR-Cas(例如Cas9)系统，PAM位于紧邻靶标区域的3’。I型CRISPR-Cas系统的PAM位于靶标链的5’。没有已知的用于III型CRISPR-Cas系统的PAM。Makarova等人描述了CRISPR系统的所有类别、类型和亚型的命名法(Nature Reviews Microbiology，13：722-736(2015))。R.Barrangou(Genome Biol.16：247(2015))描述了指导结构和PAM。
典型的Cas12a PAM富含T。在一些实施方案中，典型的Cas12a PAM序列可以是5’-TTN、5’-TTTN或5’TTTV。在一些实施方案中，典型的Cas9(例如，酿脓链球菌)PAM可以是5’-NGG-3’。在一些实施方案中，可以使用非典型PAM，但效率可能较低。
本领域技术人员可以通过已建立的实验和计算方法来确定额外的PAM序列。因此，例如，实验方法包括靶向侧接有所有可能的核苷酸序列的序列并识别不经历靶向的序列成员，例如通过靶标质粒DNA的转化(Esvelt等人2013.Nat.Methods 10：1116-1121；Jiang等人2013.Nat.Biotechnol.31：233-239)。在一些方面，计算方法可以包括对天然间隔区执行BLAST搜索以鉴定噬菌体或质粒中的原始靶DNA序列，并比对这些序列以确定与靶序列相邻的保守序列(Briner和Barrangou.2014.Appl.Environ.Microbiol.80：994-1001；Mojica等人2009.Microbiology 155：733-740)。
在一些实施方案中，本发明提供了包含本发明的核酸构建体(例如，本发明的编辑系统的一种或多种组分)的表达盒和/或载体。在一些实施方案中，可以提供包含本发明的核酸构建体和/或一种或多种指导核酸的表达盒和/或载体。在一些实施方案中，编码碱基编辑器的本发明的核酸构建体(例如，包含CRISPR-Cas效应蛋白和脱氨酶结构域(例如，融合蛋白)的构建体)或用于碱基编辑的组分(例如，融合至肽标签或亲和多肽的CRISPR-Cas效应蛋白，融合至肽标签或亲和多肽的脱氨酶结构域，和/或融合至肽标签或亲和多肽的UGI)可以包含在与包含一种或多种指导核酸的表达盒或载体相同或分开的表达盒或载体上。当编码碱基编辑器的核酸构建体或用于碱基编辑的组分包含在与包含指导核酸的表达盒或载体分开的表达盒或载体上时，可以使靶核酸接触(例如，提供有)编码碱基编辑器的表达盒或载体或用于碱基编辑的组分，以任何互相顺序接触，以及引导核酸，例如，在提供包含引导核酸的表达盒之前、同时或之后(例如，与靶核酸接触)。
本发明的融合蛋白可以包含融合至肽标签或与肽标签相互作用的亲和多肽的序列特异性DNA结合结构域、CRISPR-Cas效应蛋白和/或脱氨酶，如本领域已知的，用于将脱氨酶募集至靶核酸。募集方法还可包括连接至RNA募集基序的指导核酸和融合至能够与RNA募集基序相互作用的亲和多肽的脱氨酶，从而将脱氨酶募集至靶核酸。备选地，化学相互作用可用于将多肽(例如脱氨酶)募集至靶核酸。
如本文所述，“肽标签”可用于募集一种或多种多肽。肽标签可以是能够被相应的亲和多肽结合的任何多肽。肽标签也可以称为“表位”，当以多个拷贝提供时，称为“多聚化表位”。示例性肽标签可包括但不限于GCN4肽标签(例如Sun-Tag)、c-Myc亲和标签、HA亲和标签、His亲和标签、S亲和标签、甲硫氨酸-His亲和标签、RGD-His亲和标签、FLAG八肽、strep标签或strep标签II、V5标签和/或VSV-G表位。在一些实施方案中，肽标签还可以包括在由SH2结构域识别的特定序列环境中的磷酸化酪氨酸、含有被14-3-3蛋白识别的磷酸丝氨酸的特征性共有序列、被SH3结构域识别的富含脯氨酸的肽基序、PDZ蛋白相互作用结构域或PDZ信号序列和来自植物的AGO钩子基序(AGO hook motif)。肽标签在WO2018/136783和美国专利申请公开号2017/0219596中公开，通过引用将它们公开的肽标签并入本文。可用于本发明的肽标签可包括但不限于SEQ ID NO：65和SEQ ID NO：66。可用于肽标签的亲和多肽包括但不限于SEQ ID NO：67。
任何可以连接至多肽并且对于其存在可以连接至另一多肽的相应亲和多肽的表位可用于本发明作为肽标签。在一些实施方案中，肽标签可包含1或2或更多个拷贝的肽标签(例如，重复单元、多聚化表位(例如，串联重复))(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或更多个重复单元)。在一些实施方案中，与肽标签相互作用/结合的亲和多肽可以是抗体。在一些实施方案中，抗体可以是scFv抗体。在一些实施方案中，结合肽标签的亲和多肽可以是合成的(例如，进化用于亲和相互作用)，包括，但不限于，亲和体(affibody)、抗运载蛋白(anticalin)、单抗体和/或DARPin(参见，例如，Sha等人，Protein Sci.26(5)：910-924(2017))；Gilbreth(Curr Opin Struc Biol 22(4)：413-420(2013))，美国专利号9,982,053，每一个都通过引用将与亲和体、抗运载蛋白、单抗体和/或DARPins相关的教导整体并入。
在一些实施方案中，可以将指导核酸连接至RNA募集基序，并且可以将待募集的多肽(例如脱氨酶)融合至结合RNA募集基序的亲和多肽，其中指导物结合靶核酸而RNA募集基序结合亲和多肽，从而将多肽募集至指导物并使靶核酸与多肽(例如脱氨酶)接触。在一些实施方案中，可以将两种或更多种多肽募集至指导核酸，从而使靶核酸与两种或更多种多肽(例如，脱氨酶)接触。
在本发明的一些实施方案中，可以将指导RNA连接至一个或两个或更多个RNA募集基序(例如1、2、3、4、5、6、7、8、9、10或更多个基序；例如，至少10至约25个基序)，任选地其中两个或更多个RNA募集基序可以是相同的RNA募集基序或不同的RNA募集基序。在一些实施方案中，RNA募集基序和相应的亲和多肽可以包括但不限于：端粒酶Ku结合基序(例如，Ku结合发夹)和Ku的亲和多肽(例如，Ku异二聚体)，端粒酶Sm7结合基序和Sm7的亲和多肽，MS2噬菌体操纵子茎环和MS2外壳蛋白(MCP)的亲和多肽，PP7噬菌体操纵子茎环和PP7外壳蛋白(PCP)的亲和多肽，SfMu噬菌体Com茎环和Com RNA结合蛋白的亲和多肽，PUF结合位点(PBS)和Pumilio/fem-3mRNA结合因子(PUF)的亲和多肽，和/或合成的RNA-适配体和适配体配体作为相应的亲和多肽。在一些实施方案中，RNA募集基序和相应的亲和多肽可以是MS2噬菌体操纵子茎环和亲和多肽MS2外壳蛋白(MCP)。在一些实施方案中，RNA募集基序和相应的亲和多肽可以是PUF结合位点(PBS)和亲和多肽Pumilio/fem-3mRNA结合因子(PUF)。可用于本发明的示例性RNA募集基序和相应的亲和多肽可包括但不限于SEQ ID NO：68-78。
在一些实施方案中，用于募集多肽和核酸的组分可包括通过化学相互作用起作用的组分，其可包括但不限于雷帕霉素诱导的FRB-FKBP二聚化；生物素-链霉亲和素；SNAP标签；Halo标签；CLIP标签；化合物诱导的DmrA-DmrC异二聚体；双功能配体(例如，两种蛋白质结合化学物质融合在一起；例如二氢叶酸还原酶(DHFR)。
肽标签可以包含或存在于肽标签的一个拷贝或2个或更多个拷贝中(例如，多聚化肽标签或多聚化表位)(例如，约2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、9、20、21、22、23、24或25个或更多个肽标签)。当多聚化时，肽标签可以直接彼此融合，或者它们可以通过一个或多个氨基酸(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或更多个氨基酸，任选约3至约10、约4至约10、约5至约10、约5至约15，或约5至约20个氨基酸等，以及其中的任何值或范围)彼此连接。因此，在一些实施方案中，本发明的CRISPR-Cas效应蛋白可以包含融合至一个肽标签或者融合至两个或更多个肽标签的CRISPR-Cas效应蛋白结构域，任选其中两个或更多个肽标签通过一个或多个氨基酸残基彼此融合。在一些实施方案中，可用于本发明的肽标签可以是单拷贝的GCN4肽标签或表位或者可以是多聚化GCN4表位，其包含约2至约25或更多个拷贝的肽标签(例如，约2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或更多个拷贝的GCN4表位或其中的任何范围)。
在一些实施方案中，可以将肽标签融合至CRISPR-Cas多肽或结构域。在一些实施方案中，可以将肽标签融合或连接至CRISPR-Cas效应蛋白的C末端以形成CRISPR-Cas融合蛋白。在一些实施方案中，可以将肽标签融合或连接至CRISPR-Cas效应蛋白的N末端以形成CRISPR-Cas融合蛋白。在一些实施方案中，可以将肽标签融合在CRISPR-Cas效应蛋白内(例如，肽标签可以在CRISPR-Cas效应蛋白的环区中)。在一些实施方案中，可以将肽标签融合至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
在一些实施方案中，当肽标签包含多于一个肽标签时，可以优化每个肽标签的数量和间距以最大化肽标签的占据并使例如脱氨酶结构域彼此之间的空间干扰最小化。
“亲和多肽”(例如，“募集多肽”)是指能够结合其相应的肽标签、肽标签或RNA募集基序的任何多肽。肽标签的亲和多肽可以是例如分别特异性结合肽标签的抗体和/或单链抗体。在一些实施方案中，针对肽标签的抗体可以是但不限于scFv抗体。在一些实施方案中，可以将亲和多肽融合或连接至脱氨酶(例如胞嘧啶脱氨酶或腺嘌呤脱氨酶)的N末端。在一些实施方案中，亲和多肽在细胞或细胞提取物的还原条件下是稳定的。
本发明的核酸构建体和/或指导核酸可以包含在如本文所述的一种或多种表达盒中。在一些实施方案中，本发明的核酸构建体可以包含在与包含指导核酸和/或募集指导核酸的表达盒或载体相同或分开的表达盒或载体中。
当与指导核酸和募集指导核酸组合使用时，本发明的核酸构建体(以及包含其的表达盒和载体)可用于修饰靶核酸和/或其表达。靶核酸可以在靶核酸与指导核酸/募集指导核酸(和/或包含其的表达盒和载体)接触之前、同时或之后与本发明的核酸构建体和/或包含该构建体的表达盒和/或载体接触。
本发明进一步提供了使用本发明的核酸构建体和/或包含其的表达盒和/或载体来修饰靶核酸的方法。所述方法可以在体内系统(例如，在细胞中或在生物体中)或在体外系统(例如，无细胞)中进行。本发明的方法、组合物和/或系统可以任选地以半随机方式产生和/或提供等位基因多样性。在一些实施方案中，本发明的方法包括使用和/或基于修饰的靶核酸来确定期望的或优选的表型。本发明的方法可以提供一种或多种修饰的靶核酸，并且可以对一种或多种修饰的靶核酸分析期望或优选表型。
在一些实施方案中，本发明提供修饰靶核酸的方法，所述方法包括使靶核酸与以下接触：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)、胞嘧啶脱氨酶和腺嘌呤脱氨酶，其中CRISPR-Cas效应蛋白和胞嘧啶脱氨酶和/或腺嘌呤脱氨酶形成复合物或包含在复合物中。在一些实施方案中，CRISPR-Cas效应蛋白包含指导核酸或复合物进一步包含指导核酸。可以将胞嘧啶脱氨酶和腺嘌呤脱氨酶融合在一起和/或可以将胞嘧啶脱氨酶和腺嘌呤脱氨酶之一或两者融合至CRISPR-Cas效应蛋白。在一些实施方案中，胞嘧啶脱氨酶和腺嘌呤脱氨酶不同时存在于复合物中，而是可以各自在短时间内和/或连续地分别与CRISPR-Cas效应蛋白存在于复合物中。在一些实施方案中，胞嘧啶脱氨酶和CRISPR-Cas效应蛋白在第一复合物中并且腺嘌呤脱氨酶和CRISPR-Cas效应蛋白在第二复合物中，任选地其中第一复合物和第二复合物包括相同或不同的指导核酸。在一些实施方案中，将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶不融合至Cas9。在一些实施方案中，CRISPR-Cas效应蛋白是V型CRISPR-Cas效应蛋白(例如，Cpf1)。在一些实施方案中，靶核酸在基因的非编码区(例如启动子区)中和/或在基因的编码区中。
在一些实施方案中，本发明的方法和/或包含CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶的复合物可以并行地和/或同时地修饰靶核酸，因为包含CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶的试剂的单次递送可以提供和/或导致靶核酸中存在的胞苷和腺嘌呤碱基被修饰(例如，C至T和A至G)。靶核酸的并行和/或同时修饰可以发生在对应于足以导致两种类型编辑(即C至T和A至G)的试剂的单次递送的时间段内。在一些实施方案中，C至T和A至G的编辑发生在从起始于将试剂递送到细胞、组织和/或生物体的时间到细胞、组织和/或生物体被筛选用于编辑的时间的时间段内，其中只有单次递送试剂到细胞、组织和/或生物体。方法和/或单次递送可以进一步包括糖基化酶抑制剂(例如，UGI)和/或MCP或其部分，任选地包括肽标签。在一些实施方案中，胞嘧啶脱氨酶和腺嘌呤脱氨酶两者被募集到靶核酸并提供与CRISPR-Cas效应蛋白的单一复合物。胞嘧啶脱氨酶和腺嘌呤脱氨酶可各自使用相同或不同的募集策略(例如本文所述的那些)被募集至CRISPR-Cas效应蛋白。
本发明的方法和/或包含CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶的复合物可以提供和/或导致与目前的诱变方法例如Cas9介导的诱变(例如，Cas9介导的启动子诱变，TadA融合至Cas9的N末端，和/或pmCDA1融合至Cas9的C末端)相比增加的等位基因数量。在一些实施方案中，本发明的方法和/或包含CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶的复合物可以提供和/或导致2个或更多个(例如，2、3、4、5、6、7、8、9、10、20或更多个)不同的修饰的靶核酸/靶核酸位点。
在一些实施方案中，可以使用RNA募集基序来募集胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。在一些实施方案中，指导核酸包含如本文所述的RNA募集基序，任选地其中RNA募集基序是MS2发夹。胞嘧啶脱氨酶和/或腺嘌呤脱氨酶可包含RNA募集基序的相应亲和多肽，例如MCP或其部分。可以将如本文所述的糖基化酶抑制剂(例如，UGI)融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。在一些实施方案中，糖基化酶抑制剂以反式提供。如本文所用的“反式”是指与CRISPR-Cas效应蛋白和脱氨酶分开地表达组分(例如，化合物，例如糖基化酶抑制剂)，任选地在相同的盒中使用其自身的启动子或在细胞中使用单独的表达盒。例如，在一些实施方案中，指导RNA包含至少一个MS2发夹，并且将结合至MS2发夹的MS2加帽蛋白(MCP)或其部分单独或作为单一融合物融合至腺嘌呤和胞苷脱氨酶。糖基化酶抑制剂(例如，UGI)可以作为如本文所述的融合物或以反式提供。因此，腺嘌呤和胞苷脱氨酶可以任选同时被募集至指导RNA和/或靶核酸，并且可以在脱氨时间框架和/或脱氨窗口内进行C至T和A至G的编辑(例如，靶核酸中通常观察到碱基编辑的子序列)。
在一些实施方案中，可以将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。例如，在一些实施方案中，将胞嘧啶脱氨酶和腺嘌呤脱氨酶之一融合至CRISPR-Cas效应蛋白，而另一个通过使用诸如RNA募集基序的募集策略进行募集。
在一些实施方案中，将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶并且将腺嘌呤脱氨酶通过RNA募集基序例如MS2发夹募集至复合物。例如，腺嘌呤脱氨酶可包含(MCP)或其部分(例如，可以将腺嘌呤脱氨酶和MCP或其部分融合在一起)，因为MCP或其部分能够和/或结合MS2发夹。在一些实施方案中，将CRISPR-Cas效应蛋白融合至腺嘌呤脱氨酶并且胞嘧啶脱氨酶通过RNA募集基序例如MS2发夹募集至复合物。例如，胞嘧啶脱氨酶可包含(MCP)或其部分(例如，可以将胞嘧啶脱氨酶和MCP或其部分融合在一起)，因为MCP或其部分能够和/或结合MS2发夹。
在一些实施方案中，CRISPR-Cas效应蛋白包含如本文所述的肽标签。肽标签可以是SunTag和/或可以包含一个或多个(例如1、2、3、4或更多个)GCN4表位。腺嘌呤脱氨酶和/或胞嘧啶脱氨酶可包含如本文所述的能够结合肽标签的亲和多肽(例如，scFv)。在一些实施方案中，将腺嘌呤脱氨酶和/或胞嘧啶脱氨酶与亲和多肽融合在一起。因此，可以使用亲和多肽将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶募集至CRISPR-Cas效应蛋白和/或靶核酸。例如，可以将CRISPR-Cas效应蛋白的N或C末端融合至包含多个GCN4表位的SunTag，可以单独或作为单一融合物将识别GCN4的scFv融合至腺嘌呤脱氨酶和/或胞嘧啶脱氨酶。糖基化酶抑制剂(例如，UGI)可以作为融合物或以反式提供。可以任选同时地将腺嘌呤脱氨酶和胞嘧啶脱氨酶募集至靶核酸，并且可以在脱氨时间框架和/或脱氨窗口内进行C和A编辑(例如，靶核酸中的子序列，其中碱基编辑是通常观察到)。
在一些实施方案中，CRISPR-Cas效应蛋白包含如本文所述的肽标签，并且可以将CRISPR-Cas效应蛋白融合至腺嘌呤脱氨酶和/或胞嘧啶脱氨酶。肽标签可以是SunTag和/或可以包含一个或多个(例如1、2、3、4或更多个)GCN4表位。在一些实施方案中，将腺嘌呤脱氨酶和胞嘧啶脱氨酶之一融合至CRISPR-Cas效应蛋白，并且腺嘌呤脱氨酶和胞嘧啶脱氨酶中的另一个包含如本文所述的亲和多肽(例如，scFv)，其能够结合肽标签。因此，可以使用亲和多肽将胞嘧啶脱氨酶和腺嘌呤脱氨酶之一募集至CRISPR-Cas效应蛋白和/或靶核酸。例如，可以将CRISPR-Cas效应蛋白的N或C末端融合至包含多个GCN4表位的SunTag，而可以将另一个末端融合至腺嘌呤脱氨酶结构域或胞嘧啶脱氨酶结构域，并且可以将识别GCN4的scFv融合至腺嘌呤脱氨酶或胞嘧啶脱氨酶，这取决于融合至CRISPR-Cas效应蛋白的情况。糖基化酶抑制剂(例如，UGI)可以作为融合物或以反式提供。
在一些实施方案中，腺嘌呤脱氨酶和/或胞嘧啶脱氨酶可包含肽标签。肽标签可以是SunTag和/或可以包含一个或多个(例如1、2、3、4或更多个)GCN4表位。在一些实施方案中，可以将腺嘌呤脱氨酶和/或胞嘧啶脱氨酶和/或肽标签融合在一起。CRISPR-Cas效应蛋白可以包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将CRISPR-Cas效应蛋白和亲和多肽融合在一起。因此，可以使用亲和多肽将CRISPR-Cas效应蛋白募集至腺嘌呤脱氨酶和/或胞嘧啶脱氨酶和/或靶核酸。糖基化酶抑制剂(例如，UGI)可以作为融合物或反式提供。
在一些实施方案中，CRISPR-Cas效应蛋白可包含指导核酸(例如，指导RNA)，其包含RNA募集基序。例如，可以将CRISPR-Cas效应蛋白融合至包含RNA募集基序的指导RNA，任选地其中将指导RNA融合至RNA募集基序。在一些实施方案中，指导RNA可以包含一个或多个MS2发夹。RNA募集基序的相应亲和多肽，例如MCP或其部分，可以包含如本文所述的肽标签，并且相应的亲和多肽可以在接触步骤期间存在和/或也可以与靶核酸接触。胞嘧啶脱氨酶和/或腺嘌呤脱氨酶可包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶与亲和多肽融合在一起。在一些实施方案中，将胞嘧啶脱氨酶和腺嘌呤脱氨酶各自分别地融合至可以相同或不同的亲和多肽。在一些实施方案中，将胞嘧啶脱氨酶、腺嘌呤脱氨酶和亲和多肽融合在一起。在一些实施方案中，包含肽标签(例如，SunTag)的MCP或其部分可被募集至包含指导RNA的CRISPR-Cas效应蛋白，该指导RNA包括一个或多个MS2发夹，以及胞嘧啶脱氨酶和/或腺嘌呤脱氨酶包含亲和多肽(例如，scFv)并被募集至肽标签。
根据本发明的一些实施方案，本发明提供了一种修饰靶核酸的方法，该方法包括使靶核酸接触：CRISPR-Cas效应蛋白(例如，CRISPR酶)、指导核酸(例如，指导RNA)和胞嘧啶脱氨酶，其中该方法将靶核酸的胞嘧啶(C)修饰为腺嘌呤(A)、鸟嘌呤(G)或胸腺嘧啶(T)。在一些实施方案中，C以半随机方式被转化为T、G或A。在一些实施方案中，靶核酸存在于植物细胞中。CRISPR-Cas效应蛋白、指导核酸和胞嘧啶脱氨酶可形成复合物或可包含在复合物中。在一些实施方案中，复合物可缺乏糖基化酶抑制剂(例如UGI)或其结构域和/或胞嘧啶脱氨酶缺乏糖基化酶抑制剂(例如UGI)或其结构域。CRISPR-Cas效应蛋白可以是V型CRISPR-Cas效应蛋白。在一些实施方案中，CRISPR-Cas效应蛋白是Cas9(例如，dCas9或nCas9)。方法、组合物和/或系统可以提供大于约0.1％、0.5％、1％、1.25％、1.5％、1.75％、2％、2.25％、2.5％、2.75％、3％、3.25％、3.5％、3.75％、4％、4.25％、4.5％、4.75％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％或更多的碱基置换频率，任选地其中C至非T编辑(例如，C至G编辑和/或C至A编辑)的碱基置换频率为大于0.1％、0 0.5％、1％、1.25％、1.5％、1.75％、2％、2.25％、2.5％、2.75％、3％、3.25％、3.5％、3.75％、4％、4.25％、4.5％、4.75％、5％、10％、15％、20％、25％、30％或更多。在一些实施方案中，方法、组合物和/或系统可以提供大于约1％的碱基置换频率，任选地其中C至非T编辑(例如，C至G编辑和/或C至A编辑)的碱基置换频率大于约1％。本申请的发明人出人意料地发现，本发明的方法、组合物和/或系统可提供改进的碱基置换频率和与C至T变化相比C至G变化的改进比率。例如，在一些实施方案中，本发明的方法、组合物和/或系统可以提供C→G∶C→T变化的比率为约1∶1，任选地在植物中。在一些实施例中，本发明的方法、组合物和/或系统可以提供约0.1∶1、0.2∶1、0.3∶1、0.4∶1、0.5∶1、0.6∶1、0.7∶1、0.8∶1、0.9∶1、1∶1、1.1∶1、1.2∶1、1.3∶1、1.4∶1、1.5∶1的C→G∶C→T变化的比率，任选地在植物中。
胞嘧啶脱氨酶可包含MCP或其部分，任选地其中将MCP或其部分融合至胞嘧啶脱氨酶氨基酸序列的N末端。在一些实施方案中，胞嘧啶脱氨酶包含Cas9结构域，任选地其中将胞嘧啶脱氨酶融合至Cas9结构域。在一些实施方案中，胞嘧啶脱氨酶包含失活的LbCpf1(dLbCpf1)，任选地其中将胞嘧啶脱氨酶融合至dLbCpf1。胞嘧啶脱氨酶可以是密码子优化的。在一些实施方案中，胞嘧啶脱氨酶经密码子优化用于单子叶植物表达和/或经密码子优化用于双子叶植物表达。
在一些实施方案中，本发明的方法、组合物和/或系统可以提供和/或产生脱碱基位点(abasic site)。脱碱基位点可用作跨损伤DNA合成的模板。在聚合过程中，可以在脱碱基位点对面掺入任何核苷酸，因为糖环缺少可以在聚合期间参与碱基配对的DNA碱基。因此，在一些实施方案中，可以以半随机方式将靶C转换为T、G或A。在一些实施方案中，靶核酸可以与尿嘧啶N-糖基化酶(UNG)接触。UNG可以存在于其中存在靶核酸的细胞中。在一些实施方案中，糖基化酶结构域(例如，UNG结构域)可以通过共价和/或非共价相互作用、任选地通过抗体-表位相互作用和/或RNA结合基序-MS2而募集至靶核酸相互作用。
在一些实施方案中，胞嘧啶脱氨酶可以是rAPOBEC1、APOBEC3A、APOBEC3B、hAID和pmCDA1中的一种或多种，并且可以任选地将胞嘧啶脱氨酶融合至亲和多肽，例如MCP或其部分。如本领域技术人员将理解的，不同的胞嘧啶脱氨酶可以产生不同水平的碱基编辑以及不同核苷酸组成中的产物碱基谱；因此，可以选择胞嘧啶脱氨酶用于在靶核酸位点处的所需编辑窗口。胞嘧啶脱氨酶可以通过共价和/或非共价相互作用、任选地通过抗体-表位相互作用和/或RNA结合基序-MS2相互作用而募集至靶核酸。在一些实施方案中，胞嘧啶脱氨酶可包含(例如，融合至)MCP或其部分。可以将MCP或其部分融合至胞嘧啶脱氨酶的N末端或脱氨酶的C末端。在一些实施方案中，指导核酸可以包含一种或多种RNA募集基序(例如，一种或多种MS2发夹)。在一些实施方案中，可以将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶。在一些实施方案中，CRISPR-Cas效应蛋白可以包含肽标签并且胞嘧啶脱氨酶可以包含能够结合肽标签的亲和多肽，或者胞嘧啶脱氨酶可以包含肽标签并且CRISPR-Cas效应蛋白可以包含能够结合肽标签的亲和多肽。
本发明的方法可以包括调节CRISPR-Cas效应蛋白的DNA结合亲和力。在胞嘧啶碱基编辑期间，胞苷通过胞苷脱氨基作用转化为尿苷。因此，尿苷/尿嘧啶是一种中间产物。在一些实施方案中，与不符合本发明的方法、组合物和/或系统相比(例如，与在一些实施方案中，与包括包含UGI和/或包含UGI的胞嘧啶脱氨酶的复合物的方法、组合物和/或系统相比)，本发明的方法、组合物和/或系统可以增加尿苷/尿嘧啶中间体的存在期。在一些实施方案中，本发明的指导核酸与靶核酸具有低于完全的互补性，例如小于100％的互补性(例如，小于99％、98％、97％、96％、95％、94％、93％、92％、91％、90％等)，与使用具有100％互补性的指导核酸的方法中的尿苷/尿嘧啶中间体的存在期相比，这可以增加尿苷/尿嘧啶中间体的存在期。在一些实施方案中，本发明的CRISPR-Cas效应蛋白(例如Cas9)与靶核酸的相互作用减弱，与使用与靶核酸没有减弱相互作用的CRISPR-Cas效应蛋白的尿苷/尿嘧啶中间体的存在期相比，这可以产生脱碱基位点和/或增加尿苷/尿嘧啶中间体的存在期。在一些实施方案中，方法可以包括从尿嘧啶N-糖基化酶阻断尿苷/尿嘧啶中间体直到DNA复制期间和/或之后。例如，在一些实施方案中，CRISPR-Cas效应蛋白和/或胞嘧啶脱氨酶可以保留在靶位点，这可以屏蔽它从UNG产生尿苷/尿嘧啶中间体，直到复合物在DNA复制期间溶解，如它可能会导致一个有利的情况，即在DNA复制期间产生的脱碱基位点可以优先用作DNA聚合酶的模板。在一些实施方案中，本发明的方法可以包括调节(例如，增加或减少)CRISPR-Cas效应蛋白在靶核酸处的停留时间。
在一些实施方案中，方法包括在存在AP核酸内切酶I(APE1)抑制剂的情况下进行接触步骤和/或进一步包括使靶核酸接触APE1抑制剂。一种或多种APE1抑制剂可以存在于本发明的方法、组合物和/或系统中。在一些实施方案中，APE1抑制剂是有机化合物或核酸(例如，siRNA)。示例性APE1抑制剂包括但不限于Curr Mol Pharmacol.2012Jan；5(1)：14-35；Mol Pharmacol.，2008，73，669-677；Madhusudan等人Nucleic Acids Research，2005，Vol.33，No.15 4711-4724；和J.Med.Chem.，2009，52，20-32中描述的那些，每一篇都通过引用整体并入本文。在一些实施方案中，APE1抑制剂包含CRT0044876。本发明的方法可以包括抑制APE1，任选地在至少一部分接触步骤和/或碱基编辑期间抑制APE1。在一些实施方案中，siRNA可用于抑制细胞APE1。
在一些实施方案中，本发明的方法包括抑制或减少插入缺失形成，任选地与不存在APE1抑制剂和/或siRNA的插入缺失形成量相比。在一些实施方案中，本发明的方法可以提供具有小于约50％、45％、40％、35％、30％、25％、20％、15％、10％、5％、4％、3％、2％、1％或0.5％的包含插入缺失的修饰的靶核酸的修饰的靶核酸。在一些实施方案中，本发明的方法可以通过减少产生的插入缺失的量来提高碱基多样化率。
本发明的方法可以包括调节一种或多种细胞通路。在一些实施方案中，本发明的方法可以减少非同源末端连接(NHEJ)，任选地通过抑制DNA连接酶IV和/或通过DNA-PKcs。在一些实施方案中，方法包括在存在DNA连接酶IV抑制剂和/或DNA-PKcs抑制剂的情况下进行接触步骤，和/或方法还包括使靶核酸与DNA连接酶IV抑制剂和/或DNA-PKcs抑制剂接触。在一些实施方案中，DNA连接酶IV抑制剂和/或DNA-PKcs抑制剂可以在本发明方法中的碱基编辑和/或碱基多样化事件期间存在。示例性的DNA连接酶IV抑制剂包括但不限于Scr7、L189和在Cancer Res.2008May 1；68(9)：3169-77中描述的那些，其通过引用整体并入本文。在一些实施方案中，DNA连接酶IV抑制剂可以是Scr7。在CRISPR/Cas9介导的基因组编辑期间，使用Scr7已显示增加HDR并减少NHEJ(Nat Biotechnol.2015May；33(5)：538-542.；FEBS J.2015Nov；282(22)：4289-94.)。示例性DNA-PKcs抑制剂包括但不限于NU7026、KU-0060648、NU7441、IC86621和在Sci Rep.2019Feb 12；9(1)：1847；Genome Med.2015Aug 27；7：93；和Mol Cell Biol.2011Apr；31(8)：1719-33中描述的那些，其各自通过引用整体并入本文。在一些实施方案中，本发明的方法可以抑制NHEJ，任选地在碱基编辑或碱基多样化期间，并且可以增加或改善碱基编辑和/或碱基多样化和/或可以减少插入缺失形成。
在一些实施方案中，方法可以包括抑制NHEJ通路中的一种或多种蛋白质，这可以导致该方法期间产生的插入缺失的量减少。在一些实施方案中，方法可以包括调节CRISPR介导的插入缺失率和/或同源定向修复(HDR)率。可抑制NHEJ通路中的一种或多种蛋白质和/或调节CRISPR介导的插入缺失和/或同源定向修复(HDR)率的示例性化合物包括但不限于FEBS J.2015Nov；282(22)：4289-94中描述的那些，其通过引用整体并入本文。
在一些实施方案中，本发明的方法可以促进或增加聚合介导的脱碱基位点修复。在一些实施方案中，方法包括在存在外源聚合酶的情况下进行接触步骤和/或进一步包括使靶核酸接触外源聚合酶。外源聚合酶可以通过将DNA聚合酶带至靶核酸来增加和/或强制在脱碱基位点上的聚合。外源聚合酶可以通过包含CRISPR-Cas效应蛋白、指导核酸和胞嘧啶脱氨酶的复合物募集至靶核酸，或者可以通过不同的复合物募集至靶核酸。在一些实施方案中，可以将外源聚合酶融合至CRISPR-Cas效应蛋白(例如，V型CRISPR-Cas效应蛋白)，任选地其中将外源聚合酶融合至Cas9(例如，dCas9或nCas9)。外源聚合酶可以进行密码子优化，任选地进行密码子优化以在植物中表达。在一些实施方案中，聚合酶的过表达和/或能够跨脱碱基位点(包括涉及跨损伤旁路的那些，例如Rev1)有活性的聚合酶的募集可以上调导致碱基多样化的通路。可用于本发明的方法、组合物和/或系统的示例性聚合酶包括但不限于人Rev1、酵母Rev1、人聚合酶iota、人聚合酶kappa、工程化聚合酶3A10(NatBiotechnol.2007Aug；25(8)：939-43)、人类引物酶/聚合酶PRIMPOL(Mol Cell.2013Nov21；52(4)：541-53)、噬菌体聚合酶B35DNAP(Proc Natl Acad Sci U S A.2015Jul 7；112(27)：E3476-84)、转座子衍生聚合酶EhDNAPolB2(PLoS One.2012；7(11)：e49964)、细菌T4DNA聚合酶和/或硫叶菌(Sulfolobus solfataricus)P2 DNA聚合酶IV(Dpo4)。
在一些实施方案中，CRISPR-Cas效应蛋白包含如本文所述的肽标签。在一些实施方案中，肽标签包含SunTag和/或肽标签包含一个或多个(例如，1、2、3、4或更多个)GCN4表位。胞嘧啶脱氨酶可以包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将胞嘧啶脱氨酶和亲和多肽融合在一起。因此，可以使用亲和多肽通过与融合至CRISPR-Cas效应蛋白的肽标签结合将胞嘧啶脱氨酶募集至CRISPR-Cas效应蛋白和/或靶核酸。
在一些实施方案中，胞嘧啶脱氨酶包含如本文所述的肽标签。在一些实施方案中，肽标签包含SunTag和/或肽标签包含一个或多个(例如，1、2、3、4或更多个)GCN4表位。CRISPR-Cas效应蛋白可以包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将CRISPR-Cas效应蛋白和亲和多肽融合在一起。在一些实施方案中，使用亲和多肽将CRISPR-Cas效应蛋白募集至靶核酸。
本发明的方法可以包括使靶核酸接触CRISPR Cas效应蛋白、脱氨酶和/或其融合蛋白和/或目标多肽，和/或靶核酸可以接触编码CRISPR Cas效应蛋白、脱氨酶和/或其融合蛋白和/或目标多肽的多核苷酸，该多肽可以任选地包含在如本文所述的一个或多个表达盒和/或载体中，所述表达盒和/或载体任选地包含一种或多种指导核酸。
如本文所述，本发明的核酸和/或包含其的表达盒和/或载体可以经密码子优化以在生物体中表达。可用于本发明的生物体可以是核酸修饰对其有用的任何生物体或其细胞。生物体可包括但不限于任何动物(例如哺乳动物)、任何植物、任何真菌、任何古细菌或任何细菌。在一些实施方案中，生物体可以是植物或其细胞。
在一些实施方案中，经优化以在植物中表达的本发明的核酸构建体、表达盒或载体可以与包含相同多核苷酸但没有经密码子优化以在植物中表达的核酸构建体、表达盒或载体约70％至100％相同(例如，约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或100％)。
可以使用本发明的核酸构建体修饰任何植物或植物部分的靶核酸。可以使用本发明的核酸构建体修饰任何植物(或植物群，例如，属或更高级分类)，包括：被子植物，裸子植物，单子叶植物，双子叶植物，C3、C4、CAM植物，苔藓植物，蕨类植物和/或拟蕨植物，微藻和/或大型藻类。可用于本发明的植物和/或植物部分可以是任何植物物种/品种/栽培品种的植物和/或植物部分。如本文所用，术语“植物部分”包括但不限于胚、花粉、胚珠、种子、叶、茎、幼苗、花、枝条、果实、籽粒、穗、穗轴、外壳、梗、根、根尖、花药、植物细胞(包括在植物和/或植物部分中完整的植物细胞)、植物原生质体、植物组织、植物细胞组织培养物、植物愈伤组织、植物团块等。如本文所用，“幼苗”是指地上部分，包括叶和茎。此外，如本文所用，“植物细胞”是指植物的结构和生理单元，其包括细胞壁，也可以指原生质体。植物细胞可以是分离的单细胞的形式，或者可以是培养的细胞，或者可以是更高组织单元(例如植物组织或植物器官)的一部分。
可用于本发明的植物的非限制性实例包括：草坪草(例如早熟禾、翦股颖、黑麦草、羊茅)，羽毛芦苇草，簇绒草，芒草，芦竹，柳枝稷，蔬菜作物，包括朝鲜蓟，大头菜，芝麻菜，韭菜，芦笋，生菜(如头、叶、长叶)，马兰加，瓜(如甜瓜、西瓜、crenshaw、蜜瓜、哈密瓜)，芸苔属作物(如抱子甘蓝、卷心菜、花椰菜、西兰花、羽衣甘蓝、甘蓝、大白菜、白菜)，刺菜蓟(cardoni)，胡萝卜，napa，秋葵，洋葱，芹菜，欧芹，鹰嘴豆，欧洲防风草，菊苣，辣椒，马铃薯，葫芦科(如西葫芦、黄瓜、密生西葫芦、倭瓜、南瓜、蜜瓜、西瓜，哈密瓜)，萝卜，干洋葱，芜菁甘蓝，茄子，婆罗门参，茅菜(escarole)，青葱，菊苣，大蒜，菠菜，大葱，倭瓜，青菜，甜菜(糖用甜菜和饲料甜菜)，红薯，甜菜，辣根，番茄，芜菁和香料；水果作物，例如苹果、杏、樱桃、油桃、桃、梨、梅子、李子、樱桃、榅桲、无花果、坚果(例如，栗子、山核桃、开心果、榛子、开心果、花生、核桃、澳洲坚果、杏仁等)，柑橘(例如，小柑桔、金橘、橙子、葡萄柚、橘子、柑橘、柠檬、酸橙等)、蓝莓、黑树莓、波森莓、蔓越莓、醋栗、鹅莓、罗甘莓、覆盆子、草莓、黑莓、葡萄(葡萄酒和餐食)、鳄梨、香蕉、猕猴桃、柿子、石榴、菠萝、热带水果、柚子、甜瓜、芒果、木瓜和荔枝，田间作物植物例如三叶草、苜蓿、提摩西草、月见草、草地泡泡草、玉米/玉蜀黍(田间、甜味、爆米花)、hops、荷荷巴、荞麦、红花、藜麦、小麦、水稻、大麦、黑麦、小米、高粱、燕麦、黑小麦、高粱、烟草、木棉，豆科植物(豆类)(例如，绿色和干燥的)、小扁豆、豌豆、大豆)，和油料植物(油菜、卡诺拉油菜、芥末、罂粟、橄榄、向日葵、椰子、蓖麻油植物、可可豆、花生、油棕)、浮萍、拟南芥，纤维植物(棉花、亚麻、大麻、黄麻)、大麻(例如，大麻(Cannabis sativa)、印度大麻(Cannabis indica)和粗大麻(Cannabis ruderalis))、月桂科(肉桂、樟脑)，或者例如咖啡、甘蔗、茶和天然橡胶植物等植物；和/或花坛植物，例如开花植物、仙人掌、多肉植物和/或观赏植物(例如玫瑰、郁金香、紫罗兰)，以及树木，例如林木(阔叶树和常青树，如针叶树；例如，榆树、白蜡树、橡树、枫树、冷杉、云杉、雪松、松树、桦树、柏树、桉树、柳树)，以及灌木和其他苗木。在一些实施方案中，本发明的核酸构建体和/或编码其的表达盒和/或载体可用于修饰玉蜀黍、大豆、小麦、卡诺拉油菜、水稻、番茄、胡椒、向日葵、覆盆子、黑莓、黑树莓和/或樱桃。
在一些实施方案中，本发明提供了包含本发明的多肽、多核苷酸、核酸构建体、表达盒或载体的细胞(例如，植物细胞、动物细胞、细菌细胞、古细菌细胞等)。
本发明进一步包括用于实施本发明方法的一个或多个试剂盒。本发明的试剂盒可以包括试剂、缓冲剂和用于混合、测量、分类、标记等的装置，以及适用于修饰靶核酸的说明书等。
在一些实施方案中，本发明提供了一种试剂盒，其包括一种或多种本发明的核酸构建体，和/或如本文所述包含其的表达盒和/或载体和/或细胞，以及其使用的可选说明书。在一些实施方案中，试剂盒可进一步包括CRISPR-Cas指导核酸(对应于由本发明的多核苷酸编码的CRISPR-Cas效应蛋白)和/或包含其的表达盒和/或载体和/或细胞。在一些实施方案中，可以在与本发明的一种或多种核酸构建体相同的表达盒和/或载体上提供指导核酸。在一些实施方案中，可以在与包含本发明的一种或多种核酸构建体的表达盒或载体分开的表达盒或载体上提供指导核酸。
因此，在一些实施方案中，提供了包括核酸构建体的试剂盒，所述核酸构建体包含(a)本文提供的一种或多种多核苷酸和(b)驱动(a)的一种或多种多核苷酸表达的启动子。在一些实施方案中，试剂盒还可包括编码指导核酸的核酸构建体，其中构建体包含用于将与靶核酸序列相同或互补的核酸序列克隆到指导核酸的骨架中的克隆位点。
在一些实施方案中，本发明的核酸构建体可以是可编码在所编码的多核苷酸内的一个或多个内含子的mRNA。在一些实施方案中，本发明的核酸构建体和/或包含其的表达盒和/或载体可以进一步编码一种或多种用于鉴定转化体的可选择标志物(例如，编码抗生素抗性基因、除草剂抗性基因等的核酸)。本发明的多肽、多核苷酸、核酸构建体、表达盒、载体、组合物、试剂盒、系统和/或细胞可以包含SEQ ID NO：1-112中的一个或多个的序列的全部或部分。在一些实施方案中，本发明的多肽、多核苷酸、核酸构建体、表达盒、载体、组合物、试剂盒、系统和/或细胞可以包含SEQ ID NO：1-112中的一个或多个的序列的至少约20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多连续氨基酸。
现在将参考以下实施例描述本发明。应当理解，这些实施例并非旨在将权利要求的范围限制于本发明，而是旨在作为某些实施方案的示例。本领域技术人员想到的示例性方法的任何变化都旨在落入本发明的范围内。
实施例
实施例1：用于使用募集进行C和A编辑的MS2/MCP系统。
在该系统中，递送CRISPR-Cas效应蛋白(例如酶)、胞嘧啶脱氨酶、腺嘌呤脱氨酶和指导RNA。将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶结构域(CBE)或腺嘌呤脱氨酶结构域(ABE)，另一种脱氨酶使用MS2发夹被募集至靶核酸。在HEK293T细胞中，转染编码CBE或ABE、MCP-C-脱氨酶或MCP-A-脱氨酶(互补CBE或ABE)和含有MS2发夹的指导RNA的质粒。3d后，收获细胞并使用高通量测序进行分析(图1)。
例如，使用BE4Max和MCP-2xTadA靶向HEK2基因座(表1)。大部分细胞群具有编辑了的C和A两者(表1)。此外，以高频率获得了包含多个突变的几个等位基因(表1)。
表1.一个版本的并发碱基编辑器靶向样品的等位基因频率图。
Figure BDA0003846855640000631
Figure BDA0003846855640000641
实施例2：用于C和A编辑的SunTag系统。
将CRISPR-Cas效应蛋白(例如酶)的N或C末端融合至包含多个GCN4表位的SunTag。将识别GCN4的单链可变片段抗体(scFv)单独地或作为单一融合物融合至腺嘌呤和胞苷脱氨酶，但在本实施例中单独融合至腺嘌呤和胞苷脱氨酶。UGI可以作为融合物或以反式提供，但在本实施例中以反式提供。结合后，两种脱氨酶将同时募集至靶位点，并在脱氨窗口内执行C和A编辑(例如，通常观察到碱基编辑的靶位点中的子序列)。这样的系统用于两种不同的指导RNA。在这些基因座上，观察到靶向的C和A的稳健多样化，如图2所示。观察到窗口中C和A的稳健多样化(图2)。
实施例3：用于C和A编辑的TREE系统。
在TREE系统中，CRISPR-Cas效应蛋白(例如酶)包含用MS2发夹修饰的指导RNA。然后，通过融合至MCP蛋白(称为“分支”)，将SunTag表位募集至MS2发夹。最后，通过融合至结合SunTag的抗体，将目标蛋白质募集至SunTag。在HEK293T细胞中使用nCas9(D10A)或enCas9(D10A)、MCP-SunTag、scFv-APOBEC1和scFv-2xTadA来利用TREE系统。它导致窗口中腺嘌呤和胞苷残基两者的诱变(图3)。如图3所示，观察到多样化。
实施例4：多样化的脱氨酶筛选。
对已显示有作为Cas9融合物功能的五种脱氨酶分析碱基多样化功能：rAPOBECl、APOBEC3A、APOBEC3B、hAID、pmCDA1。将它们在N末端融合至MCP(MS2加帽蛋白)，并通过使用融合至2x MS2发夹的gRNA而募集至Cas9切口酶(D10A)。针对HEK293T细胞中的几个基因组位点对它们进行测定。通过高通量测序来分析碱基转化谱，结果如图4所示。
APOBEC1、APOBEC3A和pmCDA1在碱基编辑窗口内稳健地将C转化为G、T和A核苷酸(图4)。每个脱氨酶结构域产生不同水平的碱基编辑以及不同核苷酸组成的产物碱基谱(图4)。此外，与APOBEC1或APOBEC3A相比，pmCDA1更喜欢编辑远离PAM位点的胞苷，因此可以为靶位点的所需编辑窗口选择不同的酶(图4)。这是使用APOBEC3B、pmCDA1脱氨酶来诱导非C到T碱基变化的首次演示。
AP核酸内切酶I(APE1)是碱基切除修复通路中的一种酶，在脱碱基位点处切割磷酸二酯键，在碱基编辑的链中产生切口。当与切口非碱基编辑的链的Cas9切口酶结合使用时，导致双链断裂(DSB)，引起插入缺失。在缺乏UGI的构建体中，碱基多样化通常伴随着插入缺失。例如，在上述所有靶位点中，约5-20％的产物包含插入缺失，这降低碱基多样化的效率(图5)。
实施例5：细胞通路的调节——APE1抑制剂
使用CRT0044876抑制APE1(方案1)，CRT0044876是一种有效且众所周知的APE1抑制剂。
方案1：CRT0044876的化学结构。
Figure BDA0003846855640000651
为了确定这种化合物是否能改善碱基多样化谱，在CRT0044876存在的情况下，用融合至Cas9切口酶(D10A)的AID或pmCDA1处理HEK293T细胞。3d后，收获细胞并通过高通量测序(HTS)进行分析。在100μM和200μM浓度下，CRT0044876导致多个靶位点间产生的插入缺失的量显著减少，尽管也观察到碱基多样化率有所下降(图6)。
实施例6：细胞通路的调节-siRNA
可以通过siRNA抑制细胞APE1。将通过使用RNAi方法抑制APE1。我们将在转染编码碱基多样化构建体的质粒之前或期间，转染靶向内源性APE1的siRNA。孵育后，将收获细胞并通过HTS进行分析。
实施例7：DNA-PKcs抑制剂和/或DNA连接酶IV抑制剂
抑制DNA-PKcs(例如，NU7026和/或KU-0060648)和/或抑制DNA连接酶IV(例如，Scr7)的化合物将以不同的剂量应用于HEK293T细胞。随后将编码碱基多样化构建体的质粒转染到细胞中。3d孵育后，将通过HTS分析细胞以评估在靶位点处的碱基多样化率。
实施例8：人体细胞测试方法
真核HEK293T(ATCC CRL-3216)细胞在补充有10％(v/v)FBS(FBS)的Dulbecco’sModified Eagle’s Medium plus GlutaMax(ThermoFisher)中于37℃和5％CO2下培养。Cas和逆转录酶组分是使用固态合成进行合成的，并随后克隆到质粒中在CMV启动子后。将指导RNA克隆在人类U6启动子后。HEK293T细胞接种在48孔胶原蛋白涂覆的BioCoat板(Coming)上。细胞在～70％的汇合度进行转染。根据制造商的方案，每孔使用1.5μl的Lipofectamine3000(ThermoFisher Scientific)转染750ng的CRISPR质粒和250ng的crRNA表达质粒。3天后获得来自转染细胞的基因组DNA，并使用高通量Illumina扩增子测序来检测和定量插入缺失。
实施例9：使用ABE8腺嘌呤脱氨酶的并行碱基编辑(CUBE)
ABE8.20m使用经过工程化和进化的TadA酶(TadA8.20m)，其具有改善的腺嘌呤编辑活性(Gaudelli等人Nat Biotechnol.2020Jul；38(7)：892-900)。在本实验中，掺入了ABE8中使用的脱氨酶，以进一步改善CUBE活性。测试了APOBEC3A(A3A)与进化的TadA8.20m到切口酶Cas9和尿嘧啶糖基化酶抑制剂(UGI)的不同融合组合。测试的融合蛋白具有SEQID NO：79-83中任一个的序列。TadA*表示上一代腺嘌呤脱氨酶(Gaudelli等人.Nature.2017Nov 23；551(7681)：464-471)。这些构建体在HEK293T细胞中用多个间隔区进行了测试。HEK293T测试方法如实施例8所述进行。测试的间隔区序列具有SEQ ID NO：84-93中任一个的序列。观察到用测试的间隔区在碱基编辑窗口内对腺嘌呤和胞嘧啶两者进行的稳健编辑(图7-16)。如图7-16所示，并行碱基编辑使得能够转换间隔区中的腺嘌呤和胞嘧啶两者。
实施例10：Gam蛋白的胞嘧啶碱基多样化(CBD)
Gam是一种结合双链DNA断裂的蛋白质(Shee等人eLife 2013；2：e01222)，其包含SEQ ID NO：100的序列。通过结合双链断裂(DSB)末端，已显示其在基因编辑实验期间减少插入缺失产物的数量(Komor等人Sci Adv.2017Aug 30；3(8)：eaao4774)。在这个实施例中，将Gam蛋白的至少一部分要么融合至CBD构建体，要么在细胞中表达以改善碱基多样化活性。转染的质粒编码CBD和Gam构建体，并指导RNA靶向HEK293T细胞内源基因组中的各个位点。所测试的融合蛋白具有FEQ ID NO：94-99中任一项的序列，并且本实验中使用的间隔区序列具有SEQ ID NO：101-110中任一项的序列。HEK293T测试方法如实施例8所述进行。3天后，使用高通量测序分析编辑结果。据观察，Gam蛋白可与CBD酶一起使用以使胞嘧啶碱基多样化(图17-26)。将Gam要么在N端融合至CBD酶，要么作为单独的分子添加，表示为“+Gam”。如图17-26所示，胞嘧啶碱基多样化可以由具有和不具有Gam的CBD构建体介导。图27显示了带有或不带有Gam的CBD构建体生成的插入缺失。
实施例11：具有Cas12a CBD的大豆(Glycine Max)中的胞嘧啶碱基多样化
使用农杆菌介导的T-DNA转化将APOBEC3A-dCas12a(SEQ ID NO：111)转化到大豆植物中。靶序列具有SEQ ID NO：112的序列。在选择稳定的转化体后，在转化后5周对叶子进行取样。从叶子样品中提取DNA，然后使用Illumina高通量测序对编辑进行分析。表2显示了APOBEC3A-dCas12a在大豆植物中的胞嘧啶多样化活性。
表2.
Figure BDA0003846855640000671
Figure BDA0003846855640000681
以上是对本发明的示例性说明，不应被解释为对本发明的限制。本发明由以下权利要求限定，其中包括权利要求的等同物。
序列表
<110> 成对植物服务股份有限公司（Pairwise Plants Services, Inc.）
Kim, Yongjoo
Hummel, Aaron
<120> 用于碱基多样化的组合物、系统和方法
<130> 1499.9.WO
<150> US 62/967,737
<151> 2020-01-30
<160> 113
<170> PatentIn version 3.5
<210> 1
<211> 1592
<212> DNA
<213> 蒺藜苜蓿（Medicago truncatula）
<400> 1
actgttaata atttttaaac gtcagcgcac taaaaaaacg aaaagacgga cacgtgaaaa 60
taaaaaacac acactagttt atgacgcaat actattttac ttatgatttg ggtacattag 120
acaaaaccgt gaaagagatg tatcagctat gaaacctgta tacttcaata cagagactta 180
ctcatatcgg atacgtacgc acgaagtatc atattaatta ttttaatttt taataaatat 240
tttatcggat acttatgtga tactctacat atacacaagg atatttctaa gatactttat 300
agatacgtat cctagaaaaa catgaagagt aaaaaagtga gacaatgttg taaaaattca 360
ttataaatgt atatgattca attttagata tgcatcagta taattgattc tcgatgaaac 420
acttaaaatt atatttcttg tggaagaacg tagcgagaga ggtgattcag ttagacaaca 480
ttaaataaaa ttaatgttaa gttcttttaa tgatgtttct ctcaatatca catcatatga 540
aaatgtaata tgatttataa gaaaattttt aaaaaattta ttttaataat cacatgtact 600
attttttaaa aattgtatct tttataataa tacaataata aagagtaatc agtgttaatt 660
tttcttcaaa tataagtttt attataaatc attgttaacg tatcataagt cattaccgta 720
tcgtatctta attttttttt aaaaaccgct aattcacgta cccgtattgt attgtacccg 780
cacctgtatc acaatcgatc ttagttagaa gaattgtctc gaggcggtgc aagacagcat 840
ataatagacg tggactctct tataccaaac gttgtcgtat cacaaagggt taggtaacaa 900
gtcacagttt gtccacgtgt cacgttttaa ttggaagagg tgccgttggc gtaatataac 960
agccaatcga tttttgctat aaaagcaaat caggtaaact aaacttcttc attcttttct 1020
tccccatcgc tacaaaaccg gttcctttgg aaaagagatt cattcaaacc tagcacccaa 1080
ttccgtttca aggtataatc tactttctat tcttcgatta ttttattatt attagctact 1140
atcgtttaat cgatcttttc ttttgatccg tcaaatttaa attcaattag ggttttgttc 1200
ttttctttca tctgattgaa atccttctga attgaaccgt ttacttgatt ttactgttta 1260
ttgtatgatt taatcctttg tttttcaaag acagtcttta gattgtgatt aggggttcat 1320
ataaattttt agatttggat ttttgtattg tatgattcaa aaaatacgtc ctttaattag 1380
attagtacat ggatattttt tacccgattt attgattgtc agggagaatt tgatgagcaa 1440
gtttttttga tgtctgttgt aaattgaatt gattataatt gctgatctgc tgcttccagt 1500
tttcataacc catattcttt taaccttgtt gtacacacaa tgaaaaattg gtgattgatt 1560
catttgtttt tctttgtttt ggattataca gg 1592
<210> 2
<211> 2000
<212> DNA
<213> 玉蜀黍（Zea mays）
<400> 2
gtcgtgcccc tctctagaga taaagagcat tgcatgtcta aagtataaaa aattaccaca 60
tatttttttg tcacacttat ttgaagtgta gtttatctat ctctatacat atatttaaac 120
ttcactctac aaataatata gtctataata ctaaaataat attagtgttt tagaggatca 180
tataaataaa ctgctagaca tggtctaaag gataattgaa tattttgaca atctacagtt 240
ttatcttttt agtgtgcatg tgatctctct gttttttttg caaatagctt gacctatata 300
atacttcatc cattttatta gtacatccat ttaggattta gggttgatgg tttctataga 360
ctaattttta gtacatccat tttattcttt ttagtctcta aattttttaa aactaaaact 420
ctattttagt tttttattta ataatttaga tataaaatga aataaaataa attgactaca 480
aataaaacaa atacccttta agaaataaaa aaactaagca aacatttttc ttgtttcgag 540
tagataatga caggctgttc aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc 600
agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtag ctgcctctgg 660
acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt 720
gcgtggcgga gcggcagacg tgaggcggca cggcaggcgg cctcttcctc ctctcacggc 780
accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc 840
gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttcg ttcggagcgc 900
acacacacgc aaccagatct cccccaaatc cagccgtcgg cacctccgct tcaaggtacg 960
ccgctcatcc tccccccccc cctctctcta ccttctctag atcggcgatc cggtccatgg 1020
ttagggcccg gtagttctac ttctgttcat gtttgtgtta gagcaaacat gttcatgttc 1080
atgtttgtga tgatgtggtc tggttgggcg gtcgttctag atcggagtag gatactgttt 1140
caagctacct ggtggattta ttaattttgt atctgtatgt gtgtgccata catcttcata 1200
gttacgagtt taagatgatg gatggaaata tcgatctagg ataggtatac atgttgatgc 1260
gggttttact gatgcatata cagagatgct ttttttctcg cttggttgtg atgatatggt 1320
ctggttgggc ggtcgttcta gatcggagta gaatactgtt tcaaactacc tggtggattt 1380
attaaaggat aaagggtcgt tctagatcgg agtagaatac tgtttcaaac tacctggtgg 1440
atttattaaa ggatctgtat gtatgtgcct acatcttcat agttacgagt ttaagatgat 1500
ggatggaaat atcgatctag gataggtata catgttgatg cgggttttac tgatgcatat 1560
acagagatgc tttttttcgc ttggttgtga tgatgtggtc tggttgggcg gtcgttctag 1620
atcggagtag aatactgttt caaactacct ggtggattta ttaattttgt atctttatgt 1680
gtgtgccata catcttcata gttacgagtt taagatgatg gatggaaata ttgatctagg 1740
ataggtatac atgttgatgt gggttttact gatgcatata catgatggca tatgcggcat 1800
ctattcatat gctctaacct tgagtaccta tctattataa taaacaagta tgttttataa 1860
ttattttgat cttgatatac ttggatgatg gcatatgcag cagctatatg tggatttttt 1920
agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcc gatgctcacc 1980
ctgttgtttg gtgatacttc 2000
<210> 3
<211> 1227
<212> PRT
<213> 未知
<220>
<223> 毛螺菌科（Lachnospiraceae）细菌
<400> 3
Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu
1 5 10 15
Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn
20 25 30
Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly
35 40 45
Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val
50 55 60
Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe
65 70 75 80
Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu
85 90 95
Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu
100 105 110
Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro
115 120 125
Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn
130 135 140
Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met
145 150 155 160
Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn
165 170 175
Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val
180 185 190
Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile
195 200 205
Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe
210 215 220
Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile
225 230 235 240
Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu
245 250 255
Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe
260 265 270
Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe
275 280 285
Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg
290 295 300
Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu
305 310 315 320
Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe
325 330 335
Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly
340 345 350
Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile
355 360 365
His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg
370 375 380
Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln
385 390 395 400
Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile
405 410 415
Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu
420 425 430
Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn
435 440 445
Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser
450 455 460
Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn
465 470 475 480
Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu
485 490 495
Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln
500 505 510
Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln
515 520 525
Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr
530 535 540
Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys
545 550 555 560
Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly Asn
565 570 575
Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu
580 585 590
Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser
595 600 605
Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp
610 615 620
Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp
625 630 635 640
Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe
645 650 655
Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val
660 665 670
Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu
675 680 685
Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr
690 695 700
Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr
705 710 715 720
Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg
725 730 735
Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys
740 745 750
Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn
755 760 765
Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys
770 775 780
Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala
785 790 795 800
Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val Arg
805 810 815
Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Ala Arg
820 825 830
Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly Asn
835 840 845
Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly
850 855 860
Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys
865 870 875 880
Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys
885 890 895
Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu
900 905 910
Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser
915 920 925
Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln Lys
930 935 940
Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys
945 950 955 960
Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr
965 970 975
Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile
980 985 990
Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly
995 1000 1005
Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser
1010 1015 1020
Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu
1025 1030 1035
Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg
1040 1045 1050
Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly
1055 1060 1065
Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe
1070 1075 1080
Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe
1085 1090 1095
Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu
1100 1105 1110
Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala
1115 1120 1125
Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg
1130 1135 1140
Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly
1145 1150 1155
Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile
1160 1165 1170
Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg
1175 1180 1185
Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu
1190 1195 1200
Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu
1205 1210 1215
Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 4
<211> 1307
<212> PRT
<213> 氨基酸球菌属物种（Acidaminococcus sp.）
<400> 4
Met Thr Gln Phe Glu Gly Phe Thr Asn Leu Tyr Gln Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Lys His Ile Gln
20 25 30
Glu Gln Gly Phe Ile Glu Glu Asp Lys Ala Arg Asn Asp His Tyr Lys
35 40 45
Glu Leu Lys Pro Ile Ile Asp Arg Ile Tyr Lys Thr Tyr Ala Asp Gln
50 55 60
Cys Leu Gln Leu Val Gln Leu Asp Trp Glu Asn Leu Ser Ala Ala Ile
65 70 75 80
Asp Ser Tyr Arg Lys Glu Lys Thr Glu Glu Thr Arg Asn Ala Leu Ile
85 90 95
Glu Glu Gln Ala Thr Tyr Arg Asn Ala Ile His Asp Tyr Phe Ile Gly
100 105 110
Arg Thr Asp Asn Leu Thr Asp Ala Ile Asn Lys Arg His Ala Glu Ile
115 120 125
Tyr Lys Gly Leu Phe Lys Ala Glu Leu Phe Asn Gly Lys Val Leu Lys
130 135 140
Gln Leu Gly Thr Val Thr Thr Thr Glu His Glu Asn Ala Leu Leu Arg
145 150 155 160
Ser Phe Asp Lys Phe Thr Thr Tyr Phe Ser Gly Phe Tyr Glu Asn Arg
165 170 175
Lys Asn Val Phe Ser Ala Glu Asp Ile Ser Thr Ala Ile Pro His Arg
180 185 190
Ile Val Gln Asp Asn Phe Pro Lys Phe Lys Glu Asn Cys His Ile Phe
195 200 205
Thr Arg Leu Ile Thr Ala Val Pro Ser Leu Arg Glu His Phe Glu Asn
210 215 220
Val Lys Lys Ala Ile Gly Ile Phe Val Ser Thr Ser Ile Glu Glu Val
225 230 235 240
Phe Ser Phe Pro Phe Tyr Asn Gln Leu Leu Thr Gln Thr Gln Ile Asp
245 250 255
Leu Tyr Asn Gln Leu Leu Gly Gly Ile Ser Arg Glu Ala Gly Thr Glu
260 265 270
Lys Ile Lys Gly Leu Asn Glu Val Leu Asn Leu Ala Ile Gln Lys Asn
275 280 285
Asp Glu Thr Ala His Ile Ile Ala Ser Leu Pro His Arg Phe Ile Pro
290 295 300
Leu Phe Lys Gln Ile Leu Ser Asp Arg Asn Thr Leu Ser Phe Ile Leu
305 310 315 320
Glu Glu Phe Lys Ser Asp Glu Glu Val Ile Gln Ser Phe Cys Lys Tyr
325 330 335
Lys Thr Leu Leu Arg Asn Glu Asn Val Leu Glu Thr Ala Glu Ala Leu
340 345 350
Phe Asn Glu Leu Asn Ser Ile Asp Leu Thr His Ile Phe Ile Ser His
355 360 365
Lys Lys Leu Glu Thr Ile Ser Ser Ala Leu Cys Asp His Trp Asp Thr
370 375 380
Leu Arg Asn Ala Leu Tyr Glu Arg Arg Ile Ser Glu Leu Thr Gly Lys
385 390 395 400
Ile Thr Lys Ser Ala Lys Glu Lys Val Gln Arg Ser Leu Lys His Glu
405 410 415
Asp Ile Asn Leu Gln Glu Ile Ile Ser Ala Ala Gly Lys Glu Leu Ser
420 425 430
Glu Ala Phe Lys Gln Lys Thr Ser Glu Ile Leu Ser His Ala His Ala
435 440 445
Ala Leu Asp Gln Pro Leu Pro Thr Thr Leu Lys Lys Gln Glu Glu Lys
450 455 460
Glu Ile Leu Lys Ser Gln Leu Asp Ser Leu Leu Gly Leu Tyr His Leu
465 470 475 480
Leu Asp Trp Phe Ala Val Asp Glu Ser Asn Glu Val Asp Pro Glu Phe
485 490 495
Ser Ala Arg Leu Thr Gly Ile Lys Leu Glu Met Glu Pro Ser Leu Ser
500 505 510
Phe Tyr Asn Lys Ala Arg Asn Tyr Ala Thr Lys Lys Pro Tyr Ser Val
515 520 525
Glu Lys Phe Lys Leu Asn Phe Gln Met Pro Thr Leu Ala Ser Gly Trp
530 535 540
Asp Val Asn Lys Glu Lys Asn Asn Gly Ala Ile Leu Phe Val Lys Asn
545 550 555 560
Gly Leu Tyr Tyr Leu Gly Ile Met Pro Lys Gln Lys Gly Arg Tyr Lys
565 570 575
Ala Leu Ser Phe Glu Pro Thr Glu Lys Thr Ser Glu Gly Phe Asp Lys
580 585 590
Met Tyr Tyr Asp Tyr Phe Pro Asp Ala Ala Lys Met Ile Pro Lys Cys
595 600 605
Ser Thr Gln Leu Lys Ala Val Thr Ala His Phe Gln Thr His Thr Thr
610 615 620
Pro Ile Leu Leu Ser Asn Asn Phe Ile Glu Pro Leu Glu Ile Thr Lys
625 630 635 640
Glu Ile Tyr Asp Leu Asn Asn Pro Glu Lys Glu Pro Lys Lys Phe Gln
645 650 655
Thr Ala Tyr Ala Lys Lys Thr Gly Asp Gln Lys Gly Tyr Arg Glu Ala
660 665 670
Leu Cys Lys Trp Ile Asp Phe Thr Arg Asp Phe Leu Ser Lys Tyr Thr
675 680 685
Lys Thr Thr Ser Ile Asp Leu Ser Ser Leu Arg Pro Ser Ser Gln Tyr
690 695 700
Lys Asp Leu Gly Glu Tyr Tyr Ala Glu Leu Asn Pro Leu Leu Tyr His
705 710 715 720
Ile Ser Phe Gln Arg Ile Ala Glu Lys Glu Ile Met Asp Ala Val Glu
725 730 735
Thr Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Lys
740 745 750
Gly His His Gly Lys Pro Asn Leu His Thr Leu Tyr Trp Thr Gly Leu
755 760 765
Phe Ser Pro Glu Asn Leu Ala Lys Thr Ser Ile Lys Leu Asn Gly Gln
770 775 780
Ala Glu Leu Phe Tyr Arg Pro Lys Ser Arg Met Lys Arg Met Ala His
785 790 795 800
Arg Leu Gly Glu Lys Met Leu Asn Lys Lys Leu Lys Asp Gln Lys Thr
805 810 815
Pro Ile Pro Asp Thr Leu Tyr Gln Glu Leu Tyr Asp Tyr Val Asn His
820 825 830
Arg Leu Ser His Asp Leu Ser Asp Glu Ala Arg Ala Leu Leu Pro Asn
835 840 845
Val Ile Thr Lys Glu Val Ser His Glu Ile Ile Lys Asp Arg Arg Phe
850 855 860
Thr Ser Asp Lys Phe Phe Phe His Val Pro Ile Thr Leu Asn Tyr Gln
865 870 875 880
Ala Ala Asn Ser Pro Ser Lys Phe Asn Gln Arg Val Asn Ala Tyr Leu
885 890 895
Lys Glu His Pro Glu Thr Pro Ile Ile Gly Ile Asp Arg Gly Glu Arg
900 905 910
Asn Leu Ile Tyr Ile Thr Val Ile Asp Ser Thr Gly Lys Ile Leu Glu
915 920 925
Gln Arg Ser Leu Asn Thr Ile Gln Gln Phe Asp Tyr Gln Lys Lys Leu
930 935 940
Asp Asn Arg Glu Lys Glu Arg Val Ala Ala Arg Gln Ala Trp Ser Val
945 950 955 960
Val Gly Thr Ile Lys Asp Leu Lys Gln Gly Tyr Leu Ser Gln Val Ile
965 970 975
His Glu Ile Val Asp Leu Met Ile His Tyr Gln Ala Val Val Val Leu
980 985 990
Glu Asn Leu Asn Phe Gly Phe Lys Ser Lys Arg Thr Gly Ile Ala Glu
995 1000 1005
Lys Ala Val Tyr Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu
1010 1015 1020
Asn Cys Leu Val Leu Lys Asp Tyr Pro Ala Glu Lys Val Gly Gly
1025 1030 1035
Val Leu Asn Pro Tyr Gln Leu Thr Asp Gln Phe Thr Ser Phe Ala
1040 1045 1050
Lys Met Gly Thr Gln Ser Gly Phe Leu Phe Tyr Val Pro Ala Pro
1055 1060 1065
Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Phe Val Asp Pro Phe
1070 1075 1080
Val Trp Lys Thr Ile Lys Asn His Glu Ser Arg Lys His Phe Leu
1085 1090 1095
Glu Gly Phe Asp Phe Leu His Tyr Asp Val Lys Thr Gly Asp Phe
1100 1105 1110
Ile Leu His Phe Lys Met Asn Arg Asn Leu Ser Phe Gln Arg Gly
1115 1120 1125
Leu Pro Gly Phe Met Pro Ala Trp Asp Ile Val Phe Glu Lys Asn
1130 1135 1140
Glu Thr Gln Phe Asp Ala Lys Gly Thr Pro Phe Ile Ala Gly Lys
1145 1150 1155
Arg Ile Val Pro Val Ile Glu Asn His Arg Phe Thr Gly Arg Tyr
1160 1165 1170
Arg Asp Leu Tyr Pro Ala Asn Glu Leu Ile Ala Leu Leu Glu Glu
1175 1180 1185
Lys Gly Ile Val Phe Arg Asp Gly Ser Asn Ile Leu Pro Lys Leu
1190 1195 1200
Leu Glu Asn Asp Asp Ser His Ala Ile Asp Thr Met Val Ala Leu
1205 1210 1215
Ile Arg Ser Val Leu Gln Met Arg Asn Ser Asn Ala Ala Thr Gly
1220 1225 1230
Glu Asp Tyr Ile Asn Ser Pro Val Arg Asp Leu Asn Gly Val Cys
1235 1240 1245
Phe Asp Ser Arg Phe Gln Asn Pro Glu Trp Pro Met Asp Ala Asp
1250 1255 1260
Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Gln Leu Leu Leu
1265 1270 1275
Asn His Leu Lys Glu Ser Lys Asp Leu Lys Leu Gln Asn Gly Ile
1280 1285 1290
Ser Asn Gln Asp Trp Leu Ala Tyr Ile Gln Glu Leu Arg Asn
1295 1300 1305
<210> 5
<211> 1241
<212> PRT
<213> Butyrivibrio proteoclasticus
<400> 5
Met Leu Leu Tyr Glu Asn Tyr Thr Lys Arg Asn Gln Ile Thr Lys Ser
1 5 10 15
Leu Arg Leu Glu Leu Arg Pro Gln Gly Lys Thr Leu Arg Asn Ile Lys
20 25 30
Glu Leu Asn Leu Leu Glu Gln Asp Lys Ala Ile Tyr Ala Leu Leu Glu
35 40 45
Arg Leu Lys Pro Val Ile Asp Glu Gly Ile Lys Asp Ile Ala Arg Asp
50 55 60
Thr Leu Lys Asn Cys Glu Leu Ser Phe Glu Lys Leu Tyr Glu His Phe
65 70 75 80
Leu Ser Gly Asp Lys Lys Ala Tyr Ala Lys Glu Ser Glu Arg Leu Lys
85 90 95
Lys Glu Ile Val Lys Thr Leu Ile Lys Asn Leu Pro Glu Gly Ile Gly
100 105 110
Lys Ile Ser Glu Ile Asn Ser Ala Lys Tyr Leu Asn Gly Val Leu Tyr
115 120 125
Asp Phe Ile Asp Lys Thr His Lys Asp Ser Glu Glu Lys Gln Asn Ile
130 135 140
Leu Ser Asp Ile Leu Glu Thr Lys Gly Tyr Leu Ala Leu Phe Ser Lys
145 150 155 160
Phe Leu Thr Ser Arg Ile Thr Thr Leu Glu Gln Ser Met Pro Lys Arg
165 170 175
Val Ile Glu Asn Phe Glu Ile Tyr Ala Ala Asn Ile Pro Lys Met Gln
180 185 190
Asp Ala Leu Glu Arg Gly Ala Val Ser Phe Ala Ile Glu Tyr Glu Ser
195 200 205
Ile Cys Ser Val Asp Tyr Tyr Asn Gln Ile Leu Ser Gln Glu Asp Ile
210 215 220
Asp Ser Tyr Asn Arg Leu Ile Ser Gly Ile Met Asp Glu Asp Gly Ala
225 230 235 240
Lys Glu Lys Gly Ile Asn Gln Thr Ile Ser Glu Lys Asn Ile Lys Ile
245 250 255
Lys Ser Glu His Leu Glu Glu Lys Pro Phe Arg Ile Leu Lys Gln Leu
260 265 270
His Lys Gln Ile Leu Glu Glu Arg Glu Lys Ala Phe Thr Ile Asp His
275 280 285
Ile Asp Ser Asp Glu Glu Val Val Gln Val Thr Lys Glu Ala Phe Glu
290 295 300
Gln Thr Lys Glu Gln Trp Glu Asn Ile Lys Lys Ile Asn Gly Phe Tyr
305 310 315 320
Ala Lys Asp Pro Gly Asp Ile Thr Leu Phe Ile Val Val Gly Pro Asn
325 330 335
Gln Thr His Val Leu Ser Gln Leu Ile Tyr Gly Glu His Asp Arg Ile
340 345 350
Arg Leu Leu Leu Glu Glu Tyr Glu Lys Asn Thr Leu Glu Val Leu Pro
355 360 365
Arg Arg Thr Lys Ser Glu Asp Ala Arg Tyr Asp Lys Phe Val Asn Ala
370 375 380
Val Pro Lys Lys Val Ala Lys Glu Ser His Thr Phe Asp Gly Leu Gln
385 390 395 400
Lys Met Thr Gly Asp Asp Arg Leu Phe Ile Leu Tyr Arg Asp Glu Leu
405 410 415
Ala Arg Asn Tyr Met Arg Ile Lys Glu Ala Tyr Gly Thr Phe Glu Arg
420 425 430
Asp Ile Leu Lys Ser Arg Arg Gly Ile Lys Gly Asn Arg Asp Val Gln
435 440 445
Glu Ser Leu Val Ser Phe Tyr Asp Glu Leu Thr Lys Phe Arg Ser Ala
450 455 460
Leu Arg Ile Ile Asn Ser Gly Asn Asp Glu Lys Ala Asp Pro Ile Phe
465 470 475 480
Tyr Asn Thr Phe Asp Gly Ile Phe Glu Lys Ala Asn Arg Thr Tyr Lys
485 490 495
Ala Glu Asn Leu Cys Arg Asn Tyr Val Thr Lys Ser Pro Ala Asp Asp
500 505 510
Ala Arg Ile Met Ala Ser Cys Leu Gly Thr Pro Ala Arg Leu Arg Thr
515 520 525
His Trp Trp Asn Gly Glu Glu Asn Phe Ala Ile Asn Asp Val Ala Met
530 535 540
Ile Arg Arg Gly Asp Glu Tyr Tyr Tyr Phe Val Leu Thr Pro Asp Val
545 550 555 560
Lys Pro Val Asp Leu Lys Thr Lys Asp Glu Thr Asp Ala Gln Ile Phe
565 570 575
Val Gln Arg Lys Gly Ala Lys Ser Phe Leu Gly Leu Pro Lys Ala Leu
580 585 590
Phe Lys Cys Ile Leu Glu Pro Tyr Phe Glu Ser Pro Glu His Lys Asn
595 600 605
Asp Lys Asn Cys Val Ile Glu Glu Tyr Val Ser Lys Pro Leu Thr Ile
610 615 620
Asp Arg Arg Ala Tyr Asp Ile Phe Lys Asn Gly Thr Phe Lys Lys Thr
625 630 635 640
Asn Ile Gly Ile Asp Gly Leu Thr Glu Glu Lys Phe Lys Asp Asp Cys
645 650 655
Arg Tyr Leu Ile Asp Val Tyr Lys Glu Phe Ile Ala Val Tyr Thr Arg
660 665 670
Tyr Ser Cys Phe Asn Met Ser Gly Leu Lys Arg Ala Asp Glu Tyr Asn
675 680 685
Asp Ile Gly Glu Phe Phe Ser Asp Val Asp Thr Arg Leu Cys Thr Met
690 695 700
Glu Trp Ile Pro Val Ser Phe Glu Arg Ile Asn Asp Met Val Asp Lys
705 710 715 720
Lys Glu Gly Leu Leu Phe Leu Val Arg Ser Met Phe Leu Tyr Asn Arg
725 730 735
Pro Arg Lys Pro Tyr Glu Arg Thr Phe Ile Gln Leu Phe Ser Asp Ser
740 745 750
Asn Met Glu His Thr Ser Met Leu Leu Asn Ser Arg Ala Met Ile Gln
755 760 765
Tyr Arg Ala Ala Ser Leu Pro Arg Arg Val Thr His Lys Lys Gly Ser
770 775 780
Ile Leu Val Ala Leu Arg Asp Ser Asn Gly Glu His Ile Pro Met His
785 790 795 800
Ile Arg Glu Ala Ile Tyr Lys Met Lys Asn Asn Phe Asp Ile Ser Ser
805 810 815
Glu Asp Phe Ile Met Ala Lys Ala Tyr Leu Ala Glu His Asp Val Ala
820 825 830
Ile Lys Lys Ala Asn Glu Asp Ile Ile Arg Asn Arg Arg Tyr Thr Glu
835 840 845
Asp Lys Phe Phe Leu Ser Leu Ser Tyr Thr Lys Asn Ala Asp Ile Ser
850 855 860
Ala Arg Thr Leu Asp Tyr Ile Asn Asp Lys Val Glu Glu Asp Thr Gln
865 870 875 880
Asp Ser Arg Met Ala Val Ile Val Thr Arg Asn Leu Lys Asp Leu Thr
885 890 895
Tyr Val Ala Val Val Asp Glu Lys Asn Asn Val Leu Glu Glu Lys Ser
900 905 910
Leu Asn Glu Ile Asp Gly Val Asn Tyr Arg Glu Leu Leu Lys Glu Arg
915 920 925
Thr Lys Ile Lys Tyr His Asp Lys Thr Arg Leu Trp Gln Tyr Asp Val
930 935 940
Ser Ser Lys Gly Leu Lys Glu Ala Tyr Val Glu Leu Ala Val Thr Gln
945 950 955 960
Ile Ser Lys Leu Ala Thr Lys Tyr Asn Ala Val Val Val Val Glu Ser
965 970 975
Met Ser Ser Thr Phe Lys Asp Lys Phe Ser Phe Leu Asp Glu Gln Ile
980 985 990
Phe Lys Ala Phe Glu Ala Arg Leu Cys Ala Arg Met Ser Asp Leu Ser
995 1000 1005
Phe Asn Thr Ile Lys Glu Gly Glu Ala Gly Ser Ile Ser Asn Pro
1010 1015 1020
Ile Gln Val Ser Asn Asn Asn Gly Asn Ser Tyr Gln Asp Gly Val
1025 1030 1035
Ile Tyr Phe Leu Asn Asn Ala Tyr Thr Arg Thr Leu Cys Pro Asp
1040 1045 1050
Thr Gly Phe Val Asp Val Phe Asp Lys Thr Arg Leu Ile Thr Met
1055 1060 1065
Gln Ser Lys Arg Gln Phe Phe Ala Lys Met Lys Asp Ile Arg Ile
1070 1075 1080
Asp Asp Gly Glu Met Leu Phe Thr Phe Asn Leu Glu Glu Tyr Pro
1085 1090 1095
Thr Lys Arg Leu Leu Asp Arg Lys Glu Trp Thr Val Lys Ile Ala
1100 1105 1110
Gly Asp Gly Ser Tyr Phe Asp Lys Asp Lys Gly Glu Tyr Val Tyr
1115 1120 1125
Val Asn Asp Ile Val Arg Glu Gln Ile Ile Pro Ala Leu Leu Glu
1130 1135 1140
Asp Lys Ala Val Phe Asp Gly Asn Met Ala Glu Lys Phe Leu Asp
1145 1150 1155
Lys Thr Ala Ile Ser Gly Lys Ser Val Glu Leu Ile Tyr Lys Trp
1160 1165 1170
Phe Ala Asn Ala Leu Tyr Gly Ile Ile Thr Lys Lys Asp Gly Glu
1175 1180 1185
Lys Ile Tyr Arg Ser Pro Ile Thr Gly Thr Glu Ile Asp Val Ser
1190 1195 1200
Lys Asn Thr Thr Tyr Asn Phe Gly Lys Lys Phe Met Phe Lys Gln
1205 1210 1215
Glu Tyr Arg Gly Asp Gly Asp Phe Leu Asp Ala Phe Leu Asn Tyr
1220 1225 1230
Met Gln Ala Gln Asp Ile Ala Val
1235 1240
<210> 6
<211> 1238
<212> PRT
<213> Candidatus Methanoplasma termitum
<400> 6
Met Asn Asn Tyr Asp Glu Phe Thr Lys Leu Tyr Pro Ile Gln Lys Thr
1 5 10 15
Ile Arg Phe Glu Leu Lys Pro Gln Gly Arg Thr Met Glu His Leu Glu
20 25 30
Thr Phe Asn Phe Phe Glu Glu Asp Arg Asp Arg Ala Glu Lys Tyr Lys
35 40 45
Ile Leu Lys Glu Ala Ile Asp Glu Tyr His Lys Lys Phe Ile Asp Glu
50 55 60
His Leu Thr Asn Met Ser Leu Asp Trp Asn Ser Leu Lys Gln Ile Ser
65 70 75 80
Glu Lys Tyr Tyr Lys Ser Arg Glu Glu Lys Asp Lys Lys Val Phe Leu
85 90 95
Ser Glu Gln Lys Arg Met Arg Gln Glu Ile Val Ser Glu Phe Lys Lys
100 105 110
Asp Asp Arg Phe Lys Asp Leu Phe Ser Lys Lys Leu Phe Ser Glu Leu
115 120 125
Leu Lys Glu Glu Ile Tyr Lys Lys Gly Asn His Gln Glu Ile Asp Ala
130 135 140
Leu Lys Ser Phe Asp Lys Phe Ser Gly Tyr Phe Ile Gly Leu His Glu
145 150 155 160
Asn Arg Lys Asn Met Tyr Ser Asp Gly Asp Glu Ile Thr Ala Ile Ser
165 170 175
Asn Arg Ile Val Asn Glu Asn Phe Pro Lys Phe Leu Asp Asn Leu Gln
180 185 190
Lys Tyr Gln Glu Ala Arg Lys Lys Tyr Pro Glu Trp Ile Ile Lys Ala
195 200 205
Glu Ser Ala Leu Val Ala His Asn Ile Lys Met Asp Ile Val Phe Ser
210 215 220
Leu Glu Tyr Phe Asn Lys Val Leu Asn Gln Glu Gly Ile Gln Arg Tyr
225 230 235 240
Asn Leu Ala Leu Gly Gly Tyr Val Thr Lys Ser Gly Glu Lys Met Met
245 250 255
Gly Leu Asn Asp Ala Leu Asn Leu Ala His Gln Ser Glu Lys Ser Ser
260 265 270
Lys Gly Arg Ile His Met Thr Pro Leu Phe Lys Gln Ile Leu Ser Glu
275 280 285
Lys Glu Ser Phe Ser Tyr Ile Pro Asp Val Phe Thr Glu Asp Ser Gln
290 295 300
Leu Leu Pro Ser Ile Gly Gly Phe Phe Ala Gln Ile Glu Asn Asp Lys
305 310 315 320
Asp Gly Asn Ile Phe Asp Arg Ala Leu Glu Leu Ile Ser Ser Tyr Ala
325 330 335
Glu Tyr Asp Thr Glu Arg Ile Tyr Ile Arg Gln Ala Asp Ile Asn Arg
340 345 350
Val Ser Asn Val Ile Phe Gly Glu Trp Gly Thr Leu Gly Gly Leu Met
355 360 365
Arg Glu Tyr Lys Ala Asp Ser Ile Asn Asp Ile Asn Leu Glu Arg Thr
370 375 380
Cys Lys Lys Val Asp Lys Trp Leu Asp Ser Lys Glu Phe Ala Leu Ser
385 390 395 400
Asp Val Leu Glu Ala Ile Asp Arg Thr Gly Asn Asn Asp Ala Phe Asn
405 410 415
Glu Tyr Ile Ser Lys Met Arg Thr Ala Arg Glu Lys Ile Asp Ala Ala
420 425 430
Arg Lys Glu Met Lys Phe Ile Ser Glu Lys Ile Ser Gly Asp Glu Glu
435 440 445
Ser Ile His Ile Ile Lys Thr Leu Leu Asp Ser Val Gln Gln Phe Leu
450 455 460
His Phe Phe Asn Leu Phe Lys Ala Arg Gln Asp Ile Pro Leu Asp Gly
465 470 475 480
Ala Phe Tyr Ala Glu Phe Asp Glu Val His Ser Lys Leu Phe Ala Ile
485 490 495
Val Pro Leu Tyr Asn Lys Val Arg Asn Tyr Leu Thr Lys Asn Asn Leu
500 505 510
Asn Thr Lys Lys Ile Lys Leu Asn Phe Lys Asn Pro Thr Leu Ala Asn
515 520 525
Gly Trp Asp Gln Asn Lys Val Tyr Asp Tyr Ala Ser Leu Ile Phe Leu
530 535 540
Arg Asp Gly Asn Tyr Tyr Leu Gly Ile Ile Asn Pro Lys Arg Lys Lys
545 550 555 560
Asn Ile Lys Phe Glu Gln Gly Ser Gly Asn Gly Pro Phe Tyr Arg Lys
565 570 575
Met Val Tyr Lys Gln Ile Pro Gly Pro Asn Lys Asn Leu Arg Pro Val
580 585 590
Phe Leu Thr Ser Thr Lys Gly Lys Lys Glu Tyr Lys Pro Ser Lys Glu
595 600 605
Ile Ile Glu Gly Tyr Glu Ala Asp Lys His Ile Arg Gly Asp Lys Phe
610 615 620
Asp Leu Asp Phe Cys His Lys Leu Ile Asp Phe Phe Lys Glu Ser Ile
625 630 635 640
Glu Lys His Lys Asp Trp Ser Lys Phe Asn Phe Tyr Phe Ser Pro Thr
645 650 655
Glu Ser Tyr Gly Asp Ile Ser Glu Phe Tyr Leu Asp Val Glu Lys Gln
660 665 670
Gly Tyr Arg Met His Phe Glu Asn Ile Ser Ala Glu Thr Ile Asp Glu
675 680 685
Tyr Val Glu Lys Gly Asp Leu Phe Leu Phe Gln Ile Tyr Asn Lys Asp
690 695 700
Phe Val Lys Ala Ala Thr Gly Lys Lys Asp Met His Thr Ile Tyr Trp
705 710 715 720
Asn Ala Ala Phe Ser Pro Glu Asn Leu Gln Asp Val Val Val Lys Leu
725 730 735
Asn Gly Glu Ala Glu Leu Phe Tyr Arg Asp Lys Ser Asp Ile Lys Glu
740 745 750
Ile Val His Arg Glu Gly Glu Ile Leu Val Asn Arg Thr Tyr Asn Gly
755 760 765
Arg Thr Pro Val Pro Asp Lys Ile His Lys Lys Leu Thr Asp Tyr His
770 775 780
Asn Gly Arg Thr Lys Asp Leu Gly Glu Ala Lys Glu Tyr Leu Asp Lys
785 790 795 800
Val Arg Tyr Phe Lys Ala His Tyr Asp Ile Thr Lys Asp Arg Arg Tyr
805 810 815
Leu Asn Asp Lys Ile Tyr Phe His Val Pro Leu Thr Leu Asn Phe Lys
820 825 830
Ala Asn Gly Lys Lys Asn Leu Asn Lys Met Val Ile Glu Lys Phe Leu
835 840 845
Ser Asp Glu Lys Ala His Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn
850 855 860
Leu Leu Tyr Tyr Ser Ile Ile Asp Arg Ser Gly Lys Ile Ile Asp Gln
865 870 875 880
Gln Ser Leu Asn Val Ile Asp Gly Phe Asp Tyr Arg Glu Lys Leu Asn
885 890 895
Gln Arg Glu Ile Glu Met Lys Asp Ala Arg Gln Ser Trp Asn Ala Ile
900 905 910
Gly Lys Ile Lys Asp Leu Lys Glu Gly Tyr Leu Ser Lys Ala Val His
915 920 925
Glu Ile Thr Lys Met Ala Ile Gln Tyr Asn Ala Ile Val Val Met Glu
930 935 940
Glu Leu Asn Tyr Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln
945 950 955 960
Ile Tyr Gln Lys Phe Glu Asn Met Leu Ile Asp Lys Met Asn Tyr Leu
965 970 975
Val Phe Lys Asp Ala Pro Asp Glu Ser Pro Gly Gly Val Leu Asn Ala
980 985 990
Tyr Gln Leu Thr Asn Pro Leu Glu Ser Phe Ala Lys Leu Gly Lys Gln
995 1000 1005
Thr Gly Ile Leu Phe Tyr Val Pro Ala Ala Tyr Thr Ser Lys Ile
1010 1015 1020
Asp Pro Thr Thr Gly Phe Val Asn Leu Phe Asn Thr Ser Ser Lys
1025 1030 1035
Thr Asn Ala Gln Glu Arg Lys Glu Phe Leu Gln Lys Phe Glu Ser
1040 1045 1050
Ile Ser Tyr Ser Ala Lys Asp Gly Gly Ile Phe Ala Phe Ala Phe
1055 1060 1065
Asp Tyr Arg Lys Phe Gly Thr Ser Lys Thr Asp His Lys Asn Val
1070 1075 1080
Trp Thr Ala Tyr Thr Asn Gly Glu Arg Met Arg Tyr Ile Lys Glu
1085 1090 1095
Lys Lys Arg Asn Glu Leu Phe Asp Pro Ser Lys Glu Ile Lys Glu
1100 1105 1110
Ala Leu Thr Ser Ser Gly Ile Lys Tyr Asp Gly Gly Gln Asn Ile
1115 1120 1125
Leu Pro Asp Ile Leu Arg Ser Asn Asn Asn Gly Leu Ile Tyr Thr
1130 1135 1140
Met Tyr Ser Ser Phe Ile Ala Ala Ile Gln Met Arg Val Tyr Asp
1145 1150 1155
Gly Lys Glu Asp Tyr Ile Ile Ser Pro Ile Lys Asn Ser Lys Gly
1160 1165 1170
Glu Phe Phe Arg Thr Asp Pro Lys Arg Arg Glu Leu Pro Ile Asp
1175 1180 1185
Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Arg Gly Glu Leu
1190 1195 1200
Thr Met Arg Ala Ile Ala Glu Lys Phe Asp Pro Asp Ser Glu Lys
1205 1210 1215
Met Ala Lys Leu Glu Leu Lys His Lys Asp Trp Phe Glu Phe Met
1220 1225 1230
Gln Thr Arg Gly Asp
1235
<210> 7
<211> 1281
<212> PRT
<213> 挑剔真杆菌（Eubacterium eligens）
<400> 7
Met Asn Gly Asn Arg Ser Ile Val Tyr Arg Glu Phe Val Gly Val Ile
1 5 10 15
Pro Val Ala Lys Thr Leu Arg Asn Glu Leu Arg Pro Val Gly His Thr
20 25 30
Gln Glu His Ile Ile Gln Asn Gly Leu Ile Gln Glu Asp Glu Leu Arg
35 40 45
Gln Glu Lys Ser Thr Glu Leu Lys Asn Ile Met Asp Asp Tyr Tyr Arg
50 55 60
Glu Tyr Ile Asp Lys Ser Leu Ser Gly Val Thr Asp Leu Asp Phe Thr
65 70 75 80
Leu Leu Phe Glu Leu Met Asn Leu Val Gln Ser Ser Pro Ser Lys Asp
85 90 95
Asn Lys Lys Ala Leu Glu Lys Glu Gln Ser Lys Met Arg Glu Gln Ile
100 105 110
Cys Thr His Leu Gln Ser Asp Ser Asn Tyr Lys Asn Ile Phe Asn Ala
115 120 125
Lys Leu Leu Lys Glu Ile Leu Pro Asp Phe Ile Lys Asn Tyr Asn Gln
130 135 140
Tyr Asp Val Lys Asp Lys Ala Gly Lys Leu Glu Thr Leu Ala Leu Phe
145 150 155 160
Asn Gly Phe Ser Thr Tyr Phe Thr Asp Phe Phe Glu Lys Arg Lys Asn
165 170 175
Val Phe Thr Lys Glu Ala Val Ser Thr Ser Ile Ala Tyr Arg Ile Val
180 185 190
His Glu Asn Ser Leu Ile Phe Leu Ala Asn Met Thr Ser Tyr Lys Lys
195 200 205
Ile Ser Glu Lys Ala Leu Asp Glu Ile Glu Val Ile Glu Lys Asn Asn
210 215 220
Gln Asp Lys Met Gly Asp Trp Glu Leu Asn Gln Ile Phe Asn Pro Asp
225 230 235 240
Phe Tyr Asn Met Val Leu Ile Gln Ser Gly Ile Asp Phe Tyr Asn Glu
245 250 255
Ile Cys Gly Val Val Asn Ala His Met Asn Leu Tyr Cys Gln Gln Thr
260 265 270
Lys Asn Asn Tyr Asn Leu Phe Lys Met Arg Lys Leu His Lys Gln Ile
275 280 285
Leu Ala Tyr Thr Ser Thr Ser Phe Glu Val Pro Lys Met Phe Glu Asp
290 295 300
Asp Met Ser Val Tyr Asn Ala Val Asn Ala Phe Ile Asp Glu Thr Glu
305 310 315 320
Lys Gly Asn Ile Ile Gly Lys Leu Lys Asp Ile Val Asn Lys Tyr Asp
325 330 335
Glu Leu Asp Glu Lys Arg Ile Tyr Ile Ser Lys Asp Phe Tyr Glu Thr
340 345 350
Leu Ser Cys Phe Met Ser Gly Asn Trp Asn Leu Ile Thr Gly Cys Val
355 360 365
Glu Asn Phe Tyr Asp Glu Asn Ile His Ala Lys Gly Lys Ser Lys Glu
370 375 380
Glu Lys Val Lys Lys Ala Val Lys Glu Asp Lys Tyr Lys Ser Ile Asn
385 390 395 400
Asp Val Asn Asp Leu Val Glu Lys Tyr Ile Asp Glu Lys Glu Arg Asn
405 410 415
Glu Phe Lys Asn Ser Asn Ala Lys Gln Tyr Ile Arg Glu Ile Ser Asn
420 425 430
Ile Ile Thr Asp Thr Glu Thr Ala His Leu Glu Tyr Asp Asp His Ile
435 440 445
Ser Leu Ile Glu Ser Glu Glu Lys Ala Asp Glu Met Lys Lys Arg Leu
450 455 460
Asp Met Tyr Met Asn Met Tyr His Trp Ala Lys Ala Phe Ile Val Asp
465 470 475 480
Glu Val Leu Asp Arg Asp Glu Met Phe Tyr Ser Asp Ile Asp Asp Ile
485 490 495
Tyr Asn Ile Leu Glu Asn Ile Val Pro Leu Tyr Asn Arg Val Arg Asn
500 505 510
Tyr Val Thr Gln Lys Pro Tyr Asn Ser Lys Lys Ile Lys Leu Asn Phe
515 520 525
Gln Ser Pro Thr Leu Ala Asn Gly Trp Ser Gln Ser Lys Glu Phe Asp
530 535 540
Asn Asn Ala Ile Ile Leu Ile Arg Asp Asn Lys Tyr Tyr Leu Ala Ile
545 550 555 560
Phe Asn Ala Lys Asn Lys Pro Asp Lys Lys Ile Ile Gln Gly Asn Ser
565 570 575
Asp Lys Lys Asn Asp Asn Asp Tyr Lys Lys Met Val Tyr Asn Leu Leu
580 585 590
Pro Gly Ala Asn Lys Met Leu Pro Lys Val Phe Leu Ser Lys Lys Gly
595 600 605
Ile Glu Thr Phe Lys Pro Ser Asp Tyr Ile Ile Ser Gly Tyr Asn Ala
610 615 620
His Lys His Ile Lys Thr Ser Glu Asn Phe Asp Ile Ser Phe Cys Arg
625 630 635 640
Asp Leu Ile Asp Tyr Phe Lys Asn Ser Ile Glu Lys His Ala Glu Trp
645 650 655
Arg Lys Tyr Glu Phe Lys Phe Ser Ala Thr Asp Ser Tyr Ser Asp Ile
660 665 670
Ser Glu Phe Tyr Arg Glu Val Glu Met Gln Gly Tyr Arg Ile Asp Trp
675 680 685
Thr Tyr Ile Ser Glu Ala Asp Ile Asn Lys Leu Asp Glu Glu Gly Lys
690 695 700
Ile Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ala Glu Asn Ser Thr
705 710 715 720
Gly Lys Glu Asn Leu His Thr Met Tyr Phe Lys Asn Ile Phe Ser Glu
725 730 735
Glu Asn Leu Asp Lys Ile Ile Lys Leu Asn Gly Gln Ala Glu Leu Phe
740 745 750
Tyr Arg Arg Ala Ser Val Lys Asn Pro Val Lys His Lys Lys Asp Ser
755 760 765
Val Leu Val Asn Lys Thr Tyr Lys Asn Gln Leu Asp Asn Gly Asp Val
770 775 780
Val Arg Ile Pro Ile Pro Asp Asp Ile Tyr Asn Glu Ile Tyr Lys Met
785 790 795 800
Tyr Asn Gly Tyr Ile Lys Glu Ser Asp Leu Ser Glu Ala Ala Lys Glu
805 810 815
Tyr Leu Asp Lys Val Glu Val Arg Thr Ala Gln Lys Asp Ile Val Lys
820 825 830
Asp Tyr Arg Tyr Thr Val Asp Lys Tyr Phe Ile His Thr Pro Ile Thr
835 840 845
Ile Asn Tyr Lys Val Thr Ala Arg Asn Asn Val Asn Asp Met Val Val
850 855 860
Lys Tyr Ile Ala Gln Asn Asp Asp Ile His Val Ile Gly Ile Asp Arg
865 870 875 880
Gly Glu Arg Asn Leu Ile Tyr Ile Ser Val Ile Asp Ser His Gly Asn
885 890 895
Ile Val Lys Gln Lys Ser Tyr Asn Ile Leu Asn Asn Tyr Asp Tyr Lys
900 905 910
Lys Lys Leu Val Glu Lys Glu Lys Thr Arg Glu Tyr Ala Arg Lys Asn
915 920 925
Trp Lys Ser Ile Gly Asn Ile Lys Glu Leu Lys Glu Gly Tyr Ile Ser
930 935 940
Gly Val Val His Glu Ile Ala Met Leu Ile Val Glu Tyr Asn Ala Ile
945 950 955 960
Ile Ala Met Glu Asp Leu Asn Tyr Gly Phe Lys Arg Gly Arg Phe Lys
965 970 975
Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Ser Met Leu Ile Asn Lys
980 985 990
Leu Asn Tyr Phe Ala Ser Lys Glu Lys Ser Val Asp Glu Pro Gly Gly
995 1000 1005
Leu Leu Lys Gly Tyr Gln Leu Thr Tyr Val Pro Asp Asn Ile Lys
1010 1015 1020
Asn Leu Gly Lys Gln Cys Gly Val Ile Phe Tyr Val Pro Ala Ala
1025 1030 1035
Phe Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Ile Ser Ala Phe
1040 1045 1050
Asn Phe Lys Ser Ile Ser Thr Asn Ala Ser Arg Lys Gln Phe Phe
1055 1060 1065
Met Gln Phe Asp Glu Ile Arg Tyr Cys Ala Glu Lys Asp Met Phe
1070 1075 1080
Ser Phe Gly Phe Asp Tyr Asn Asn Phe Asp Thr Tyr Asn Ile Thr
1085 1090 1095
Met Gly Lys Thr Gln Trp Thr Val Tyr Thr Asn Gly Glu Arg Leu
1100 1105 1110
Gln Ser Glu Phe Asn Asn Ala Arg Arg Thr Gly Lys Thr Lys Ser
1115 1120 1125
Ile Asn Leu Thr Glu Thr Ile Lys Leu Leu Leu Glu Asp Asn Glu
1130 1135 1140
Ile Asn Tyr Ala Asp Gly His Asp Ile Arg Ile Asp Met Glu Lys
1145 1150 1155
Met Asp Glu Asp Lys Lys Ser Glu Phe Phe Ala Gln Leu Leu Ser
1160 1165 1170
Leu Tyr Lys Leu Thr Val Gln Met Arg Asn Ser Tyr Thr Glu Ala
1175 1180 1185
Glu Glu Gln Glu Asn Gly Ile Ser Tyr Asp Lys Ile Ile Ser Pro
1190 1195 1200
Val Ile Asn Asp Glu Gly Glu Phe Phe Asp Ser Asp Asn Tyr Lys
1205 1210 1215
Glu Ser Asp Asp Lys Glu Cys Lys Met Pro Lys Asp Ala Asp Ala
1220 1225 1230
Asn Gly Ala Tyr Cys Ile Ala Leu Lys Gly Leu Tyr Glu Val Leu
1235 1240 1245
Lys Ile Lys Ser Glu Trp Thr Glu Asp Gly Phe Asp Arg Asn Cys
1250 1255 1260
Leu Lys Leu Pro His Ala Glu Trp Leu Asp Phe Ile Gln Asn Lys
1265 1270 1275
Arg Tyr Glu
1280
<210> 8
<211> 1300
<212> PRT
<213> 新凶手弗朗西丝氏菌（Francisella novicida）
<400> 8
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 Val Asn 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> 9
<211> 1206
<212> PRT
<213> 未知
<220>
<223> 毛螺菌科细菌
<400> 9
Met Tyr Tyr Glu Ser Leu Thr Lys Gln Tyr Pro Val Ser Lys Thr Ile
1 5 10 15
Arg Asn Glu Leu Ile Pro Ile Gly Lys Thr Leu Asp Asn Ile Arg Gln
20 25 30
Asn Asn Ile Leu Glu Ser Asp Val Lys Arg Lys Gln Asn Tyr Glu His
35 40 45
Val Lys Gly Ile Leu Asp Glu Tyr His Lys Gln Leu Ile Asn Glu Ala
50 55 60
Leu Asp Asn Cys Thr Leu Pro Ser Leu Lys Ile Ala Ala Glu Ile Tyr
65 70 75 80
Leu Lys Asn Gln Lys Glu Val Ser Asp Arg Glu Asp Phe Asn Lys Thr
85 90 95
Gln Asp Leu Leu Arg Lys Glu Val Val Glu Lys Leu Lys Ala His Glu
100 105 110
Asn Phe Thr Lys Ile Gly Lys Lys Asp Ile Leu Asp Leu Leu Glu Lys
115 120 125
Leu Pro Ser Ile Ser Glu Asp Asp Tyr Asn Ala Leu Glu Ser Phe Arg
130 135 140
Asn Phe Tyr Thr Tyr Phe Thr Ser Tyr Asn Lys Val Arg Glu Asn Leu
145 150 155 160
Tyr Ser Asp Lys Glu Lys Ser Ser Thr Val Ala Tyr Arg Leu Ile Asn
165 170 175
Glu Asn Phe Pro Lys Phe Leu Asp Asn Val Lys Ser Tyr Arg Phe Val
180 185 190
Lys Thr Ala Gly Ile Leu Ala Asp Gly Leu Gly Glu Glu Glu Gln Asp
195 200 205
Ser Leu Phe Ile Val Glu Thr Phe Asn Lys Thr Leu Thr Gln Asp Gly
210 215 220
Ile Asp Thr Tyr Asn Ser Gln Val Gly Lys Ile Asn Ser Ser Ile Asn
225 230 235 240
Leu Tyr Asn Gln Lys Asn Gln Lys Ala Asn Gly Phe Arg Lys Ile Pro
245 250 255
Lys Met Lys Met Leu Tyr Lys Gln Ile Leu Ser Asp Arg Glu Glu Ser
260 265 270
Phe Ile Asp Glu Phe Gln Ser Asp Glu Val Leu Ile Asp Asn Val Glu
275 280 285
Ser Tyr Gly Ser Val Leu Ile Glu Ser Leu Lys Ser Ser Lys Val Ser
290 295 300
Ala Phe Phe Asp Ala Leu Arg Glu Ser Lys Gly Lys Asn Val Tyr Val
305 310 315 320
Lys Asn Asp Leu Ala Lys Thr Ala Met Ser Val Ile Val Phe Glu Asn
325 330 335
Trp Arg Thr Phe Asp Asp Leu Leu Asn Gln Glu Tyr Asp Leu Ala Asn
340 345 350
Glu Asn Lys Lys Lys Asp Asp Lys Tyr Phe Glu Lys Arg Gln Lys Glu
355 360 365
Leu Lys Lys Asn Lys Ser Tyr Ser Leu Glu His Leu Cys Asn Leu Ser
370 375 380
Glu Asp Ser Cys Asn Leu Ile Glu Asn Tyr Ile His Gln Ile Ser Asp
385 390 395 400
Asp Ile Glu Asn Ile Ile Ile Asn Asn Glu Thr Phe Leu Arg Ile Val
405 410 415
Ile Asn Glu His Asp Arg Ser Arg Lys Leu Ala Lys Asn Arg Lys Ala
420 425 430
Val Lys Ala Ile Lys Asp Phe Leu Asp Ser Ile Lys Val Leu Glu Arg
435 440 445
Glu Leu Lys Leu Ile Asn Ser Ser Gly Gln Glu Leu Glu Lys Asp Leu
450 455 460
Ile Val Tyr Ser Ala His Glu Glu Leu Leu Val Glu Leu Lys Gln Val
465 470 475 480
Asp Ser Leu Tyr Asn Met Thr Arg Asn Tyr Leu Thr Lys Lys Pro Phe
485 490 495
Ser Thr Glu Lys Val Lys Leu Asn Phe Asn Arg Ser Thr Leu Leu Asn
500 505 510
Gly Trp Asp Arg Asn Lys Glu Thr Asp Asn Leu Gly Val Leu Leu Leu
515 520 525
Lys Asp Gly Lys Tyr Tyr Leu Gly Ile Met Asn Thr Ser Ala Asn Lys
530 535 540
Ala Phe Val Asn Pro Pro Val Ala Lys Thr Glu Lys Val Phe Lys Lys
545 550 555 560
Val Asp Tyr Lys Leu Leu Pro Val Pro Asn Gln Met Leu Pro Lys Val
565 570 575
Phe Phe Ala Lys Ser Asn Ile Asp Phe Tyr Asn Pro Ser Ser Glu Ile
580 585 590
Tyr Ser Asn Tyr Lys Lys Gly Thr His Lys Lys Gly Asn Met Phe Ser
595 600 605
Leu Glu Asp Cys His Asn Leu Ile Asp Phe Phe Lys Glu Ser Ile Ser
610 615 620
Lys His Glu Asp Trp Ser Lys Phe Gly Phe Lys Phe Asp Thr Gln Ala
625 630 635 640
Ser Tyr Asn Asp Ile Ser Glu Phe Tyr Arg Glu Val Glu Lys Gln Gly
645 650 655
Tyr Lys Leu Thr Tyr Thr Asp Ile Asp Glu Thr Tyr Ile Asn Asp Leu
660 665 670
Ile Glu Arg Asn Glu Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe
675 680 685
Ser Met Tyr Ser Lys Gly Lys Leu Asn Leu His Thr Leu Tyr Phe Met
690 695 700
Met Leu Phe Asp Gln Arg Asn Ile Asp Asp Val Val Tyr Lys Leu Asn
705 710 715 720
Gly Glu Ala Glu Val Phe Tyr Arg Pro Ala Ser Ile Ser Glu Asp Glu
725 730 735
Leu Ile Ile His Lys Ala Gly Glu Glu Ile Lys Asn Lys Asn Pro Asn
740 745 750
Arg Ala Arg Thr Lys Glu Thr Ser Thr Phe Ser Tyr Asp Ile Val Lys
755 760 765
Asp Lys Arg Tyr Ser Lys Asp Lys Phe Thr Leu His Ile Pro Ile Thr
770 775 780
Met Asn Phe Gly Val Asp Glu Val Lys Arg Phe Asn Asp Ala Val Asn
785 790 795 800
Ser Ala Ile Arg Ile Asp Glu Asn Val Asn Val Ile Gly Ile Asp Arg
805 810 815
Gly Glu Arg Asn Leu Leu Tyr Val Val Val Ile Asp Ser Lys Gly Asn
820 825 830
Ile Leu Glu Gln Ile Ser Leu Asn Ser Ile Ile Asn Lys Glu Tyr Asp
835 840 845
Ile Glu Thr Asp Tyr His Ala Leu Leu Asp Glu Arg Glu Gly Gly Arg
850 855 860
Asp Lys Ala Arg Lys Asp Trp Asn Thr Val Glu Asn Ile Arg Asp Leu
865 870 875 880
Lys Ala Gly Leu Tyr Leu Gln Val Val Asn Val Val Ala Lys Leu Val
885 890 895
Leu Lys Tyr Asn Ala Ile Ile Cys Leu Glu Asp Leu Asn Phe Gly Phe
900 905 910
Lys Arg Gly Arg Gln Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu
915 920 925
Lys Met Leu Ile Asp Lys Leu Asn Tyr Leu Val Ile Asp Lys Ser Arg
930 935 940
Glu Gln Thr Ser Pro Lys Glu Leu Gly Gly Ala Leu Asn Ala Leu Gln
945 950 955 960
Leu Thr Ser Lys Phe Lys Ser Phe Lys Glu Leu Gly Lys Gln Ser Gly
965 970 975
Val Ile Tyr Tyr Val Pro Ala Tyr Leu Thr Ser Lys Ile Asp Pro Thr
980 985 990
Thr Gly Phe Ala Asn Leu Phe Tyr Met Lys Cys Glu Asn Val Glu Lys
995 1000 1005
Ser Lys Arg Phe Phe Asp Gly Phe Asp Phe Ile Arg Phe Asn Ala
1010 1015 1020
Leu Glu Asn Val Phe Glu Phe Gly Phe Asp Tyr Arg Ser Phe Thr
1025 1030 1035
Gln Arg Ala Cys Gly Ile Asn Ser Lys Trp Thr Val Cys Thr Asn
1040 1045 1050
Gly Glu Arg Ile Ile Lys Tyr Arg Asn Pro Asp Lys Asn Asn Met
1055 1060 1065
Phe Asp Glu Lys Val Val Val Val Thr Asp Glu Met Lys Asn Leu
1070 1075 1080
Phe Glu Gln Tyr Lys Ile Pro Tyr Glu Asp Gly Arg Asn Val Lys
1085 1090 1095
Asp Met Ile Ile Ser Asn Glu Glu Ala Glu Phe Tyr Arg Arg Leu
1100 1105 1110
Tyr Arg Leu Leu Gln Gln Thr Leu Gln Met Arg Asn Ser Thr Ser
1115 1120 1125
Asp Gly Thr Arg Asp Tyr Ile Ile Ser Pro Val Lys Asn Lys Arg
1130 1135 1140
Glu Ala Tyr Phe Asn Ser Glu Leu Ser Asp Gly Ser Val Pro Lys
1145 1150 1155
Asp Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly Leu
1160 1165 1170
Trp Val Leu Glu Gln Ile Arg Gln Lys Ser Glu Gly Glu Lys Ile
1175 1180 1185
Asn Leu Ala Met Thr Asn Ala Glu Trp Leu Glu Tyr Ala Gln Thr
1190 1195 1200
His Leu Leu
1205
<210> 10
<211> 1233
<212> PRT
<213> 未知
<220>
<223> 毛螺菌科细菌
<400> 10
Met Asp Tyr Gly Asn Gly Gln Phe Glu Arg Arg Ala Pro Leu Thr Lys
1 5 10 15
Thr Ile Thr Leu Arg Leu Lys Pro Ile Gly Glu Thr Arg Glu Thr Ile
20 25 30
Arg Glu Gln Lys Leu Leu Glu Gln Asp Ala Ala Phe Arg Lys Leu Val
35 40 45
Glu Thr Val Thr Pro Ile Val Asp Asp Cys Ile Arg Lys Ile Ala Asp
50 55 60
Asn Ala Leu Cys His Phe Gly Thr Glu Tyr Asp Phe Ser Cys Leu Gly
65 70 75 80
Asn Ala Ile Ser Lys Asn Asp Ser Lys Ala Ile Lys Lys Glu Thr Glu
85 90 95
Lys Val Glu Lys Leu Leu Ala Lys Val Leu Thr Glu Asn Leu Pro Asp
100 105 110
Gly Leu Arg Lys Val Asn Asp Ile Asn Ser Ala Ala Phe Ile Gln Asp
115 120 125
Thr Leu Thr Ser Phe Val Gln Asp Asp Ala Asp Lys Arg Val Leu Ile
130 135 140
Gln Glu Leu Lys Gly Lys Thr Val Leu Met Gln Arg Phe Leu Thr Thr
145 150 155 160
Arg Ile Thr Ala Leu Thr Val Trp Leu Pro Asp Arg Val Phe Glu Asn
165 170 175
Phe Asn Ile Phe Ile Glu Asn Ala Glu Lys Met Arg Ile Leu Leu Asp
180 185 190
Ser Pro Leu Asn Glu Lys Ile Met Lys Phe Asp Pro Asp Ala Glu Gln
195 200 205
Tyr Ala Ser Leu Glu Phe Tyr Gly Gln Cys Leu Ser Gln Lys Asp Ile
210 215 220
Asp Ser Tyr Asn Leu Ile Ile Ser Gly Ile Tyr Ala Asp Asp Glu Val
225 230 235 240
Lys Asn Pro Gly Ile Asn Glu Ile Val Lys Glu Tyr Asn Gln Gln Ile
245 250 255
Arg Gly Asp Lys Asp Glu Ser Pro Leu Pro Lys Leu Lys Lys Leu His
260 265 270
Lys Gln Ile Leu Met Pro Val Glu Lys Ala Phe Phe Val Arg Val Leu
275 280 285
Ser Asn Asp Ser Asp Ala Arg Ser Ile Leu Glu Lys Ile Leu Lys Asp
290 295 300
Thr Glu Met Leu Pro Ser Lys Ile Ile Glu Ala Met Lys Glu Ala Asp
305 310 315 320
Ala Gly Asp Ile Ala Val Tyr Gly Ser Arg Leu His Glu Leu Ser His
325 330 335
Val Ile Tyr Gly Asp His Gly Lys Leu Ser Gln Ile Ile Tyr Asp Lys
340 345 350
Glu Ser Lys Arg Ile Ser Glu Leu Met Glu Thr Leu Ser Pro Lys Glu
355 360 365
Arg Lys Glu Ser Lys Lys Arg Leu Glu Gly Leu Glu Glu His Ile Arg
370 375 380
Lys Ser Thr Tyr Thr Phe Asp Glu Leu Asn Arg Tyr Ala Glu Lys Asn
385 390 395 400
Val Met Ala Ala Tyr Ile Ala Ala Val Glu Glu Ser Cys Ala Glu Ile
405 410 415
Met Arg Lys Glu Lys Asp Leu Arg Thr Leu Leu Ser Lys Glu Asp Val
420 425 430
Lys Ile Arg Gly Asn Arg His Asn Thr Leu Ile Val Lys Asn Tyr Phe
435 440 445
Asn Ala Trp Thr Val Phe Arg Asn Leu Ile Arg Ile Leu Arg Arg Lys
450 455 460
Ser Glu Ala Glu Ile Asp Ser Asp Phe Tyr Asp Val Leu Asp Asp Ser
465 470 475 480
Val Glu Val Leu Ser Leu Thr Tyr Lys Gly Glu Asn Leu Cys Arg Ser
485 490 495
Tyr Ile Thr Lys Lys Ile Gly Ser Asp Leu Lys Pro Glu Ile Ala Thr
500 505 510
Tyr Gly Ser Ala Leu Arg Pro Asn Ser Arg Trp Trp Ser Pro Gly Glu
515 520 525
Lys Phe Asn Val Lys Phe His Thr Ile Val Arg Arg Asp Gly Arg Leu
530 535 540
Tyr Tyr Phe Ile Leu Pro Lys Gly Ala Lys Pro Val Glu Leu Glu Asp
545 550 555 560
Met Asp Gly Asp Ile Glu Cys Leu Gln Met Arg Lys Ile Pro Asn Pro
565 570 575
Thr Ile Phe Leu Pro Lys Leu Val Phe Lys Asp Pro Glu Ala Phe Phe
580 585 590
Arg Asp Asn Pro Glu Ala Asp Glu Phe Val Phe Leu Ser Gly Met Lys
595 600 605
Ala Pro Val Thr Ile Thr Arg Glu Thr Tyr Glu Ala Tyr Arg Tyr Lys
610 615 620
Leu Tyr Thr Val Gly Lys Leu Arg Asp Gly Glu Val Ser Glu Glu Glu
625 630 635 640
Tyr Lys Arg Ala Leu Leu Gln Val Leu Thr Ala Tyr Lys Glu Phe Leu
645 650 655
Glu Asn Arg Met Ile Tyr Ala Asp Leu Asn Phe Gly Phe Lys Asp Leu
660 665 670
Glu Glu Tyr Lys Asp Ser Ser Glu Phe Ile Lys Gln Val Glu Thr His
675 680 685
Asn Thr Phe Met Cys Trp Ala Lys Val Ser Ser Ser Gln Leu Asp Asp
690 695 700
Leu Val Lys Ser Gly Asn Gly Leu Leu Phe Glu Ile Trp Ser Glu Arg
705 710 715 720
Leu Glu Ser Tyr Tyr Lys Tyr Gly Asn Glu Lys Val Leu Arg Gly Tyr
725 730 735
Glu Gly Val Leu Leu Ser Ile Leu Lys Asp Glu Asn Leu Val Ser Met
740 745 750
Arg Thr Leu Leu Asn Ser Arg Pro Met Leu Val Tyr Arg Pro Lys Glu
755 760 765
Ser Ser Lys Pro Met Val Val His Arg Asp Gly Ser Arg Val Val Asp
770 775 780
Arg Phe Asp Lys Asp Gly Lys Tyr Ile Pro Pro Glu Val His Asp Glu
785 790 795 800
Leu Tyr Arg Phe Phe Asn Asn Leu Leu Ile Lys Glu Lys Leu Gly Glu
805 810 815
Lys Ala Arg Lys Ile Leu Asp Asn Lys Lys Val Lys Val Lys Val Leu
820 825 830
Glu Ser Glu Arg Val Lys Trp Ser Lys Phe Tyr Asp Glu Gln Phe Ala
835 840 845
Val Thr Phe Ser Val Lys Lys Asn Ala Asp Cys Leu Asp Thr Thr Lys
850 855 860
Asp Leu Asn Ala Glu Val Met Glu Gln Tyr Ser Glu Ser Asn Arg Leu
865 870 875 880
Ile Leu Ile Arg Asn Thr Thr Asp Ile Leu Tyr Tyr Leu Val Leu Asp
885 890 895
Lys Asn Gly Lys Val Leu Lys Gln Arg Ser Leu Asn Ile Ile Asn Asp
900 905 910
Gly Ala Arg Asp Val Asp Trp Lys Glu Arg Phe Arg Gln Val Thr Lys
915 920 925
Asp Arg Asn Glu Gly Tyr Asn Glu Trp Asp Tyr Ser Arg Thr Ser Asn
930 935 940
Asp Leu Lys Glu Val Tyr Leu Asn Tyr Ala Leu Lys Glu Ile Ala Glu
945 950 955 960
Ala Val Ile Glu Tyr Asn Ala Ile Leu Ile Ile Glu Lys Met Ser Asn
965 970 975
Ala Phe Lys Asp Lys Tyr Ser Phe Leu Asp Asp Val Thr Phe Lys Gly
980 985 990
Phe Glu Thr Lys Lys Leu Ala Lys Leu Ser Asp Leu His Phe Arg Gly
995 1000 1005
Ile Lys Asp Gly Glu Pro Cys Ser Phe Thr Asn Pro Leu Gln Leu
1010 1015 1020
Cys Gln Asn Asp Ser Asn Lys Ile Leu Gln Asp Gly Val Ile Phe
1025 1030 1035
Met Val Pro Asn Ser Met Thr Arg Ser Leu Asp Pro Asp Thr Gly
1040 1045 1050
Phe Ile Phe Ala Ile Asn Asp His Asn Ile Arg Thr Lys Lys Ala
1055 1060 1065
Lys Leu Asn Phe Leu Ser Lys Phe Asp Gln Leu Lys Val Ser Ser
1070 1075 1080
Glu Gly Cys Leu Ile Met Lys Tyr Ser Gly Asp Ser Leu Pro Thr
1085 1090 1095
His Asn Thr Asp Asn Arg Val Trp Asn Cys Cys Cys Asn His Pro
1100 1105 1110
Ile Thr Asn Tyr Asp Arg Glu Thr Lys Lys Val Glu Phe Ile Glu
1115 1120 1125
Glu Pro Val Glu Glu Leu Ser Arg Val Leu Glu Glu Asn Gly Ile
1130 1135 1140
Glu Thr Asp Thr Glu Leu Asn Lys Leu Asn Glu Arg Glu Asn Val
1145 1150 1155
Pro Gly Lys Val Val Asp Ala Ile Tyr Ser Leu Val Leu Asn Tyr
1160 1165 1170
Leu Arg Gly Thr Val Ser Gly Val Ala Gly Gln Arg Ala Val Tyr
1175 1180 1185
Tyr Ser Pro Val Thr Gly Lys Lys Tyr Asp Ile Ser Phe Ile Gln
1190 1195 1200
Ala Met Asn Leu Asn Arg Lys Cys Asp Tyr Tyr Arg Ile Gly Ser
1205 1210 1215
Lys Glu Arg Gly Glu Trp Thr Asp Phe Val Ala Gln Leu Ile Asn
1220 1225 1230
<210> 11
<211> 1227
<212> PRT
<213> 未知
<220>
<223> 毛螺菌科细菌
<400> 11
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 Asn Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu
805 810 815
Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile
820 825 830
Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys
835 840 845
Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe
850 855 860
Asn Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys
865 870 875 880
Glu Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn
885 890 895
Ile Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile
900 905 910
Cys Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu
915 920 925
Asn Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr
930 935 940
Gln Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp
945 950 955 960
Lys Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln
965 970 975
Ile Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly
980 985 990
Phe Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser
995 1000 1005
Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala
1010 1015 1020
Asp 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 Ser Asn Lys Glu Trp Leu
1205 1210 1215
Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 12
<211> 1264
<212> PRT
<213> 稻田氏钩端螺旋体（Leptospira inadai）
<400> 12
Met Glu Asp Tyr Ser Gly Phe Val Asn Ile Tyr Ser Ile Gln Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Lys Pro Val Gly Lys Thr Leu Glu His Ile Glu
20 25 30
Lys Lys Gly Phe Leu Lys Lys Asp Lys Ile Arg Ala Glu Asp Tyr Lys
35 40 45
Ala Val Lys Lys Ile Ile Asp Lys Tyr His Arg Ala Tyr Ile Glu Glu
50 55 60
Val Phe Asp Ser Val Leu His Gln Lys Lys Lys Lys Asp Lys Thr Arg
65 70 75 80
Phe Ser Thr Gln Phe Ile Lys Glu Ile Lys Glu Phe Ser Glu Leu Tyr
85 90 95
Tyr Lys Thr Glu Lys Asn Ile Pro Asp Lys Glu Arg Leu Glu Ala Leu
100 105 110
Ser Glu Lys Leu Arg Lys Met Leu Val Gly Ala Phe Lys Gly Glu Phe
115 120 125
Ser Glu Glu Val Ala Glu Lys Tyr Asn Lys Asn Leu Phe Ser Lys Glu
130 135 140
Leu Ile Arg Asn Glu Ile Glu Lys Phe Cys Glu Thr Asp Glu Glu Arg
145 150 155 160
Lys Gln Val Ser Asn Phe Lys Ser Phe Thr Thr Tyr Phe Thr Gly Phe
165 170 175
His Ser Asn Arg Gln Asn Ile Tyr Ser Asp Glu Lys Lys Ser Thr Ala
180 185 190
Ile Gly Tyr Arg Ile Ile His Gln Asn Leu Pro Lys Phe Leu Asp Asn
195 200 205
Leu Lys Ile Ile Glu Ser Ile Gln Arg Arg Phe Lys Asp Phe Pro Trp
210 215 220
Ser Asp Leu Lys Lys Asn Leu Lys Lys Ile Asp Lys Asn Ile Lys Leu
225 230 235 240
Thr Glu Tyr Phe Ser Ile Asp Gly Phe Val Asn Val Leu Asn Gln Lys
245 250 255
Gly Ile Asp Ala Tyr Asn Thr Ile Leu Gly Gly Lys Ser Glu Glu Ser
260 265 270
Gly Glu Lys Ile Gln Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Arg Gln
275 280 285
Lys Asn Asn Ile Asp Arg Lys Asn Pro Leu Asn Val Lys Ile Leu Phe
290 295 300
Lys Gln Ile Leu Gly Asp Arg Glu Thr Lys Ser Phe Ile Pro Glu Ala
305 310 315 320
Phe Pro Asp Asp Gln Ser Val Leu Asn Ser Ile Thr Glu Phe Ala Lys
325 330 335
Tyr Leu Lys Leu Asp Lys Lys Lys Lys Ser Ile Ile Ala Glu Leu Lys
340 345 350
Lys Phe Leu Ser Ser Phe Asn Arg Tyr Glu Leu Asp Gly Ile Tyr Leu
355 360 365
Ala Asn Asp Asn Ser Leu Ala Ser Ile Ser Thr Phe Leu Phe Asp Asp
370 375 380
Trp Ser Phe Ile Lys Lys Ser Val Ser Phe Lys Tyr Asp Glu Ser Val
385 390 395 400
Gly Asp Pro Lys Lys Lys Ile Lys Ser Pro Leu Lys Tyr Glu Lys Glu
405 410 415
Lys Glu Lys Trp Leu Lys Gln Lys Tyr Tyr Thr Ile Ser Phe Leu Asn
420 425 430
Asp Ala Ile Glu Ser Tyr Ser Lys Ser Gln Asp Glu Lys Arg Val Lys
435 440 445
Ile Arg Leu Glu Ala Tyr Phe Ala Glu Phe Lys Ser Lys Asp Asp Ala
450 455 460
Lys Lys Gln Phe Asp Leu Leu Glu Arg Ile Glu Glu Ala Tyr Ala Ile
465 470 475 480
Val Glu Pro Leu Leu Gly Ala Glu Tyr Pro Arg Asp Arg Asn Leu Lys
485 490 495
Ala Asp Lys Lys Glu Val Gly Lys Ile Lys Asp Phe Leu Asp Ser Ile
500 505 510
Lys Ser Leu Gln Phe Phe Leu Lys Pro Leu Leu Ser Ala Glu Ile Phe
515 520 525
Asp Glu Lys Asp Leu Gly Phe Tyr Asn Gln Leu Glu Gly Tyr Tyr Glu
530 535 540
Glu Ile Asp Ile Ser Gly His Leu Tyr Asn Lys Val Arg Asn Tyr Leu
545 550 555 560
Thr Gly Lys Ile Tyr Ser Lys Glu Lys Phe Lys Leu Asn Phe Glu Asn
565 570 575
Ser Thr Leu Leu Lys Gly Trp Asp Glu Asn Arg Glu Val Ala Asn Leu
580 585 590
Cys Val Ile Phe Arg Glu Asp Gln Lys Tyr Tyr Leu Gly Val Met Asp
595 600 605
Lys Glu Asn Asn Thr Ile Leu Ser Asp Ile Pro Lys Val Lys Pro Asn
610 615 620
Glu Leu Phe Tyr Glu Lys Met Val Tyr Lys Leu Ile Pro Thr Pro His
625 630 635 640
Met Gln Leu Pro Arg Ile Ile Phe Ser Ser Asp Asn Leu Ser Ile Tyr
645 650 655
Asn Pro Ser Lys Ser Ile Leu Lys Ile Arg Glu Ala Lys Ser Phe Lys
660 665 670
Glu Gly Lys Asn Phe Lys Leu Lys Asp Cys His Lys Phe Ile Asp Phe
675 680 685
Tyr Lys Glu Ser Ile Ser Lys Asn Glu Asp Trp Ser Arg Phe Asp Phe
690 695 700
Lys Phe Ser Lys Thr Ser Ser Tyr Glu Asn Ile Ser Glu Phe Tyr Arg
705 710 715 720
Glu Val Glu Arg Gln Gly Tyr Asn Leu Asp Phe Lys Lys Val Ser Lys
725 730 735
Phe Tyr Ile Asp Ser Leu Val Glu Asp Gly Lys Leu Tyr Leu Phe Gln
740 745 750
Ile Tyr Asn Lys Asp Phe Ser Ile Phe Ser Lys Gly Lys Pro Asn Leu
755 760 765
His Thr Ile Tyr Phe Arg Ser Leu Phe Ser Lys Glu Asn Leu Lys Asp
770 775 780
Val Cys Leu Lys Leu Asn Gly Glu Ala Glu Met Phe Phe Arg Lys Lys
785 790 795 800
Ser Ile Asn Tyr Asp Glu Lys Lys Lys Arg Glu Gly His His Pro Glu
805 810 815
Leu Phe Glu Lys Leu Lys Tyr Pro Ile Leu Lys Asp Lys Arg Tyr Ser
820 825 830
Glu Asp Lys Phe Gln Phe His Leu Pro Ile Ser Leu Asn Phe Lys Ser
835 840 845
Lys Glu Arg Leu Asn Phe Asn Leu Lys Val Asn Glu Phe Leu Lys Arg
850 855 860
Asn Lys Asp Ile Asn Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu
865 870 875 880
Leu Tyr Leu Val Met Ile Asn Gln Lys Gly Glu Ile Leu Lys Gln Thr
885 890 895
Leu Leu Asp Ser Met Gln Ser Gly Lys Gly Arg Pro Glu Ile Asn Tyr
900 905 910
Lys Glu Lys Leu Gln Glu Lys Glu Ile Glu Arg Asp Lys Ala Arg Lys
915 920 925
Ser Trp Gly Thr Val Glu Asn Ile Lys Glu Leu Lys Glu Gly Tyr Leu
930 935 940
Ser Ile Val Ile His Gln Ile Ser Lys Leu Met Val Glu Asn Asn Ala
945 950 955 960
Ile Val Val Leu Glu Asp Leu Asn Ile Gly Phe Lys Arg Gly Arg Gln
965 970 975
Lys Val Glu Arg Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp
980 985 990
Lys Leu Asn Phe Leu Val Phe Lys Glu Asn Lys Pro Thr Glu Pro Gly
995 1000 1005
Gly Val Leu Lys Ala Tyr Gln Leu Thr Asp Glu Phe Gln Ser Phe
1010 1015 1020
Glu Lys Leu Ser Lys Gln Thr Gly Phe Leu Phe Tyr Val Pro Ser
1025 1030 1035
Trp Asn Thr Ser Lys Ile Asp Pro Arg Thr Gly Phe Ile Asp Phe
1040 1045 1050
Leu His Pro Ala Tyr Glu Asn Ile Glu Lys Ala Lys Gln Trp Ile
1055 1060 1065
Asn Lys Phe Asp Ser Ile Arg Phe Asn Ser Lys Met Asp Trp Phe
1070 1075 1080
Glu Phe Thr Ala Asp Thr Arg Lys Phe Ser Glu Asn Leu Met Leu
1085 1090 1095
Gly Lys Asn Arg Val Trp Val Ile Cys Thr Thr Asn Val Glu Arg
1100 1105 1110
Tyr Phe Thr Ser Lys Thr Ala Asn Ser Ser Ile Gln Tyr Asn Ser
1115 1120 1125
Ile Gln Ile Thr Glu Lys Leu Lys Glu Leu Phe Val Asp Ile Pro
1130 1135 1140
Phe Ser Asn Gly Gln Asp Leu Lys Pro Glu Ile Leu Arg Lys Asn
1145 1150 1155
Asp Ala Val Phe Phe Lys Ser Leu Leu Phe Tyr Ile Lys Thr Thr
1160 1165 1170
Leu Ser Leu Arg Gln Asn Asn Gly Lys Lys Gly Glu Glu Glu Lys
1175 1180 1185
Asp Phe Ile Leu Ser Pro Val Val Asp Ser Lys Gly Arg Phe Phe
1190 1195 1200
Asn Ser Leu Glu Ala Ser Asp Asp Glu Pro Lys Asp Ala Asp Ala
1205 1210 1215
Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Leu Met Asn Leu Leu
1220 1225 1230
Val Leu Asn Glu Thr Lys Glu Glu Asn Leu Ser Arg Pro Lys Trp
1235 1240 1245
Lys Ile Lys Asn Lys Asp Trp Leu Glu Phe Val Trp Glu Arg Asn
1250 1255 1260
Arg
<210> 13
<211> 1373
<212> PRT
<213> 牛眼莫拉氏菌（Moraxella bovoculi）
<400> 13
Met Leu Phe Gln Asp Phe Thr His Leu Tyr Pro Leu Ser Lys Thr Val
1 5 10 15
Arg Phe Glu Leu Phe Ile Asp Arg Thr Leu Glu His Ile His Ala Lys
20 25 30
Asn Phe Leu Ser Gln Asp Glu Thr Met Ala Asp Met His Gln Lys Val
35 40 45
Lys Val Ile Leu Asp Asp Tyr His Arg Asp Phe Ile Ala Asp Met Met
50 55 60
Gly Glu Val Lys Leu Thr Lys Leu Ala Glu Phe Tyr Asp Val Tyr Leu
65 70 75 80
Lys Phe Arg Lys Asn Pro Lys Asp Asp Glu Leu Gln Lys Ala Gln Leu
85 90 95
Lys Asp Leu Gln Ala Val Leu Arg Lys Glu Ile Val Lys Pro Ile Gly
100 105 110
Asn Gly Gly Lys Tyr Lys Ala Gly Tyr Asp Arg Leu Phe Gly Ala Lys
115 120 125
Leu Phe Lys Asp Gly Lys Glu Leu Gly Asp Leu Ala Lys Phe Val Ile
130 135 140
Ala Gln Glu Gly Glu Ser Ser Pro Lys Leu Ala His Leu Ala His Phe
145 150 155 160
Glu Lys Phe Ser Thr Tyr Phe Thr Gly Phe His Asp Asn Arg Lys Asn
165 170 175
Met Tyr Ser Asp Glu Asp Lys His Thr Ala Ile Ala Tyr Arg Leu Ile
180 185 190
His Glu Asn Leu Pro Arg Phe Ile Asp Asn Leu Gln Ile Leu Thr Thr
195 200 205
Ile Lys Gln Lys His Ser Ala Leu Tyr Asp Gln Ile Ile Asn Glu Leu
210 215 220
Thr Ala Ser Gly Leu Asp Val Ser Leu Ala Ser His Leu Asp Gly Tyr
225 230 235 240
His Lys Leu Leu Thr Gln Glu Gly Ile Thr Ala Tyr Asn Thr Leu Leu
245 250 255
Gly Gly Ile Ser Gly Glu Ala Gly Ser Pro Lys Ile Gln Gly Ile Asn
260 265 270
Glu Leu Ile Asn Ser His His Asn Gln His Cys His Lys Ser Glu Arg
275 280 285
Ile Ala Lys Leu Arg Pro Leu His Lys Gln Ile Leu Ser Asp Gly Met
290 295 300
Ser Val Ser Phe Leu Pro Ser Lys Phe Ala Asp Asp Ser Glu Met Cys
305 310 315 320
Gln Ala Val Asn Glu Phe Tyr Arg His Tyr Ala Asp Val Phe Ala Lys
325 330 335
Val Gln Ser Leu Phe Asp Gly Phe Asp Asp His Gln Lys Asp Gly Ile
340 345 350
Tyr Val Glu His Lys Asn Leu Asn Glu Leu Ser Lys Gln Ala Phe Gly
355 360 365
Asp Phe Ala Leu Leu Gly Arg Val Leu Asp Gly Tyr Tyr Val Asp Val
370 375 380
Val Asn Pro Glu Phe Asn Glu Arg Phe Ala Lys Ala Lys Thr Asp Asn
385 390 395 400
Ala Lys Ala Lys Leu Thr Lys Glu Lys Asp Lys Phe Ile Lys Gly Val
405 410 415
His Ser Leu Ala Ser Leu Glu Gln Ala Ile Glu His Tyr Thr Ala Arg
420 425 430
His Asp Asp Glu Ser Val Gln Ala Gly Lys Leu Gly Gln Tyr Phe Lys
435 440 445
His Gly Leu Ala Gly Val Asp Asn Pro Ile Gln Lys Ile His Asn Asn
450 455 460
His Ser Thr Ile Lys Gly Phe Leu Glu Arg Glu Arg Pro Ala Gly Glu
465 470 475 480
Arg Ala Leu Pro Lys Ile Lys Ser Gly Lys Asn Pro Glu Met Thr Gln
485 490 495
Leu Arg Gln Leu Lys Glu Leu Leu Asp Asn Ala Leu Asn Val Ala His
500 505 510
Phe Ala Lys Leu Leu Thr Thr Lys Thr Thr Leu Asp Asn Gln Asp Gly
515 520 525
Asn Phe Tyr Gly Glu Phe Gly Val Leu Tyr Asp Glu Leu Ala Lys Ile
530 535 540
Pro Thr Leu Tyr Asn Lys Val Arg Asp Tyr Leu Ser Gln Lys Pro Phe
545 550 555 560
Ser Thr Glu Lys Tyr Lys Leu Asn Phe Gly Asn Pro Thr Leu Leu Asn
565 570 575
Gly Trp Asp Leu Asn Lys Glu Lys Asp Asn Phe Gly Val Ile Leu Gln
580 585 590
Lys Asp Gly Cys Tyr Tyr Leu Ala Leu Leu Asp Lys Ala His Lys Lys
595 600 605
Val Phe Asp Asn Ala Pro Asn Thr Gly Lys Ser Ile Tyr Gln Lys Met
610 615 620
Ile Tyr Lys Tyr Leu Glu Val Arg Lys Gln Phe Pro Lys Val Phe Phe
625 630 635 640
Ser Lys Glu Ala Ile Ala Ile Asn Tyr His Pro Ser Lys Glu Leu Val
645 650 655
Glu Ile Lys Asp Lys Gly Arg Gln Arg Ser Asp Asp Glu Arg Leu Lys
660 665 670
Leu Tyr Arg Phe Ile Leu Glu Cys Leu Lys Ile His Pro Lys Tyr Asp
675 680 685
Lys Lys Phe Glu Gly Ala Ile Gly Asp Ile Gln Leu Phe Lys Lys Asp
690 695 700
Lys Lys Gly Arg Glu Val Pro Ile Ser Glu Lys Asp Leu Phe Lys Asp
705 710 715 720
Ile Asn Gly Ile Phe Ser Ser Lys Pro Lys Leu Glu Met Glu Asp Phe
725 730 735
Phe Ile Gly Glu Phe Lys Arg Tyr Asn Pro Ser Gln Asp Leu Val Asp
740 745 750
Gln Tyr Asn Ile Tyr Lys Lys Ile Asp Ser Asn Asp Asn Arg Lys Lys
755 760 765
Glu Asn Phe Tyr Asn Asn His Pro Lys Phe Lys Lys Asp Leu Val Arg
770 775 780
Tyr Tyr Tyr Glu Ser Met Cys Lys His Glu Glu Trp Glu Glu Ser Phe
785 790 795 800
Glu Phe Ser Lys Lys Leu Gln Asp Ile Gly Cys Tyr Val Asp Val Asn
805 810 815
Glu Leu Phe Thr Glu Ile Glu Thr Arg Arg Leu Asn Tyr Lys Ile Ser
820 825 830
Phe Cys Asn Ile Asn Ala Asp Tyr Ile Asp Glu Leu Val Glu Gln Gly
835 840 845
Gln Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Pro Lys Ala
850 855 860
His Gly Lys Pro Asn Leu His Thr Leu Tyr Phe Lys Ala Leu Phe Ser
865 870 875 880
Glu Asp Asn Leu Ala Asp Pro Ile Tyr Lys Leu Asn Gly Glu Ala Gln
885 890 895
Ile Phe Tyr Arg Lys Ala Ser Leu Asp Met Asn Glu Thr Thr Ile His
900 905 910
Arg Ala Gly Glu Val Leu Glu Asn Lys Asn Pro Asp Asn Pro Lys Lys
915 920 925
Arg Gln Phe Val Tyr Asp Ile Ile Lys Asp Lys Arg Tyr Thr Gln Lys
930 935 940
Asp Phe Met Leu His Val Pro Ile Thr Met Asn Phe Gly Val Gln Gly
945 950 955 960
Met Thr Ile Lys Glu Phe Asn Lys Lys Val Asn Gln Ser Ile Gln Gln
965 970 975
Tyr Asp Glu Val Asn Val Ile Gly Ile Asp Arg Gly Glu Arg His Leu
980 985 990
Leu Tyr Leu Thr Val Ile Asn Ser Lys Gly Glu Ile Leu Glu Gln Cys
995 1000 1005
Ser Leu Asn Asp Ile Thr Thr Ala Ser Ala Asn Gly Thr Gln Met
1010 1015 1020
Thr Thr Pro Tyr His Lys Ile Leu Asp Lys Arg Glu Ile Glu Arg
1025 1030 1035
Leu Asn Ala Arg Val Gly Trp Gly Glu Ile Glu Thr Ile Lys Glu
1040 1045 1050
Leu Lys Ser Gly Tyr Leu Ser His Val Val His Gln Ile Ser Gln
1055 1060 1065
Leu Met Leu Lys Tyr Asn Ala Ile Val Val Leu Glu Asp Leu Asn
1070 1075 1080
Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Ile Tyr
1085 1090 1095
Gln Asn Phe Glu Asn Ala Leu Ile Lys Lys Leu Asn His Leu Val
1100 1105 1110
Leu Lys Asp Lys Ala Asp Asp Glu Ile Gly Ser Tyr Lys Asn Ala
1115 1120 1125
Leu Gln Leu Thr Asn Asn Phe Thr Asp Leu Lys Ser Ile Gly Lys
1130 1135 1140
Gln Thr Gly Phe Leu Phe Tyr Val Pro Ala Trp Asn Thr Ser Lys
1145 1150 1155
Ile Asp Pro Glu Thr Gly Phe Val Asp Leu Leu Lys Pro Arg Tyr
1160 1165 1170
Glu Asn Ile Gln Ala Ser Gln Ala Phe Phe Gly Lys Phe Asp Lys
1175 1180 1185
Ile Cys Tyr Asn Ala Asp Lys Asp Tyr Phe Glu Phe His Ile Asp
1190 1195 1200
Tyr Ala Lys Phe Thr Asp Lys Ala Lys Asn Ser Arg Gln Ile Trp
1205 1210 1215
Thr Ile Cys Ser His Gly Asp Lys Arg Tyr Val Tyr Asp Lys Thr
1220 1225 1230
Ala Asn Gln Asn Lys Gly Ala Ala Lys Gly Ile Asn Val Asn Asp
1235 1240 1245
Ile Leu Lys Ser Leu Phe Ala Arg His His Ile Asn Glu Lys Gln
1250 1255 1260
Pro Asn Leu Val Met Asp Ile Cys Gln Asn Asn Asp Lys Glu Phe
1265 1270 1275
His Lys Ser Leu Met Tyr Leu Leu Lys Thr Leu Leu Ala Leu Arg
1280 1285 1290
Tyr Ser Asn Ala Ser Ser Asp Glu Asp Phe Ile Leu Ser Pro Val
1295 1300 1305
Ala Asn Asp Glu Gly Val Phe Phe Asn Ser Ala Leu Ala Asp Asp
1310 1315 1320
Thr Gln Pro Gln Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala
1325 1330 1335
Leu Lys Gly Leu Trp Leu Leu Asn Glu Leu Lys Asn Ser Asp Asp
1340 1345 1350
Leu Asn Lys Val Lys Leu Ala Ile Asp Asn Gln Thr Trp Leu Asn
1355 1360 1365
Phe Ala Gln Asn Arg
1370
<210> 14
<211> 1352
<212> PRT
<213> 未知
<220>
<223> Parcubacteria细菌
<400> 14
Met Glu Asn Ile Phe Asp Gln Phe Ile Gly Lys Tyr Ser Leu Ser Lys
1 5 10 15
Thr Leu Arg Phe Glu Leu Lys Pro Val Gly Lys Thr Glu Asp Phe Leu
20 25 30
Lys Ile Asn Lys Val Phe Glu Lys Asp Gln Thr Ile Asp Asp Ser Tyr
35 40 45
Asn Gln Ala Lys Phe Tyr Phe Asp Ser Leu His Gln Lys Phe Ile Asp
50 55 60
Ala Ala Leu Ala Ser Asp Lys Thr Ser Glu Leu Ser Phe Gln Asn Phe
65 70 75 80
Ala Asp Val Leu Glu Lys Gln Asn Lys Ile Ile Leu Asp Lys Lys Arg
85 90 95
Glu Met Gly Ala Leu Arg Lys Arg Asp Lys Asn Ala Val Gly Ile Asp
100 105 110
Arg Leu Gln Lys Glu Ile Asn Asp Ala Glu Asp Ile Ile Gln Lys Glu
115 120 125
Lys Glu Lys Ile Tyr Lys Asp Val Arg Thr Leu Phe Asp Asn Glu Ala
130 135 140
Glu Ser Trp Lys Thr Tyr Tyr Gln Glu Arg Glu Val Asp Gly Lys Lys
145 150 155 160
Ile Thr Glu Ser Lys Ala Asp Leu Lys Gln Lys Gly Ala Asp Phe Leu
165 170 175
Thr Ala Ala Gly Ile Leu Lys Val Leu Lys Tyr Glu Phe Pro Glu Glu
180 185 190
Lys Glu Lys Glu Phe Gln Ala Lys Asn Gln Pro Ser Leu Phe Val Glu
195 200 205
Glu Lys Glu Asn Pro Gly Gln Lys Arg Tyr Ile Phe Asp Ser Phe Asp
210 215 220
Lys Phe Ala Gly Tyr Leu Thr Lys Phe Gln Gln Thr Lys Lys Asn Leu
225 230 235 240
Tyr Ala Ala Asp Gly Thr Ser Thr Ala Val Ala Thr Arg Ile Ala Asp
245 250 255
Asn Phe Ile Ile Phe His Gln Asn Thr Lys Val Phe Arg Asp Lys Tyr
260 265 270
Lys Asn Asn His Thr Asp Leu Gly Phe Asp Glu Glu Asn Ile Phe Glu
275 280 285
Ile Glu Arg Tyr Lys Asn Cys Leu Leu Gln Arg Glu Ile Glu His Ile
290 295 300
Lys Asn Glu Asn Ser Tyr Asn Lys Ile Ile Gly Arg Ile Asn Lys Lys
305 310 315 320
Ile Lys Glu Tyr Arg Asp Gln Lys Ala Lys Asp Thr Lys Leu Thr Lys
325 330 335
Ser Asp Phe Pro Phe Phe Lys Asn Leu Asp Lys Gln Ile Leu Gly Glu
340 345 350
Val Glu Lys Glu Lys Gln Leu Ile Glu Lys Thr Arg Glu Lys Thr Glu
355 360 365
Glu Asp Val Leu Ile Glu Arg Phe Lys Glu Phe Ile Glu Asn Asn Glu
370 375 380
Glu Arg Phe Thr Ala Ala Lys Lys Leu Met Asn Ala Phe Cys Asn Gly
385 390 395 400
Glu Phe Glu Ser Glu Tyr Glu Gly Ile Tyr Leu Lys Asn Lys Ala Ile
405 410 415
Asn Thr Ile Ser Arg Arg Trp Phe Val Ser Asp Arg Asp Phe Glu Leu
420 425 430
Lys Leu Pro Gln Gln Lys Ser Lys Asn Lys Ser Glu Lys Asn Glu Pro
435 440 445
Lys Val Lys Lys Phe Ile Ser Ile Ala Glu Ile Lys Asn Ala Val Glu
450 455 460
Glu Leu Asp Gly Asp Ile Phe Lys Ala Val Phe Tyr Asp Lys Lys Ile
465 470 475 480
Ile Ala Gln Gly Gly Ser Lys Leu Glu Gln Phe Leu Val Ile Trp Lys
485 490 495
Tyr Glu Phe Glu Tyr Leu Phe Arg Asp Ile Glu Arg Glu Asn Gly Glu
500 505 510
Lys Leu Leu Gly Tyr Asp Ser Cys Leu Lys Ile Ala Lys Gln Leu Gly
515 520 525
Ile Phe Pro Gln Glu Lys Glu Ala Arg Glu Lys Ala Thr Ala Val Ile
530 535 540
Lys Asn Tyr Ala Asp Ala Gly Leu Gly Ile Phe Gln Met Met Lys Tyr
545 550 555 560
Phe Ser Leu Asp Asp Lys Asp Arg Lys Asn Thr Pro Gly Gln Leu Ser
565 570 575
Thr Asn Phe Tyr Ala Glu Tyr Asp Gly Tyr Tyr Lys Asp Phe Glu Phe
580 585 590
Ile Lys Tyr Tyr Asn Glu Phe Arg Asn Phe Ile Thr Lys Lys Pro Phe
595 600 605
Asp Glu Asp Lys Ile Lys Leu Asn Phe Glu Asn Gly Ala Leu Leu Lys
610 615 620
Gly Trp Asp Glu Asn Lys Glu Tyr Asp Phe Met Gly Val Ile Leu Lys
625 630 635 640
Lys Glu Gly Arg Leu Tyr Leu Gly Ile Met His Lys Asn His Arg Lys
645 650 655
Leu Phe Gln Ser Met Gly Asn Ala Lys Gly Asp Asn Ala Asn Arg Tyr
660 665 670
Gln Lys Met Ile Tyr Lys Gln Ile Ala Asp Ala Ser Lys Asp Val Pro
675 680 685
Arg Leu Leu Leu Thr Ser Lys Lys Ala Met Glu Lys Phe Lys Pro Ser
690 695 700
Gln Glu Ile Leu Arg Ile Lys Lys Glu Lys Thr Phe Lys Arg Glu Ser
705 710 715 720
Lys Asn Phe Ser Leu Arg Asp Leu His Ala Leu Ile Glu Tyr Tyr Arg
725 730 735
Asn Cys Ile Pro Gln Tyr Ser Asn Trp Ser Phe Tyr Asp Phe Gln Phe
740 745 750
Gln Asp Thr Gly Lys Tyr Gln Asn Ile Lys Glu Phe Thr Asp Asp Val
755 760 765
Gln Lys Tyr Gly Tyr Lys Ile Ser Phe Arg Asp Ile Asp Asp Glu Tyr
770 775 780
Ile Asn Gln Ala Leu Asn Glu Gly Lys Met Tyr Leu Phe Glu Val Val
785 790 795 800
Asn Lys Asp Ile Tyr Asn Thr Lys Asn Gly Ser Lys Asn Leu His Thr
805 810 815
Leu Tyr Phe Glu His Ile Leu Ser Ala Glu Asn Leu Asn Asp Pro Val
820 825 830
Phe Lys Leu Ser Gly Met Ala Glu Ile Phe Gln Arg Gln Pro Ser Val
835 840 845
Asn Glu Arg Glu Lys Ile Thr Thr Gln Lys Asn Gln Cys Ile Leu Asp
850 855 860
Lys Gly Asp Arg Ala Tyr Lys Tyr Arg Arg Tyr Thr Glu Lys Lys Ile
865 870 875 880
Met Phe His Met Ser Leu Val Leu Asn Thr Gly Lys Gly Glu Ile Lys
885 890 895
Gln Val Gln Phe Asn Lys Ile Ile Asn Gln Arg Ile Ser Ser Ser Asp
900 905 910
Asn Glu Met Arg Val Asn Val Ile Gly Ile Asp Arg Gly Glu Lys Asn
915 920 925
Leu Leu Tyr Tyr Ser Val Val Lys Gln Asn Gly Glu Ile Ile Glu Gln
930 935 940
Ala Ser Leu Asn Glu Ile Asn Gly Val Asn Tyr Arg Asp Lys Leu Ile
945 950 955 960
Glu Arg Glu Lys Glu Arg Leu Lys Asn Arg Gln Ser Trp Lys Pro Val
965 970 975
Val Lys Ile Lys Asp Leu Lys Lys Gly Tyr Ile Ser His Val Ile His
980 985 990
Lys Ile Cys Gln Leu Ile Glu Lys Tyr Ser Ala Ile Val Val Leu Glu
995 1000 1005
Asp Leu Asn Met Arg Phe Lys Gln Ile Arg Gly Gly Ile Glu Arg
1010 1015 1020
Ser Val Tyr Gln Gln Phe Glu Lys Ala Leu Ile Asp Lys Leu Gly
1025 1030 1035
Tyr Leu Val Phe Lys Asp Asn Arg Asp Leu Arg Ala Pro Gly Gly
1040 1045 1050
Val Leu Asn Gly Tyr Gln Leu Ser Ala Pro Phe Val Ser Phe Glu
1055 1060 1065
Lys Met Arg Lys Gln Thr Gly Ile Leu Phe Tyr Thr Gln Ala Glu
1070 1075 1080
Tyr Thr Ser Lys Thr Asp Pro Ile Thr Gly Phe Arg Lys Asn Val
1085 1090 1095
Tyr Ile Ser Asn Ser Ala Ser Leu Asp Lys Ile Lys Glu Ala Val
1100 1105 1110
Lys Lys Phe Asp Ala Ile Gly Trp Asp Gly Lys Glu Gln Ser Tyr
1115 1120 1125
Phe Phe Lys Tyr Asn Pro Tyr Asn Leu Ala Asp Glu Lys Tyr Lys
1130 1135 1140
Asn Ser Thr Val Ser Lys Glu Trp Ala Ile Phe Ala Ser Ala Pro
1145 1150 1155
Arg Ile Arg Arg Gln Lys Gly Glu Asp Gly Tyr Trp Lys Tyr Asp
1160 1165 1170
Arg Val Lys Val Asn Glu Glu Phe Glu Lys Leu Leu Lys Val Trp
1175 1180 1185
Asn Phe Val Asn Pro Lys Ala Thr Asp Ile Lys Gln Glu Ile Ile
1190 1195 1200
Lys Lys Ile Lys Ala Gly Asp Leu Gln Gly Glu Lys Glu Leu Asp
1205 1210 1215
Gly Arg Leu Arg Asn Phe Trp His Ser Phe Ile Tyr Leu Phe Asn
1220 1225 1230
Leu Val Leu Glu Leu Arg Asn Ser Phe Ser Leu Gln Ile Lys Ile
1235 1240 1245
Lys Ala Gly Glu Val Ile Ala Val Asp Glu Gly Val Asp Phe Ile
1250 1255 1260
Ala Ser Pro Val Lys Pro Phe Phe Thr Thr Pro Asn Pro Tyr Ile
1265 1270 1275
Pro Ser Asn Leu Cys Trp Leu Ala Val Glu Asn Ala Asp Ala Asn
1280 1285 1290
Gly Ala Tyr Asn Ile Ala Arg Lys Gly Val Met Ile Leu Lys Lys
1295 1300 1305
Ile Arg Glu His Ala Lys Lys Asp Pro Glu Phe Lys Lys Leu Pro
1310 1315 1320
Asn Leu Phe Ile Ser Asn Ala Glu Trp Asp Glu Ala Ala Arg Asp
1325 1330 1335
Trp Gly Lys Tyr Ala Gly Thr Thr Ala Leu Asn Leu Asp His
1340 1345 1350
<210> 15
<211> 1260
<212> PRT
<213> 狗口腔卟啉单胞菌（Porphyromonas crevioricanis）
<400> 15
Met Asp Ser Leu Lys Asp Phe Thr Asn Leu Tyr Pro Val Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Lys Pro Val Gly Lys Thr Leu Glu Asn Ile Glu
20 25 30
Lys Ala Gly Ile Leu Lys Glu Asp Glu His Arg Ala Glu Ser Tyr Arg
35 40 45
Arg Val Lys Lys Ile Ile Asp Thr Tyr His Lys Val Phe Ile Asp Ser
50 55 60
Ser Leu Glu Asn Met Ala Lys Met Gly Ile Glu Asn Glu Ile Lys Ala
65 70 75 80
Met Leu Gln Ser Phe Cys Glu Leu Tyr Lys Lys Asp His Arg Thr Glu
85 90 95
Gly Glu Asp Lys Ala Leu Asp Lys Ile Arg Ala Val Leu Arg Gly Leu
100 105 110
Ile Val Gly Ala Phe Thr Gly Val Cys Gly Arg Arg Glu Asn Thr Val
115 120 125
Gln Asn Glu Lys Tyr Glu Ser Leu Phe Lys Glu Lys Leu Ile Lys Glu
130 135 140
Ile Leu Pro Asp Phe Val Leu Ser Thr Glu Ala Glu Ser Leu Pro Phe
145 150 155 160
Ser Val Glu Glu Ala Thr Arg Ser Leu Lys Glu Phe Asp Ser Phe Thr
165 170 175
Ser Tyr Phe Ala Gly Phe Tyr Glu Asn Arg Lys Asn Ile Tyr Ser Thr
180 185 190
Lys Pro Gln Ser Thr Ala Ile Ala Tyr Arg Leu Ile His Glu Asn Leu
195 200 205
Pro Lys Phe Ile Asp Asn Ile Leu Val Phe Gln Lys Ile Lys Glu Pro
210 215 220
Ile Ala Lys Glu Leu Glu His Ile Arg Ala Asp Phe Ser Ala Gly Gly
225 230 235 240
Tyr Ile Lys Lys Asp Glu Arg Leu Glu Asp Ile Phe Ser Leu Asn Tyr
245 250 255
Tyr Ile His Val Leu Ser Gln Ala Gly Ile Glu Lys Tyr Asn Ala Leu
260 265 270
Ile Gly Lys Ile Val Thr Glu Gly Asp Gly Glu Met Lys Gly Leu Asn
275 280 285
Glu His Ile Asn Leu Tyr Asn Gln Gln Arg Gly Arg Glu Asp Arg Leu
290 295 300
Pro Leu Phe Arg Pro Leu Tyr Lys Gln Ile Leu Ser Asp Arg Glu Gln
305 310 315 320
Leu Ser Tyr Leu Pro Glu Ser Phe Glu Lys Asp Glu Glu Leu Leu Arg
325 330 335
Ala Leu Lys Glu Phe Tyr Asp His Ile Ala Glu Asp Ile Leu Gly Arg
340 345 350
Thr Gln Gln Leu Met Thr Ser Ile Ser Glu Tyr Asp Leu Ser Arg Ile
355 360 365
Tyr Val Arg Asn Asp Ser Gln Leu Thr Asp Ile Ser Lys Lys Met Leu
370 375 380
Gly Asp Trp Asn Ala Ile Tyr Met Ala Arg Glu Arg Ala Tyr Asp His
385 390 395 400
Glu Gln Ala Pro Lys Arg Ile Thr Ala Lys Tyr Glu Arg Asp Arg Ile
405 410 415
Lys Ala Leu Lys Gly Glu Glu Ser Ile Ser Leu Ala Asn Leu Asn Ser
420 425 430
Cys Ile Ala Phe Leu Asp Asn Val Arg Asp Cys Arg Val Asp Thr Tyr
435 440 445
Leu Ser Thr Leu Gly Gln Lys Glu Gly Pro His Gly Leu Ser Asn Leu
450 455 460
Val Glu Asn Val Phe Ala Ser Tyr His Glu Ala Glu Gln Leu Leu Ser
465 470 475 480
Phe Pro Tyr Pro Glu Glu Asn Asn Leu Ile Gln Asp Lys Asp Asn Val
485 490 495
Val Leu Ile Lys Asn Leu Leu Asp Asn Ile Ser Asp Leu Gln Arg Phe
500 505 510
Leu Lys Pro Leu Trp Gly Met Gly Asp Glu Pro Asp Lys Asp Glu Arg
515 520 525
Phe Tyr Gly Glu Tyr Asn Tyr Ile Arg Gly Ala Leu Asp Gln Val Ile
530 535 540
Pro Leu Tyr Asn Lys Val Arg Asn Tyr Leu Thr Arg Lys Pro Tyr Ser
545 550 555 560
Thr Arg Lys Val Lys Leu Asn Phe Gly Asn Ser Gln Leu Leu Ser Gly
565 570 575
Trp Asp Arg Asn Lys Glu Lys Asp Asn Ser Cys Val Ile Leu Arg Lys
580 585 590
Gly Gln Asn Phe Tyr Leu Ala Ile Met Asn Asn Arg His Lys Arg Ser
595 600 605
Phe Glu Asn Lys Met Leu Pro Glu Tyr Lys Glu Gly Glu Pro Tyr Phe
610 615 620
Glu Lys Met Asp Tyr Lys Phe Leu Pro Asp Pro Asn Lys Met Leu Pro
625 630 635 640
Lys Val Phe Leu Ser Lys Lys Gly Ile Glu Ile Tyr Lys Pro Ser Pro
645 650 655
Lys Leu Leu Glu Gln Tyr Gly His Gly Thr His Lys Lys Gly Asp Thr
660 665 670
Phe Ser Met Asp Asp Leu His Glu Leu Ile Asp Phe Phe Lys His Ser
675 680 685
Ile Glu Ala His Glu Asp Trp Lys Gln Phe Gly Phe Lys Phe Ser Asp
690 695 700
Thr Ala Thr Tyr Glu Asn Val Ser Ser Phe Tyr Arg Glu Val Glu Asp
705 710 715 720
Gln Gly Tyr Lys Leu Ser Phe Arg Lys Val Ser Glu Ser Tyr Val Tyr
725 730 735
Ser Leu Ile Asp Gln Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys
740 745 750
Asp Phe Ser Pro Cys Ser Lys Gly Thr Pro Asn Leu His Thr Leu Tyr
755 760 765
Trp Arg Met Leu Phe Asp Glu Arg Asn Leu Ala Asp Val Ile Tyr Lys
770 775 780
Leu Asp Gly Lys Ala Glu Ile Phe Phe Arg Glu Lys Ser Leu Lys Asn
785 790 795 800
Asp His Pro Thr His Pro Ala Gly Lys Pro Ile Lys Lys Lys Ser Arg
805 810 815
Gln Lys Lys Gly Glu Glu Ser Leu Phe Glu Tyr Asp Leu Val Lys Asp
820 825 830
Arg Arg Tyr Thr Met Asp Lys Phe Gln Phe His Val Pro Ile Thr Met
835 840 845
Asn Phe Lys Cys Ser Ala Gly Ser Lys Val Asn Asp Met Val Asn Ala
850 855 860
His Ile Arg Glu Ala Lys Asp Met His Val Ile Gly Ile Asp Arg Gly
865 870 875 880
Glu Arg Asn Leu Leu Tyr Ile Cys Val Ile Asp Ser Arg Gly Thr Ile
885 890 895
Leu Asp Gln Ile Ser Leu Asn Thr Ile Asn Asp Ile Asp Tyr His Asp
900 905 910
Leu Leu Glu Ser Arg Asp Lys Asp Arg Gln Gln Glu His Arg Asn Trp
915 920 925
Gln Thr Ile Glu Gly Ile Lys Glu Leu Lys Gln Gly Tyr Leu Ser Gln
930 935 940
Ala Val His Arg Ile Ala Glu Leu Met Val Ala Tyr Lys Ala Val Val
945 950 955 960
Ala Leu Glu Asp Leu Asn Met Gly Phe Lys Arg Gly Arg Gln Lys Val
965 970 975
Glu Ser Ser Val Tyr Gln Gln Phe Glu Lys Gln Leu Ile Asp Lys Leu
980 985 990
Asn Tyr Leu Val Asp Lys Lys Lys Arg Pro Glu Asp Ile Gly Gly Leu
995 1000 1005
Leu Arg Ala Tyr Gln Phe Thr Ala Pro Phe Lys Ser Phe Lys Glu
1010 1015 1020
Met Gly Lys Gln Asn Gly Phe Leu Phe Tyr Ile Pro Ala Trp Asn
1025 1030 1035
Thr Ser Asn Ile Asp Pro Thr Thr Gly Phe Val Asn Leu Phe His
1040 1045 1050
Val Gln Tyr Glu Asn Val Asp Lys Ala Lys Ser Phe Phe Gln Lys
1055 1060 1065
Phe Asp Ser Ile Ser Tyr Asn Pro Lys Lys Asp Trp Phe Glu Phe
1070 1075 1080
Ala Phe Asp Tyr Lys Asn Phe Thr Lys Lys Ala Glu Gly Ser Arg
1085 1090 1095
Ser Met Trp Ile Leu Cys Thr His Gly Ser Arg Ile Lys Asn Phe
1100 1105 1110
Arg Asn Ser Gln Lys Asn Gly Gln Trp Asp Ser Glu Glu Phe Ala
1115 1120 1125
Leu Thr Glu Ala Phe Lys Ser Leu Phe Val Arg Tyr Glu Ile Asp
1130 1135 1140
Tyr Thr Ala Asp Leu Lys Thr Ala Ile Val Asp Glu Lys Gln Lys
1145 1150 1155
Asp Phe Phe Val Asp Leu Leu Lys Leu Phe Lys Leu Thr Val Gln
1160 1165 1170
Met Arg Asn Ser Trp Lys Glu Lys Asp Leu Asp Tyr Leu Ile Ser
1175 1180 1185
Pro Val Ala Gly Ala Asp Gly Arg Phe Phe Asp Thr Arg Glu Gly
1190 1195 1200
Asn Lys Ser Leu Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr Asn
1205 1210 1215
Ile Ala Leu Lys Gly Leu Trp Ala Leu Arg Gln Ile Arg Gln Thr
1220 1225 1230
Ser Glu Gly Gly Lys Leu Lys Leu Ala Ile Ser Asn Lys Glu Trp
1235 1240 1245
Leu Gln Phe Val Gln Glu Arg Ser Tyr Glu Lys Asp
1250 1255 1260
<210> 16
<211> 1324
<212> PRT
<213> 解糖胨普雷沃氏菌（Prevotella disiens）
<400> 16
Met Glu Asn Tyr Gln Glu Phe Thr Asn Leu Phe Gln Leu Asn Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Lys Pro Ile Gly Lys Thr Cys Glu Leu Leu Glu
20 25 30
Glu Gly Lys Ile Phe Ala Ser Gly Ser Phe Leu Glu Lys Asp Lys Val
35 40 45
Arg Ala Asp Asn Val Ser Tyr Val Lys Lys Glu Ile Asp Lys Lys His
50 55 60
Lys Ile Phe Ile Glu Glu Thr Leu Ser Ser Phe Ser Ile Ser Asn Asp
65 70 75 80
Leu Leu Lys Gln Tyr Phe Asp Cys Tyr Asn Glu Leu Lys Ala Phe Lys
85 90 95
Lys Asp Cys Lys Ser Asp Glu Glu Glu Val Lys Lys Thr Ala Leu Arg
100 105 110
Asn Lys Cys Thr Ser Ile Gln Arg Ala Met Arg Glu Ala Ile Ser Gln
115 120 125
Ala Phe Leu Lys Ser Pro Gln Lys Lys Leu Leu Ala Ile Lys Asn Leu
130 135 140
Ile Glu Asn Val Phe Lys Ala Asp Glu Asn Val Gln His Phe Ser Glu
145 150 155 160
Phe Thr Ser Tyr Phe Ser Gly Phe Glu Thr Asn Arg Glu Asn Phe Tyr
165 170 175
Ser Asp Glu Glu Lys Ser Thr Ser Ile Ala Tyr Arg Leu Val His Asp
180 185 190
Asn Leu Pro Ile Phe Ile Lys Asn Ile Tyr Ile Phe Glu Lys Leu Lys
195 200 205
Glu Gln Phe Asp Ala Lys Thr Leu Ser Glu Ile Phe Glu Asn Tyr Lys
210 215 220
Leu Tyr Val Ala Gly Ser Ser Leu Asp Glu Val Phe Ser Leu Glu Tyr
225 230 235 240
Phe Asn Asn Thr Leu Thr Gln Lys Gly Ile Asp Asn Tyr Asn Ala Val
245 250 255
Ile Gly Lys Ile Val Lys Glu Asp Lys Gln Glu Ile Gln Gly Leu Asn
260 265 270
Glu His Ile Asn Leu Tyr Asn Gln Lys His Lys Asp Arg Arg Leu Pro
275 280 285
Phe Phe Ile Ser Leu Lys Lys Gln Ile Leu Ser Asp Arg Glu Ala Leu
290 295 300
Ser Trp Leu Pro Asp Met Phe Lys Asn Asp Ser Glu Val Ile Asp Ala
305 310 315 320
Leu Lys Gly Phe Tyr Ile Glu Asp Gly Phe Glu Asn Asn Val Leu Thr
325 330 335
Pro Leu Ala Thr Leu Leu Ser Ser Leu Asp Lys Tyr Asn Leu Asn Gly
340 345 350
Ile Phe Ile Arg Asn Asn Glu Ala Leu Ser Ser Leu Ser Gln Asn Val
355 360 365
Tyr Arg Asn Phe Ser Ile Asp Glu Ala Ile Asp Ala Gln Asn Ala Glu
370 375 380
Leu Gln Thr Phe Asn Asn Tyr Glu Leu Ile Ala Asn Ala Leu Arg Ala
385 390 395 400
Lys Ile Lys Lys Glu Thr Lys Gln Gly Arg Lys Ser Phe Glu Lys Tyr
405 410 415
Glu Glu Tyr Ile Asp Lys Lys Val Lys Ala Ile Asp Ser Leu Ser Ile
420 425 430
Gln Glu Ile Asn Glu Leu Val Glu Asn Tyr Val Ser Glu Phe Asn Ser
435 440 445
Asn Ser Gly Asn Met Pro Arg Lys Val Glu Asp Tyr Phe Ser Leu Met
450 455 460
Arg Lys Gly Asp Phe Gly Ser Asn Asp Leu Ile Glu Asn Ile Lys Thr
465 470 475 480
Lys Leu Ser Ala Ala Glu Lys Leu Leu Gly Thr Lys Tyr Gln Glu Thr
485 490 495
Ala Lys Asp Ile Phe Lys Lys Asp Glu Asn Ser Lys Leu Ile Lys Glu
500 505 510
Leu Leu Asp Ala Thr Lys Gln Phe Gln His Phe Ile Lys Pro Leu Leu
515 520 525
Gly Thr Gly Glu Glu Ala Asp Arg Asp Leu Val Phe Tyr Gly Asp Phe
530 535 540
Leu Pro Leu Tyr Glu Lys Phe Glu Glu Leu Thr Leu Leu Tyr Asn Lys
545 550 555 560
Val Arg Asn Arg Leu Thr Gln Lys Pro Tyr Ser Lys Asp Lys Ile Arg
565 570 575
Leu Cys Phe Asn Lys Pro Lys Leu Met Thr Gly Trp Val Asp Ser Lys
580 585 590
Thr Glu Lys Ser Asp Asn Gly Thr Gln Tyr Gly Gly Tyr Leu Phe Arg
595 600 605
Lys Lys Asn Glu Ile Gly Glu Tyr Asp Tyr Phe Leu Gly Ile Ser Ser
610 615 620
Lys Ala Gln Leu Phe Arg Lys Asn Glu Ala Val Ile Gly Asp Tyr Glu
625 630 635 640
Arg Leu Asp Tyr Tyr Gln Pro Lys Ala Asn Thr Ile Tyr Gly Ser Ala
645 650 655
Tyr Glu Gly Glu Asn Ser Tyr Lys Glu Asp Lys Lys Arg Leu Asn Lys
660 665 670
Val Ile Ile Ala Tyr Ile Glu Gln Ile Lys Gln Thr Asn Ile Lys Lys
675 680 685
Ser Ile Ile Glu Ser Ile Ser Lys Tyr Pro Asn Ile Ser Asp Asp Asp
690 695 700
Lys Val Thr Pro Ser Ser Leu Leu Glu Lys Ile Lys Lys Val Ser Ile
705 710 715 720
Asp Ser Tyr Asn Gly Ile Leu Ser Phe Lys Ser Phe Gln Ser Val Asn
725 730 735
Lys Glu Val Ile Asp Asn Leu Leu Lys Thr Ile Ser Pro Leu Lys Asn
740 745 750
Lys Ala Glu Phe Leu Asp Leu Ile Asn Lys Asp Tyr Gln Ile Phe Thr
755 760 765
Glu Val Gln Ala Val Ile Asp Glu Ile Cys Lys Gln Lys Thr Phe Ile
770 775 780
Tyr Phe Pro Ile Ser Asn Val Glu Leu Glu Lys Glu Met Gly Asp Lys
785 790 795 800
Asp Lys Pro Leu Cys Leu Phe Gln Ile Ser Asn Lys Asp Leu Ser Phe
805 810 815
Ala Lys Thr Phe Ser Ala Asn Leu Arg Lys Lys Arg Gly Ala Glu Asn
820 825 830
Leu His Thr Met Leu Phe Lys Ala Leu Met Glu Gly Asn Gln Asp Asn
835 840 845
Leu Asp Leu Gly Ser Gly Ala Ile Phe Tyr Arg Ala Lys Ser Leu Asp
850 855 860
Gly Asn Lys Pro Thr His Pro Ala Asn Glu Ala Ile Lys Cys Arg Asn
865 870 875 880
Val Ala Asn Lys Asp Lys Val Ser Leu Phe Thr Tyr Asp Ile Tyr Lys
885 890 895
Asn Arg Arg Tyr Met Glu Asn Lys Phe Leu Phe His Leu Ser Ile Val
900 905 910
Gln Asn Tyr Lys Ala Ala Asn Asp Ser Ala Gln Leu Asn Ser Ser Ala
915 920 925
Thr Glu Tyr Ile Arg Lys Ala Asp Asp Leu His Ile Ile Gly Ile Asp
930 935 940
Arg Gly Glu Arg Asn Leu Leu Tyr Tyr Ser Val Ile Asp Met Lys Gly
945 950 955 960
Asn Ile Val Glu Gln Asp Ser Leu Asn Ile Ile Arg Asn Asn Asp Leu
965 970 975
Glu Thr Asp Tyr His Asp Leu Leu Asp Lys Arg Glu Lys Glu Arg Lys
980 985 990
Ala Asn Arg Gln Asn Trp Glu Ala Val Glu Gly Ile Lys Asp Leu Lys
995 1000 1005
Lys Gly Tyr Leu Ser Gln Ala Val His Gln Ile Ala Gln Leu Met
1010 1015 1020
Leu Lys Tyr Asn Ala Ile Ile Ala Leu Glu Asp Leu Gly Gln Met
1025 1030 1035
Phe Val Thr Arg Gly Gln Lys Ile Glu Lys Ala Val Tyr Gln Gln
1040 1045 1050
Phe Glu Lys Ser Leu Val Asp Lys Leu Ser Tyr Leu Val Asp Lys
1055 1060 1065
Lys Arg Pro Tyr Asn Glu Leu Gly Gly Ile Leu Lys Ala Tyr Gln
1070 1075 1080
Leu Ala Ser Ser Ile Thr Lys Asn Asn Ser Asp Lys Gln Asn Gly
1085 1090 1095
Phe Leu Phe Tyr Val Pro Ala Trp Asn Thr Ser Lys Ile Asp Pro
1100 1105 1110
Val Thr Gly Phe Thr Asp Leu Leu Arg Pro Lys Ala Met Thr Ile
1115 1120 1125
Lys Glu Ala Gln Asp Phe Phe Gly Ala Phe Asp Asn Ile Ser Tyr
1130 1135 1140
Asn Asp Lys Gly Tyr Phe Glu Phe Glu Thr Asn Tyr Asp Lys Phe
1145 1150 1155
Lys Ile Arg Met Lys Ser Ala Gln Thr Arg Trp Thr Ile Cys Thr
1160 1165 1170
Phe Gly Asn Arg Ile Lys Arg Lys Lys Asp Lys Asn Tyr Trp Asn
1175 1180 1185
Tyr Glu Glu Val Glu Leu Thr Glu Glu Phe Lys Lys Leu Phe Lys
1190 1195 1200
Asp Ser Asn Ile Asp Tyr Glu Asn Cys Asn Leu Lys Glu Glu Ile
1205 1210 1215
Gln Asn Lys Asp Asn Arg Lys Phe Phe Asp Asp Leu Ile Lys Leu
1220 1225 1230
Leu Gln Leu Thr Leu Gln Met Arg Asn Ser Asp Asp Lys Gly Asn
1235 1240 1245
Asp Tyr Ile Ile Ser Pro Val Ala Asn Ala Glu Gly Gln Phe Phe
1250 1255 1260
Asp Ser Arg Asn Gly Asp Lys Lys Leu Pro Leu Asp Ala Asp Ala
1265 1270 1275
Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly Leu Trp Asn Ile Arg
1280 1285 1290
Gln Ile Lys Gln Thr Lys Asn Lys Asp Asp Leu Asn Leu Ser Ile
1295 1300 1305
Ser Ser Thr Glu Trp Leu Asp Phe Val Arg Glu Lys Pro Tyr Leu
1310 1315 1320
Lys
<210> 17
<211> 1484
<212> PRT
<213> 未知
<220>
<223> Peregrinibacteria细菌
<220>
<221> misc_feature
<222> (1073)..(1073)
<223> Xaa可以是任何天然存在的氨基酸
<400> 17
Met Ser Asn Phe Phe Lys Asn Phe Thr Asn Leu Tyr Glu Leu Ser Lys
1 5 10 15
Thr Leu Arg Phe Glu Leu Lys Pro Val Gly Asp Thr Leu Thr Asn Met
20 25 30
Lys Asp His Leu Glu Tyr Asp Glu Lys Leu Gln Thr Phe Leu Lys Asp
35 40 45
Gln Asn Ile Asp Asp Ala Tyr Gln Ala Leu Lys Pro Gln Phe Asp Glu
50 55 60
Ile His Glu Glu Phe Ile Thr Asp Ser Leu Glu Ser Lys Lys Ala Lys
65 70 75 80
Glu Ile Asp Phe Ser Glu Tyr Leu Asp Leu Phe Gln Glu Lys Lys Glu
85 90 95
Leu Asn Asp Ser Glu Lys Lys Leu Arg Asn Lys Ile Gly Glu Thr Phe
100 105 110
Asn Lys Ala Gly Glu Lys Trp Lys Lys Glu Lys Tyr Pro Gln Tyr Glu
115 120 125
Trp Lys Lys Gly Ser Lys Ile Ala Asn Gly Ala Asp Ile Leu Ser Cys
130 135 140
Gln Asp Met Leu Gln Phe Ile Lys Tyr Lys Asn Pro Glu Asp Glu Lys
145 150 155 160
Ile Lys Asn Tyr Ile Asp Asp Thr Leu Lys Gly Phe Phe Thr Tyr Phe
165 170 175
Gly Gly Phe Asn Gln Asn Arg Ala Asn Tyr Tyr Glu Thr Lys Lys Glu
180 185 190
Ala Ser Thr Ala Val Ala Thr Arg Ile Val His Glu Asn Leu Pro Lys
195 200 205
Phe Cys Asp Asn Val Ile Gln Phe Lys His Ile Ile Lys Arg Lys Lys
210 215 220
Asp Gly Thr Val Glu Lys Thr Glu Arg Lys Thr Glu Tyr Leu Asn Ala
225 230 235 240
Tyr Gln Tyr Leu Lys Asn Asn Asn Lys Ile Thr Gln Ile Lys Asp Ala
245 250 255
Glu Thr Glu Lys Met Ile Glu Ser Thr Pro Ile Ala Glu Lys Ile Phe
260 265 270
Asp Val Tyr Tyr Phe Ser Ser Cys Leu Ser Gln Lys Gln Ile Glu Glu
275 280 285
Tyr Asn Arg Ile Ile Gly His Tyr Asn Leu Leu Ile Asn Leu Tyr Asn
290 295 300
Gln Ala Lys Arg Ser Glu Gly Lys His Leu Ser Ala Asn Glu Lys Lys
305 310 315 320
Tyr Lys Asp Leu Pro Lys Phe Lys Thr Leu Tyr Lys Gln Ile Gly Cys
325 330 335
Gly Lys Lys Lys Asp Leu Phe Tyr Thr Ile Lys Cys Asp Thr Glu Glu
340 345 350
Glu Ala Asn Lys Ser Arg Asn Glu Gly Lys Glu Ser His Ser Val Glu
355 360 365
Glu Ile Ile Asn Lys Ala Gln Glu Ala Ile Asn Lys Tyr Phe Lys Ser
370 375 380
Asn Asn Asp Cys Glu Asn Ile Asn Thr Val Pro Asp Phe Ile Asn Tyr
385 390 395 400
Ile Leu Thr Lys Glu Asn Tyr Glu Gly Val Tyr Trp Ser Lys Ala Ala
405 410 415
Met Asn Thr Ile Ser Asp Lys Tyr Phe Ala Asn Tyr His Asp Leu Gln
420 425 430
Asp Arg Leu Lys Glu Ala Lys Val Phe Gln Lys Ala Asp Lys Lys Ser
435 440 445
Glu Asp Asp Ile Lys Ile Pro Glu Ala Ile Glu Leu Ser Gly Leu Phe
450 455 460
Gly Val Leu Asp Ser Leu Ala Asp Trp Gln Thr Thr Leu Phe Lys Ser
465 470 475 480
Ser Ile Leu Ser Asn Glu Lys Leu Lys Ile Ile Thr Asp Ser Gln Thr
485 490 495
Pro Ser Glu Ala Leu Leu Lys Met Ile Phe Asn Asp Ile Glu Lys Asn
500 505 510
Met Glu Ser Phe Leu Lys Glu Thr Asn Asp Ile Ile Thr Leu Lys Lys
515 520 525
Tyr Lys Gly Asn Lys Glu Gly Thr Glu Lys Ile Lys Gln Trp Phe Asp
530 535 540
Tyr Thr Leu Ala Ile Asn Arg Met Leu Lys Tyr Phe Leu Val Lys Glu
545 550 555 560
Asn Lys Ile Lys Gly Asn Ser Leu Asp Thr Asn Ile Ser Glu Ala Leu
565 570 575
Lys Thr Leu Ile Tyr Ser Asp Asp Ala Glu Trp Phe Lys Trp Tyr Asp
580 585 590
Ala Leu Arg Asn Tyr Leu Thr Gln Lys Pro Gln Asp Glu Ala Lys Glu
595 600 605
Asn Lys Leu Lys Leu Asn Phe Asp Asn Pro Ser Leu Ala Gly Gly Trp
610 615 620
Asp Val Asn Lys Glu Cys Ser Asn Phe Cys Val Ile Leu Lys Asp Lys
625 630 635 640
Asn Glu Lys Lys Tyr Leu Ala Met Ile Lys Lys Gly Glu Asn Thr Leu
645 650 655
Phe Gln Lys Glu Trp Thr Glu Gly Arg Gly Lys Asn Leu Thr Lys Lys
660 665 670
Ser Asn Pro Leu Phe Glu Ile Asn Asn Cys Glu Ile Leu Ser Lys Met
675 680 685
Glu Tyr Asp Phe Trp Ala Asp Val Ser Lys Met Ile Pro Lys Cys Ser
690 695 700
Thr Gln Leu Lys Ala Val Val Asn His Phe Lys Gln Ser Asp Asn Glu
705 710 715 720
Phe Ile Phe Pro Ile Gly Tyr Lys Val Thr Ser Gly Glu Lys Phe Arg
725 730 735
Glu Glu Cys Lys Ile Ser Lys Gln Asp Phe Glu Leu Asn Asn Lys Val
740 745 750
Phe Asn Lys Asn Glu Leu Ser Val Thr Ala Met Arg Tyr Asp Leu Ser
755 760 765
Ser Thr Gln Glu Lys Gln Tyr Ile Lys Ala Phe Gln Lys Glu Tyr Trp
770 775 780
Glu Leu Leu Phe Lys Gln Glu Lys Arg Asp Thr Lys Leu Thr Asn Asn
785 790 795 800
Glu Ile Phe Asn Glu Trp Ile Asn Phe Cys Asn Lys Lys Tyr Ser Glu
805 810 815
Leu Leu Ser Trp Glu Arg Lys Tyr Lys Asp Ala Leu Thr Asn Trp Ile
820 825 830
Asn Phe Cys Lys Tyr Phe Leu Ser Lys Tyr Pro Lys Thr Thr Leu Phe
835 840 845
Asn Tyr Ser Phe Lys Glu Ser Glu Asn Tyr Asn Ser Leu Asp Glu Phe
850 855 860
Tyr Arg Asp Val Asp Ile Cys Ser Tyr Lys Leu Asn Ile Asn Thr Thr
865 870 875 880
Ile Asn Lys Ser Ile Leu Asp Arg Leu Val Glu Glu Gly Lys Leu Tyr
885 890 895
Leu Phe Glu Ile Lys Asn Gln Asp Ser Asn Asp Gly Lys Ser Ile Gly
900 905 910
His Lys Asn Asn Leu His Thr Ile Tyr Trp Asn Ala Ile Phe Glu Asn
915 920 925
Phe Asp Asn Arg Pro Lys Leu Asn Gly Glu Ala Glu Ile Phe Tyr Arg
930 935 940
Lys Ala Ile Ser Lys Asp Lys Leu Gly Ile Val Lys Gly Lys Lys Thr
945 950 955 960
Lys Asn Gly Thr Trp Ile Ile Lys Asn Tyr Arg Phe Ser Lys Glu Lys
965 970 975
Phe Ile Leu His Val Pro Ile Thr Leu Asn Phe Cys Ser Asn Asn Glu
980 985 990
Tyr Val Asn Asp Ile Val Asn Thr Lys Phe Tyr Asn Phe Ser Asn Leu
995 1000 1005
His Phe Leu Gly Ile Asp Arg Gly Glu Lys His Leu Ala Tyr Tyr
1010 1015 1020
Ser Leu Val Asn Lys Asn Gly Glu Ile Val Asp Gln Gly Thr Leu
1025 1030 1035
Asn Leu Pro Phe Thr Asp Lys Asp Gly Asn Gln Arg Ser Ile Lys
1040 1045 1050
Lys Glu Lys Tyr Phe Tyr Asn Lys Gln Glu Asp Lys Trp Glu Ala
1055 1060 1065
Lys Glu Val Asp Xaa Trp Asn Tyr Asn Asp Leu Leu Asp Ala Met
1070 1075 1080
Ala Ser Asn Arg Asp Met Ala Arg Lys Asn Trp Gln Arg Ile Gly
1085 1090 1095
Thr Ile Lys Glu Ala Lys Asn Gly Tyr Val Ser Leu Val Ile Arg
1100 1105 1110
Lys Ile Ala Asp Leu Ala Val Asn Asn Glu Arg Pro Ala Phe Ile
1115 1120 1125
Val Leu Glu Asp Leu Asn Thr Gly Phe Lys Arg Ser Arg Gln Lys
1130 1135 1140
Ile Asp Lys Ser Val Tyr Gln Lys Phe Glu Leu Ala Leu Ala Lys
1145 1150 1155
Lys Leu Asn Phe Leu Val Asp Lys Asn Ala Lys Arg Asp Glu Ile
1160 1165 1170
Gly Ser Pro Thr Lys Ala Leu Gln Leu Thr Pro Pro Val Asn Asn
1175 1180 1185
Tyr Gly Asp Ile Glu Asn Lys Lys Gln Ala Gly Ile Met Leu Tyr
1190 1195 1200
Thr Arg Ala Asn Tyr Thr Ser Gln Thr Asp Pro Ala Thr Gly Trp
1205 1210 1215
Arg Lys Thr Ile Tyr Leu Lys Ala Gly Pro Glu Glu Thr Thr Tyr
1220 1225 1230
Lys Lys Asp Gly Lys Ile Lys Asn Lys Ser Val Lys Asp Gln Ile
1235 1240 1245
Ile Glu Thr Phe Thr Asp Ile Gly Phe Asp Gly Lys Asp Tyr Tyr
1250 1255 1260
Phe Glu Tyr Asp Lys Gly Glu Phe Val Asp Glu Lys Thr Gly Glu
1265 1270 1275
Ile Lys Pro Lys Lys Trp Arg Leu Tyr Ser Gly Glu Asn Gly Lys
1280 1285 1290
Ser Leu Asp Arg Phe Arg Gly Glu Arg Glu Lys Asp Lys Tyr Glu
1295 1300 1305
Trp Lys Ile Asp Lys Ile Asp Ile Val Lys Ile Leu Asp Asp Leu
1310 1315 1320
Phe Val Asn Phe Asp Lys Asn Ile Ser Leu Leu Lys Gln Leu Lys
1325 1330 1335
Glu Gly Val Glu Leu Thr Arg Asn Asn Glu His Gly Thr Gly Glu
1340 1345 1350
Ser Leu Arg Phe Ala Ile Asn Leu Ile Gln Gln Ile Arg Asn Thr
1355 1360 1365
Gly Asn Asn Glu Arg Asp Asn Asp Phe Ile Leu Ser Pro Val Arg
1370 1375 1380
Asp Glu Asn Gly Lys His Phe Asp Ser Arg Glu Tyr Trp Asp Lys
1385 1390 1395
Glu Thr Lys Gly Glu Lys Ile Ser Met Pro Ser Ser Gly Asp Ala
1400 1405 1410
Asn Gly Ala Phe Asn Ile Ala Arg Lys Gly Ile Ile Met Asn Ala
1415 1420 1425
His Ile Leu Ala Asn Ser Asp Ser Lys Asp Leu Ser Leu Phe Val
1430 1435 1440
Ser Asp Glu Glu Trp Asp Leu His Leu Asn Asn Lys Thr Glu Trp
1445 1450 1455
Lys Lys Gln Leu Asn Ile Phe Ser Ser Arg Lys Ala Met Ala Lys
1460 1465 1470
Arg Lys Lys Lys Arg Pro Ala Ala Thr Lys Lys
1475 1480
<210> 18
<211> 1245
<212> PRT
<213> Porphyromonas macacae
<400> 18
Met Lys Thr Gln His Phe Phe Glu Asp Phe Thr Ser Leu Tyr Ser Leu
1 5 10 15
Ser Lys Thr Ile Arg Phe Glu Leu Lys Pro Ile Gly Lys Thr Leu Glu
20 25 30
Asn Ile Lys Lys Asn Gly Leu Ile Arg Arg Asp Glu Gln Arg Leu Asp
35 40 45
Asp Tyr Glu Lys Leu Lys Lys Val Ile Asp Glu Tyr His Glu Asp Phe
50 55 60
Ile Ala Asn Ile Leu Ser Ser Phe Ser Phe Ser Glu Glu Ile Leu Gln
65 70 75 80
Ser Tyr Ile Gln Asn Leu Ser Ile Ser Glu Ala Arg Ala Lys Ile Glu
85 90 95
Lys Thr Met Arg Asp Thr Leu Ala Lys Ala Phe Ser Glu Asp Glu Arg
100 105 110
Tyr Lys Ser Ile Phe Lys Lys Glu Leu Val Lys Lys Asp Ile Pro Val
115 120 125
Trp Cys Pro Ala Tyr Lys Ser Leu Cys Lys Lys Phe Asp Asn Phe Thr
130 135 140
Thr Ser Leu Val Pro Phe His Glu Asn Arg Lys Asn Leu Tyr Thr Ser
145 150 155 160
Asn Glu Ile Thr Ala Ser Ile Pro Tyr Arg Ile Val His Val Asn Leu
165 170 175
Pro Lys Phe Ile Gln Asn Ile Glu Ala Leu Cys Glu Leu Gln Lys Lys
180 185 190
Met Gly Ala Asp Leu Tyr Leu Glu Met Met Glu Asn Leu Arg Asn Val
195 200 205
Trp Pro Ser Phe Val Lys Thr Pro Asp Asp Leu Cys Asn Leu Lys Thr
210 215 220
Tyr Asn His Leu Met Val Gln Ser Ser Ile Ser Glu Tyr Asn Arg Phe
225 230 235 240
Val Gly Gly Tyr Ser Thr Glu Asp Gly Thr Lys His Gln Gly Ile Asn
245 250 255
Glu Trp Ile Asn Ile Tyr Arg Gln Arg Asn Lys Glu Met Arg Leu Pro
260 265 270
Gly Leu Val Phe Leu His Lys Gln Ile Leu Ala Lys Val Asp Ser Ser
275 280 285
Ser Phe Ile Ser Asp Thr Leu Glu Asn Asp Asp Gln Val Phe Cys Val
290 295 300
Leu Arg Gln Phe Arg Lys Leu Phe Trp Asn Thr Val Ser Ser Lys Glu
305 310 315 320
Asp Asp Ala Ala Ser Leu Lys Asp Leu Phe Cys Gly Leu Ser Gly Tyr
325 330 335
Asp Pro Glu Ala Ile Tyr Val Ser Asp Ala His Leu Ala Thr Ile Ser
340 345 350
Lys Asn Ile Phe Asp Arg Trp Asn Tyr Ile Ser Asp Ala Ile Arg Arg
355 360 365
Lys Thr Glu Val Leu Met Pro Arg Lys Lys Glu Ser Val Glu Arg Tyr
370 375 380
Ala Glu Lys Ile Ser Lys Gln Ile Lys Lys Arg Gln Ser Tyr Ser Leu
385 390 395 400
Ala Glu Leu Asp Asp Leu Leu Ala His Tyr Ser Glu Glu Ser Leu Pro
405 410 415
Ala Gly Phe Ser Leu Leu Ser Tyr Phe Thr Ser Leu Gly Gly Gln Lys
420 425 430
Tyr Leu Val Ser Asp Gly Glu Val Ile Leu Tyr Glu Glu Gly Ser Asn
435 440 445
Ile Trp Asp Glu Val Leu Ile Ala Phe Arg Asp Leu Gln Val Ile Leu
450 455 460
Asp Lys Asp Phe Thr Glu Lys Lys Leu Gly Lys Asp Glu Glu Ala Val
465 470 475 480
Ser Val Ile Lys Lys Ala Leu Asp Ser Ala Leu Arg Leu Arg Lys Phe
485 490 495
Phe Asp Leu Leu Ser Gly Thr Gly Ala Glu Ile Arg Arg Asp Ser Ser
500 505 510
Phe Tyr Ala Leu Tyr Thr Asp Arg Met Asp Lys Leu Lys Gly Leu Leu
515 520 525
Lys Met Tyr Asp Lys Val Arg Asn Tyr Leu Thr Lys Lys Pro Tyr Ser
530 535 540
Ile Glu Lys Phe Lys Leu His Phe Asp Asn Pro Ser Leu Leu Ser Gly
545 550 555 560
Trp Asp Lys Asn Lys Glu Leu Asn Asn Leu Ser Val Ile Phe Arg Gln
565 570 575
Asn Gly Tyr Tyr Tyr Leu Gly Ile Met Thr Pro Lys Gly Lys Asn Leu
580 585 590
Phe Lys Thr Leu Pro Lys Leu Gly Ala Glu Glu Met Phe Tyr Glu Lys
595 600 605
Met Glu Tyr Lys Gln Ile Ala Glu Pro Met Leu Met Leu Pro Lys Val
610 615 620
Phe Phe Pro Lys Lys Thr Lys Pro Ala Phe Ala Pro Asp Gln Ser Val
625 630 635 640
Val Asp Ile Tyr Asn Lys Lys Thr Phe Lys Thr Gly Gln Lys Gly Phe
645 650 655
Asn Lys Lys Asp Leu Tyr Arg Leu Ile Asp Phe Tyr Lys Glu Ala Leu
660 665 670
Thr Val His Glu Trp Lys Leu Phe Asn Phe Ser Phe Ser Pro Thr Glu
675 680 685
Gln Tyr Arg Asn Ile Gly Glu Phe Phe Asp Glu Val Arg Glu Gln Ala
690 695 700
Tyr Lys Val Ser Met Val Asn Val Pro Ala Ser Tyr Ile Asp Glu Ala
705 710 715 720
Val Glu Asn Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys Asp Phe
725 730 735
Ser Pro Tyr Ser Lys Gly Ile Pro Asn Leu His Thr Leu Tyr Trp Lys
740 745 750
Ala Leu Phe Ser Glu Gln Asn Gln Ser Arg Val Tyr Lys Leu Cys Gly
755 760 765
Gly Gly Glu Leu Phe Tyr Arg Lys Ala Ser Leu His Met Gln Asp Thr
770 775 780
Thr Val His Pro Lys Gly Ile Ser Ile His Lys Lys Asn Leu Asn Lys
785 790 795 800
Lys Gly Glu Thr Ser Leu Phe Asn Tyr Asp Leu Val Lys Asp Lys Arg
805 810 815
Phe Thr Glu Asp Lys Phe Phe Phe His Val Pro Ile Ser Ile Asn Tyr
820 825 830
Lys Asn Lys Lys Ile Thr Asn Val Asn Gln Met Val Arg Asp Tyr Ile
835 840 845
Ala Gln Asn Asp Asp Leu Gln His Gly Ile Asp Arg Gly Glu Arg Asn
850 855 860
Leu Leu Tyr Ile Ser Arg Ile Asp Thr Arg Gly Asn Leu Leu Glu Gln
865 870 875 880
Phe Ser Leu Asn Val Ile Glu Ser Asp Lys Gly Asp Leu Arg Thr Asp
885 890 895
Tyr Gln Lys Ile Leu Gly Asp Arg Glu Gln Glu Arg Leu Arg Arg Arg
900 905 910
Gln Glu Trp Lys Ser Ile Glu Ser Ile Lys Asp Leu Lys Asp Gly Tyr
915 920 925
Met Ser Gln Val Val His Lys Ile Cys Asn Met Val Val Glu His Lys
930 935 940
Ala Ile Val Val Leu Glu Asn Leu Asn Leu Ser Phe Met Lys Gly Arg
945 950 955 960
Lys Lys Val Glu Lys Ser Val Tyr Glu Lys Phe Glu Arg Met Leu Val
965 970 975
Asp Lys Leu Asn Tyr Leu Val Val Asp Lys Lys Asn Leu Ser Asn Glu
980 985 990
Pro Gly Gly Leu Tyr Ala Ala Tyr Gln Leu Thr Asn Pro Leu Phe Ser
995 1000 1005
Phe Glu Glu Leu His Arg Tyr Pro Gln Ser Gly Ile Leu Phe Phe
1010 1015 1020
Val Asp Pro Trp Asn Thr Ser Leu Thr Asp Pro Ser Thr Gly Phe
1025 1030 1035
Val Asn Leu Leu Gly Arg Ile Asn Tyr Thr Asn Val Gly Asp Ala
1040 1045 1050
Arg Lys Phe Phe Asp Arg Phe Asn Ala Ile Arg Tyr Asp Gly Lys
1055 1060 1065
Gly Asn Ile Leu Phe Asp Leu Asp Leu Ser Arg Phe Asp Val Arg
1070 1075 1080
Val Glu Thr Gln Arg Lys Leu Trp Thr Leu Thr Thr Phe Gly Ser
1085 1090 1095
Arg Ile Ala Lys Ser Lys Lys Ser Gly Lys Trp Met Val Glu Arg
1100 1105 1110
Ile Glu Asn Leu Ser Leu Cys Phe Leu Glu Leu Phe Glu Gln Phe
1115 1120 1125
Asn Ile Gly Tyr Arg Val Glu Lys Asp Leu Lys Lys Ala Ile Leu
1130 1135 1140
Ser Gln Asp Arg Lys Glu Phe Tyr Val Arg Leu Ile Tyr Leu Phe
1145 1150 1155
Asn Leu Met Met Gln Ile Arg Asn Ser Asp Gly Glu Glu Asp Tyr
1160 1165 1170
Ile Leu Ser Pro Ala Leu Asn Glu Lys Asn Leu Gln Phe Asp Ser
1175 1180 1185
Arg Leu Ile Glu Ala Lys Asp Leu Pro Val Asp Ala Asp Ala Asn
1190 1195 1200
Gly Ala Tyr Asn Val Ala Arg Lys Gly Leu Met Val Val Gln Arg
1205 1210 1215
Ile Lys Arg Gly Asp His Glu Ser Ile His Arg Ile Gly Arg Ala
1220 1225 1230
Gln Trp Leu Arg Tyr Val Gln Glu Gly Ile Val Glu
1235 1240 1245
<210> 19
<211> 1250
<212> PRT
<213> 史密斯氏菌属物种（Smithella sp.）
<400> 19
Met Gln Thr Leu Phe Glu Asn Phe Thr Asn Gln Tyr Pro Val Ser Lys
1 5 10 15
Thr Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Lys Asp Phe Ile
20 25 30
Glu Gln Lys Gly Leu Leu Lys Lys Asp Glu Asp Arg Ala Glu Lys Tyr
35 40 45
Lys Lys Val Lys Asn Ile Ile Asp Glu Tyr His Lys Asp Phe Ile Glu
50 55 60
Lys Ser Leu Asn Gly Leu Lys Leu Asp Gly Leu Glu Lys Tyr Lys Thr
65 70 75 80
Leu Tyr Leu Lys Gln Glu Lys Asp Asp Lys Asp Lys Lys Ala Phe Asp
85 90 95
Lys Glu Lys Glu Asn Leu Arg Lys Gln Ile Ala Asn Ala Phe Arg Asn
100 105 110
Asn Glu Lys Phe Lys Thr Leu Phe Ala Lys Glu Leu Ile Lys Asn Asp
115 120 125
Leu Met Ser Phe Ala Cys Glu Glu Asp Lys Lys Asn Val Lys Glu Phe
130 135 140
Glu Ala Phe Thr Thr Tyr Phe Thr Gly Phe His Gln Asn Arg Ala Asn
145 150 155 160
Met Tyr Val Ala Asp Glu Lys Arg Thr Ala Ile Ala Ser Arg Leu Ile
165 170 175
His Glu Asn Leu Pro Lys Phe Ile Asp Asn Ile Lys Ile Phe Glu Lys
180 185 190
Met Lys Lys Glu Ala Pro Glu Leu Leu Ser Pro Phe Asn Gln Thr Leu
195 200 205
Lys Asp Met Lys Asp Val Ile Lys Gly Thr Thr Leu Glu Glu Ile Phe
210 215 220
Ser Leu Asp Tyr Phe Asn Lys Thr Leu Thr Gln Ser Gly Ile Asp Ile
225 230 235 240
Tyr Asn Ser Val Ile Gly Gly Arg Thr Pro Glu Glu Gly Lys Thr Lys
245 250 255
Ile Lys Gly Leu Asn Glu Tyr Ile Asn Thr Asp Phe Asn Gln Lys Gln
260 265 270
Thr Asp Lys Lys Lys Arg Gln Pro Lys Phe Lys Gln Leu Tyr Lys Gln
275 280 285
Ile Leu Ser Asp Arg Gln Ser Leu Ser Phe Ile Ala Glu Ala Phe Lys
290 295 300
Asn Asp Thr Glu Ile Leu Glu Ala Ile Glu Lys Phe Tyr Val Asn Glu
305 310 315 320
Leu Leu His Phe Ser Asn Glu Gly Lys Ser Thr Asn Val Leu Asp Ala
325 330 335
Ile Lys Asn Ala Val Ser Asn Leu Glu Ser Phe Asn Leu Thr Lys Met
340 345 350
Tyr Phe Arg Ser Gly Ala Ser Leu Thr Asp Val Ser Arg Lys Val Phe
355 360 365
Gly Glu Trp Ser Ile Ile Asn Arg Ala Leu Asp Asn Tyr Tyr Ala Thr
370 375 380
Thr Tyr Pro Ile Lys Pro Arg Glu Lys Ser Glu Lys Tyr Glu Glu Arg
385 390 395 400
Lys Glu Lys Trp Leu Lys Gln Asp Phe Asn Val Ser Leu Ile Gln Thr
405 410 415
Ala Ile Asp Glu Tyr Asp Asn Glu Thr Val Lys Gly Lys Asn Ser Gly
420 425 430
Lys Val Ile Ala Asp Tyr Phe Ala Lys Phe Cys Asp Asp Lys Glu Thr
435 440 445
Asp Leu Ile Gln Lys Val Asn Glu Gly Tyr Ile Ala Val Lys Asp Leu
450 455 460
Leu Asn Thr Pro Cys Pro Glu Asn Glu Lys Leu Gly Ser Asn Lys Asp
465 470 475 480
Gln Val Lys Gln Ile Lys Ala Phe Met Asp Ser Ile Met Asp Ile Met
485 490 495
His Phe Val Arg Pro Leu Ser Leu Lys Asp Thr Asp Lys Glu Lys Asp
500 505 510
Glu Thr Phe Tyr Ser Leu Phe Thr Pro Leu Tyr Asp His Leu Thr Gln
515 520 525
Thr Ile Ala Leu Tyr Asn Lys Val Arg Asn Tyr Leu Thr Gln Lys Pro
530 535 540
Tyr Ser Thr Glu Lys Ile Lys Leu Asn Phe Glu Asn Ser Thr Leu Leu
545 550 555 560
Gly Gly Trp Asp Leu Asn Lys Glu Thr Asp Asn Thr Ala Ile Ile Leu
565 570 575
Arg Lys Asp Asn Leu Tyr Tyr Leu Gly Ile Met Asp Lys Arg His Asn
580 585 590
Arg Ile Phe Arg Asn Val Pro Lys Ala Asp Lys Lys Asp Phe Cys Tyr
595 600 605
Glu Lys Met Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro
610 615 620
Lys Val Phe Phe Ser Gln Ser Arg Ile Gln Glu Phe Thr Pro Ser Ala
625 630 635 640
Lys Leu Leu Glu Asn Tyr Ala Asn Glu Thr His Lys Lys Gly Asp Asn
645 650 655
Phe Asn Leu Asn His Cys His Lys Leu Ile Asp Phe Phe Lys Asp Ser
660 665 670
Ile Asn Lys His Glu Asp Trp Lys Asn Phe Asp Phe Arg Phe Ser Ala
675 680 685
Thr Ser Thr Tyr Ala Asp Leu Ser Gly Phe Tyr His Glu Val Glu His
690 695 700
Gln Gly Tyr Lys Ile Ser Phe Gln Ser Val Ala Asp Ser Phe Ile Asp
705 710 715 720
Asp Leu Val Asn Glu Gly Lys Leu Tyr Leu Phe Gln Ile Tyr Asn Lys
725 730 735
Asp Phe Ser Pro Phe Ser Lys Gly Lys Pro Asn Leu His Thr Leu Tyr
740 745 750
Trp Lys Met Leu Phe Asp Glu Asn Asn Leu Lys Asp Val Val Tyr Lys
755 760 765
Leu Asn Gly Glu Ala Glu Val Phe Tyr Arg Lys Lys Ser Ile Ala Glu
770 775 780
Lys Asn Thr Thr Ile His Lys Ala Asn Glu Ser Ile Ile Asn Lys Asn
785 790 795 800
Pro Asp Asn Pro Lys Ala Thr Ser Thr Phe Asn Tyr Asp Ile Val Lys
805 810 815
Asp Lys Arg Tyr Thr Ile Asp Lys Phe Gln Phe His Ile Pro Ile Thr
820 825 830
Met Asn Phe Lys Ala Glu Gly Ile Phe Asn Met Asn Gln Arg Val Asn
835 840 845
Gln Phe Leu Lys Ala Asn Pro Asp Ile Asn Ile Ile Gly Ile Asp Arg
850 855 860
Gly Glu Arg His Leu Leu Tyr Tyr Ala Leu Ile Asn Gln Lys Gly Lys
865 870 875 880
Ile Leu Lys Gln Asp Thr Leu Asn Val Ile Ala Asn Glu Lys Gln Lys
885 890 895
Val Asp Tyr His Asn Leu Leu Asp Lys Lys Glu Gly Asp Arg Ala Thr
900 905 910
Ala Arg Gln Glu Trp Gly Val Ile Glu Thr Ile Lys Glu Leu Lys Glu
915 920 925
Gly Tyr Leu Ser Gln Val Ile His Lys Leu Thr Asp Leu Met Ile Glu
930 935 940
Asn Asn Ala Ile Ile Val Met Glu Asp Leu Asn Phe Gly Phe Lys Arg
945 950 955 960
Gly Arg Gln Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met
965 970 975
Leu Ile Asp Lys Leu Asn Tyr Leu Val Asp Lys Asn Lys Lys Ala Asn
980 985 990
Glu Leu Gly Gly Leu Leu Asn Ala Phe Gln Leu Ala Asn Lys Phe Glu
995 1000 1005
Ser Phe Gln Lys Met Gly Lys Gln Asn Gly Phe Ile Phe Tyr Val
1010 1015 1020
Pro Ala Trp Asn Thr Ser Lys Thr Asp Pro Ala Thr Gly Phe Ile
1025 1030 1035
Asp Phe Leu Lys Pro Arg Tyr Glu Asn Leu Asn Gln Ala Lys Asp
1040 1045 1050
Phe Phe Glu Lys Phe Asp Ser Ile Arg Leu Asn Ser Lys Ala Asp
1055 1060 1065
Tyr Phe Glu Phe Ala Phe Asp Phe Lys Asn Phe Thr Glu Lys Ala
1070 1075 1080
Asp Gly Gly Arg Thr Lys Trp Thr Val Cys Thr Thr Asn Glu Asp
1085 1090 1095
Arg Tyr Gln Trp Asn Arg Ala Leu Asn Asn Asn Arg Gly Ser Gln
1100 1105 1110
Glu Lys Tyr Asp Ile Thr Ala Glu Leu Lys Ser Leu Phe Asp Gly
1115 1120 1125
Lys Val Asp Tyr Lys Ser Gly Lys Asp Leu Lys Gln Gln Ile Ala
1130 1135 1140
Ser Gln Glu Ser Ala Asp Phe Phe Lys Ala Leu Met Lys Asn Leu
1145 1150 1155
Ser Ile Thr Leu Ser Leu Arg His Asn Asn Gly Glu Lys Gly Asp
1160 1165 1170
Asn Glu Gln Asp Tyr Ile Leu Ser Pro Val Ala Asp Ser Lys Gly
1175 1180 1185
Arg Phe Phe Asp Ser Arg Lys Ala Asp Asp Asp Met Pro Lys Asn
1190 1195 1200
Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys Gly Leu Trp
1205 1210 1215
Cys Leu Glu Gln Ile Ser Lys Thr Asp Asp Leu Lys Lys Val Lys
1220 1225 1230
Leu Ala Ile Ser Asn Lys Glu Trp Leu Glu Phe Val Gln Thr Leu
1235 1240 1245
Lys Gly
1250
<210> 20
<211> 3987
<212> DNA
<213> 人工的
<220>
<223> Cas12a多核苷酸
<400> 20
atggccggga gcaagaagcg ccggataaag caggacacgc agttcgaggg cttcaccaac 60
ctgtaccaag tctccaagac gctccggttc gagcttatcc cgcaagggaa gaccctgaaa 120
cacatccagg aacaaggttt catcgaggag gacaaggccc gcaacgacca ctacaaggag 180
ctcaagccca taatcgatcg gatctacaag acgtacgccg accagtgcct ccaactggtg 240
cagctcgact gggagaacct gagcgccgcc attgacagct accgcaagga aaagacggag 300
gagacgcgca acgcccttat tgaggagcaa gccacctacc gcaacgccat ccacgactac 360
ttcatcgggc gcaccgacaa cctgacggac gcgatcaaca agcgccacgc ggaaatctac 420
aagggccttt tcaaggccga gctcttcaac gggaaggtcc taaaacagct cgggactgtc 480
acgacaaccg agcatgagaa cgccctcctt cgcagcttcg acaagttcac cacatacttc 540
tcgggcttct accggaaccg caagaacgtt ttcagcgccg aggacatctc caccgccatc 600
ccgcacagga tcgtccagga caacttcccc aagttcaagg agaactgcca catcttcacg 660
cgcctgatta cagccgtacc ttcacttcgt gagcacttcg agaacgtcaa aaaggccatc 720
gggatcttcg tctccacgtc catcgaggag gtattctctt tcccgttcta taaccagctc 780
ctgacccaga cgcagatcga cctctacaac cagctactgg gcggcatcag ccgggaggcc 840
gggaccgaga aaataaaggg cctcaacgaa gttctcaacc tggccatcca gaagaacgac 900
gagaccgcgc atatcatcgc atccctgccg catcgcttca ttcctttgtt caagcagata 960
ttgagcgacc ggaacaccct ctcgttcatc ctcgaagaat tcaagagcga cgaggaggtc 1020
attcagtctt tctgcaagta caagacgctc ctacggaatg agaatgtgct ggagaccgcg 1080
gaggcactct tcaatgagct gaactccatt gacctgaccc acatcttcat tagccacaag 1140
aaactggaga cgatctccag cgccctgtgc gaccactggg acactctccg caacgccctc 1200
tacgaacgcc ggatctccga acttaccggc aagataacta agtcggctaa ggagaaggtg 1260
caacggagcc tcaagcacga ggacatcaac cttcaggaaa tcatctcagc cgcgggcaag 1320
gagctgagcg aggcgtttaa gcagaaaaca tcggagatac tgagccacgc gcacgcggcc 1380
ctggatcaac cgctgccgac gactctcaag aagcaagagg agaaggaaat ccttaagtcc 1440
cagctcgact cgctgctcgg cctctatcac ttgctcgact ggttcgcggt tgatgagtcc 1500
aacgaggtgg acccggagtt ctccgcgcgc ctcacgggta ttaagctgga gatggagcca 1560
agcttaagct tctacaacaa ggcccgcaac tacgcgacca aaaaaccgta ctcagtcgag 1620
aaattcaagc tgaatttcca gatgcctaca ttggcgaggg ggtgggacgt gaaccgcgag 1680
aagaacaatg gagccatcct gttcgtcaaa aatgggttgt actacctggg catcatgccc 1740
aagcagaagg gccgttacaa ggccctgtca ttcgagccta ccgagaagac ctcggagggc 1800
ttcgacaaga tgtactacga ctatttcccg gacgccgcca agatgatccc gaagtgctcc 1860
acgcagctca aagccgtcac ggcccacttc cagacgcata ccacgccgat acttctgagc 1920
aacaacttca ttgagccgct agagatcacg aaggagatat acgacctaaa caaccccgaa 1980
aaggagccca agaagttcca gacagcctac gctaagaaga caggtgatca gaagggatat 2040
agggaggcac tctgcaagtg gatcgacttc acgcgcgact tcctgtcgaa atatacaaag 2100
acgaccagca ttgacctaag ttctctccgc ccatcctccc agtacaagga tctgggcgag 2160
tattatgcgg agctgaaccc attgctgtac cacatcagct tccagaggat cgccgagaag 2220
gagattatgg acgcggtgga gacggggaaa ctatacctgt tccaaatata taacaaggac 2280
ttcgctaaag ggcaccacgg gaagcccaac ctgcacacac tctactggac gggcttgttt 2340
tcgccagaaa atttggccaa gacttcgatc aagctcaacg gccaggcgga gttgttttac 2400
cgtcccaagt ctcgcatgaa gcgcatggcg catcgcctcg gagagaaaat gcttaacaag 2460
aagctcaagg atcagaagac gcccatacct gatacgttgt accaggaatt gtacgactac 2520
gtgaaccacc gcctatcgca cgacctctca gacgaggccc gcgccctcct cccaaacgtg 2580
attactaagg aggtttccca tgaaataatc aaggaccgac ggttcaccag cgacaaattt 2640
tttttccacg tgcctatcac gctcaattac caggcggcca actccccatc gaagttcaac 2700
cagcgcgtga acgcctacct taaggagcac ccggagaccc caatcatcgg gatcgaccgt 2760
ggcgagcgga acctgatcta tattacggtg atcgatagca ccgggaagat cctggagcag 2820
cgctccctga acacaatcca gcagtttgac taccagaaga aactcgacaa ccgggagaag 2880
gagcgcgtcg cagcccggca agcatggagt gtggtcggca ccataaagga cctgaaacag 2940
ggttacctaa gtcaagttat ccacgagatc gttgacctga tgatacacta tcaagccgta 3000
gtcgtgctgg agaacctcaa cttcgggttt aagtccaagc gcaccggcat cgcggagaag 3060
gcggtgtacc agcagttcga gaagatgctg atcgacaagc tgaactgcct ggtgctcaag 3120
gactaccctg cggagaaggt cggcggggtc ttgaacccgt accagctaac cgaccagttc 3180
acgagcttcg ccaaaatggg cacgcagtcc ggattcttgt tttatgtccc ggctccatat 3240
acaagtaaga tcgacccgct gacagggttt gttgacccat tcgtgtggaa gaccatcaag 3300
aaccacgaga gcaggaaaca cttcttagag ggcttcgact tcctgcatta cgacgttaag 3360
acaggcgact tcatcctgca cttcaagatg aaccgcaacc tgtcgttcca gaggggcctg 3420
cccggcttca tgcccgcctg ggatatcgtc tttgagaaga atgagacgca gttcgacgcg 3480
aaggggacgc cgttcatcgc tggaaagcgg atcgtgccgg tcatcgagaa ccaccgcttc 3540
acgggtcgct accgagattt ataccccgcc aacgaactaa ttgcgctgct ggaggagaag 3600
gggatcgtgt tccgagatgg cagcaacatt ctcccgaagc tgctggagaa cgacgactcg 3660
cacgctattg acacgatggt cgccctcata cggagcgtgc ttcagatgcg gaacagtaac 3720
gctgccacgg gcgaggacta cattaactcc cccgtccgcg acctcaacgg ggtctgcttc 3780
gatagccgct tccagaaccc ggagtggcct atggatgcgg acgcgaacgg ggcctaccac 3840
atcgccctca agggccaact cctgctcaac cacttgaagg aaagcaaaga cctcaaattg 3900
cagaatggca tcagtaacca ggactggctc gcgtacatcc aggaactgag aaacgggtcc 3960
aagaagcggc gtatcaagca agattga 3987
<210> 21
<211> 3987
<212> DNA
<213> 人工的
<220>
<223> Cas12a多核苷酸
<400> 21
atggcgggaa gcaaaaagcg ccggattaag caagacacgc agttcgaggg cttcacgaac 60
ctctaccaag tcagcaagac cctccggttc gagctgatac cacagggaaa gacgctcaag 120
cacatccagg aacagggctt catcgaggag gacaaggcgc gcaacgacca ctacaaggag 180
ttgaaaccga tcatcgaccg catctacaag acgtacgccg accagtgcct ccagctcgtg 240
cagctcgact gggagaacct ctccgccgcc attgactcgt accggaagga gaagactgag 300
gagacccgca acgccctgat cgaggagcaa gcaacctacc ggaacgccat ccacgactac 360
ttcatcggcc gcaccgacaa cctcaccgac gcgatcaaca agcggcacgc ggagatatac 420
aaagggctgt tcaaggcgga gctgttcaac ggcaaggtgc tcaagcagct agggacggtg 480
accacgaccg agcacgagaa cgcgctcctc cgcagcttcg acaagttcac cacctacttc 540
agcggcttct accggaaccg caagaatgtg ttcagcgcgg aggacatcag cacggccatc 600
ccgcaccgca tcgtccagga caacttcccg aagttcaagg agaactgcca catcttcacc 660
cgcctgataa ccgccgtccc ctccctgcgg gagcacttcg agaacgtcaa aaaggcaatt 720
gggatcttcg tctcgaccag cattgaggag gtgttcagct tccccttcta caaccagctc 780
ctcacccaga cgcagatcga cctgtacaat cagttgctcg gcgggataag ccgcgaggcg 840
ggaaccgaaa aaatcaaggg gctgaacgaa gtgttgaacc tcgccatcca gaagaacgac 900
gagaccgcgc acatcatcgc ctccctgccc caccggttca tcccgctgtt caagcagatc 960
ctctctgacc ggaacaccct gtccttcatt cttgaggagt tcaagtcgga cgaggaggtc 1020
atccagagct tctgcaagta caagacgctg ctacggaacg agaacgtgct ggagacggcg 1080
gaggcactgt tcaacgagct aaacagcatc gacctcacgc acatcttcat cagtcacaag 1140
aaactggaga ccatctcctc cgcgctgtgc gaccactggg acacgctcag gaacgcgctc 1200
tacgagcgcc gaatcagtga gctgacgggc aagatcacga agtccgcgaa ggagaaggtg 1260
cagcggtccc tcaagcacga ggacatcaac ctccaggaga tcatctcagc ggctgggaaa 1320
gagctgtccg aggcgttcaa gcagaaaacg agcgaaatcc tgtcccacgc gcacgcggcc 1380
ctggatcagc ctctgccgac gaccctcaag aaacaagaag aaaaggaaat cctcaagtcg 1440
cagctcgact cgctgctggg cctgtaccat ctcctcgact ggttcgccgt ggacgagagc 1500
aacgaggtgg accccgagtt ctccgcgcgg cttacgggga tcaagctgga gatggagccc 1560
agcctgtcct tctacaacaa ggcgcgcaac tacgccacca agaagcccta cagcgtggag 1620
aagttcaagc tcaacttcca gatgcccact ctcgcacgtg ggtgggacgt caaccgcgaa 1680
aaaaataatg gggcgatcct gttcgtcaag aacggcctgt actacttggg catcatgccg 1740
aaacagaagg gccgctacaa ggccctgagc ttcgaaccga ccgagaaaac gagcgagggg 1800
ttcgacaaga tgtactacga ctacttcccc gacgccgcga agatgattcc aaagtgctcc 1860
acgcagctta aggccgtgac ggcccacttc cagacgcaca cgaccccgat cctcctcagc 1920
aacaacttca tcgagcccct ggagatcacg aaggagatat acgacctgaa caacccggag 1980
aaggagccca agaaattcca gaccgcctac gccaagaaga caggcgacca aaagggttac 2040
agggaggccc tctgcaagtg gatcgacttc actagggact tcctgtccaa gtacaccaag 2100
actacctcta tcgacctgtc cagcctccgc ccgtcgtccc agtacaaaga tttgggcgag 2160
tattacgcgg agctgaaccc actgctctac cacatcagct tccagcgcat cgcggagaag 2220
gagatcatgg acgcagtgga gacgggcaag ctatacctat ttcagatata caacaaagac 2280
ttcgctaagg gacaccacgg caagcctaac ctgcacaccc tctactggac ggggctcttc 2340
agcccggaga acctcgccaa gacctcgatc aagctcaacg gccaggccga gctgttctac 2400
cggcccaagt cccgcatgaa gcggatggcc caccggctcg gggagaaaat gctcaacaag 2460
aaattgaagg accaaaaaac gccgataccc gacaccctat accaggagct gtacgactat 2520
gtgaaccacc gcctgagcca cgacctcagc gacgaggcgc gggccctcct gccgaacgtc 2580
atcacaaagg aggtcagcca cgagatcatc aaggaccggc gcttcacctc cgacaagttt 2640
ttctttcacg tgcccatcac gctcaactac caggccgcca actcgccgtc caagttcaac 2700
cagcgcgtga acgcctacct caaggagcac cccgagaccc cgatcatcgg gattgaccga 2760
ggggagcgga acctcatcta catcaccgtc atcgacagca ccgggaagat ccttgaacag 2820
cggtcgctca acaccatcca gcagttcgac taccagaaga aactcgacaa ccgggagaag 2880
gagagagtgg cggcccgcca ggcttggtcc gtcgtcggga cgattaagga cttgaaacaa 2940
ggttacctgt cgcaagtgat ccacgagatc gttgacctga tgatccacta ccaagccgtc 3000
gtggtcctgg agaacctcaa cttcggcttc aagagcaaac gaaccggcat cgcggagaag 3060
gccgtgtacc agcagttcga aaaaatgctg atcgacaagc tgaactgcct cgtgctcaag 3120
gactaccccg ctgagaaggt cggcggggtg ctgaacccgt accagctcac tgaccagttc 3180
accagcttcg caaagatggg cacccagtcc ggcttcctgt tctacgtgcc tgcgccatac 3240
acctcgaaga tcgacccgct caccgggttc gtggacccct tcgtctggaa gaccatcaag 3300
aaccacgaga gccgcaagca cttcctggag ggcttcgact tcctccacta cgacgtcaag 3360
accggggact tcatcctgca cttcaagatg aaccgcaacc tcagtttcca gcgcggcctg 3420
ccggggttca tgcccgcttg ggatatagtc ttcgagaaga atgagacgca gttcgacgcg 3480
aagggcaccc cgttcatcgc cgggaagcgc atcgtgccgg tcatcgagaa ccaccggttc 3540
accgggcgct accgcgacct atacccggcg aacgagttga tcgccctcct ggaggagaag 3600
ggcatcgtgt tccgcgacgg ctccaacatc ctcccgaagc tgctcgaaaa cgacgactcc 3660
cacgccatcg acacgatggt cgcgctgatc cggtcggtgc tccagatgcg gaactccaac 3720
gccgcgacgg gcgaggacta catcaacagt ccggtccgcg atctgaacgg cgtctgcttc 3780
gactcccggt tccagaaccc cgagtggccg atggacgcgg acgcgaacgg cgcataccac 3840
atcgccctaa aagggcaatt gctgctcaac cacctcaagg aatccaaaga cctaaagctc 3900
cagaacggca tctccaacca ggactggctg gcgtacatcc aggaactgcg gaacgggagc 3960
aaaaaacgtc ggatcaagca agattga 3987
<210> 22
<211> 3987
<212> DNA
<213> 人工的
<220>
<223> Cas12a多核苷酸
<400> 22
atggcgggct ccaagaaacg ccggattaag caagataccc agttcgaggg gttcacgaac 60
ctctaccaag tgagcaagac cctccgattc gaactgattc ctcaggggaa gaccctcaag 120
cacatccagg agcaagggtt catcgaggag gacaaggcgc ggaacgacca ctacaaggaa 180
ctcaaaccca tcatcgaccg catctacaag acctacgccg atcagtgcct ccagctcgtg 240
cagttggact gggagaacct cagcgcggcc attgactcct accggaagga gaaaacggag 300
gagacgcgca acgcgctcat cgaggaacag gcaacctatc gcaacgccat ccacgactac 360
ttcatcggga ggactgacaa cctcactgac gcgattaaca agcgccacgc ggagatatac 420
aagggactct tcaaagcgga gctgtttaac ggcaaggttc tcaagcaact cggcactgtg 480
accacgaccg agcatgagaa cgccctgctc cgctccttcg acaagttcac cacctacttc 540
tccgggttct accgcaaccg caagaatgtc ttcagcgcgg aggacatcag cacggccatt 600
ccacatcgaa tcgtccaaga taacttcccg aagttcaagg agaactgcca catcttcacc 660
cgactcatta ctgctgtacc gtcgttacgc gaacacttcg agaacgtcaa gaaggcaatt 720
ggaatcttcg tctctacgtc aatagaggag gtgttcagct tccctttcta caaccagctc 780
cttacgcaga cccagataga cctgtacaat cagctcctcg gtgggatcag ccgggaggcg 840
gggactgaga agattaaagg gctcaacgag gtcttgaacc tggccatcca aaaaaacgat 900
gagacggcgc acatcatcgc ctcgctgccc caccggttca tcccgctgtt caagcagatc 960
ctcagtgaca ggaacacctt gagctttatc ctagaggagt tcaagagcga cgaggaggtg 1020
atccagagct tctgcaagta caaaaccctg ctgaggaacg agaacgtcct ggagacggcg 1080
gaggcgctgt tcaacgagct gaactctatc gacttaactc acatattcat ctcgcacaag 1140
aagctggaga ctattagctc tgcactctgc gaccactggg acaccctccg caacgcgctc 1200
tacgagcgcc gcatctcgga gctgaccggg aagatcacca aatccgcgaa ggaaaaggtc 1260
cagcgttccc tcaaacacga ggatattaac ttacaggaga ttatctcagc ggctgggaag 1320
gagttgtcag aggcgttcaa gcagaaaact tccgagatcc tgagccacgc gcacgcagcg 1380
ctcgaccagc ctctgcccac caccctcaaa aagcaggaag aaaaagagat cctcaagagc 1440
cagttggact ccctgctggg gctctatcac cttctcgact ggttcgccgt cgatgagtcg 1500
aacgaggtgg accccgagtt ctccgcccgg ctgaccggca tcaagctaga gatggagccg 1560
tccctcagct tctacaataa ggcccgcaac tacgcgacca aaaaacccta cagcgtggag 1620
aagttcaagc tgaacttcca gatgccgacc ttagcacgcg gttgggacgt aaacagggag 1680
aagaacaatg gagccatcct gttcgtcaag aacgggcttt actacctcgg gataatgccc 1740
aagcagaagg gccgctacaa ggccctttcc ttcgagccga cggagaaaac ctccgagggg 1800
ttcgacaaga tgtactacga ctacttcccc gacgccgcca agatgatccc gaagtgctca 1860
acgcagctaa aagccgtgac cgcccacttc cagacccaca cgacgccgat cctgctgagc 1920
aacaacttca tcgagcccct tgagatcact aaggagatat acgacctgaa caaccccgag 1980
aaggagccca agaagtttca aaccgcctac gccaaaaaaa ctggcgacca aaagggctac 2040
agggaggcgc tgtgtaagtg gatcgacttc acacgcgact tcctttcgaa gtatacgaag 2100
acaacctcta ttgacctgag cagcctgcgt cctagctccc agtacaaaga tttgggcgag 2160
tactacgcgg agcttaatcc actactctac cacatctcat tccagcgcat cgctgagaag 2220
gaaatcatgg acgcggtgga gacaggcaaa ctgtacctct tccagatata caacaaagac 2280
ttcgctaagg ggcaccacgg gaagcccaac cttcatacgc tctactggac gggcctattc 2340
agccccgaaa atctggccaa gacctccatc aagctgaacg gccaagcgga gctgttctac 2400
agacccaaga gccggatgaa gcggatggcc cacaggctcg gcgagaaaat gcttaacaaa 2460
aagttgaagg accagaaaac ccctatcccc gacaccctct accaggaact gtacgactac 2520
gtgaaccaca ggctctcgca cgacctttcc gacgaggccc gtgccctact cccgaacgtc 2580
attaccaaag aggtttcgca cgagatcatc aaggaccggc ggttcacgag cgacaagttt 2640
ttctttcacg tccccatcac ccttaactac caggcggcca actccccatc caagttcaac 2700
cagcgtgtga atgcctacct caaggagcac ccagagaccc cgatcattgg gatcgaccgg 2760
ggcgagcgga acctgatcta catcaccgtc atcgactcga cgggcaagat tcttgagcag 2820
agatcgttga ataccataca gcagttcgac taccagaaga aactcgacaa ccgcgagaag 2880
gagcgcgtgg cggcccgcca ggcgtggtcc gtcgttggga cgattaagga cttgaaacaa 2940
ggttatctgt cccaagtcat ccacgagatc gttgatctga tgatccacta tcaggcagtg 3000
gtggtgctgg agaatctcaa cttcggcttc aagagtaagc ggacgggaat cgccgagaag 3060
gccgtgtacc agcagttcga gaagatgctg atcgacaagc tcaactgcct tgtgctgaaa 3120
gactacccgg ccgagaaggt cggcggcgtc ctcaacccgt accaacttac cgaccagttc 3180
acctccttcg ccaagatggg cactcagtcc gggttcttgt tctacgtccc cgcaccttac 3240
acctctaaga tcgaccctct gactggcttc gtagatccat tcgtgtggaa gaccattaag 3300
aaccacgaga gccgcaagca cttcctggag ggcttcgact tcctgcacta cgacgtgaag 3360
accggggact tcatccttca cttcaagatg aaccggaacc tcagcttcca gcggggcctg 3420
ccggggttca tgcccgcctg ggacatcgtg ttcgagaaga acgagaccca gttcgacgcg 3480
aagggcacgc ccttcatcgc cgggaagcgt atcgtgccgg tgatcgagaa ccatcgtttc 3540
acgggtcgct accgtgacct ctacccggcg aacgagctta tcgcactcct ggaggagaag 3600
ggcatcgtct tccgggacgg ctccaacatc ctcccgaaac tgctggaaaa cgacgactct 3660
cacgccatcg acacgatggt ggccctcatc cggtccgtgc tccaaatgcg gaacagcaac 3720
gccgccaccg gtgaggacta catcaacagc ccggtccggg atctgaacgg ggtgtgcttc 3780
gattcgcggt tccagaatcc tgagtggccg atggacgcgg atgcaaacgg ggcgtaccac 3840
atcgcgctca agggccagtt acttctgaac caccttaagg agtctaaaga tttgaaactc 3900
cagaacggga tctcgaacca ggactggctg gcctacatcc aagagttgcg gaacggcagc 3960
aagaagcggc ggattaagca agattag 3987
<210> 23
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 23
gacaagaagt acagcatcgg gctggcgatc gggaccaact ccgtcggctg ggctgtgatt 60
accgacgagt acaaggtgcc atccaagaag ttcaaggtcc tcggcaacac tgaccggcac 120
agcattaaga agaacctgat tggggcgctg ctgttcgatt cgggggagac tgcggaggcg 180
accaggctga agcggactgc gcgccggagg tacaccagga ggaagaatcg gatctgctac 240
ctccaggaga ttttctcgaa tgagatggcc aaggtggacg attccttctt ccatcgcctg 300
gaggagtcgt tcctcgttga ggaggacaag aagcatgaga ggcatcccat tttcgggaat 360
atcgttgacg aggtggctta ccatgagaag tacccgacca tctaccatct gcggaagaag 420
ctcgtcgatt cgaccgataa ggccgacctg cggctgatct acctggccct cgcgcacatg 480
attaagttcc ggggccattt cctcatcgag ggcgacctca acccggacaa ctcggacgtg 540
gataagctct tcattcagct cgtgcagaca tacaaccagc tcttcgagga gaatcccatt 600
aacgcctcgg gggtcgacgc taaggctatt ctctcggctc ggctgtcgaa gtcgcgccgg 660
ctggagaatc tcattgccca gctcccaggc gagaagaaga acggcctctt cggcaacctg 720
attgccctgt cgctggggct cacaccgaat ttcaagtcga acttcgacct cgccgaggac 780
gctaagctcc agctcagcaa ggatacttac gatgatgacc tcgataacct gctcgcccag 840
attggggatc agtacgcgga tctgttcctc gcggccaaga atctcagcga tgctattctc 900
ctgtcggaca ttctccgcgt caacacagag attactaagg ccccactgtc ggcgagcatg 960
attaagaggt acgatgagca tcatcaggac ctgacactgc tcaaggcgct ggtccggcag 1020
cagctccccg agaagtacaa ggagattttc ttcgatcagt caaagaatgg gtacgcgggc 1080
tacattgatg gcggcgcgtc ccaggaggag ttctacaagt tcattaagcc catcctggag 1140
aagatggacg ggaccgagga gctgctggtg aagctcaatc gggaggacct gctccggaag 1200
cagcgcacat tcgacaatgg ctcgattcct caccagattc acctgggcga gctgcacgcc 1260
attctccgca ggcaggagga cttctacccg ttcctcaagg acaaccgcga gaagatcgag 1320
aagatcctga ccttccggat tccatactac gtggggccgc tcgcgcgggg gaactcccgg 1380
ttcgcgtgga tgactcgcaa gtccgaagaa acgattacac cgtggaattt cgaggaggtc 1440
gtcgacaagg gcgctagtgc gcagtcattc attgagagga tgaccaattt cgataagaac 1500
ctgcctaacg agaaggtgct gccgaagcat tcgctgctct acgagtactt caccgtttac 1560
aatgagctga ccaaggtgaa gtatgtgact gagggcatga ggaagccagc gttcctgagc 1620
ggcgagcaga agaaggctat cgtggacctg ctcttcaaga ctaaccggaa ggtgactgtg 1680
aagcagctca aggaggacta cttcaagaag attgagtgct tcgattccgt tgagattagc 1740
ggggtggagg atcggttcaa tgcttcgctc gggacatacc acgatctcct gaagatcatt 1800
aaggataagg acttcctcga caacgaggag aacgaggaca ttctcgaaga tattgtcctg 1860
accctcaccc tcttcgagga tcgggagatg atcgaggaga ggctcaagac atacgctcat 1920
ctgttcgatg ataaggtcat gaagcagctg aagcgcaggc ggtacacagg gtgggggcgg 1980
ctgagccgga agctgatcaa cgggattcgg gataagcagt ccgggaagac aattctcgac 2040
ttcctcaagt ccgacgggtt cgctaaccgg aacttcatgc agctcattca tgatgactcg 2100
ctgacattca aggaggatat tcagaaggcg caggtttcgg ggcagggcga ctcgctccac 2160
gagcatattg cgaatctggc gggctccccc gcgattaaga agggcattct gcaaaccgtc 2220
aaggtggttg atgagctggt caaggtcatg gggcggcata agccagagaa tattgtcatc 2280
gagatggcgc gggagaatca gaccacacag aaggggcaga agaactcacg ggagcggatg 2340
aagcgcatcg aggagggcat caaggagctg gggtcgcaga tcctgaagga gcatcccgtg 2400
gagaacactc agctgcaaaa tgagaagctg tacctctact acctccagaa cgggagggac 2460
atgtatgtgg atcaggagct ggatattaat aggctgagcg attacgatgt cgaccacatt 2520
gtcccacagt cgttcctgaa ggacgacagc attgacaaca aggtgctgac ccgctcggat 2580
aagaacaggg gcaagagcga taatgttcca agcgaggagg ttgtgaagaa gatgaagaac 2640
tactggcggc agctcctgaa cgcgaagctc atcacacagc ggaagttcga caacctcacc 2700
aaggctgagc gcgggggcct gagcgagctg gacaaggcgg ggttcattaa gaggcagctg 2760
gtcgagacac ggcagattac aaagcatgtt gcgcagattc tcgattcccg gatgaacacc 2820
aagtacgatg agaacgataa gctgattcgg gaggtcaagg taattaccct gaagtccaag 2880
ctggtgtccg acttcaggaa ggacttccag ttctacaagg ttcgggagat caacaactac 2940
caccacgcgc atgatgccta cctcaacgcg gtcgtgggga ccgctctcat caagaagtac 3000
ccaaagctgg agtcagagtt cgtctacggg gattacaagg tttacgacgt gcggaagatg 3060
atcgctaaga gcgagcagga gattggcaag gctaccgcta agtacttctt ctactccaac 3120
atcatgaact tcttcaagac agagattacc ctcgcgaatg gcgagatccg gaagaggccc 3180
ctcatcgaga caaatgggga gacaggggag attgtctggg ataaggggcg ggatttcgcg 3240
accgtccgga aggtcctgtc gatgccccag gttaatattg tcaagaagac tgaggtccag 3300
actggcggct tctcaaagga gtcgattctc ccaaagagga actccgataa gctcattgct 3360
cggaagaagg attgggaccc caagaagtac gggggattcg actcccccac tgttgcttac 3420
tctgttctgg ttgttgctaa ggtggagaag gggaagtcga agaagctgaa gagcgtgaag 3480
gagctgctcg ggattacaat tatggagagg tcatccttcg agaagaatcc catcgacttc 3540
ctggaggcca agggctacaa ggaggtgaag aaggacctga ttattaagct gcccaagtac 3600
tcgctcttcg agctggagaa tgggcggaag cggatgctgg cgtccgcggg ggagctgcaa 3660
aaggggaacg agctggcgct cccctccaag tatgtgaact tcctctacct ggcgtcgcac 3720
tacgagaagc tgaaggggtc cccagaggat aatgagcaga agcagctctt cgtcgagcag 3780
cataagcact acctggacga gattatcgag cagattagcg agttctcgaa gcgggtcatc 3840
ctcgcggatg cgaacctgga taaggtgctc agcgcctaca ataagcaccg ggacaagccg 3900
attcgggagc aggcggagaa tattattcac ctcttcacac tcaccaacct cggggcacca 3960
gctgcgttca agtacttcga cactactatc gaccggaagc ggtacacctc gacgaaggag 4020
gtgctcgacg ccaccctcat tcaccagtcg atcacaggcc tgtacgagac acggattgac 4080
ctgtcccagc tcgggggcga c 4101
<210> 24
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 24
gacaagaagt actccattgg cctggcgatt gggacaaact cggtggggtg ggccgtgatt 60
acggatgagt acaaggttcc aagcaagaag ttcaaggtcc tcgggaacac agatcggcat 120
tcgattaaga agaatctcat tggggcgctc ctcttcgact cgggggagac agcggaggct 180
accaggctca agcggacagc caggcggcgg tacacaaggc ggaagaatcg catctgctac 240
ctccaggaga ttttctcgaa tgagatggcg aaggtggacg acagcttctt ccatcggctg 300
gaggagtcct tcctggtgga ggaggataag aagcacgaga ggcatccaat tttcgggaac 360
atcgtggacg aggttgcgta ccatgagaag taccctacaa tctaccatct gcggaagaag 420
ctggttgact ccacagacaa ggcggacctg aggctgatct acctcgctct ggcccacatg 480
attaagttcc gcgggcattt cctgatcgag ggggacctga atcccgacaa ttcggatgtg 540
gacaagctct tcatccagct ggtgcagacc tacaaccagc tgttcgagga gaatcccatc 600
aatgcgtcgg gcgttgacgc taaggccatt ctgtccgcta ggctgtcgaa gagcaggagg 660
ctggagaacc tgatcgccca gctgccaggc gagaagaaga atgggctctt cgggaatctg 720
attgcgctct ccctggggct gacaccgaac ttcaagagca atttcgatct ggctgaggac 780
gcgaagctcc agctctcgaa ggacacttac gacgatgacc tcgataacct cctcgcgcag 840
atcggggacc agtacgctga tctcttcctc gccgctaaga acctctcgga tgctatcctg 900
ctctccgaca ttctccgggt taataccgag attacaaagg ccccactgtc ggcgtccatg 960
atcaagcggt acgatgagca tcatcaggat ctcaccctgc tcaaggccct cgtgcggcag 1020
cagctgcccg agaagtacaa ggagattttc ttcgaccaga gcaagaatgg gtacgctggc 1080
tacattgacg gcggggcctc acaggaggag ttctacaagt tcatcaagcc aatcctggag 1140
aagatggatg ggacagagga gctgctggtg aagctcaacc gggaggatct gctcaggaag 1200
cagcggacgt tcgacaacgg gtcgattccc catcagatcc acctggggga gctgcacgcg 1260
atcctgcgcc ggcaggagga tttctaccct ttcctgaagg ataatcggga gaagatcgag 1320
aagattctca ccttccggat tccctactac gtcgggccac tcgcgcgggg caatagcagg 1380
ttcgcctgga tgacacggaa gagcgaggag acaatcaccc cctggaactt cgaggaggtt 1440
gtcgacaagg gggcgtccgc ccagtcattc attgagcgga tgaccaattt cgacaagaat 1500
ctgccaaatg agaaggttct cccaaagcat agcctcctct acgagtactt cactgtttac 1560
aacgagctga ccaaggtgaa gtatgtgacc gagggcatgc ggaagcccgc gttcctgtcc 1620
ggcgagcaga agaaggccat tgtggacctc ctgttcaaga ccaatcgcaa ggtcacagtc 1680
aagcagctca aggaggatta cttcaagaag atcgagtgct tcgactcggt tgagattagc 1740
ggggtggagg atcggttcaa cgcgagcctc ggcacttacc acgacctcct gaagatcatc 1800
aaggataagg acttcctcga caacgaggag aacgaggata ttctggagga catcgtgctc 1860
accctgacgc tgttcgagga tcgggagatg atcgaggagc gcctgaagac ctacgctcat 1920
ctcttcgatg ataaggtcat gaagcagctg aagaggaggc ggtacaccgg gtggggccgc 1980
ctgagcagga agctcattaa cgggatcagg gacaagcaga gcggcaagac catcctggac 2040
ttcctcaaga gcgatggctt cgccaaccgg aatttcatgc agctcatcca cgacgactcc 2100
ctcaccttca aggaggacat tcagaaggct caggtcagcg gccagggcga ctcgctgcat 2160
gagcacatcg ctaacctggc gggcagccca gccatcaaga agggcatcct ccagacagtg 2220
aaggtcgtgg atgagctggt gaaggtcatg ggccggcata agcccgagaa tattgtgatt 2280
gagatggcgc gggagaatca gaccactcag aagggccaga agaactcgcg ggagcgcatg 2340
aagaggatcg aggaggggat taaggagctg ggcagccaga ttctcaagga gcaccccgtg 2400
gagaataccc agctccagaa cgagaagctg tacctctact acctccagaa tgggcgggac 2460
atgtatgttg atcaggagct ggacatcaat cgcctctcgg attacgacgt ggaccacatc 2520
gtgccccaga gcttcctgaa ggatgatagc atcgacaata aggtcctgac ccgctccgac 2580
aagaatcgcg gcaagagcga caacgtgccg agcgaggagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgcgaagctc attacacagc ggaagttcga taacctgacg 2700
aaggcggaga ggggcggcct ctccgagctg gacaaggcgg gcttcattaa gaggcagctc 2760
gtggagactc gccagatcac caagcacgtg gctcagatcc tcgatagccg gatgaatacg 2820
aagtacgatg agaatgacaa gctcatccgg gaggtgaagg taatcaccct gaagtcaaag 2880
ctcgttagcg atttccggaa ggacttccag ttctacaagg tgcgggagat taacaactac 2940
catcatgcgc acgatgcgta cctcaatgcg gtggtgggca cagccctgat taagaagtac 3000
cccaagctgg agagcgagtt cgtctacggg gactacaagg tgtacgatgt tcggaagatg 3060
atcgccaaga gcgagcagga gattgggaag gccaccgcta agtacttctt ctactcgaat 3120
attatgaatt tcttcaagac cgagatcaca ctcgctaatg gggagattcg gaagcggccc 3180
ctcatcgaga ctaacgggga gactggcgag attgtgtggg acaaggggcg cgacttcgct 3240
accgtgcgca aggtcctctc gatgccccag gttaatattg ttaagaagac agaggtgcag 3300
acgggcgggt tctccaagga gtctatcctg ccgaagcgga actcggacaa gctgatcgcc 3360
cgcaagaagg attgggaccc caagaagtac gggggattcg atagcccaac cgtggcttac 3420
agcgtcctgg tggtcgccaa ggttgagaag gggaagtcga agaagctcaa gagcgttaag 3480
gagctgctgg gcatcaccat catggagcgg tccagcttcg agaagaatcc tatcgacttc 3540
ctggaggcta aggggtacaa ggaggtcaag aaggacctga tcattaagct gcccaagtac 3600
tctctgttcg agctggagaa cgggaggaag cggatgctgg cgtctgctgg cgagctacag 3660
aagggcaatg agctggcgct cccctcgaag tatgtcaact tcctctacct ggcttcccat 3720
tacgagaagc tgaagggctc gcccgaggat aatgagcaga agcagctctt cgtggagcag 3780
cacaagcact acctcgacga gatcattgag cagatttcgg agttctcgaa gcgggtcatt 3840
ctcgcggacg cgaacctcga caaggtcctc tcggcgtaca acaagcaccg ggacaagccc 3900
atccgggagc aggccgagaa cattatccac ctcttcacac tgaccaacct cggcgctccc 3960
gccgcgttca agtacttcga caccaccatt gaccgcaaga gatacacatc caccaaggag 4020
gtgctggacg cgaccctcat ccaccagagc atcacaggcc tctacgagac acggatcgac 4080
ctctcgcagc tcgggggcga t 4101
<210> 25
<211> 4092
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 25
gacaagaagt actcgatcgg cctggcgatt ggcacaaaca gcgtggggtg ggctgtgatc 60
actgatgagt acaaggtgcc atcgaagaag ttcaaggtgc tggggaatac agaccggcat 120
tcgatcaaga agaatctcat tggcgctctc ctcttcgatt ccggcgagac tgctgaggcg 180
acccgcctga agcgcaccgc ccggcggcgc tacactcggc ggaagaatag gatttgctac 240
ctccaggaga ttttctcgaa tgagatggcc aaggtggatg acagcttctt ccaccgcctg 300
gaggagtcgt tcctggtcga ggaggacaag aagcatgagc ggcaccctat cttcgggaat 360
atcgttgatg aggtcgccta ccacgagaag taccccacta tctaccatct ccgcaagaag 420
ctcgtggaca gcacagataa ggccgacctc cgcctgatct acctcgccct cgcgcacatg 480
attaagttcc gggggcactt cctcattgag ggggatctga atcccgataa ctccgacgtg 540
gacaagctgt tcatccagct ggtgcagaca tacaaccagc tgttcgagga gaatcccatc 600
aacgcgagcg gcgtggacgc taaggccatt ctgtcggcta ggctctcgaa gtcgaggcgg 660
ctggagaacc tgattgcgca gctccccggc gagaagaaga acgggctgtt cgggaatctc 720
atcgccctct ccctcggcct cacaccaaac ttcaagagca atttcgacct ggctgaggac 780
gctaagctgc aactctcaaa ggatacatac gatgacgacc tggacaatct cctggctcag 840
atcggcgacc agtacgctga cctgttcctc gcggccaaga atctgtcgga cgcgattctc 900
ctcagcgaca tcctgcgcgt caataccgag attacgaagg ctccactgtc tgcgtcaatg 960
attaagcggt acgatgagca tcaccaggat ctgaccctcc tgaaggcgct cgtgcggcag 1020
cagctgcccg agaagtacaa ggagattttc ttcgatcaga gcaagaatgg ctacgccggc 1080
tacatcgacg ggggcgcgag ccaggaggag ttctacaagt tcatcaagcc catcctggag 1140
aagatggacg gcaccgagga gctactcgtg aagctcaatc gggaggatct cctccggaag 1200
cagcggacat tcgataacgg gtctatccca caccagatcc acctcggcga gctgcatgcg 1260
attctgcggc ggcaggagga tttctaccct ttcctgaagg acaaccggga gaagatcgag 1320
aagatcctca cattccggat tccatactac gtcggccccc tggcgagggg caatagccgg 1380
ttcgcgtgga tgacaaggaa gtccgaggag actattaccc cgtggaattt cgaggaggtg 1440
gttgacaagg gcgcttccgc gcagagcttc attgagcgga tgacaaactt cgacaagaat 1500
ctccccaacg agaaggtcct gccgaagcat agcctcctgt acgagtactt caccgtctac 1560
aatgagctaa ctaaggtcaa gtatgtgaca gagggcatga ggaagccagc cttcctctca 1620
ggcgagcaga agaaggccat tgtggacctc ctgttcaaga caaaccgcaa ggtgacagtg 1680
aagcagctga aggaggatta cttcaagaag attgagtgct tcgactcagt ggagatttca 1740
ggcgtggagg atcggttcaa cgcgagcctg gggacttacc acgacctgct gaagattatt 1800
aaggacaagg acttcctgga taacgaggag aatgaggaca tcctggagga tattgtgctc 1860
accctcaccc tgttcgagga cagggagatg attgaggaga ggctcaagac ctacgcgcac 1920
ctgttcgatg acaaggtcat gaagcagctg aagaggcggc gctacactgg gtggggccgc 1980
ctgtcgcgga agctgatcaa cggcattcgg gataagcagt ccgggaagac cattctggat 2040
ttcctgaagt cggacggctt cgccaacagg aatttcatgc agctgatcca cgacgactcc 2100
ctcaccttca aggaggacat tcagaaggcc caggttagcg gccaggggga ctcactccac 2160
gagcatattg ccaatctggc cggctctcca gctatcaaga agggcatcct gcaaacagtt 2220
aaggttgttg acgagctggt taaggtcatg gggcggcata agcccgagaa cattgtcatc 2280
gagatggctc gggagaacca gacaactcag aagggccaga agaactccag ggagcgcatg 2340
aagcggattg aggagggcat taaggagctg gggtcccaga tcctcaagga gcaccctgtc 2400
gagaacactc agctgcaaaa cgagaagctc tacctgtact acctccagaa cgggcgggat 2460
atgtatgtgg atcaggagct ggacatcaac aggctctccg actacgacgt ggatcacatt 2520
gtcccacagt ctttcctcaa ggatgattcc atcgacaaca aggtgctgac gcgcagcgac 2580
aagaataggg ggaagtcgga caacgttccg agcgaggagg tcgtgaagaa gatgaagaat 2640
tactggaggc agctcctgaa tgcgaagctg atcactcaga ggaagttcga caatctgaca 2700
aaggcggaga ggggcgggct ctcggagctg gataaggcgg gcttcatcaa gcggcagctc 2760
gttgaaaccc ggcagatcac caagcatgtc gcccagatcc tcgatagccg catgaacacc 2820
aagtacgatg agaacgacaa gctcattcgg gaggttaagg tcattacgct gaagtccaag 2880
ctcgtcagcg acttcaggaa ggatttccag ttctacaagg ttcgggagat taacaactac 2940
caccacgcgc atgatgcgta cctgaacgct gttgtcggca ctgctctcat caagaagtac 3000
ccaaagctgg agtccgagtt cgtctacggg gactacaagg tctacgatgt ccggaagatg 3060
atcgccaagt cggagcagga gatcgggaag gctactgcga agtacttctt ctacagcaac 3120
attatgaatt tcttcaagac ggagattacg ctggcgaacg gggagattag gaagaggccc 3180
ctcattgaga ctaatgggga gacaggcgag attgtttggg acaagggccg cgacttcgcg 3240
actgtgcgga aggtcctgtc catgccacag gtgaatattg ttaagaagac agaggtgcag 3300
actgggggct tctcgaagga gagcattctc ccaaagcgga acagcgataa gctcatcgcg 3360
cgcaagaagg attgggaccc taagaagtac ggcggcttcg attctcccac tgtggcctac 3420
tccgttctcg tggttgccaa ggttgagaag gggaagtcga agaagctgaa gtcggtcaag 3480
gagctgctcg ggattacaat catggagcgg agcagcttcg agaagaaccc tattgatttc 3540
ctggaggcca agggctacaa ggaggttaag aaggatctca ttatcaagct ccctaagtac 3600
tctctgttcg agctggagaa tggccggaag aggatgctgg cctcggctgg cgagctacag 3660
aaggggaatg agctggccct cccgtcgaag tatgtgaatt tcctgtacct cgcgtcgcac 3720
tacgagaagc tcaagggcag cccggaggat aatgagcaga agcagctctt cgtggagcag 3780
cataagcact acctggacga gatcattgag cagatcagcg agttctcgaa gcgggttatt 3840
ctggctgatg ctaacctgga caaggttctg agcgcctaca ataagcatcg cgacaagccg 3900
attcgcgagc aggcggagaa tattatccac ctgttcaccc tcactaacct cggggctccc 3960
gcggccttca agtacttcga taccacaata gataggaagc ggtacacctc gacgaaggag 4020
gtcctcgacg ccacactcat ccatcagtcg attacaggcc tgtacgagac acggattgac 4080
ctctcgcagc tg 4092
<210> 26
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 26
gacaagaagt attccatagg cctggctatc ggcaccaaca gcgtgggctg ggccgtcatc 60
accgacgagt acaaagtgcc gagtaaaaag ttcaaagtgc tcggcaacac cgaccgccac 120
tccataaaga aaaacctgat cggggcgctc ctgttcgaca gcggcgagac ggcggaggcc 180
acccgcttga aacgcacggc ccgacggcgc tacacgcggc gcaagaaccg gatctgttac 240
ctacaggaga ttttctctaa cgagatggcg aaggtggacg actcgttctt tcaccgcctc 300
gaagagtcct tcctcgtgga ggaggacaag aaacacgagc gccacccgat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag tacccgacca tctaccacct ccggaagaaa 420
ctcgtggaca gcacggacaa ggccgacctg aggctcatct acctcgccct ggcgcacatg 480
attaagttcc ggggccactt cctgatcgag ggcgacctga acccggacaa cagcgacgtg 540
gacaagctgt tcatccagct agtccagacc tacaaccagc ttttcgagga aaaccccatc 600
aacgccagcg gggtggacgc gaaggcgatc ctgtccgccc ggctgagcaa gtcccggcgg 660
ctggagaacc tcatcgcgca gttgcccggc gagaagaaga acgggctgtt cgggaacctg 720
atcgccctct ccctggggct caccccgaac ttcaagtcca acttcgacct cgccgaggac 780
gccaaactac agctgagcaa ggacacctac gacgacgacc tcgacaacct gctggcccag 840
atcggggacc agtacgcaga cctgttcctc gccgccaaga acctctccga cgccatcctg 900
ctgtcggaca tcctgcgggt gaacacggag atcacgaagg ccccgctctc ggcctcgatg 960
attaaacgct acgacgagca ccaccaggac ttgaccctcc tcaaggcgct ggtccgccag 1020
cagcttcccg agaagtacaa ggaaatcttt ttcgatcaga gcaagaacgg gtacgccggg 1080
tacatcgacg gcggggcgtc ccaggaggag ttctacaagt tcatcaagcc catcctggag 1140
aaaatggacg ggaccgagga gctgctcgtg aagctcaacc gcgaagattt gctccgcaag 1200
cagcgcacgt tcgacaacgg gtcgatcccg caccagatcc acctgggcga gctgcacgcg 1260
atcctcaggc gtcaggaaga cttctacccc ttcctcaagg acaaccgcga gaagatagag 1320
aagattctga ccttcagaat tccttattac gtgggcccgc tggctcgggg caactcgcgc 1380
ttcgcctgga tgacgcgcaa gtccgaggag accatcaccc cgtggaactt cgaggaggtg 1440
gtggataagg gtgcctcggc ccagtccttc atcgagcgga tgaccaactt cgacaagaac 1500
ctgccgaacg agaaggtgct ccccaagcac agcctgctct acgaatattt cacggtgtac 1560
aacgagctga cgaaggtcaa gtacgtgacc gagggaatga ggaaacctgc attcctctcc 1620
ggggagcaga agaaagccat agtcgacctc ctgttcaaga ccaaccggaa ggtcaccgtc 1680
aagcagctca aggaggacta cttcaagaag atcgagtgct tcgattcagt ggagatcagc 1740
ggcgtcgagg accggttcaa cgccagcctg ggcacctacc acgacctgct caagatcatc 1800
aaggacaagg acttcctcga caacgaggag aacgaggaca tcctggagga catcgtgctg 1860
accctgacgc tcttcgagga ccgcgagatg atcgaggagc gcctcaagac ctacgcccac 1920
ctgttcgacg acaaggtgat gaagcagctc aagcggcgga gatatactgg gtggggccgc 1980
ctctcccgga agctcattaa cggtatcagg gataagcagt ccgggaagac gatcctcgac 2040
ttcctcaagt cggacgggtt cgccaaccgc aacttcatgc agctcatcca cgacgactcc 2100
ctgacgttca aggaggacat ccagaaggcc caagtgtctg gtcaaggtga ctcgctccac 2160
gagcacatcg ccaacctcgc gggcagcccg gccatcaaga agggaatact ccagaccgtc 2220
aaggtggtgg acgagctggt gaaggtcatg ggccgccaca agccggagaa catcgtcatc 2280
gagatggcgc gggagaacca gaccacgcag aaggggcaga aaaatagccg tgagcgcatg 2340
aagcgcatcg aggaggggat taaggagttg ggcagccaga tcctcaagga gcaccctgtg 2400
gagaacacgc agttgcaaaa cgagaagctc tacctgtact acctccagaa cgggagggat 2460
atgtacgtgg accaagaact ggacatcaac cgcctgtccg actacgacgt ggaccacatc 2520
gtgccgcaga gcttcctcaa ggacgacagc atcgacaaca aggtgctcac ccggtccgac 2580
aagaatcggg gcaagtccga caacgtgccc agcgaggagg tcgtcaaaaa gatgaaaaac 2640
tactggcgac aactactgaa cgccaagctc atcacccagc gcaagttcga caacctcaca 2700
aaagccgagc gcggcgggtt gagcgagctg gacaaggccg ggttcatcaa gcgccagctc 2760
gtcgagacgc gccagatcac gaagcacgtc gcgcagatac tcgacagccg gatgaacacc 2820
aagtacgacg agaacgacaa gctcatccgg gaggtgaagg tcatcaccct caagtcgaag 2880
ctcgtgagcg acttccgcaa ggacttccag ttctacaagg tccgggagat caacaactac 2940
caccacgccc acgatgctta tcttaacgcc gtggtgggga cggccctcat taagaaatac 3000
ccgaagctgg agtcggagtt cgtgtacggc gactacaagg tgtacgacgt caggaagatg 3060
atcgccaagt ccgaacagga gatcgggaag gccacggcga aatacttctt ctacagcaac 3120
atcatgaact tcttcaagac cgagatcacc ctcgccaacg gcgagatccg caagcgcccg 3180
ctcatcgaga cgaacgggga gaccggcgag atcgtctggg acaaggggcg cgacttcgcc 3240
actgtgcgga aggtgctgtc gatgccccag gtcaacatcg tcaagaagac ggaggtccag 3300
acgggcgggt tcagcaagga gagcatcctg ccgaagcgca acagcgacaa gctgatcgcc 3360
cgcaaaaagg actgggatcc aaaaaagtac ggcggcttcg acagccccac cgtcgcctac 3420
agcgtcctcg tcgtcgctaa agtcgagaag ggcaagtcca aaaagctcaa gagcgtcaag 3480
gagctgctcg ggatcaccat catggagcgg tccagcttcg agaagaaccc aattgatttc 3540
ctggaggcga agggctacaa ggaggtcaag aaagacctca tcataaagct gccgaagtac 3600
tcactcttcg agctggagaa cgggcgcaag cggatgctgg cgtcggccgg agagctccaa 3660
aagggcaacg agctggcgct gccgagcaag tacgtgaact tcctctacct ggcgtcccac 3720
tacgagaagc tcaagggcag tccagaggat aacgagcaga agcagctatt cgtggagcag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gcgcgtcatc 3840
ctggcggacg ccaacctgga caaggtgctg tccgcgtaca acaagcaccg cgacaagccg 3900
atccgcgagc aagccgagaa catcatccac ctgttcaccc tcacgaacct cggggcaccc 3960
gccgccttca aatatttcga cacgaccatc gaccgcaagc gctacaccag cacgaaggag 4020
gtgctcgacg ccaccctgat ccaccagagc atcaccgggc tgtacgagac ccgcatcgac 4080
ctctcgcagc tcggcgggga c 4101
<210> 27
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 27
gacaagaagt acagtattgg attggccatc gggacgaaca gcgtgggctg ggccgtcatc 60
accgacgagt acaaggtgcc atccaagaag tttaaggttc tggggaatac cgaccgccac 120
tcgatcaaga aaaatctcat cggggcgctg cttttcgaca gcggcgagac ggcggaagcg 180
acgcggctca agcggacggc tcgtcgccgt tacacccggc gtaagaaccg catctgttac 240
ctccaggaga tattcagcaa cgagatggcg aaggtggacg actccttttt ccaccgtctt 300
gaggagtcct tcctggtcga ggaggacaag aagcacgagc gccacccgat cttcgggaac 360
atcgtggacg aggtggccta ccacgagaag taccccacga tctaccacct ccgcaaaaaa 420
ctcgtggact caactgacaa ggccgatttg aggcttatct acctcgccct cgcccacatg 480
attaagttcc gtgggcactt cctaatcgag ggtgacctca accccgacaa ctctgacgtg 540
gacaagctgt tcatccagct tgtgcagacc tacaatcagc tctttgagga gaatccgatc 600
aacgcatctg gtgtggacgc aaaggccatc ctcagcgcgc ggctgagcaa gtctaggcgg 660
ttggagaacc tgatcgccca actgcccggc gagaagaaaa atggcctctt cggcaacctg 720
atcgccctgt cgctggggct cacgccgaac ttcaagagta actttgacct ggcggaggac 780
gctaagctcc agctatctaa ggacacatac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctcttccta gccgccaaga acctgtccga cgccatcctc 900
ctcagcgaca tcctgcgcgt gaacacggag atcacgaagg ctccgctcag cgcctccatg 960
attaagcggt acgacgagca ccaccaagac ctaactttac tcaaagccct cgtgcggcag 1020
cagcttcccg agaagtacaa agagatattt tttgatcagt ccaagaacgg ttatgcgggc 1080
tacatcgacg gcggcgcgag ccaggaggag ttctacaagt tcatcaagcc catcctggag 1140
aagatggacg gcacggagga gctgctcgtg aagctcaacc gtgaagacct cctgcgaaag 1200
cagcgaacct tcgacaacgg ttcgatcccg caccagatcc acctcgggga gctgcacgcc 1260
atcctgaggc gacaggagga cttctaccct ttcctaaagg acaaccgcga gaagattgaa 1320
aaaatcctga cgtttcgcat accctactac gtcggcccgc tggcgcgcgg caactcccgg 1380
ttcgcctgga tgacccgtaa gagcgaggag acgatcaccc cgtggaactt cgaggaggtc 1440
gtggacaagg gcgcgagcgc gcagagcttc atcgagcgca tgaccaactt cgacaagaac 1500
ctcccgaacg agaaggtgct cccaaagcac tccctcctgt acgagtattt caccgtgtac 1560
aacgagttga caaaggtgaa gtacgtgacg gagggaatgc ggaagcctgc gttcctctcg 1620
ggcgagcaga agaaggcaat cgtggacctg ctcttcaaga ccaaccggaa ggtgacggtg 1680
aagcagctca aggaggacta cttcaaaaaa atcgagtgct tcgactccgt ggagataagc 1740
ggcgtggagg accgattcaa cgcctccctc ggcacctacc acgacctcct taagatcatc 1800
aaggacaagg acttcctgga caacgaggag aacgaggaca tcctggagga catcgtgctc 1860
accctgaccc tcttcgagga ccgggagatg atcgaggagc gcctcaagac gtacgcccac 1920
ttgttcgacg acaaggtgat gaagcagctc aagcggcggc gatacaccgg gtggggccgc 1980
ctatcccgca aacttatcaa cggcatccgc gacaagcagt ccggcaagac gatcctggat 2040
ttcctcaagt cggacgggtt cgccaaccgg aacttcatgc agctcatcca cgacgacagc 2100
ctcacgttca aggaggacat ccagaaggcc caagtgagcg gtcaagggga cagcctccac 2160
gagcacattg cgaaccttgc tgggagccct gcgatcaaga aggggatatt gcaaaccgtg 2220
aaggtcgtgg acgagttggt gaaggtcatg gggcgacaca agcccgagaa catcgtgatc 2280
gagatggcca gggaaaatca gaccacgcag aagggccaaa aaaacagccg cgagcggatg 2340
aagcggatcg aggagggcat caaggagctg gggtcgcaga tcctcaagga gcacccggtg 2400
gagaacacgc agctccagaa cgagaagctg tacctctatt acctacagaa cgggcgggat 2460
atgtacgtgg accaggagct agacatcaac cgcctgtccg actacgacgt ggaccatatc 2520
gtcccgcagt cgttcttgaa ggacgacagc atcgacaaca aggtgctcac aagatcggat 2580
aagaatcgag gcaagtccga caacgtgccc tcggaggagg tggtcaagaa aatgaaaaac 2640
tactggcggc agttgctgaa cgccaagctc attacgcagc ggaagttcga caacctgacg 2700
aaggctgaac gtggtgggct cagcgagcta gacaaggcgg ggttcatcaa gcggcagctc 2760
gtcgagaccc ggcagatcac caagcacgtg gcgcagatcc tggactcgcg catgaacacc 2820
aagtacgacg agaacgacaa gctcatccgt gaggtgaagg tcatcaccct taagtctaag 2880
ctggtcagtg acttccgcaa ggacttccag ttctacaagg tccgggagat caacaactac 2940
caccacgcgc acgacgccta cctcaacgcg gtggtgggga cggcgcttat taagaaatat 3000
cccaagctgg aaagcgagtt cgtttacggc gactacaagg tgtacgacgt ccgcaagatg 3060
atcgcaaagt cggaacagga aatcggaaag gcgacggcca aatatttctt ttactccaac 3120
atcatgaatt tttttaagac ggagatcacc ctggcgaacg gggagatccg caagcggccc 3180
ctcatcgaga ccaacgggga gacgggcgag atcgtctggg acaagggccg ggacttcgcc 3240
accgtgcgga aggtgctttc tatgcctcaa gtcaatatcg tcaaaaagac agaggtgcag 3300
accggcgggt tcagcaagga gtctatcctg ccgaagcgca actcggacaa gctcatcgcg 3360
cgcaagaaag actgggaccc caaaaaatat ggcgggttcg actcgccgac cgtcgcctac 3420
agcgtcctcg tggtggctaa ggtcgagaag ggcaagagca aaaagctaaa gtcggtgaag 3480
gagctgctgg gcatcaccat catggagcgc tcgtctttcg agaagaatcc aatcgacttc 3540
ctagaggcga aggggtacaa ggaggtcaaa aaggatctta tcatcaaact gccgaagtac 3600
agtctgttcg agctggagaa cgggcggaag cggatgctgg ctagtgcggg cgagttgcag 3660
aagggcaacg agttggcact gccctccaag tacgtgaact tcctgtacct ggcctcccac 3720
tacgagaagc tcaaggggag ccccgaggac aacgagcaga agcagctatt cgtcgagcag 3780
cacaagcact acctggacga gatcatcgag cagatcagtg agttctccaa gcgggtcatc 3840
ctcgcggacg ccaacctgga caaggtgctg agcgcgtaca acaagcacag ggacaagcca 3900
atcagggaac aggccgagaa catcatccac ctgttcaccc tgaccaacct gggtgcaccg 3960
gctgccttca agtactttga cacgaccatc gaccggaagc gctacacctc cacgaaggag 4020
gtgctggacg ccacgctgat ccaccagagc atcaccgggc tctacgagac acggatcgac 4080
ctgagccagc ttggcgggga c 4101
<210> 28
<211> 4092
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 28
gacaaaaagt attccattgg actcgctatc ggcacgaaca gcgtcgggtg ggcggtcatc 60
actgacgagt acaaggtgcc gagcaagaag tttaaggtgc tgggaaacac cgacaggcac 120
tcgatcaaga aaaatcttat cggggcccta ctcttcgact ccggagaaac cgccgaggcc 180
acccggttga agcgcacggc ccgccgtcgc tacaccaggc gcaagaaccg gatctgctac 240
ctccaggaga tattcagcaa tgagatggcg aaggtggacg actcgttttt tcacaggcta 300
gaggagtctt tcctcgtgga ggaggacaag aaacacgagc gccaccccat cttcggcaac 360
atcgtggatg aggtggcata tcacgagaag tacccaacca tctaccacct ccgcaaaaag 420
ctcgtggact ctaccgacaa ggccgacctc cgtctgatct acctcgcgct ggcccacatg 480
attaagttcc gaggacactt tctgatcgag ggcgacctga acccagacaa cagcgacgtg 540
gacaagctgt tcatccaact tgtccagacc tacaatcagc tcttcgagga gaaccctatc 600
aacgcctcgg gcgtggacgc gaaggccatc ctgtccgccc gcctgagcaa gtcgcggcgg 660
ctggagaacc tgatcgccca gctccccggc gaaaaaaaga acggcctctt cggcaacctc 720
atcgcgttgt cgctggggct caccccgaac ttcaagtcca acttcgacct ggccgaggac 780
gctaaactcc agctctcgaa ggatacctac gacgacgacc tcgacaacct gctggcccag 840
atcggcgacc agtacgcgga ccttttcctg gcggccaaga acctgagcga cgcgatcctc 900
cttagcgaca tactccgtgt gaacaccgag atcacgaagg ccccgctctc cgcgtccatg 960
attaagcgct acgacgagca ccaccaagac cttaccctgc ttaaggcgct ggtcaggcag 1020
cagttaccgg agaagtacaa ggagatcttt tttgatcaat ctaagaacgg ttacgccggg 1080
tacatcgacg gcggcgcgtc ccaggaggag ttctacaagt tcatcaagcc gatcttggag 1140
aaaatggacg ggaccgagga gctgctcgtg aagctcaacc gcgaagacct cctccgcaag 1200
cagcgcacct tcgacaacgg gagcatcccg caccagatcc acctgggaga gctgcacgcg 1260
atcctgcgga gacaagagga cttctacccc ttcctcaagg acaaccggga gaagattgaa 1320
aaaatactta cttttcgtat cccgtactac gtcgggcccc ttgcgagggg caactccaga 1380
ttcgcgtgga tgacccgcaa gtccgaggag accatcaccc cgtggaactt cgaggaggtg 1440
gtggacaagg gcgcgtcggc ccagtcgttc atcgagcgca tgaccaactt cgacaagaac 1500
cttccgaacg agaaggtgct cccgaagcac agcctgctct acgaatattt tactgtgtac 1560
aacgagctga cgaaggtcaa gtacgttacg gaggggatga ggaagcccgc cttcctctcc 1620
ggcgagcaga agaaagccat tgtggatctc ctgttcaaga ccaaccgcaa ggtgacggtg 1680
aaacagctca aagaggacta cttcaagaag atcgagtgct tcgactccgt agagatcagc 1740
ggggtcgagg accgcttcaa cgcctcgctg ggcacgtacc acgacctgct aaagattatc 1800
aaggacaaag acttcctaga caatgaggag aacgaggaca ttctggagga catcgtgctg 1860
actctgacgc tgttcgaaga ccgcgagatg atcgaggagc ggcttaagac gtacgcccac 1920
ctgttcgacg acaaggtgat gaagcagttg aaacggcggc gctacaccgg gtggggccgc 1980
ctctcccgca agctcatcaa cggcatccgc gacaagcagt cggggaagac gatcctggac 2040
ttcctcaaga gcgacggctt cgccaaccga aacttcatgc agctaatcca cgacgacagc 2100
ctgacgttca aggaggacat ccagaaggcc caagtgagcg gccagggaga ctcgctacac 2160
gagcatatcg ccaacctggc tggcagcccg gcgattaaga aaggaatcct ccaaaccgtc 2220
aaagtggtgg acgagctggt gaaggtgatg ggccgccaca agcccgagaa cattgtgatc 2280
gagatggcgc gggagaacca gacgacgcag aagggccaaa aaaatagcag ggaaaggatg 2340
aagcgaatag aggaggggat caaggagctg gggagccaga ttctcaaaga gcacccggtc 2400
gagaacacac agctccagaa cgagaagctg tacctctact acctccaaaa cggccgcgat 2460
atgtacgtgg accaggaact agacatcaac cggctgagcg actatgacgt ggaccacatc 2520
gtgccgcagt ccttcctcaa ggacgactcg attgacaaca aagtgctcac tagatccgac 2580
aagaacagag gcaagagcga taacgtcccg tcggaggagg tcgtcaagaa aatgaaaaac 2640
tactggcggc agctcctaaa cgccaagctc atcacgcagc gtaagttcga caacctgacg 2700
aaggcggagc ggggcgggct gagcgagctg gacaaagcgg ggttcatcaa gcggcagctc 2760
gttgagacgc ggcagatcac aaagcacgtc gcgcaaatcc tcgactcccg catgaacacc 2820
aagtacgacg agaacgacaa gctcatccgg gaggtgaagg tcattaccct taaatcgaag 2880
ctcgtcagcg actttcgtaa ggacttccag ttctacaagg tcagagagat caacaactac 2940
caccacgccc acgacgccta tctgaacgcc gtggtgggca ccgcgcttat taagaagtac 3000
cccaagctgg agtccgagtt cgtgtacggc gactacaagg tttatgacgt caggaagatg 3060
atcgccaagt cggaacagga gatcggaaaa gctaccgcca aatatttctt ctatagcaac 3120
atcatgaact tcttcaaaac cgagatcacc ctcgccaacg gcgagatccg gaagcgcccg 3180
ctcatcgaga ccaacgggga gaccggggag atcgtctggg acaaggggcg ggacttcgct 3240
actgtccgaa aggtgctctc catgccacaa gtgaatatcg tcaagaaaac agaggtgcag 3300
accggagggt tcagtaagga gtccatcctg cccaagcgga actccgacaa gctaattgct 3360
cgcaaaaagg attgggatcc taaaaaatat ggcggcttcg actcgcccac ggtcgcctac 3420
tctgtgctgg tcgtggcgaa ggtggagaag ggcaagtcca agaagctcaa gagcgtcaag 3480
gagctgctgg ggatcacgat catggagcgt agttcgtttg agaagaatcc catcgacttc 3540
ctggaggcta agggctacaa ggaggtcaaa aaggacctca tcattaagct gccgaagtac 3600
agcctcttcg agctggagaa cgggcggaag cgtatgctcg cctccgctgg ggagttacaa 3660
aaggggaacg agctggcgct gccgtctaag tacgtcaact tcctgtacct ggcctcccac 3720
tacgagaagc tcaaggggtc gccggaggac aacgagcaga agcagctctt cgtagagcag 3780
cacaagcact acctggacga gatcatcgag cagatttcag agttctcaaa gcgggtcatc 3840
ctcgccgacg ccaacctgga caaggtgctc tcggcctaca acaagcaccg ggacaagccg 3900
atccgcgaac aggccgaaaa catcatccac ctgttcacgc tcaccaacct cggtgccccg 3960
gcggccttca agtactttga cacgaccatc gaccggaagc gctatacctc gacgaaggag 4020
gtgctggacg ccaccctgat ccaccagtcc atcaccgggc tttacgagac ccggatcgac 4080
ctctcgcagc ta 4092
<210> 29
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 29
gacaagaagt atagtattgg actcgccatc ggaaccaact ctgtggggtg ggctgttatt 60
acagatgaat ataaggtgcc atccaaaaag tttaaagttc tgggcaatac tgatagacac 120
tcaatcaaga agaatctgat aggtgcactt ctgtttgata gtggagagac tgccgaggca 180
accagactta aaaggactgc aagaagaaga tataccagaa gaaagaatag gatttgctat 240
ttgcaggaaa tcttcagcaa cgaaatggcc aaggttgatg actcattttt ccataggttg 300
gaggagagtt ttcttgtgga ggaagataag aagcacgaaa gacacccaat tttcgggaat 360
atagtggacg aggtggctta tcatgagaag tatcccacta tctaccacct gagaaagaaa 420
cttgtggact caaccgataa ggctgatctt aggcttatat acttggccct tgcacatatg 480
atcaaattca ggggccattt tcttatcgaa ggcgatctta atcccgataa ctcagatgtg 540
gacaagctgt ttatacaact tgtgcaaacc tacaatcaac tcttcgagga gaatcccatt 600
aacgcctccg gcgtggatgc aaaagccata ctgtcagcca gactgagcaa aagtaggaga 660
ctggagaatc ttatagccca actgcccggt gaaaagaaga atgggctctt cggaaatctg 720
atcgctcttt cattggggtt gacacccaac tttaagagta actttgactt ggcagaagat 780
gcaaagttgc agctcagtaa agacacatat gacgatgacc ttgacaatct cttggcacaa 840
ataggggatc aatacgctga ccttttcctc gctgccaaga acctcagcga cgctatactg 900
ttgtccgaca ttcttagggt taataccgaa attacaaagg cccctcttag tgcaagtatg 960
atcaaaaggt atgatgagca tcaccaagac cttacactgc tgaaggctct ggttagacag 1020
caactccctg aaaagtataa ggaaatattc ttcgaccaaa gtaagaacgg gtacgccggt 1080
tatattgatg ggggcgcaag tcaagaagaa ttttacaaat tcatcaagcc aattcttgaa 1140
aagatggacg ggactgagga attgctggtg aaactgaata gagaggacct tcttagaaaa 1200
cagaggacat ttgacaatgg gtccatccca caccagattc atctggggga actccacgca 1260
atattgagga gacaagaaga cttttaccca ttccttaagg ataatagaga gaaaatcgaa 1320
aaaatcctga ctttcaggat tccttactat gttgggccac tggccagggg gaactcaaga 1380
ttcgcttgga tgacaaggaa gtcagaagaa accataaccc cttggaattt tgaagaggtg 1440
gttgataagg gggcatcagc ccagtctttc atagagagga tgaccaactt tgataaaaat 1500
cttccaaatg agaaggtttt gccaaaacat agtcttttgt acgagtactt tactgtttat 1560
aacgaattga ccaaggtgaa gtatgtgacc gagggaatga ggaagccagc atttttgtcc 1620
ggggagcaaa agaaagcaat cgttgatctt ctcttcaaga ccaacagaaa agtgaccgtg 1680
aaacaactga aggaagacta cttcaaaaag atagaatgtt tcgattcagt ggaaattagc 1740
ggtgttgaag acaggttcaa tgcttcattg ggtacttacc acgacctgtt gaagataatc 1800
aaagacaagg actttctcga taatgaggag aacgaagaca tcttggaaga cattgtgctt 1860
acactcactt tgtttgagga cagggaaatg attgaggaaa gactcaaaac ttacgctcat 1920
ttgtttgatg ataaggttat gaaacaacta aaaagaagaa ggtacaccgg ctggggaaga 1980
ttgagtagga aactgatcaa cggtattaga gataaacaat ccggaaagac tatcctcgat 2040
ttccttaaga gtgatggctt tgcaaatagg aattttatgc agctgattca tgacgactca 2100
cttaccttca aagaagacat ccaaaaagct caggtgtctg ggcaaggcga cagtctgcat 2160
gaacatatag ctaacttggc tgggagtccc gccatcaaga aggggatact tcaaacagtt 2220
aaagttgtgg acgaattggt gaaggtaatg ggaaggcaca agcctgaaaa tatagtgata 2280
gaaatggcaa gggaaaatca aacaacccag aagggacaga agaacagtag ggaaaggatg 2340
aaaaggatag aagaggggat caaagagctt ggtagccaga tcctcaagga acatccagtg 2400
gagaataccc aacttcaaaa cgagaaactc tatttgtact acttgcagaa cggaagagat 2460
atgtatgtgg accaagagct tgatattaac aggctgagcg attatgacgt tgaccacata 2520
gtgccccaat cattcctcaa ggatgactct attgataata aggtgctgac aaggagtgac 2580
aagaatagag ggaaatccga caacgttcca tccgaggaag ttgtgaagaa gatgaagaac 2640
tactggaggc agttgctgaa cgctaagctc attacccaga ggaaattcga taacctgacc 2700
aaagcagaga gaggcgggct gagcgaactc gataaagcag gtttcatcaa gagacaactc 2760
gtggagacta ggcaaattac taagcacgtg gctcaaatac tcgacagcag gatgaacaca 2820
aagtacgacg agaacgacaa gctcattaga gaggttaagg ttattactct gaaaagtaaa 2880
ttggttagcg atttcagaaa ggatttccaa ttctataagg ttagagagat caacaattat 2940
catcatgcac atgatgccta tctgaatgct gtggttggta cagcccttat caagaagtac 3000
cctaagctag agagcgagtt tgtgtacgga gattataagg tgtatgatgt gaggaaaatg 3060
atcgctaaaa gtgagcaaga gattggaaag gctaccgcca aatacttctt ttattccaat 3120
attatgaatt tcttcaagac agaaatcacc ctggctaacg gcgagataag gaagaggccg 3180
cttatcgaaa ctaatgggga gacaggcgaa atagtgtggg acaaagggag ggatttcgca 3240
actgtgagga aggttttgag catgcctcag gtgaatatcg ttaagaaaac cgaagttcaa 3300
actggagggt tctctaagga aagcattctc cccaagagga actccgacaa gctgattgct 3360
agaaagaaag actgggaccc caagaagtat ggcggattcg actcacccac tgtggcatat 3420
agcgttctcg tggtggcaaa ggttgaaaag ggtaaatcca aaaaactcaa atccgtgaag 3480
gaactccttg gcataactat tatggaaagg agtagctttg aaaagaatcc catcgacttt 3540
ctcgaagcta agggctataa ggaagttaag aaggacctta taatcaaact tccaaaatac 3600
tccctttttg agttggaaaa cggcagaaag agaatgttgg ccagtgccgg ggagcttcaa 3660
aagggcaacg aactggctct gcctagcaaa tatgtgaact ttttgtatct ggcatcacac 3720
tacgagaaac ttaaaggctc tcctgaggac aacgagcaaa aacagctctt tgttgaacag 3780
cataagcact acctcgacga gattattgag cagatcagcg agttctcaaa gagagttatt 3840
ctggctgacg ctaatcttga caaggttttg tccgcttaca acaaacacag ggataagcca 3900
atcagggagc aggcagaaaa cataatccat ctctttaccc tgacaaacct cggtgccccc 3960
gctgctttca agtattttga tactaccatt gacaggaaga gatatacttc cactaaggaa 4020
gtgctcgacg caaccctcat acaccaaagt atcacaggcc tctatgaaac taggatagat 4080
ttgtctcaac ttgggggcga t 4101
<210> 30
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 30
gacaaaaagt attccatcgg gcttgctatc ggaaccaact ctgtggggtg ggcagttatt 60
accgacgaat acaaggtgcc cagcaagaag tttaaggttc tggggaacac agatagacat 120
agcataaaga aaaacctgat aggcgcactg ttgttcgact ccggggaaac agccgaagct 180
accaggctga agagaactgc aagaagaagg tacaccagaa gaaaaaacag aatatgttat 240
ctccaagaga ttttctctaa cgagatggcc aaggtggacg actcattctt tcacagactg 300
gaagaatctt tccttgtgga agaagataag aaacacgaga ggcaccctat ttttggcaat 360
atcgtggatg aggtggctta ccacgaaaaa taccctacaa tataccacct caggaaaaaa 420
ttggttgata gtacagacaa ggccgacctc aggctcatct atttggccct ggcccatatg 480
attaaattca gggggcactt tctcatcgag ggagatttga accccgacaa cagtgatgtt 540
gataagctct ttattcagct cgtgcagact tacaatcagt tgtttgagga aaaccccatt 600
aatgcttccg gggtggacgc caaggcaatc ctttctgcaa gactctcaaa gtcaaggaga 660
ctcgaaaatc tgatagcaca gcttccagga gagaagaaga acgggctctt tggaaacctg 720
atcgctctgt cactcggact cacacccaat ttcaaaagca attttgattt ggcagaggac 780
gctaagctgc aactcagtaa ggatacctac gacgatgact tggataatct gctcgcacaa 840
attggggacc agtatgcaga cctgtttctc gcagctaaga acttgagtga cgccatattg 900
ctcagtgaca tcctcagggt taataccgag attacaaaag ctccactctc tgcaagcatg 960
atcaagaggt atgacgagca ccatcaagac ctgacactcc ttaaggcgtt ggttaggcag 1020
caacttcctg aaaagtataa ggaaatcttc ttcgatcaaa gcaaaaacgg ctacgccggc 1080
tatatagacg ggggagcatc ccaagaagaa ttttataagt tcataaaacc tatattggag 1140
aagatggacg ggacagagga attgctcgtg aaactgaaca gggaggatct cctcaggaag 1200
caaaggacct tcgacaatgg ctccatccca catcagattc acctcggcga actgcacgca 1260
atactgagaa gacaagagga cttttatcct ttcctgaagg acaacaggga gaaaatcgag 1320
aaaatcttga cattcagaat cccatactac gttgggcctc tggccagagg taacagtagg 1380
ttcgcctgga tgactaggaa atcagaggag actattacac cctggaactt tgaagaagtt 1440
gttgataagg gagcttcagc acaatcattc atcgaaagaa tgacaaactt tgacaaaaat 1500
ctgcctaatg agaaagtgct cccaaaacat tccctgctgt atgagtattt taccgtttat 1560
aacgagctta ccaaggtgaa atacgttact gaaggtatga gaaagccagc ttttctttca 1620
ggggagcaaa agaaggctat cgtggatctt ctctttaaga ccaacagaaa ggttaccgtg 1680
aagcagctta aggaagacta ctttaaaaag atcgagtgtt ttgactcagt ggaaataagc 1740
ggtgttgaag atagattcaa cgcatccttg ggaacttatc atgatcttct taagataatc 1800
aaggataaag actttctcga caacgaggaa aacgaagata tactggagga catagttctg 1860
acacttactt tgttcgagga tagggagatg atcgaggaaa gactgaaaac atatgctcac 1920
cttttcgacg acaaagttat gaaacaactc aagagaagga gatatacagg gtgggggaga 1980
ttgagcagga aactgattaa tggtatcaga gacaaacagt caggaaaaac aatactcgac 2040
tttttgaaat cagacgggtt cgcaaatagg aatttcatgc agcttataca cgacgattca 2100
cttactttta aagaggacat tcaaaaggct caagttagtg gacaaggtga ctccctccac 2160
gaacacatcg caaatctcgc tggcagccct gcaattaaga agggtatact ccagacagtt 2220
aaggttgttg acgagctggt taaagtgatg ggaagacaca aacccgagaa catagtgata 2280
gagatggcca gggaaaacca aaccactcaa aaagggcaga aaaattccag agagaggatg 2340
aaaaggattg aagaaggtat caaggagctg ggtagccaaa ttctgaaaga acatcctgtg 2400
gaaaacactc aactccagaa tgagaaactc tatctgtact atctgcaaaa tgggagagat 2460
atgtatgtgg accaggaact ggacataaac aggctctcag attacgatgt ggatcatatc 2520
gtgccacagt cctttcttaa ggatgatagc atcgacaata aggtgcttac caggtccgac 2580
aagaacaggg gaaagtcaga taacgtgcct tctgaagaag ttgttaaaaa gatgaagaac 2640
tactggagac agctgcttaa cgctaagctc ataacacaga ggaagtttga caacttgacc 2700
aaggccgaga gaggcggact ctcagaattg gataaggcag ggttcataaa aaggcagctg 2760
gtggaaacaa ggcagataac taaacatgtg gctcagatcc tcgatagtag gatgaataca 2820
aaatacgatg agaacgacaa gctcataagg gaggttaaag tgataactct gaaatccaaa 2880
ctggttagcg attttaggaa ggatttccag ttttacaaag ttagggagat caacaattat 2940
catcacgccc acgatgccta cttgaacgca gttgtgggta ctgcacttat caaaaagtac 3000
cctaagctgg aatccgagtt tgtttatgga gactataagg tgtacgacgt tagaaaaatg 3060
attgcaaagt cagagcagga gatagggaaa gccactgcaa aatatttctt ttatagcaat 3120
atcatgaatt tctttaagac agaaatcaca ctggccaatg gggaaataag gaagaggccc 3180
ctgatcgaaa ctaatggcga gacaggggag attgtgtggg ataaaggtag ggactttgca 3240
acagtgagga aagtgctgag catgccccaa gttaatatcg ttaaaaagac cgaggttcaa 3300
acagggggct ttagtaagga aagcattttg cccaagagga atagtgacaa attgattgct 3360
aggaaaaaag attgggaccc caaaaagtat ggcggatttg atagccccac tgttgcttac 3420
tccgtgctcg tggttgcaaa ggtggagaag ggaaagagca agaaactgaa gtcagttaag 3480
gaactccttg gtatcactat catggaaaga agctcctttg agaagaaccc tattgacttc 3540
ctggaggcta aagggtacaa agaggttaag aaagacctta tcattaaatt gcccaaatat 3600
agtcttttcg agcttgaaaa cggaagaaag aggatgcttg catccgctgg cgaattgcaa 3660
aagggcaatg agcttgctct cccttccaag tatgtgaact tcctttatct tgcctcacac 3720
tatgaaaaac tcaaaggttc acccgaagac aacgaacaaa agcaactatt tgtggaacaa 3780
cacaagcact acctggacga aatcattgag caaatttctg agttttcaaa aagggtaatc 3840
ttggctgacg caaatctcga caaagttttg tcagcttaca acaaacatag agataagcca 3900
attagagagc aagctgagaa tatcatccat ctgtttaccc tgactaacct tggagcgcct 3960
gctgctttta aatatttcga caccacaatc gacaggaaga ggtacactag cactaaggaa 4020
gttctcgacg ccaccctcat ccaccagagt attacaggcc tgtacgagac aagaattgat 4080
ctttctcaac ttggtggtga c 4101
<210> 31
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 31
gataagaagt actcaatcgg tctggcaatc ggaaccaact ctgtgggttg ggcagtgatt 60
acagatgagt ataaggtgcc aagcaaaaaa ttcaaggtgc tgggtaatac cgacagacac 120
agcattaaga agaatttgat tggagcactc ctctttgact caggggaaac agcagaggca 180
acaaggctga agaggacagc aaggcggagg tacacaaggc ggaaaaacag gatatgctac 240
ctccaggaaa tctttagcaa cgagatggct aaagtggatg atagcttttt ccatagactc 300
gaagaatcct ttcttgttga agaggacaaa aagcatgaaa ggcatcccat cttcggcaat 360
atagttgatg aggttgcata ccatgagaag taccccacaa tctaccacct cagaaagaaa 420
cttgtggact ccacagataa agcagacctg aggctcatat acctcgcact cgcacacatg 480
atcaagttca gagggcactt tctcatcgaa ggtgacctga atccagataa ttcagatgtg 540
gataaactgt ttatacagct ggtgcaaaca tacaaccaac ttttcgagga aaacccaatc 600
aatgcctccg gtgttgatgc aaaggccatc ctgtcagcaa gactcagcaa aagcaggcgg 660
ctcgaaaacc tcatcgccca gcttcccggt gaaaagaaga acgggctctt tggtaatctc 720
atcgcattga gccttggtct tactccaaac ttcaagagca attttgatct ggcagaggat 780
gctaaactgc aactctcaaa ggacacatat gacgatgacc ttgacaatct gttggcccag 840
atcggggacc aatatgcaga cctcttcctg gccgcaaaga atctgtcaga tgcaatcctc 900
ttgtccgaca tactgagagt taacactgag atcacaaagg cacctctgtc cgcctccatg 960
attaagagat acgatgagca tcaccaggat ctgactttgc tcaaagccct cgttagacag 1020
cagttgccag aaaagtacaa agaaatattc tttgatcaat caaaaaacgg atatgcaggg 1080
tacatcgacg gtggggcaag ccaggaagag ttctacaaat tcatcaaacc tatcctggaa 1140
aagatggatg ggacagaaga gctgctggtt aagctgaata gggaagacct cctcagaaag 1200
cagaggacat ttgataacgg gagcatccct catcaaatcc acctcggtga actccatgct 1260
atcctgagaa ggcaggaaga cttttatcca tttttgaagg acaataggga gaaaatcgaa 1320
aaaatcctga cattcagaat cccatactac gttggtcctc tggcaagagg taacagtagg 1380
ttcgcatgga tgacaaggaa aagcgaggag acaatcacac cctggaattt tgaggaagtt 1440
gttgacaagg gtgccagcgc acaatccttt atcgaaagaa tgacaaattt cgacaagaat 1500
ctgcctaacg aaaaggttct cccaaagcat tcactcctgt acgaatattt tacagtttat 1560
aacgaactga ctaaagttaa atacgttacc gagggtatga ggaagccagc attcctttcc 1620
ggggaacaga agaaagctat tgtggacctc ctgttcaaga caaatagaaa agtgacagtt 1680
aagcaactca aagaggatta cttcaaaaag atcgaatgtt ttgactctgt ggagatcagc 1740
ggggtggagg atagattcaa cgccagcctg ggtacatatc atgatctcct gaaaatcatt 1800
aaagacaagg acttccttga caacgaggag aacgaggaca ttctggaaga cattgttctg 1860
accctcacac tctttgagga tagggagatg attgaggaaa gactgaagac ctacgcccac 1920
ctctttgacg ataaagtgat gaaacagctc aagagaagaa ggtatacagg ttgggggaga 1980
ctgagcagga agttgatcaa tgggattagg gacaaacagt ccgggaaaac aatcctcgat 2040
tttctgaagt cagacggttt cgcaaacaga aattttatgc agctcattca cgatgacagc 2100
ttgacattca aggaagacat ccaaaaggct caagtgagcg gccaagggga tagcctccac 2160
gagcatattg caaatctggc aggttcacca gccatcaaaa agggcatact tcagacagtt 2220
aaggttgtgg acgaattggt taaagttatg ggcaggcata agccagagaa tatcgttatc 2280
gaaatggcaa gggagaacca aacaactcaa aaagggcaga aaaatagcag agagaggatg 2340
aaaagaatcg aggaagggat caaggaactt gggtcccaaa tcctcaagga gcacccagtt 2400
gaaaatactc aactgcaaaa cgagaagctc tatctctact atctccaaaa cgggagggat 2460
atgtatgttg accaggagct ggatattaac agactgtcag attatgatgt tgatcatatc 2520
gtgccccagt cattcctgaa ggacgattcc atcgacaaca aagttctcac aaggtccgat 2580
aaaaacaggg gcaagtccga taacgttcca agcgaagaag tggtgaaaaa gatgaaaaac 2640
tattggagac aacttctgaa tgcaaagttg attactcaga gaaagtttga caacctcaca 2700
aaagcagaaa gaggcgggct tagcgaactc gataaggcag ggtttatcaa aagacagctg 2760
gttgagacaa ggcagatcac aaaacatgtg gcacagatcc ttgactcaag gatgaatacc 2820
aagtatgatg agaatgataa gttgatcagg gaggttaaag ttatcacact caaatccaaa 2880
ctggtgtcag acttcaggaa agactttcaa ttttataagg tgagggagat caataactac 2940
caccatgcac atgacgccta cctgaacgca gtggtgggta cagcattgat taaaaaatac 3000
cctaagctgg agtctgagtt tgtgtacggg gactacaagg tgtacgacgt gaggaaaatg 3060
atagccaagt ccgagcagga gatcgggaaa gcaacagcta agtatttctt ttacagtaat 3120
atcatgaatt tctttaaaac tgagattact ctggcaaacg gggagatcag gaaaagaccc 3180
ctcatcgaga ctaatggtga aacaggtgag atcgtttggg acaaggggag ggattttgct 3240
actgttagaa aagttctgag tatgccacaa gtgaatattg tgaaaaagac agaagttcag 3300
acaggtgggt tctccaaaga atccatcctg cccaagagaa attcagacaa gctcatcgca 3360
agaaagaagg actgggaccc taagaagtac ggaggatttg acagccccac cgtggcctat 3420
tccgtgcttg ttgtggcaaa ggtggagaaa gggaagagca aaaaactgaa atccgtgaaa 3480
gaactgctgg gaattaccat catggaaaga agctcctttg agaagaaccc aatcgacttc 3540
ctggaagcaa aaggatataa ggaagtgaaa aaggacctca ttatcaagct cccaaaatac 3600
tcacttttcg agttggagaa cggtagaaag aggatgctgg caagcgcagg ggaacttcag 3660
aaaggcaatg agctggcatt gccatcaaag tatgtgaact tcctctactt ggccagccat 3720
tacgagaaac ttaaaggtag cccagaagat aacgagcaaa aacagctctt tgtggaacag 3780
cataagcatt atctggatga gatcatagaa caaatctcag agttttccaa gagagttatc 3840
ctcgcagatg caaacctgga taaggttctc tcagcctata ataagcatag agacaagcca 3900
attagagagc aagcagagaa cattatccac ttgttcactc ttacaaacct gggggcacca 3960
gccgccttca aatatttcga tacaacaata gacagaaaga ggtataccag caccaaagaa 4020
gttctcgacg ccacactgat ccatcaatca atcacaggcc tttacgaaac taggatcgac 4080
ttgtcacaac tgggtgggga t 4101
<210> 32
<211> 3307
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 32
gagcaaggac acctacgacg acgacttgga caacctattg gcccagatag gtgaccagta 60
tgcagacctc ttccttgcgg ccaagaactt gagtgacgct atactgctca gtgacatcct 120
gagggtgaac actgagatca ctaaggcccc tctctctgcc tcaatgatta agcgttacga 180
cgagcatcac caggatctca ccctgcttaa ggcccttgtt cggcagcagc tccctgagaa 240
gtacaaggag atattttttg accagtctaa gaacggctac gccggttaca ttgacggtgg 300
ggcaagccag gaggagttct acaagttcat caagccgatc cttgagaaga tggacggcac 360
cgaggagcta cttgtcaagt tgaaccggga agacctgctc cggaaacagc gtacattcga 420
caacggcagc atccctcacc agatccacct gggcgaacta cacgccatcc tccgacgtca 480
ggaggacttc tatccattct tgaaagataa cagggaaaaa atcgaaaaaa tacttacgtt 540
tcgaatacct tactacgtgg ggccccttgc tcggggaaac tccagattcg catggatgac 600
caggaagtca gaggagacca tcacaccctg gaactttgag gaggtggttg acaaaggtgc 660
ttctgcccag tccttcattg agcggatgac taacttcgac aagaacctgc ccaacgagaa 720
ggtgctgcca aagcacagcc tgctctacga atactttact gtgtacaatg agctgacgaa 780
ggtgaagtac gtgacagagg ggatgcggaa gcccgctttc ctgagcggcg agcaaaaaaa 840
agcaatcgtg gacctactgt tcaagaccaa ccgaaaggtg acagtgaagc agctcaagga 900
ggactacttc aaaaaaatcg agtgcttcga ctctgttgag ataagcggcg tggaggaccg 960
attcaacgcc tcattgggaa cctatcacga cctgctcaag atcattaagg acaaggactt 1020
cctggataat gaggagaatg aggacatcct ggaggatatt gtgctgaccc ttactctatt 1080
cgaggacagg gagatgatcg aggagcgact caagacctac gctcacctgt tcgacgacaa 1140
ggttatgaag caattgaagc gtaggcgata cacggggtgg ggaagactct cccgaaaact 1200
gataaacggc atcagggaca agcagtcagg gaagacgatc ttggacttcc tgaaatccga 1260
cgggttcgcc aaccgcaact tcatgcagct cattcacgac gactcactaa cgttcaaaga 1320
ggacattcag aaggctcaag tcagtggaca aggcgactcc ctgcacgagc acattgcaaa 1380
ccttgcgggc tccccggcga ttaaaaaggg cattctccaa acggttaagg tggtggacga 1440
gctggtgaag gtgatgggcc gacacaagcc tgagaacatc gtgatcgaga tggccaggga 1500
gaaccagact acccagaagg gtcagaagaa ctctcgggaa cgtatgaagc gtattgagga 1560
ggggattaag gagttgggct ctcaaatcct caaggagcac cctgtggaga acactcagct 1620
ccaaaacgag aagctgtacc tgtactacct gcaaaacggg cgcgatatgt acgtggatca 1680
ggagttggac atcaacaggc ttagcgatta cgacgtggac cacatcgtgc cacagtcatt 1740
cttaaaggac gacagcatcg acaacaaggt tctgacgagg agcgacaaga atcgagggaa 1800
aagtgacaat gttccatccg aggaggtggt caagaaaatg aagaactatt ggcgtcagct 1860
tctgaacgcc aagctcatca cccagcggaa attcgacaac ctgactaagg ctgagcgagg 1920
cggactctcc gagcttgaca aggctggctt catcaagcgg cagttggtcg aaacccgaca 1980
gataacgaag cacgttgccc agatacttga ctcccgtatg aacaccaagt acgacgagaa 2040
cgacaagctc atcagggagg tgaaggtcat tacccttaag tccaaactcg tcagcgactt 2100
tcgtaaggac ttccagttct acaaggtgcg cgagatcaat aactaccacc acgcacacga 2160
cgcctacctg aacgcagtgg ttggaaccgc gttgattaaa aagtacccca agttggagtc 2220
ggagttcgtt tacggggact acaaggtgta cgacgttcgg aagatgatcg ccaagtctga 2280
acaggagatc gggaaagcaa ccgccaagta tttcttctat agcaacatca tgaacttctt 2340
taaaaccgag atcacacttg ccaatggcga gatccgtaag aggccgctga tcgagacaaa 2400
tggggagact ggcgagatcg tgtgggacaa gggccgcgac ttcgcaaccg ttcggaaagt 2460
cttgtccatg cctcaagtca acatcgtcaa gaagactgag gtgcaaacag gcgggttctc 2520
gaaggagtcc atactgccca agaggaactc agacaagctc atagcacgca aaaaagactg 2580
ggatccaaag aaatacggcg ggttcgactc gccgacagtc gcatactccg tgttagtggt 2640
ggctaaagtg gaaaagggga agtccaagaa gctcaagtcc gtcaaggagt tgctcgggat 2700
caccattatg gaacggtcct cattcgagaa gaatcccatt gacttcctag aggcgaaggg 2760
ctacaaagag gtcaaaaagg acctaattat taagctcccc aagtattcac tcttcgaact 2820
tgaaaatggt cgtaagcgga tgttggcaag cgctggagag cttcagaagg ggaacgagct 2880
tgcactgcct tccaagtacg tgaacttcct gtacctcgcc tctcattacg agaagttgaa 2940
gggctcaccg gaggacaacg agcagaagca gttgttcgtg gagcagcaca agcactacct 3000
cgacgagatc attgagcaga taagtgagtt cagcaaacgg gtgatccttg ccgacgctaa 3060
cctggacaag gtgctgagcg cctacaacaa gcacagagac aagccgatcc gagagcaagc 3120
ggagaacatc atacacctgt tcaccctcac gaacctcggg gctcccgcag ccttcaaata 3180
ttttgacacg accatcgacc gtaaacgcta cactagcacg aaggaggtgc tggacgctac 3240
ccttatccac cagtccatca ccggcctgta cgagacgaga atcgacttgt cgcagctcgg 3300
tggtgac 3307
<210> 33
<211> 4101
<212> DNA
<213> 未知
<220>
<223> Cas9核酸序列
<400> 33
gacaaaaaat actcaattgg tctggcaatt gggaccaaca gtgtcggatg ggccgtgatt 60
accgacgagt acaaggtgcc gtccaaaaaa ttcaaggtgc ttgggaacac cgaccgccac 120
tcgatcaaga aaaacctaat cggtgcgttg cttttcgaca gtggggagac cgccgaggca 180
acacgcttaa aacgcacagc taggaggaga tatacacggc gcaagaaccg aatatgctac 240
ttacaggaga tattctccaa tgagatggcg aaggtggacg actctttctt ccatcggctt 300
gaggaatcct tcctggtcga ggaggacaag aagcacgagc gacacccgat attcgggaac 360
atcgttgatg aggtggcgta ccacgagaag tacccaacga tataccactt acgcaagaag 420
ctcgtggact ctacggacaa ggccgacttg cgccttatct acttggcact ggcccacatg 480
attaagttcc gaggccactt ccttatcgag ggtgacctga accccgataa ctccgacgtg 540
gacaagctct tcatccaact cgtccagaca tacaaccagc tattcgagga gaatcctatc 600
aacgcctctg gggtggacgc taaagctatc ctctcagccc gcctgtcaaa gtcgaggagg 660
ttggagaacc taatcgccca gcttccaggc gagaagaaaa atgggctgtt cggaaacctt 720
atcgcactct cactgggcct aaccccgaac ttcaagtcca acttcgacct ggcagaggac 780
gcgaaattgc agttgtcgaa agacacctat gacgatgacc tggacaacct gttggcccag 840
ataggggacc agtacgccga cctgttccta gcggccaaga acctgtccga cgccatcttg 900
ctgtcggata tactgcgggt gaacaccgag atcactaaag cacctctctc cgccagcatg 960
attaagcgtt acgacgagca ccaccaagat ttgaccctgc taaaggcact tgtacggcag 1020
cagcttcccg agaagtacaa ggagatcttt ttcgaccaaa gcaagaacgg ctacgccggg 1080
tacatcgacg gaggtgccag ccaggaggag ttctacaagt tcattaagcc catcctggag 1140
aagatggacg ggactgagga actacttgtg aagctgaacc gggaagactt actacggaag 1200
cagcgtacct tcgacaacgg ttctatccca catcagatcc atcttgggga gttgcacgcg 1260
atcctgcgac gccaggagga cttttacccc ttcctgaaag acaaccgcga gaaaatcgag 1320
aagatactga ccttcagaat accttactac gtcggacccc ttgcgcgagg caactcaaga 1380
ttcgcgtgga tgaccaggaa atcagaggag accatcacac cctggaattt cgaggaggtg 1440
gttgacaagg gtgcctccgc ccagtccttt atcgaacgaa tgaccaactt cgacaagaac 1500
ttgcccaacg agaaggtgct ccccaaacac agcctcctct acgaatattt cacagtgtac 1560
aacgagctta ctaaagttaa gtatgttact gagggcatga ggaaacccgc cttcctgtca 1620
ggcgagcaga agaaagctat tgtggacctc cttttcaaga ccaaccggaa ggtgacagtg 1680
aagcagctca aggaggacta cttcaagaag atagagtgct tcgacagcgt ggagatcagc 1740
ggggtggagg acagattcaa tgcctctctc ggaacatacc acgacttgct taagatcatc 1800
aaggacaagg acttcctcga caacgaggaa aacgaggata ttctggagga tattgttctg 1860
actcttaccc tgttcgagga ccgggagatg atcgaggagc gtctcaagac ctacgcccac 1920
ctgttcgacg acaaagttat gaagcagctc aagcgtcgga gatataccgg atggggccgt 1980
ctgtctcgga agctcatcaa cgggatcagg gacaagcagt cagggaagac gatcttagac 2040
ttccttaagt ctgacggctt cgccaacagg aacttcatgc agttgatcca cgacgacagc 2100
cttaccttca aggaggacat ccagaaggcc caagtgagtg gccagggtga cagcctccac 2160
gagcatattg ctaatcttgc gggttcccca gcgattaaaa agggcatact tcaaaccgtt 2220
aaggtggtgg acgagcttgt caaggtgatg gggcgacaca agcccgagaa catcgtgatc 2280
gagatggcca gggagaacca gaccacccag aaggggcaga agaatagccg agaacgcatg 2340
aagcgcatcg aggaggggat taaggagcta gggagccaga tcctcaagga acatcccgtc 2400
gagaacaccc agctccagaa cgagaagcta tacctctact acttgcaaaa cgggagggat 2460
atgtacgtgg atcaggagtt ggacattaac cgcctaagcg actacgacgt agatcacatc 2520
gtgcctcagt cattcctcaa agacgacagc attgacaaca aagtcttgac ccgatccgac 2580
aagaaccgag gaaaatccga caatgtgccc tcagaggagg tcgtcaagaa aatgaagaac 2640
tattggaggc agctacttaa cgccaaactc ataacccagc ggaagttcga caacctgaca 2700
aaggctgagc ggggtgggct cagcgagctt gacaaggctg gcttcatcaa gcggcagttg 2760
gtggagacaa gacagataac gaagcacgtg gctcagatcc tggactctcg catgaacacg 2820
aagtacgacg agaacgacaa attgatccgc gaggtcaagg ttattacgct caagagcaaa 2880
cttgtcagcg atttccgcaa ggacttccag ttctacaagg tgagggagat taacaactac 2940
caccatgcac atgatgccta cttgaacgca gtggtgggga ccgcgcttat taaaaagtac 3000
cctaagttgg agtcagagtt cgtttatggg gactacaagg tgtacgacgt ccggaagatg 3060
attgcaaagt ctgaacagga aatcgggaag gccaccgcca aatatttctt ctacagtaac 3120
attatgaatt tttttaagac tgaaattact ctcgcaaacg gcgagatcag gaagcgtccc 3180
ctcatcgaga caaacgggga gaccggggag atagtctggg acaaggggcg ggacttcgct 3240
acggtgagga aggtgctctc gatgccacaa gtgaacatcg tcaaaaagac agaggtgcag 3300
accggtggct tctcaaagga gtcaatcctg ccaaaacgta acagcgacaa gctcatcgcc 3360
cgcaagaaag actgggaccc taagaagtat ggtgggttcg actcaccgac ggtcgcatac 3420
tccgttctgg tcgtggcaaa ggtggaaaag ggcaagtcca aaaaactgaa atccgtgaag 3480
gagttgcttg gcattaccat catggaacgc agcagcttcg agaagaaccc cattgacttc 3540
ctggaggcta aagggtacaa ggaggtcaag aaagatttaa ttattaagct acctaagtac 3600
agcttgttcg agctggagaa cggccgaaaa cgaatgctcg catccgccgg ggaacttcaa 3660
aagggcaacg agcttgcgct gccctccaag tacgtgaact tcctgtactt ggcatcccac 3720
tacgagaaac tcaagggtag cccagaggac aacgagcaga agcagctatt cgtggagcag 3780
cacaagcact acctcgacga gataatcgag cagatcagtg agttcagtaa gcgggtgata 3840
ctcgcggacg ccaacttgga caaggtgctt agtgcctaca acaagcaccg tgacaagccc 3900
atccgagaac aggctgagaa catcatccac cttttcactc tgacaaacct cggtgctccc 3960
gccgccttca aatacttcga cactaccatc gacaggaagc gctacacatc tacgaaggaa 4020
gttcttgacg ctacgcttat tcatcagtct atcacagggc tgtacgagac aaggatcgac 4080
cttagccaac tcggcgggga t 4101
<210> 34
<211> 4101
<212> DNA
<213> 人工的
<220>
<223> Cas9核酸序列
<400> 34
gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300
acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360
agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960
gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080
ctgtctcagc tgggaggtga c 4101
<210> 35
<211> 4101
<212> DNA
<213> 人工的
<220>
<223> eCas9核酸序列
<400> 35
gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520
gtgcctcaga gctttctggc cgacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctgccctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaaggcccct 3180
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300
acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360
agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960
gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080
ctgtctcagc tgggaggtga c 4101
<210> 36
<211> 4101
<212> DNA
<213> 人工的
<220>
<223> nCas9核酸序列
<400> 36
gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300
acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360
agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960
gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080
ctgtctcagc tgggaggtga c 4101
<210> 37
<211> 4101
<212> DNA
<213> 人工的
<220>
<223> nCas9核酸序列
<400> 37
gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 60
accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120
agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180
acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240
ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300
gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360
atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420
ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480
atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540
gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600
aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660
ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 720
attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780
gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840
atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900
ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960
atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020
cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080
tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140
aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200
cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260
attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320
aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380
ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440
gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500
ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560
aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1620
ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680
aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740
ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800
aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860
accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920
ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980
ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040
ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100
ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160
gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220
aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280
gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340
aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400
gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460
atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggacgccatc 2520
gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580
aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640
tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700
aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760
gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820
aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880
ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940
caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000
cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060
atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120
atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180
ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240
accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300
acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360
agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420
tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480
gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540
ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600
tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660
aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720
tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780
cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840
ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900
atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960
gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020
gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080
ctgtctcagc tgggaggtga c 4101
<210> 38
<211> 1367
<212> PRT
<213> 人工的
<220>
<223> nCas9
<400> 38
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 39
<211> 1367
<212> PRT
<213> 人工的
<220>
<223> enCas9
<400> 39
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Ala Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Ala Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Ala Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 40
<211> 228
<212> PRT
<213> Rattus norvegicus
<400> 40
Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg
1 5 10 15
Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg
20 25 30
Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser
35 40 45
Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn
50 55 60
Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg
65 70 75 80
Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser
85 90 95
Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe
100 105 110
Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln
115 120 125
Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr
130 135 140
Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro
145 150 155 160
Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu
165 170 175
Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu
180 185 190
Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala
195 200 205
Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala
210 215 220
Thr Gly Leu Lys
225
<210> 41
<211> 199
<212> PRT
<213> 智人（Homo sapiens）
<400> 41
Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His
1 5 10 15
Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr
20 25 30
Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met
35 40 45
Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys
50 55 60
Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro
65 70 75 80
Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile
85 90 95
Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala
100 105 110
Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
115 120 125
Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg
130 135 140
Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His
145 150 155 160
Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp
165 170 175
Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala
180 185 190
Ile Leu Gln Asn Gln Gly Asn
195
<210> 42
<211> 621
<212> DNA
<213> Petromyzon marinus
<400> 42
acagatgcag agtatgtgag aattcacgaa aagctggaca tctatacctt caagaagcag 60
ttctttaaca ataagaagtc tgtgagccat aggtgctacg tgctgttcga gctgaagaga 120
aggggtgaaa gaagggcatg tttttggggg tatgctgtga acaagcccca gtctggaact 180
gagagaggca ttcacgccga aattttcagc atcagaaagg tggaggaata cctgagggat 240
aaccctggac agtttacaat taattggtat tctagctggt ctccatgcgc tgactgtgcc 300
gagaagatcc tggaatggta caaccaggag ctgagaggaa atggccatac cctgaagatt 360
tgggcctgca agctgtacta tgaaaagaac gcaagaaatc agatcggact gtggaacctg 420
agggataatg gtgtggggct gaacgtgatg gtgtccgagc actatcagtg ctgtagaaag 480
attttcattc agtcctcaca taatcagctg aacgagaata gatggctgga aaagactctg 540
aagagggctg agaagagaag gtccgaactg tcaattatga tccaggtgaa gatcctgcac 600
accactaagt cacctgccgt g 621
<210> 43
<211> 160
<212> PRT
<213> 人工的
<220>
<223> FERNY脱氨酶
<400> 43
Phe Glu Arg Asn Tyr Asp Pro Arg Glu Leu Arg Lys Glu Thr Tyr Leu
1 5 10 15
Leu Tyr Glu Ile Lys Trp Gly Lys Ser Gly Lys Leu Trp Arg His Trp
20 25 30
Cys Gln Asn Asn Arg Thr Gln His Ala Glu Val Tyr Phe Leu Glu Asn
35 40 45
Ile Phe Asn Ala Arg Arg Phe Asn Pro Ser Thr His Cys Ser Ile Thr
50 55 60
Trp Tyr Leu Ser Trp Ser Pro Cys Ala Glu Cys Ser Gln Lys Ile Val
65 70 75 80
Asp Phe Leu Lys Glu His Pro Asn Val Leu Glu Ile Tyr Val Ala Arg
85 90 95
Leu Tyr Tyr His Glu Asp Glu Arg Asn Arg Gln Gly Leu Arg Asp Leu
100 105 110
Val Asn Ser Gly Val Thr Ile Arg Ile Met Asp Leu Pro Asp Tyr Asn
115 120 125
Tyr Cys Trp Lys Thr Phe Val Ser Asp Gln Gly Gly Asp Glu Asp Tyr
130 135 140
Trp Pro Gly His Phe Ala Pro Trp Ile Lys Gln Tyr Ser Leu Lys Leu
145 150 155 160
<210> 44
<211> 229
<212> PRT
<213> Rattus norvegicus
<400> 44
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys
225
<210> 45
<211> 198
<212> PRT
<213> 智人（Homo sapiens）
<400> 45
Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr
35 40 45
Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190
Phe Arg Thr Leu Gly Leu
195
<210> 46
<211> 197
<212> PRT
<213> 人工的
<220>
<223> 胞嘧啶脱氨酶
<400> 46
Met Asp Ser Leu Leu Met Asn Arg Arg Glu Phe Leu Tyr Gln Phe Lys
1 5 10 15
Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val
20 25 30
Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr
35 40 45
Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr
50 55 60
Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp
65 70 75 80
Phe Ile Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp
85 90 95
Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg
100 105 110
Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg
115 120 125
Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr
130 135 140
Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Gly Arg Thr Phe Lys
145 150 155 160
Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu
165 170 175
Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala
180 185 190
Phe Arg Thr Cys Thr
195
<210> 47
<211> 207
<212> PRT
<213> 人工的
<220>
<223> 进化的胞苷脱氨酶
<400> 47
Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp Ile Tyr Thr
1 5 10 15
Phe Lys Lys Gln Phe Ser Asn Asn Lys Lys Ser Val Ser His Arg Cys
20 25 30
Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg Ala Cys Phe
35 40 45
Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu Arg Gly Ile
50 55 60
His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr Leu Arg Asp
65 70 75 80
Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp Ser Pro Cys
85 90 95
Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln Glu Leu Arg
100 105 110
Gly Asn Gly His Thr Leu Lys Ile Trp Val Cys Lys Leu Tyr Tyr Glu
115 120 125
Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg Asp Asn Gly
130 135 140
Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys Cys Arg Lys
145 150 155 160
Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn Arg Trp Leu
165 170 175
Glu Lys Thr Leu Lys Arg Ala Glu Lys Arg Arg Ser Glu Leu Ser Ile
180 185 190
Met Phe Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro Ala Val
195 200 205
<210> 48
<211> 228
<212> PRT
<213> 人工的
<220>
<223> 进化的胞苷脱氨酶
<400> 48
Ser Ser Lys Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg
1 5 10 15
Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg
20 25 30
Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser
35 40 45
Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn
50 55 60
Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg
65 70 75 80
Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser
85 90 95
Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro Asn Val Thr Leu Phe
100 105 110
Ile Tyr Ile Ala Arg Leu Tyr His Leu Ala Asn Pro Arg Asn Arg Gln
115 120 125
Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr
130 135 140
Glu Gln Glu Ser Gly Tyr Cys Trp His Asn Phe Val Asn Tyr Ser Pro
145 150 155 160
Ser Asn Glu Ser His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu
165 170 175
Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu
180 185 190
Asn Ile Leu Arg Arg Lys Gln Ser Gln Leu Thr Ser Phe Thr Ile Ala
195 200 205
Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala
210 215 220
Thr Gly Leu Lys
225
<210> 49
<211> 162
<212> PRT
<213> 人工的
<220>
<223> 进化的胞苷脱氨酶
<400> 49
Ser Phe Glu Arg Asn Tyr Asp Pro Arg Glu Leu Arg Lys Glu Thr Tyr
1 5 10 15
Leu Leu Tyr Glu Ile Lys Trp Gly Lys Ser Gly Lys Leu Trp Arg His
20 25 30
Trp Cys Gln Asn Asn Arg Thr Gln His Ala Glu Val Tyr Phe Leu Glu
35 40 45
Asn Ile Phe Asn Ala Arg Arg Phe Asn Pro Ser Thr His Cys Ser Ile
50 55 60
Thr Trp Tyr Leu Ser Trp Ser Pro Cys Ala Glu Cys Ser Gln Lys Ile
65 70 75 80
Val Asp Phe Leu Lys Glu His Pro Asn Val Asn Leu Glu Ile Tyr Val
85 90 95
Ala Arg Leu Tyr Tyr Pro Glu Asn Glu Arg Asn Arg Gln Gly Leu Arg
100 105 110
Asp Leu Val Asn Ser Gly Val Thr Ile Arg Ile Met Asp Leu Pro Asp
115 120 125
Tyr Asn Tyr Cys Trp Lys Thr Phe Val Ser Asp Gln Gly Gly Asp Glu
130 135 140
Asp Tyr Trp Pro Gly His Phe Ala Pro Trp Ile Lys Gln Tyr Ser Leu
145 150 155 160
Lys Leu
<210> 50
<211> 166
<212> PRT
<213> Escherichia coli
<400> 50
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 51
<211> 166
<212> PRT
<213> 人工的
<220>
<223> 腺嘌呤脱氨酶
<400> 51
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 52
<211> 166
<212> PRT
<213> 人工的
<220>
<223> 腺嘌呤脱氨酶
<400> 52
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ser Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 53
<211> 166
<212> PRT
<213> 人工的
<220>
<223> 腺嘌呤脱氨酶
<400> 53
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Leu Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Arg Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 54
<211> 166
<212> PRT
<213> 人工的
<220>
<223> 腺嘌呤脱氨酶
<400> 54
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Leu Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Asn Ala Leu Leu
130 135 140
Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp
165
<210> 55
<211> 1763
<212> PRT
<213> 人工的
<220>
<223> ABE7.10
<400> 55
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
165 170 175
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
180 185 190
Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp
195 200 205
Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu
210 215 220
Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu
225 230 235 240
Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu
245 250 255
Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu
260 265 270
Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala
275 280 285
Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg
290 295 300
Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr
305 310 315 320
Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp
325 330 335
Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val
340 345 350
Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser
355 360 365
Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala
370 375 380
Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr
385 390 395 400
Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile
405 410 415
Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn
420 425 430
Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe
435 440 445
Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg
450 455 460
Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile
465 470 475 480
Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu
485 490 495
Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro
500 505 510
Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro
515 520 525
Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala
530 535 540
Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg
545 550 555 560
Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val
565 570 575
Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu
580 585 590
Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser
595 600 605
Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu
610 615 620
Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser
625 630 635 640
Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp
645 650 655
Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn
660 665 670
Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala
675 680 685
Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn
690 695 700
Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr
705 710 715 720
Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln
725 730 735
Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn
740 745 750
Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr
755 760 765
Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu
770 775 780
Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe
785 790 795 800
Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala
805 810 815
Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg
820 825 830
Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly
835 840 845
Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser
850 855 860
Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly
865 870 875 880
Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn
885 890 895
Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr
900 905 910
Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly
915 920 925
Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val
930 935 940
Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys
945 950 955 960
Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser
965 970 975
Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu
980 985 990
Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu
995 1000 1005
Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp
1010 1015 1020
Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe
1025 1030 1035
Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly
1040 1045 1050
Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
1055 1060 1065
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
1070 1075 1080
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr
1085 1090 1095
Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp
1100 1105 1110
Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile
1115 1120 1125
Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val
1130 1135 1140
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met
1145 1150 1155
Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg
1160 1165 1170
Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
1175 1180 1185
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn
1190 1195 1200
Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr
1205 1210 1215
Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val
1220 1225 1230
Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp
1235 1240 1245
Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp
1250 1255 1260
Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp
1265 1270 1275
Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp
1280 1285 1290
Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
1295 1300 1305
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
1310 1315 1320
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr
1325 1330 1335
Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu
1340 1345 1350
Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr
1355 1360 1365
Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr
1370 1375 1380
Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys
1385 1390 1395
Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val
1400 1405 1410
Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr
1415 1420 1425
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr
1430 1435 1440
Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile
1445 1450 1455
Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg
1460 1465 1470
Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn
1475 1480 1485
Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu
1490 1495 1500
Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys
1505 1510 1515
Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr
1520 1525 1530
Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys
1535 1540 1545
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1550 1555 1560
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu
1565 1570 1575
Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu
1580 1585 1590
Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met
1595 1600 1605
Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu
1610 1615 1620
Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu
1625 1630 1635
Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe
1640 1645 1650
Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
1655 1660 1665
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp
1670 1675 1680
Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg
1685 1690 1695
Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu
1700 1705 1710
Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg
1715 1720 1725
Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile
1730 1735 1740
His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser
1745 1750 1755
Gln Leu Gly Gly Asp
1760
<210> 56
<211> 1565
<212> PRT
<213> 人工的
<220>
<223> ABE8e
<400> 56
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly Ala
100 105 110
Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Ile Asn Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
165 170 175
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
180 185 190
Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile
195 200 205
Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val
210 215 220
Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile
225 230 235 240
Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala
245 250 255
Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg
260 265 270
Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala
275 280 285
Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val
290 295 300
Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val
305 310 315 320
Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg
325 330 335
Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr
340 345 350
Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu
355 360 365
Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln
370 375 380
Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala
385 390 395 400
Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser
405 410 415
Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn
420 425 430
Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn
435 440 445
Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser
450 455 460
Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly
465 470 475 480
Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala
485 490 495
Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala
500 505 510
Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp
515 520 525
Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr
530 535 540
Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile
545 550 555 560
Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile
565 570 575
Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg
580 585 590
Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro
595 600 605
His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu
610 615 620
Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile
625 630 635 640
Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn
645 650 655
Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro
660 665 670
Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe
675 680 685
Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val
690 695 700
Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu
705 710 715 720
Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe
725 730 735
Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr
740 745 750
Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
755 760 765
Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe
770 775 780
Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp
785 790 795 800
Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile
805 810 815
Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
820 825 830
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu
835 840 845
Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile
850 855 860
Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu
865 870 875 880
Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp
885 890 895
Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly
900 905 910
Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro
915 920 925
Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
930 935 940
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met
945 950 955 960
Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
965 970 975
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile
980 985 990
Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
995 1000 1005
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1010 1015 1020
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1025 1030 1035
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1040 1045 1050
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1055 1060 1065
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1070 1075 1080
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1085 1090 1095
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1100 1105 1110
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1115 1120 1125
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1130 1135 1140
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1145 1150 1155
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1160 1165 1170
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1175 1180 1185
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1190 1195 1200
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1205 1210 1215
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1220 1225 1230
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1235 1240 1245
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1250 1255 1260
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1265 1270 1275
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1280 1285 1290
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1295 1300 1305
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1310 1315 1320
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1325 1330 1335
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1340 1345 1350
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1355 1360 1365
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1370 1375 1380
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1385 1390 1395
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1400 1405 1410
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1415 1420 1425
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1430 1435 1440
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1445 1450 1455
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1460 1465 1470
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1475 1480 1485
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1490 1495 1500
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1505 1510 1515
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1520 1525 1530
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1535 1540 1545
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1550 1555 1560
Gly Asp
1565
<210> 57
<211> 1565
<212> PRT
<213> 人工的
<220>
<223> ABE8.20m
<400> 57
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile
35 40 45
Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Tyr Asp Ala Thr Leu Tyr
65 70 75 80
Ser Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Cys Arg Phe Phe Arg Met Pro Arg Arg Val Phe Asn Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
165 170 175
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
180 185 190
Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile
195 200 205
Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val
210 215 220
Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile
225 230 235 240
Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala
245 250 255
Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg
260 265 270
Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala
275 280 285
Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val
290 295 300
Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val
305 310 315 320
Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg
325 330 335
Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr
340 345 350
Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu
355 360 365
Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln
370 375 380
Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala
385 390 395 400
Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser
405 410 415
Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn
420 425 430
Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn
435 440 445
Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser
450 455 460
Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly
465 470 475 480
Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala
485 490 495
Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala
500 505 510
Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp
515 520 525
Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr
530 535 540
Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile
545 550 555 560
Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile
565 570 575
Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg
580 585 590
Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro
595 600 605
His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu
610 615 620
Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile
625 630 635 640
Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn
645 650 655
Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro
660 665 670
Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe
675 680 685
Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val
690 695 700
Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu
705 710 715 720
Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe
725 730 735
Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr
740 745 750
Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
755 760 765
Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe
770 775 780
Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp
785 790 795 800
Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile
805 810 815
Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg
820 825 830
Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu
835 840 845
Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile
850 855 860
Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu
865 870 875 880
Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp
885 890 895
Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly
900 905 910
Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro
915 920 925
Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
930 935 940
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met
945 950 955 960
Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
965 970 975
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile
980 985 990
Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
995 1000 1005
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1010 1015 1020
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1025 1030 1035
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1040 1045 1050
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1055 1060 1065
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1070 1075 1080
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1085 1090 1095
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1100 1105 1110
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1115 1120 1125
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1130 1135 1140
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1145 1150 1155
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1160 1165 1170
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1175 1180 1185
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1190 1195 1200
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1205 1210 1215
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1220 1225 1230
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1235 1240 1245
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1250 1255 1260
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1265 1270 1275
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1280 1285 1290
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1295 1300 1305
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1310 1315 1320
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1325 1330 1335
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1340 1345 1350
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1355 1360 1365
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1370 1375 1380
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1385 1390 1395
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1400 1405 1410
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1415 1420 1425
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1430 1435 1440
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1445 1450 1455
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1460 1465 1470
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1475 1480 1485
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1490 1495 1500
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1505 1510 1515
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1520 1525 1530
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1535 1540 1545
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1550 1555 1560
Gly Asp
1565
<210> 58
<211> 364
<212> PRT
<213> 人工的
<220>
<223> 腺嘌呤脱氨酶
<400> 58
Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr
1 5 10 15
Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala Val
20 25 30
Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro Ile
35 40 45
Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln
50 55 60
Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr
65 70 75 80
Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser
85 90 95
Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly Ala
100 105 110
Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg
115 120 125
Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu
130 135 140
Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys Lys
145 150 155 160
Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
165 170 175
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
180 185 190
Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp
195 200 205
Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu
210 215 220
Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu
225 230 235 240
Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu
245 250 255
Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu
260 265 270
Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala
275 280 285
Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg
290 295 300
Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr
305 310 315 320
Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp
325 330 335
Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val
340 345 350
Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp
355 360
<210> 59
<211> 167
<212> PRT
<213> 人工的
<220>
<223> 腺嘌呤脱氨酶
<400> 59
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala
35 40 45
Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Tyr Asp Ala Thr Leu
65 70 75 80
Tyr Ser Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly
100 105 110
Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Cys Arg Phe Phe Arg Met Pro Arg Arg Val Phe Asn Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Thr Asp
165
<210> 60
<211> 167
<212> PRT
<213> 人工的
<220>
<223> 腺嘌呤脱氨酶
<400> 60
Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu
1 5 10 15
Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala
20 25 30
Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala
35 40 45
Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg
50 55 60
Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu
65 70 75 80
Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His
85 90 95
Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly
100 105 110
Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His
115 120 125
Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu
130 135 140
Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys
145 150 155 160
Lys Ala Gln Ser Ser Ile Asn
165
<210> 61
<211> 83
<212> PRT
<213> 芽孢杆菌噬菌体（Bacillus phage）
<400> 61
Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val
1 5 10 15
Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile
20 25 30
Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu
35 40 45
Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
50 55 60
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile
65 70 75 80
Lys Met Leu
<210> 62
<211> 19
<212> DNA
<213> 人工的
<220>
<223> RNA间隔区
<220>
<221> misc_feature
<222> (1)..(19)
<223> wherein n is A, C, T or G
<400> 62
nnnnnnnnnn nnnnnnnnn 19
<210> 63
<211> 22
<212> DNA
<213> 人工的
<220>
<223> 靶标链
<220>
<221> misc_feature
<222> (4)..(22)
<223> wherein n is A, C, T or G
<400> 63
aaannnnnnn nnnnnnnnnn nn 22
<210> 64
<211> 22
<212> DNA
<213> 人工的
<220>
<223> 非靶标链
<220>
<221> misc_feature
<222> (4)..(22)
<223> wherein n is A, C, T or G
<400> 64
tttnnnnnnn nnnnnnnnnn nn 22
<210> 65
<211> 24
<212> PRT
<213> 人工的
<220>
<223> GCN4序列
<400> 65
Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala Arg
1 5 10 15
Leu Lys Lys Gly Ser Gly Ser Gly
20
<210> 66
<211> 241
<212> PRT
<213> 人工的
<220>
<223> GCN4序列
<400> 66
Glu Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala
1 5 10 15
Arg Leu Lys Lys Gly Ser Gly Ser Gly Glu Glu Leu Leu Ser Lys Asn
20 25 30
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Gly Ser Gly Ser
35 40 45
Gly Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala
50 55 60
Arg Leu Lys Lys Gly Ser Gly Ser Gly Glu Glu Leu Leu Ser Lys Asn
65 70 75 80
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Gly Ser Gly Ser
85 90 95
Gly Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala
100 105 110
Arg Leu Lys Lys Gly Ser Gly Ser Gly Glu Glu Leu Leu Ser Lys Asn
115 120 125
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Gly Ser Gly Ser
130 135 140
Gly Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala
145 150 155 160
Arg Leu Lys Lys Gly Ser Gly Ser Gly Glu Glu Leu Leu Ser Lys Asn
165 170 175
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Gly Ser Gly Ser
180 185 190
Gly Glu Glu Leu Leu Ser Lys Asn Tyr His Leu Glu Asn Glu Val Ala
195 200 205
Arg Leu Lys Lys Gly Ser Gly Ser Gly Glu Glu Leu Leu Ser Lys Asn
210 215 220
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Gly Ser Gly Ser
225 230 235 240
Gly
<210> 67
<211> 277
<212> PRT
<213> 人工的
<220>
<223> ScFv抗体
<400> 67
Met Gly Pro Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
1 5 10 15
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Thr Gly Ala
20 25 30
Val Thr Thr Ser Asn Tyr Ala Ser Trp Val Gln Glu Lys Pro Gly Lys
35 40 45
Leu Phe Lys Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Ala Leu
85 90 95
Trp Tyr Ser Asn His Trp Val Phe Gly Gln Gly Thr Lys Val Glu Leu
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Ser Gly Gly Gly Ser Glu Val Lys Leu Leu Glu Ser Gly Gly Gly
130 135 140
Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Val Ser Gly
145 150 155 160
Phe Ser Leu Thr Asp Tyr Gly Val Asn Trp Val Arg Gln Ala Pro Gly
165 170 175
Arg Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Asp Gly Ile Thr Asp
180 185 190
Tyr Asn Ser Ala Leu Lys Asp Arg Phe Ile Ile Ser Lys Asp Asn Gly
195 200 205
Lys Asn Thr Val Tyr Leu Gln Met Ser Lys Val Arg Ser Asp Asp Thr
210 215 220
Ala Leu Tyr Tyr Cys Val Thr Gly Leu Phe Asp Tyr Trp Gly Gln Gly
225 230 235 240
Thr Leu Val Thr Val Ser Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
245 250 255
Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
260 265 270
Gly Gly Gly Gly Ser
275
<210> 68
<211> 66
<212> DNA
<213> 贝酵母（Saccharomyces bayanus）
<400> 68
ttcttgtcgt acttatagat cgctacgtta tttcaatttt gaaaatctga gtcctgggag 60
tgcgga 66
<210> 69
<211> 605
<212> PRT
<213> 智人（Homo sapiens）
<400> 69
Met Ser Gly Trp Glu Ser Tyr Tyr Lys Thr Glu Gly Asp Glu Glu Ala
1 5 10 15
Glu Glu Glu Gln Glu Glu Asn Leu Glu Ala Ser Gly Asp Tyr Lys Tyr
20 25 30
Ser Gly Arg Asp Ser Leu Ile Phe Leu Val Asp Ala Ser Lys Ala Met
35 40 45
Phe Glu Ser Gln Ser Glu Asp Glu Leu Thr Pro Phe Asp Met Ser Ile
50 55 60
Gln Cys Ile Gln Ser Val Tyr Ile Ser Lys Ile Ile Ser Ser Asp Arg
65 70 75 80
Asp Leu Leu Ala Trp Phe Tyr Gly Thr Glu Lys Asp Lys Asn Ser Val
85 90 95
Asn Phe Lys Ile Tyr Val Leu Gln Glu Leu Asp Asn Pro Gly Ala Lys
100 105 110
Arg Ile Leu Glu Leu Asp Gln Phe Lys Gly Gln Gln Gly Gln Lys Arg
115 120 125
Phe Gln Asp Met Met Gly His Gly Ser Asp Tyr Ser Leu Ser Glu Val
130 135 140
Leu Trp Val Cys Ala Asn Leu Phe Ser Asp Val Gln Phe Lys Met Ser
145 150 155 160
His Lys Arg Ile Met Leu Phe Thr Asn Glu Asp Asn Pro His Gly Asn
165 170 175
Asp Ser Ala Lys Ala Ser Arg Ala Arg Thr Lys Ala Gly Asp Leu Arg
180 185 190
Asp Thr Gly Ile Phe Leu Asp Leu His Leu Lys Lys Pro Gly Gly Phe
195 200 205
Asp Ile Ser Leu Phe Tyr Arg Asp Ile Ile Ser Ile Ala Glu Asp Glu
210 215 220
Asp Leu Arg Val His Phe Glu Glu Ser Ser Lys Leu Glu Asp Leu Leu
225 230 235 240
Arg Lys Val Arg Ala Lys Glu Thr Arg Lys Arg Ala Leu Ser Arg Leu
245 250 255
Lys Leu Lys Leu Asn Lys Asp Ile Val Ile Ser Val Gly Ile Tyr Asn
260 265 270
Leu Val Gln Lys Ala Leu Lys Pro Pro Pro Ile Lys Leu Tyr Arg Glu
275 280 285
Thr Asn Glu Pro Val Lys Thr Lys Thr Arg Thr Phe Asn Thr Ser Thr
290 295 300
Gly Gly Leu Leu Leu Pro Ser Asp Thr Lys Arg Ser Gln Ile Tyr Gly
305 310 315 320
Ser Arg Gln Ile Ile Leu Glu Lys Glu Glu Thr Glu Glu Leu Lys Arg
325 330 335
Phe Asp Asp Pro Gly Leu Met Leu Met Gly Phe Lys Pro Leu Val Leu
340 345 350
Leu Lys Lys His His Tyr Leu Arg Pro Ser Leu Phe Val Tyr Pro Glu
355 360 365
Glu Ser Leu Val Ile Gly Ser Ser Thr Leu Phe Ser Ala Leu Leu Ile
370 375 380
Lys Cys Leu Glu Lys Glu Val Ala Ala Leu Cys Arg Tyr Thr Pro Arg
385 390 395 400
Arg Asn Ile Pro Pro Tyr Phe Val Ala Leu Val Pro Gln Glu Glu Glu
405 410 415
Leu Asp Asp Gln Lys Ile Gln Val Thr Pro Pro Gly Phe Gln Leu Val
420 425 430
Phe Leu Pro Phe Ala Asp Asp Lys Arg Lys Met Pro Phe Thr Glu Lys
435 440 445
Ile Met Ala Thr Pro Glu Gln Val Gly Lys Met Lys Ala Ile Val Glu
450 455 460
Lys Leu Arg Phe Thr Tyr Arg Ser Asp Ser Phe Glu Asn Pro Val Leu
465 470 475 480
Gln Gln His Phe Arg Asn Leu Glu Ala Leu Ala Leu Asp Leu Met Glu
485 490 495
Pro Glu Gln Ala Val Asp Leu Thr Leu Pro Lys Val Glu Ala Met Asn
500 505 510
Lys Arg Leu Gly Ser Leu Val Asp Glu Phe Lys Glu Leu Val Tyr Pro
515 520 525
Pro Asp Tyr Asn Pro Glu Gly Lys Val Thr Lys Arg Lys His Asp Asn
530 535 540
Glu Gly Ser Gly Ser Lys Arg Pro Lys Val Glu Tyr Ser Glu Glu Glu
545 550 555 560
Leu Lys Thr His Ile Ser Lys Gly Thr Leu Gly Lys Phe Thr Val Pro
565 570 575
Leu Lys Glu Ala Cys Arg Ala Tyr Gly Leu Lys Ser Gly Leu Lys Lys
580 585 590
Gln Glu Leu Leu Glu Ala Leu Thr Lys His Phe Gln Asp
595 600 605
<210> 70
<211> 482
<212> PRT
<213> 人工的
<220>
<223> 多肽
<400> 70
Met Val Arg Ser Gly Asn Lys Ala Ala Trp Leu Cys Met Asp Val Gly
1 5 10 15
Phe Thr Met Ser Asn Ser Ile Pro Gly Ile Glu Ser Pro Phe Glu Gln
20 25 30
Ala Lys Lys Val Ile Thr Met Phe Val Gln Arg Gln Val Phe Ala Glu
35 40 45
Asn Lys Asp Glu Ile Ala Leu Val Leu Phe Gly Thr Asp Gly Thr Asp
50 55 60
Asn Pro Leu Ser Gly Gly Asp Gln Tyr Gln Asn Ile Thr Val His Arg
65 70 75 80
His Leu Met Leu Pro Asp Phe Asp Leu Leu Glu Asp Ile Glu Ser Lys
85 90 95
Ile Gln Pro Gly Ser Gln Gln Ala Asp Phe Leu Asp Ala Leu Ile Val
100 105 110
Ser Met Asp Val Ile Gln His Glu Thr Ile Gly Lys Lys Phe Glu Lys
115 120 125
Arg His Ile Glu Ile Phe Thr Asp Leu Ser Ser Arg Phe Ser Lys Ser
130 135 140
Gln Leu Asp Ile Ile Ile His Ser Leu Lys Lys Cys Asp Ile Ser Glu
145 150 155 160
Arg His Ser Ile His Trp Pro Cys Arg Leu Thr Ile Gly Ser Asn Leu
165 170 175
Ser Ile Arg Ile Ala Ala Tyr Lys Ser Ile Leu Gln Glu Arg Val Lys
180 185 190
Lys Thr Thr Trp Asp Ala Lys Thr Leu Lys Lys Glu Asp Ile Gln Lys
195 200 205
Glu Thr Val Tyr Cys Leu Asn Asp Asp Asp Glu Thr Glu Val Leu Lys
210 215 220
Glu Asp Ile Ile Gln Gly Phe Arg Tyr Gly Ser Asp Ile Val Pro Phe
225 230 235 240
Ser Lys Val Asp Glu Glu Gln Met Lys Tyr Lys Ser Glu Gly Lys Cys
245 250 255
Phe Ser Val Leu Gly Phe Cys Lys Ser Ser Gln Val Gln Arg Arg Phe
260 265 270
Phe Met Gly Asn Gln Val Leu Lys Val Phe Ala Ala Arg Asp Asp Glu
275 280 285
Ala Ala Ala Val Ala Leu Ser Ser Leu Ile His Ala Leu Asp Asp Leu
290 295 300
Asp Ile Trp Ala Ile Val Arg Tyr Ala Tyr Asp Lys Arg Ala Asn Pro
305 310 315 320
Gln Val Gly Val Ala Phe Pro His Ile Lys His Asn Tyr Glu Cys Leu
325 330 335
Val Tyr Val Gln Leu Pro Phe Met Glu Asp Leu Arg Gln Tyr Met Phe
340 345 350
Ser Ser Leu Lys Asn Ser Lys Lys Tyr Ala Pro Thr Glu Ala Gln Leu
355 360 365
Asn Ala Val Asp Ala Leu Ile Asp Ser Met Ser Leu Ala Lys Lys Asp
370 375 380
Glu Lys Thr Asp Thr Leu Glu Asp Leu Phe Pro Thr Thr Lys Ile Pro
385 390 395 400
Asn Pro Arg Phe Gln Arg Leu Phe Gln Cys Leu Leu His Arg Ala Leu
405 410 415
His Pro Arg Glu Pro Leu Pro Pro Ile Gln Gln His Ile Trp Asn Met
420 425 430
Leu Asn Pro Pro Ala Glu Val Thr Thr Lys Ser Gln Ile Pro Leu Ser
435 440 445
Lys Ile Lys Thr Leu Phe Pro Leu Ile Glu Ala Lys Lys Lys Asp Gln
450 455 460
Val Thr Ala Gln Glu Ile Phe Gln Asp Asn His Glu Asp Gly Pro Thr
465 470 475 480
Ala Lys
<210> 71
<211> 10
<212> DNA
<213> Methanobacterium thermoautotrophicum
<400> 71
aatttttgga 10
<210> 72
<211> 83
<212> PRT
<213> Methanobacterium thermoautotrophicum
<400> 72
Gly Ser Val Ile Asp Val Ser Ser Gln Arg Val Asn Val Gln Arg Pro
1 5 10 15
Leu Asp Ala Leu Gly Asn Ser Leu Asn Ser Pro Val Ile Ile Lys Leu
20 25 30
Lys Gly Asp Arg Glu Phe Arg Gly Val Leu Lys Ser Phe Asp Leu His
35 40 45
Met Asn Leu Val Leu Asn Asp Ala Glu Glu Leu Glu Asp Gly Glu Val
50 55 60
Thr Arg Arg Leu Gly Thr Val Leu Ile Arg Gly Asp Asn Ile Val Tyr
65 70 75 80
Ile Ser Pro
<210> 73
<211> 25
<212> DNA
<213> 噬菌体MS2
<400> 73
gcgcacatga ggatcaccca tgtgc 25
<210> 74
<211> 116
<212> PRT
<213> 噬菌体MS2
<400> 74
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Ile Ala Glu
20 25 30
Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser Val
35 40 45
Arg Gln Ser Ser Ala Gln Asn Arg Lys Tyr Thr Ile Lys Val Glu Val
50 55 60
Pro Lys Gly Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro
65 70 75 80
Ile Phe Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln
85 90 95
Gly Leu Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn
100 105 110
Ser Gly Ile Tyr
115
<210> 75
<211> 26
<212> DNA
<213> 噬菌体PP7
<400> 75
ataaggagtt tatatggaaa ccctta 26
<210> 76
<211> 127
<212> PRT
<213> 噬菌体PP7
<400> 76
Met Ser Lys Thr Ile Val Leu Ser Val Gly Glu Ala Thr Arg Thr Leu
1 5 10 15
Thr Glu Ile Gln Ser Thr Ala Asp Arg Gln Ile Phe Glu Glu Lys Val
20 25 30
Gly Pro Leu Val Gly Arg Leu Arg Leu Thr Ala Ser Leu Arg Gln Asn
35 40 45
Gly Ala Lys Thr Ala Tyr Arg Val Asn Leu Lys Leu Asp Gln Ala Asp
50 55 60
Trp Asp Cys Ser Thr Ser Val Cys Gly Glu Leu Pro Lys Val Arg Tyr
65 70 75 80
Thr Gln Val Trp Ser His Asp Val Thr Ile Val Ala Asn Ser Thr Glu
85 90 95
Ala Ser Arg Lys Ser Leu Tyr Asp Leu Thr Lys Ser Leu Val Ala Thr
100 105 110
Ser Gln Val Glu Asp Leu Val Val Asn Leu Val Pro Leu Gly Arg
115 120 125
<210> 77
<211> 19
<212> DNA
<213> Shigella flexneri
<400> 77
ctgaatgcct gcgagcatc 19
<210> 78
<211> 62
<212> PRT
<213> 未知
<220>
<223> Shigella phage
<400> 78
Met Lys Ser Ile Arg Cys Lys Asn Cys Asn Lys Leu Leu Phe Lys Ala
1 5 10 15
Asp Ser Phe Asp His Ile Glu Ile Arg Cys Pro Arg Cys Lys Arg His
20 25 30
Ile Ile Met Leu Asn Ala Cys Glu His Pro Thr Glu Lys His Cys Gly
35 40 45
Lys Arg Glu Lys Ile Thr His Ser Asp Glu Thr Val Arg Tyr
50 55 60
<210> 79
<211> 2016
<212> PRT
<213> 人工的
<220>
<223> A3A-TadA*-nCas9(D10A)-UGI
<400> 79
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp
20 25 30
Pro His Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys
35 40 45
Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val
50 55 60
Lys Met Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu
65 70 75 80
Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu
85 90 95
Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp
100 105 110
Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val
115 120 125
Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala
130 135 140
Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met
145 150 155 160
Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe
165 170 175
Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln
180 185 190
Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu
195 200 205
Arg Ala Ile Leu Gln Asn Gln Gly Asn Gly Gly Ser Gly Gly Ser Gly
210 215 220
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
225 230 235 240
Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg
245 250 255
His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro
260 265 270
Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp
275 280 285
Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met
290 295 300
Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp
305 310 315 320
Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala
325 330 335
Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala
340 345 350
Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly
355 360 365
Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys
370 375 380
Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn
385 390 395 400
Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly
405 410 415
Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro
420 425 430
Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile
435 440 445
Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp
450 455 460
Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp
465 470 475 480
Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser
485 490 495
Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg
500 505 510
Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser
515 520 525
Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu
530 535 540
Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe
545 550 555 560
Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile
565 570 575
Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu
580 585 590
Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His
595 600 605
Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys
610 615 620
Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn
625 630 635 640
Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg
645 650 655
Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly
660 665 670
Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly
675 680 685
Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys
690 695 700
Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu
705 710 715 720
Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn
725 730 735
Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu
740 745 750
Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu
755 760 765
His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu
770 775 780
Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr
785 790 795 800
Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe
805 810 815
Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val
820 825 830
Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn
835 840 845
Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu
850 855 860
Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys
865 870 875 880
Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu
885 890 895
Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
900 905 910
Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser
915 920 925
Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro
930 935 940
Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr
945 950 955 960
Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg
965 970 975
Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu
980 985 990
Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp
995 1000 1005
Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly
1010 1015 1020
Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu
1025 1030 1035
Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
1040 1045 1050
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1055 1060 1065
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1070 1075 1080
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1085 1090 1095
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1100 1105 1110
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1115 1120 1125
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1130 1135 1140
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1145 1150 1155
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1160 1165 1170
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1175 1180 1185
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1190 1195 1200
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1205 1210 1215
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1220 1225 1230
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1235 1240 1245
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1250 1255 1260
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1265 1270 1275
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1280 1285 1290
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1295 1300 1305
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1310 1315 1320
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1325 1330 1335
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1340 1345 1350
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1355 1360 1365
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1370 1375 1380
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1385 1390 1395
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1400 1405 1410
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1415 1420 1425
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1430 1435 1440
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1445 1450 1455
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1460 1465 1470
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1475 1480 1485
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1490 1495 1500
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1505 1510 1515
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1520 1525 1530
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1535 1540 1545
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1550 1555 1560
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1565 1570 1575
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1580 1585 1590
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1595 1600 1605
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1610 1615 1620
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1625 1630 1635
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1640 1645 1650
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1655 1660 1665
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1670 1675 1680
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1685 1690 1695
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1700 1705 1710
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1715 1720 1725
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1730 1735 1740
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1745 1750 1755
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1760 1765 1770
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1775 1780 1785
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1790 1795 1800
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Gly Gly Ser Gly Gly
1805 1810 1815
Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln
1820 1825 1830
Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu
1835 1840 1845
Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr
1850 1855 1860
Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser
1865 1870 1875
Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser
1880 1885 1890
Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly
1895 1900 1905
Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr
1910 1915 1920
Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu
1925 1930 1935
Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu
1940 1945 1950
Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu
1955 1960 1965
Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile
1970 1975 1980
Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly
1985 1990 1995
Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys
2000 2005 2010
Arg Lys Val
2015
<210> 80
<211> 2016
<212> PRT
<213> 人工的
<220>
<223> A3A-TadA8.20m-nCas9(D10A)-UGI
<400> 80
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp
20 25 30
Pro His Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys
35 40 45
Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val
50 55 60
Lys Met Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu
65 70 75 80
Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu
85 90 95
Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp
100 105 110
Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val
115 120 125
Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala
130 135 140
Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met
145 150 155 160
Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe
165 170 175
Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln
180 185 190
Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu
195 200 205
Arg Ala Ile Leu Gln Asn Gln Gly Asn Gly Gly Ser Gly Gly Ser Gly
210 215 220
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
225 230 235 240
Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg
245 250 255
His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro
260 265 270
Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp
275 280 285
Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met
290 295 300
Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Tyr Asp
305 310 315 320
Ala Thr Leu Tyr Ser Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala
325 330 335
Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala
340 345 350
Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly
355 360 365
Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys
370 375 380
Ala Ala Leu Leu Cys Arg Phe Phe Arg Met Pro Arg Arg Val Phe Asn
385 390 395 400
Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly
405 410 415
Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro
420 425 430
Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile
435 440 445
Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp
450 455 460
Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp
465 470 475 480
Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser
485 490 495
Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg
500 505 510
Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser
515 520 525
Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu
530 535 540
Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe
545 550 555 560
Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile
565 570 575
Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu
580 585 590
Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His
595 600 605
Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys
610 615 620
Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn
625 630 635 640
Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg
645 650 655
Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly
660 665 670
Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly
675 680 685
Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys
690 695 700
Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu
705 710 715 720
Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn
725 730 735
Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu
740 745 750
Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu
755 760 765
His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu
770 775 780
Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr
785 790 795 800
Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe
805 810 815
Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val
820 825 830
Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn
835 840 845
Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu
850 855 860
Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys
865 870 875 880
Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu
885 890 895
Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
900 905 910
Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser
915 920 925
Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro
930 935 940
Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr
945 950 955 960
Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg
965 970 975
Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu
980 985 990
Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp
995 1000 1005
Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly
1010 1015 1020
Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu
1025 1030 1035
Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
1040 1045 1050
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1055 1060 1065
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1070 1075 1080
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1085 1090 1095
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1100 1105 1110
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1115 1120 1125
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1130 1135 1140
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1145 1150 1155
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1160 1165 1170
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1175 1180 1185
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1190 1195 1200
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1205 1210 1215
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1220 1225 1230
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1235 1240 1245
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1250 1255 1260
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1265 1270 1275
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1280 1285 1290
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1295 1300 1305
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1310 1315 1320
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1325 1330 1335
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1340 1345 1350
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1355 1360 1365
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1370 1375 1380
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1385 1390 1395
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1400 1405 1410
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1415 1420 1425
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1430 1435 1440
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1445 1450 1455
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1460 1465 1470
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1475 1480 1485
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1490 1495 1500
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1505 1510 1515
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1520 1525 1530
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1535 1540 1545
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1550 1555 1560
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1565 1570 1575
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1580 1585 1590
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1595 1600 1605
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1610 1615 1620
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1625 1630 1635
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1640 1645 1650
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1655 1660 1665
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1670 1675 1680
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1685 1690 1695
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1700 1705 1710
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1715 1720 1725
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1730 1735 1740
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1745 1750 1755
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1760 1765 1770
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1775 1780 1785
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1790 1795 1800
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Gly Gly Ser Gly Gly
1805 1810 1815
Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln
1820 1825 1830
Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu
1835 1840 1845
Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr
1850 1855 1860
Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser
1865 1870 1875
Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser
1880 1885 1890
Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly
1895 1900 1905
Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr
1910 1915 1920
Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu
1925 1930 1935
Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu
1940 1945 1950
Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu
1955 1960 1965
Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile
1970 1975 1980
Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly
1985 1990 1995
Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys
2000 2005 2010
Arg Lys Val
2015
<210> 81
<211> 2016
<212> PRT
<213> 人工的
<220>
<223> TadA8.20m-A3A-nCas9(D10A)-UGI
<400> 81
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Tyr Asp Ala
85 90 95
Thr Leu Tyr Ser Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys
115 120 125
Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Cys Arg Phe Phe Arg Met Pro Arg Arg Val Phe Asn Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Thr Asp Gly Gly Ser Gly Gly Ser Gly
180 185 190
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
195 200 205
Ser Gly Gly Ser Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met
210 215 220
Asp Pro His Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His
225 230 235 240
Lys Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser
245 250 255
Val Lys Met Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn
260 265 270
Leu Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp
275 280 285
Leu Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr
290 295 300
Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu
305 310 315 320
Val Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe
325 330 335
Ala Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln
340 345 350
Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu
355 360 365
Phe Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe
370 375 380
Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg
385 390 395 400
Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn Ser Gly Gly Ser Ser Gly
405 410 415
Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro
420 425 430
Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile
435 440 445
Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp
450 455 460
Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp
465 470 475 480
Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser
485 490 495
Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg
500 505 510
Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser
515 520 525
Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu
530 535 540
Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe
545 550 555 560
Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile
565 570 575
Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu
580 585 590
Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His
595 600 605
Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys
610 615 620
Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn
625 630 635 640
Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg
645 650 655
Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly
660 665 670
Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly
675 680 685
Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys
690 695 700
Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu
705 710 715 720
Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn
725 730 735
Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu
740 745 750
Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu
755 760 765
His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu
770 775 780
Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr
785 790 795 800
Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe
805 810 815
Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val
820 825 830
Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn
835 840 845
Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu
850 855 860
Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys
865 870 875 880
Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu
885 890 895
Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu
900 905 910
Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser
915 920 925
Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro
930 935 940
Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr
945 950 955 960
Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg
965 970 975
Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu
980 985 990
Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp
995 1000 1005
Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly
1010 1015 1020
Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu
1025 1030 1035
Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn
1040 1045 1050
Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu
1055 1060 1065
Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu
1070 1075 1080
Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
1085 1090 1095
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
1100 1105 1110
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
1115 1120 1125
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu
1130 1135 1140
Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
1145 1150 1155
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
1160 1165 1170
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu
1175 1180 1185
Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu
1190 1195 1200
Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
1205 1210 1215
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
1220 1225 1230
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1235 1240 1245
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1250 1255 1260
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1265 1270 1275
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1280 1285 1290
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1295 1300 1305
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1310 1315 1320
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1325 1330 1335
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1340 1345 1350
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1355 1360 1365
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1370 1375 1380
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1385 1390 1395
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1400 1405 1410
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1415 1420 1425
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1430 1435 1440
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1445 1450 1455
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1460 1465 1470
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1475 1480 1485
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1490 1495 1500
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1505 1510 1515
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1520 1525 1530
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1535 1540 1545
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1550 1555 1560
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1565 1570 1575
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1580 1585 1590
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1595 1600 1605
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1610 1615 1620
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1625 1630 1635
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1640 1645 1650
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1655 1660 1665
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1670 1675 1680
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1685 1690 1695
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1700 1705 1710
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1715 1720 1725
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1730 1735 1740
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1745 1750 1755
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1760 1765 1770
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1775 1780 1785
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1790 1795 1800
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Gly Gly Ser Gly Gly
1805 1810 1815
Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln
1820 1825 1830
Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu
1835 1840 1845
Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr
1850 1855 1860
Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser
1865 1870 1875
Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser
1880 1885 1890
Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly
1895 1900 1905
Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr
1910 1915 1920
Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu
1925 1930 1935
Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu
1940 1945 1950
Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu
1955 1960 1965
Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile
1970 1975 1980
Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly
1985 1990 1995
Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys
2000 2005 2010
Arg Lys Val
2015
<210> 82
<211> 2016
<212> PRT
<213> 人工的
<220>
<223> A3A-nCas9(D10A)-TadA8.20m-UGI
<400> 82
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp
20 25 30
Pro His Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys
35 40 45
Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val
50 55 60
Lys Met Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu
65 70 75 80
Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu
85 90 95
Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp
100 105 110
Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val
115 120 125
Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala
130 135 140
Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met
145 150 155 160
Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe
165 170 175
Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln
180 185 190
Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu
195 200 205
Arg Ala Ile Leu Gln Asn Gln Gly Asn Ser Gly Gly Ser Ser Gly Gly
210 215 220
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
225 230 235 240
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
245 250 255
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
260 265 270
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
275 280 285
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
290 295 300
Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
305 310 315 320
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
325 330 335
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
340 345 350
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
355 360 365
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
370 375 380
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
385 390 395 400
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
405 410 415
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
420 425 430
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
435 440 445
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
450 455 460
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
465 470 475 480
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
485 490 495
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
500 505 510
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
515 520 525
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
530 535 540
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
545 550 555 560
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
565 570 575
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
580 585 590
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
595 600 605
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
610 615 620
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
625 630 635 640
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
645 650 655
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
660 665 670
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
675 680 685
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
690 695 700
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
705 710 715 720
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
725 730 735
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
740 745 750
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
755 760 765
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
770 775 780
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
785 790 795 800
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
805 810 815
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
820 825 830
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
835 840 845
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
850 855 860
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
865 870 875 880
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
885 890 895
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
900 905 910
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
915 920 925
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
930 935 940
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
945 950 955 960
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
965 970 975
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
980 985 990
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
995 1000 1005
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys
1010 1015 1020
Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu
1025 1030 1035
Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln
1040 1045 1050
Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg
1055 1060 1065
Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp
1070 1075 1080
Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp
1085 1090 1095
Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
1100 1105 1110
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
1115 1120 1125
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg
1130 1135 1140
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu
1145 1150 1155
Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
1160 1165 1170
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn
1175 1180 1185
Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val
1190 1195 1200
Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe
1205 1210 1215
Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
1220 1225 1230
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
1235 1240 1245
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val
1250 1255 1260
Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly
1265 1270 1275
Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe
1280 1285 1290
Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg
1295 1300 1305
Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp
1310 1315 1320
Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro
1325 1330 1335
Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1340 1345 1350
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1355 1360 1365
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp
1370 1375 1380
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu
1385 1390 1395
Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1400 1405 1410
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp
1415 1420 1425
Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
1430 1435 1440
Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg
1445 1450 1455
Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu
1460 1465 1470
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1475 1480 1485
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys
1490 1495 1500
Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile
1505 1510 1515
Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
1520 1525 1530
Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys
1535 1540 1545
Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu
1550 1555 1560
Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr
1565 1570 1575
Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
1580 1585 1590
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1595 1600 1605
Asp Leu Ser Gln Leu Gly Gly Asp Gly Gly Ser Gly Gly Ser Gly
1610 1615 1620
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
1625 1630 1635
Gly Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr Trp
1640 1645 1650
Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg
1655 1660 1665
Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile
1670 1675 1680
Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala
1685 1690 1695
His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln
1700 1705 1710
Asn Tyr Arg Leu Tyr Asp Ala Thr Leu Tyr Ser Thr Phe Glu Pro
1715 1720 1725
Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg
1730 1735 1740
Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser
1745 1750 1755
Leu Met Asp Val Leu His His Pro Gly Met Asn His Arg Val Glu
1760 1765 1770
Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys
1775 1780 1785
Arg Phe Phe Arg Met Pro Arg Arg Val Phe Asn Ala Gln Lys Lys
1790 1795 1800
Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Gly Gly Ser Gly Gly
1805 1810 1815
Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln
1820 1825 1830
Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu
1835 1840 1845
Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr
1850 1855 1860
Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser
1865 1870 1875
Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser
1880 1885 1890
Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly
1895 1900 1905
Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr
1910 1915 1920
Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu
1925 1930 1935
Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu
1940 1945 1950
Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu
1955 1960 1965
Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile
1970 1975 1980
Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly
1985 1990 1995
Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys
2000 2005 2010
Arg Lys Val
2015
<210> 83
<211> 1918
<212> PRT
<213> 人工的
<220>
<223> TadA8.20m-nCas9(D10A)-A3A-UGI
<400> 83
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His
20 25 30
Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val
35 40 45
Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn
50 55 60
Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala
65 70 75 80
Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Tyr Asp Ala
85 90 95
Thr Leu Tyr Ser Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met
100 105 110
Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys
115 120 125
Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met
130 135 140
Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala
145 150 155 160
Ala Leu Leu Cys Arg Phe Phe Arg Met Pro Arg Arg Val Phe Asn Ala
165 170 175
Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly
180 185 190
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
195 200 205
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
210 215 220
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
225 230 235 240
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
245 250 255
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
260 265 270
Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
275 280 285
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
290 295 300
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
305 310 315 320
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
325 330 335
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
340 345 350
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
355 360 365
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
370 375 380
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
385 390 395 400
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
405 410 415
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
420 425 430
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
435 440 445
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
450 455 460
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
465 470 475 480
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
485 490 495
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
500 505 510
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
515 520 525
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
530 535 540
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
545 550 555 560
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
565 570 575
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
580 585 590
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
595 600 605
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
610 615 620
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
625 630 635 640
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
645 650 655
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
660 665 670
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
675 680 685
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
690 695 700
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
705 710 715 720
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
725 730 735
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
740 745 750
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
755 760 765
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
770 775 780
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
785 790 795 800
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
805 810 815
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
820 825 830
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
835 840 845
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
850 855 860
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
865 870 875 880
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
885 890 895
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
900 905 910
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
915 920 925
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
930 935 940
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
945 950 955 960
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
965 970 975
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn
980 985 990
Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly
995 1000 1005
Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln
1010 1015 1020
Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met
1025 1030 1035
Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp
1040 1045 1050
Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile
1055 1060 1065
Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser
1070 1075 1080
Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr
1085 1090 1095
Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
1100 1105 1110
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
1115 1120 1125
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile
1130 1135 1140
Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys
1145 1150 1155
Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
1160 1165 1170
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe
1175 1180 1185
Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala
1190 1195 1200
Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro
1205 1210 1215
Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
1220 1225 1230
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala
1235 1240 1245
Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys
1250 1255 1260
Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu
1265 1270 1275
Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly
1280 1285 1290
Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val
1295 1300 1305
Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys
1310 1315 1320
Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg
1325 1330 1335
Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1340 1345 1350
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1355 1360 1365
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr
1370 1375 1380
Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu
1385 1390 1395
Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
1400 1405 1410
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg
1415 1420 1425
Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala
1430 1435 1440
Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr
1445 1450 1455
Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
1460 1465 1470
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln
1475 1480 1485
Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu
1490 1495 1500
Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile
1505 1510 1515
Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn
1520 1525 1530
Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp
1535 1540 1545
Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
1550 1555 1560
Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu
1565 1570 1575
Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser
1580 1585 1590
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
1595 1600 1605
Ser Gly Gly Ser Ser Gly Gly Ser Glu Ala Ser Pro Ala Ser Gly
1610 1615 1620
Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser Asn Phe Asn
1625 1630 1635
Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu Val Glu
1640 1645 1650
Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His Arg Gly
1655 1660 1665
Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly
1670 1675 1680
Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln
1685 1690 1695
Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp
1700 1705 1710
Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe
1715 1720 1725
Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg
1730 1735 1740
Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu
1745 1750 1755
Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe
1760 1765 1770
Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe
1775 1780 1785
Gln Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly
1790 1795 1800
Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn Thr Asn Leu Ser
1805 1810 1815
Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu
1820 1825 1830
Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn
1835 1840 1845
Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser
1850 1855 1860
Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr
1865 1870 1875
Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys
1880 1885 1890
Ile Lys Met Leu Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser
1895 1900 1905
Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1910 1915
<210> 84
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp11间隔区
<400> 84
ggaatccctt ctgcagcacc 20
<210> 85
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp12间隔区
<400> 85
gaacacaaag catagactgc 20
<210> 86
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp15间隔区
<400> 86
gtcatcttag tcattacctg 20
<210> 87
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp89间隔区
<400> 87
gctccagagc cgtgcgaatg 20
<210> 88
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp90间隔区
<400> 88
gcactaccta cgtcagcacc 20
<210> 89
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp91间隔区
<400> 89
gtgttccagt ttcctttaca 20
<210> 90
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp92间隔区
<400> 90
ctgtcacagt tagctcagcc 20
<210> 91
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp93间隔区
<400> 91
ttagccaaca tacagaagtc 20
<210> 92
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp94间隔区
<400> 92
agttcccatg ttttgcttaa 20
<210> 93
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp95间隔区
<400> 93
ggatcgcttt tccgagcttc 20
<210> 94
<211> 1667
<212> PRT
<213> 人工的
<220>
<223> APOBEC1-nCas9
<400> 94
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu
20 25 30
Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg
35 40 45
Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly
50 55 60
Arg His Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val
65 70 75 80
Glu Val Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro
85 90 95
Asn Thr Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly
100 105 110
Glu Cys Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val
115 120 125
Thr Leu Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg
130 135 140
Asn Arg Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln
145 150 155 160
Ile Met Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn
165 170 175
Tyr Ser Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp
180 185 190
Val Arg Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro
195 200 205
Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe
210 215 220
Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile
225 230 235 240
Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser
245 250 255
Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser
260 265 270
Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala
275 280 285
Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys
290 295 300
Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser
305 310 315 320
Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr
325 330 335
Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg
340 345 350
Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met
355 360 365
Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu
370 375 380
Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
385 390 395 400
Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu
405 410 415
Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile
420 425 430
Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile
435 440 445
Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile
450 455 460
Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
465 470 475 480
Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys
485 490 495
Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys
500 505 510
Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro
515 520 525
Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu
530 535 540
Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile
545 550 555 560
Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp
565 570 575
Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys
580 585 590
Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln
595 600 605
Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys
610 615 620
Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
625 630 635 640
Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro
645 650 655
Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn
660 665 670
Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile
675 680 685
Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln
690 695 700
Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
705 710 715 720
Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly
725 730 735
Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr
740 745 750
Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser
755 760 765
Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys
770 775 780
Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn
785 790 795 800
Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala
805 810 815
Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys
820 825 830
Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys
835 840 845
Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
850 855 860
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
865 870 875 880
Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp
885 890 895
Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu
900 905 910
Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln
915 920 925
Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu
930 935 940
Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
945 950 955 960
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His
965 970 975
Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser
980 985 990
Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser
995 1000 1005
Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp
1010 1015 1020
Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
1025 1030 1035
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys
1040 1045 1050
Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu
1055 1060 1065
Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln
1070 1075 1080
Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg
1085 1090 1095
Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp
1100 1105 1110
Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp
1115 1120 1125
Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
1130 1135 1140
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
1145 1150 1155
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg
1160 1165 1170
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu
1175 1180 1185
Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
1190 1195 1200
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn
1205 1210 1215
Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val
1220 1225 1230
Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe
1235 1240 1245
Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
1250 1255 1260
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
1265 1270 1275
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val
1280 1285 1290
Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly
1295 1300 1305
Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe
1310 1315 1320
Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg
1325 1330 1335
Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp
1340 1345 1350
Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro
1355 1360 1365
Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1370 1375 1380
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1385 1390 1395
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp
1400 1405 1410
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu
1415 1420 1425
Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1430 1435 1440
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp
1445 1450 1455
Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
1460 1465 1470
Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg
1475 1480 1485
Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu
1490 1495 1500
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1505 1510 1515
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys
1520 1525 1530
Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile
1535 1540 1545
Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
1550 1555 1560
Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys
1565 1570 1575
Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu
1580 1585 1590
Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr
1595 1600 1605
Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
1610 1615 1620
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1625 1630 1635
Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr
1640 1645 1650
Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1655 1660 1665
<210> 95
<211> 1637
<212> PRT
<213> 人工的
<220>
<223> A3A-nCas9
<400> 95
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp
20 25 30
Pro His Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys
35 40 45
Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val
50 55 60
Lys Met Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu
65 70 75 80
Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu
85 90 95
Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp
100 105 110
Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val
115 120 125
Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala
130 135 140
Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met
145 150 155 160
Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe
165 170 175
Lys His Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln
180 185 190
Pro Trp Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu
195 200 205
Arg Ala Ile Leu Gln Asn Gln Gly Asn Ser Gly Gly Ser Ser Gly Gly
210 215 220
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
225 230 235 240
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
245 250 255
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
260 265 270
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
275 280 285
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
290 295 300
Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
305 310 315 320
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
325 330 335
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
340 345 350
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
355 360 365
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
370 375 380
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
385 390 395 400
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
405 410 415
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
420 425 430
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
435 440 445
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
450 455 460
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
465 470 475 480
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
485 490 495
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
500 505 510
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
515 520 525
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
530 535 540
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
545 550 555 560
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
565 570 575
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
580 585 590
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
595 600 605
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
610 615 620
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
625 630 635 640
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
645 650 655
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
660 665 670
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
675 680 685
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
690 695 700
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
705 710 715 720
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
725 730 735
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
740 745 750
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
755 760 765
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
770 775 780
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
785 790 795 800
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
805 810 815
Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu
820 825 830
Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile
835 840 845
Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
850 855 860
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile
865 870 875 880
Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met
885 890 895
Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg
900 905 910
Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu
915 920 925
Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
930 935 940
Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
945 950 955 960
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala
965 970 975
Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val
980 985 990
Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val
995 1000 1005
Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys
1010 1015 1020
Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu
1025 1030 1035
Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln
1040 1045 1050
Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg
1055 1060 1065
Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp
1070 1075 1080
Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp
1085 1090 1095
Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
1100 1105 1110
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
1115 1120 1125
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg
1130 1135 1140
Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu
1145 1150 1155
Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
1160 1165 1170
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn
1175 1180 1185
Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val
1190 1195 1200
Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe
1205 1210 1215
Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
1220 1225 1230
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys
1235 1240 1245
Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val
1250 1255 1260
Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly
1265 1270 1275
Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe
1280 1285 1290
Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg
1295 1300 1305
Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp
1310 1315 1320
Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro
1325 1330 1335
Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1340 1345 1350
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1355 1360 1365
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp
1370 1375 1380
Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu
1385 1390 1395
Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
1400 1405 1410
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp
1415 1420 1425
Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile
1430 1435 1440
Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg
1445 1450 1455
Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu
1460 1465 1470
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser
1475 1480 1485
His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys
1490 1495 1500
Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile
1505 1510 1515
Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
1520 1525 1530
Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys
1535 1540 1545
Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu
1550 1555 1560
Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr
1565 1570 1575
Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
1580 1585 1590
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1595 1600 1605
Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Lys Arg Thr
1610 1615 1620
Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg Lys Val
1625 1630 1635
<210> 96
<211> 1646
<212> PRT
<213> 人工的
<220>
<223> evoCDA1-nCas9
<400> 96
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp
20 25 30
Ile Tyr Thr Phe Lys Lys Gln Phe Ser Asn Asn Lys Lys Ser Val Ser
35 40 45
His Arg Cys Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg
50 55 60
Ala Cys Phe Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu
65 70 75 80
Arg Gly Ile His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr
85 90 95
Leu Arg Asp Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp
100 105 110
Ser Pro Cys Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln
115 120 125
Glu Leu Arg Gly Asn Gly His Thr Leu Lys Ile Trp Val Cys Lys Leu
130 135 140
Tyr Tyr Glu Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg
145 150 155 160
Asp Asn Gly Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys
165 170 175
Cys Arg Lys Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn
180 185 190
Arg Trp Leu Glu Lys Thr Leu Lys Arg Ala Glu Lys Arg Arg Ser Glu
195 200 205
Leu Ser Ile Met Phe Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro
210 215 220
Ala Val Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro
225 230 235 240
Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
245 250 255
Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser
260 265 270
Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys
275 280 285
Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu
290 295 300
Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg
305 310 315 320
Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile
325 330 335
Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp
340 345 350
Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys
355 360 365
Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala
370 375 380
Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val
385 390 395 400
Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala
405 410 415
His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn
420 425 430
Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr
435 440 445
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp
450 455 460
Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu
465 470 475 480
Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly
485 490 495
Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn
500 505 510
Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr
515 520 525
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala
530 535 540
Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser
545 550 555 560
Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala
565 570 575
Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu
580 585 590
Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe
595 600 605
Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala
610 615 620
Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met
625 630 635 640
Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu
645 650 655
Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His
660 665 670
Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro
675 680 685
Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg
690 695 700
Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala
705 710 715 720
Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu
725 730 735
Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met
740 745 750
Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His
755 760 765
Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val
770 775 780
Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu
785 790 795 800
Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val
805 810 815
Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe
820 825 830
Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu
835 840 845
Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu
850 855 860
Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu
865 870 875 880
Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr
885 890 895
Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg
900 905 910
Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg
915 920 925
Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly
930 935 940
Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr
945 950 955 960
Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser
965 970 975
Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys
980 985 990
Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met
995 1000 1005
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu
1010 1015 1020
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
1025 1030 1035
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu
1040 1045 1050
Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
1055 1060 1065
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1070 1075 1080
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1085 1090 1095
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1100 1105 1110
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1115 1120 1125
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1130 1135 1140
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1145 1150 1155
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1160 1165 1170
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1175 1180 1185
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1190 1195 1200
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1205 1210 1215
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1220 1225 1230
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1235 1240 1245
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1250 1255 1260
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1265 1270 1275
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1280 1285 1290
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1295 1300 1305
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1310 1315 1320
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1325 1330 1335
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1340 1345 1350
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1355 1360 1365
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1370 1375 1380
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1385 1390 1395
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1400 1405 1410
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1415 1420 1425
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1430 1435 1440
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1445 1450 1455
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1460 1465 1470
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1475 1480 1485
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1490 1495 1500
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1505 1510 1515
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1520 1525 1530
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1535 1540 1545
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1550 1555 1560
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1565 1570 1575
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1580 1585 1590
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1595 1600 1605
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1610 1615 1620
Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe
1625 1630 1635
Glu Pro Lys Lys Lys Arg Lys Val
1640 1645
<210> 97
<211> 1826
<212> PRT
<213> 人工的
<220>
<223> Gam-A3A-nCas9
<400> 97
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ala Lys Pro Ala Lys Arg Ile Lys Ser Ala Ala Ala Ala
20 25 30
Tyr Val Pro Gln Asn Arg Asp Ala Val Ile Thr Asp Ile Lys Arg Ile
35 40 45
Gly Asp Leu Gln Arg Glu Ala Ser Arg Leu Glu Thr Glu Met Asn Asp
50 55 60
Ala Ile Ala Glu Ile Thr Glu Lys Phe Ala Ala Arg Ile Ala Pro Ile
65 70 75 80
Lys Thr Asp Ile Glu Thr Leu Ser Lys Gly Val Gln Gly Trp Cys Glu
85 90 95
Ala Asn Arg Asp Glu Leu Thr Asn Gly Gly Lys Val Lys Thr Ala Asn
100 105 110
Leu Val Thr Gly Asp Val Ser Trp Arg Val Arg Pro Pro Ser Val Ser
115 120 125
Ile Arg Gly Met Asp Ala Val Met Glu Thr Leu Glu Arg Leu Gly Leu
130 135 140
Gln Arg Phe Ile Arg Thr Lys Gln Glu Ile Asn Lys Glu Ala Ile Leu
145 150 155 160
Leu Glu Pro Lys Ala Val Ala Gly Val Ala Gly Ile Thr Val Lys Ser
165 170 175
Gly Ile Glu Asp Phe Ser Ile Ile Pro Phe Glu Gln Glu Ala Gly Ile
180 185 190
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
195 200 205
Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile
210 215 220
Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu
225 230 235 240
Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp
245 250 255
Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly
260 265 270
Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser
275 280 285
Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser
290 295 300
Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe
305 310 315 320
Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile
325 330 335
Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp
340 345 350
Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His Cys
355 360 365
Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp Asp
370 375 380
Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile
385 390 395 400
Leu Gln Asn Gln Gly Asn Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
405 410 415
Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly
420 425 430
Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile
435 440 445
Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val
450 455 460
Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile
465 470 475 480
Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala
485 490 495
Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg
500 505 510
Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala
515 520 525
Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val
530 535 540
Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val
545 550 555 560
Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg
565 570 575
Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr
580 585 590
Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu
595 600 605
Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln
610 615 620
Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala
625 630 635 640
Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser
645 650 655
Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn
660 665 670
Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn
675 680 685
Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser
690 695 700
Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly
705 710 715 720
Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala
725 730 735
Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala
740 745 750
Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp
755 760 765
Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr
770 775 780
Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile
785 790 795 800
Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile
805 810 815
Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg
820 825 830
Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro
835 840 845
His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu
850 855 860
Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile
865 870 875 880
Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn
885 890 895
Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro
900 905 910
Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe
915 920 925
Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val
930 935 940
Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu
945 950 955 960
Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe
965 970 975
Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr
980 985 990
Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
995 1000 1005
Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
1010 1015 1020
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile
1025 1030 1035
Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
1040 1045 1050
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met
1055 1060 1065
Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys
1070 1075 1080
Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg
1085 1090 1095
Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly
1100 1105 1110
Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg
1115 1120 1125
Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu
1130 1135 1140
Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
1145 1150 1155
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
1160 1165 1170
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met
1175 1180 1185
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu
1190 1195 1200
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
1205 1210 1215
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu
1220 1225 1230
Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
1235 1240 1245
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1250 1255 1260
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1265 1270 1275
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1280 1285 1290
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1295 1300 1305
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1310 1315 1320
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1325 1330 1335
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1340 1345 1350
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1355 1360 1365
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1370 1375 1380
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1385 1390 1395
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1400 1405 1410
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1415 1420 1425
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1430 1435 1440
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1445 1450 1455
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1460 1465 1470
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1475 1480 1485
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1490 1495 1500
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1505 1510 1515
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1520 1525 1530
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1535 1540 1545
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1550 1555 1560
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1565 1570 1575
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1580 1585 1590
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1595 1600 1605
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1610 1615 1620
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1625 1630 1635
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1640 1645 1650
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1655 1660 1665
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1670 1675 1680
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1685 1690 1695
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1700 1705 1710
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1715 1720 1725
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1730 1735 1740
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1745 1750 1755
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1760 1765 1770
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1775 1780 1785
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1790 1795 1800
Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe
1805 1810 1815
Glu Pro Lys Lys Lys Arg Lys Val
1820 1825
<210> 98
<211> 1856
<212> PRT
<213> 人工的
<220>
<223> Gam-APOBEC1-nCas9
<400> 98
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ala Lys Pro Ala Lys Arg Ile Lys Ser Ala Ala Ala Ala
20 25 30
Tyr Val Pro Gln Asn Arg Asp Ala Val Ile Thr Asp Ile Lys Arg Ile
35 40 45
Gly Asp Leu Gln Arg Glu Ala Ser Arg Leu Glu Thr Glu Met Asn Asp
50 55 60
Ala Ile Ala Glu Ile Thr Glu Lys Phe Ala Ala Arg Ile Ala Pro Ile
65 70 75 80
Lys Thr Asp Ile Glu Thr Leu Ser Lys Gly Val Gln Gly Trp Cys Glu
85 90 95
Ala Asn Arg Asp Glu Leu Thr Asn Gly Gly Lys Val Lys Thr Ala Asn
100 105 110
Leu Val Thr Gly Asp Val Ser Trp Arg Val Arg Pro Pro Ser Val Ser
115 120 125
Ile Arg Gly Met Asp Ala Val Met Glu Thr Leu Glu Arg Leu Gly Leu
130 135 140
Gln Arg Phe Ile Arg Thr Lys Gln Glu Ile Asn Lys Glu Ala Ile Leu
145 150 155 160
Leu Glu Pro Lys Ala Val Ala Gly Val Ala Gly Ile Thr Val Lys Ser
165 170 175
Gly Ile Glu Asp Phe Ser Ile Ile Pro Phe Glu Gln Glu Ala Gly Ile
180 185 190
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
195 200 205
Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg Arg
210 215 220
Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu Arg
225 230 235 240
Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His Ser
245 250 255
Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val Asn
260 265 270
Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr Arg
275 280 285
Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys Ser
290 295 300
Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu Phe
305 310 315 320
Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg Gln
325 330 335
Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met Thr
340 345 350
Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser Pro
355 360 365
Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg Leu
370 375 380
Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys Leu
385 390 395 400
Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile Ala
405 410 415
Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp Ala
420 425 430
Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu
435 440 445
Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser
450 455 460
Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr
465 470 475 480
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser
485 490 495
Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys
500 505 510
Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala
515 520 525
Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn
530 535 540
Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val
545 550 555 560
Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu
565 570 575
Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu
580 585 590
Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys
595 600 605
Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala
610 615 620
Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp
625 630 635 640
Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val
645 650 655
Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly
660 665 670
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg
675 680 685
Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu
690 695 700
Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys
705 710 715 720
Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp
725 730 735
Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln
740 745 750
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu
755 760 765
Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu
770 775 780
Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr
785 790 795 800
Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu
805 810 815
Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly
820 825 830
Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu
835 840 845
Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp
850 855 860
Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln
865 870 875 880
Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe
885 890 895
Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr
900 905 910
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg
915 920 925
Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn
930 935 940
Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu
945 950 955 960
Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro
965 970 975
Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr
980 985 990
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser
995 1000 1005
Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
1010 1015 1020
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys
1025 1030 1035
Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg
1040 1045 1050
Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile
1055 1060 1065
Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
1070 1075 1080
Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met
1085 1090 1095
Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys
1100 1105 1110
Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg
1115 1120 1125
Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly
1130 1135 1140
Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg
1145 1150 1155
Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu
1160 1165 1170
Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
1175 1180 1185
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
1190 1195 1200
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met
1205 1210 1215
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu
1220 1225 1230
Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
1235 1240 1245
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu
1250 1255 1260
Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
1265 1270 1275
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1280 1285 1290
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
1295 1300 1305
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1310 1315 1320
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1325 1330 1335
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1340 1345 1350
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1355 1360 1365
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1370 1375 1380
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1385 1390 1395
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1400 1405 1410
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1415 1420 1425
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1430 1435 1440
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1445 1450 1455
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1460 1465 1470
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1475 1480 1485
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1490 1495 1500
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1505 1510 1515
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1520 1525 1530
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1535 1540 1545
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1550 1555 1560
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1565 1570 1575
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1580 1585 1590
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1595 1600 1605
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1610 1615 1620
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1625 1630 1635
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1640 1645 1650
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1655 1660 1665
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1670 1675 1680
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1685 1690 1695
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1700 1705 1710
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1715 1720 1725
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1730 1735 1740
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1745 1750 1755
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1760 1765 1770
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1775 1780 1785
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1790 1795 1800
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1805 1810 1815
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1820 1825 1830
Gly Asp Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe
1835 1840 1845
Glu Pro Lys Lys Lys Arg Lys Val
1850 1855
<210> 99
<211> 1835
<212> PRT
<213> 人工的
<220>
<223> Gam-evoCDA1-nCas9
<400> 99
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ala Lys Pro Ala Lys Arg Ile Lys Ser Ala Ala Ala Ala
20 25 30
Tyr Val Pro Gln Asn Arg Asp Ala Val Ile Thr Asp Ile Lys Arg Ile
35 40 45
Gly Asp Leu Gln Arg Glu Ala Ser Arg Leu Glu Thr Glu Met Asn Asp
50 55 60
Ala Ile Ala Glu Ile Thr Glu Lys Phe Ala Ala Arg Ile Ala Pro Ile
65 70 75 80
Lys Thr Asp Ile Glu Thr Leu Ser Lys Gly Val Gln Gly Trp Cys Glu
85 90 95
Ala Asn Arg Asp Glu Leu Thr Asn Gly Gly Lys Val Lys Thr Ala Asn
100 105 110
Leu Val Thr Gly Asp Val Ser Trp Arg Val Arg Pro Pro Ser Val Ser
115 120 125
Ile Arg Gly Met Asp Ala Val Met Glu Thr Leu Glu Arg Leu Gly Leu
130 135 140
Gln Arg Phe Ile Arg Thr Lys Gln Glu Ile Asn Lys Glu Ala Ile Leu
145 150 155 160
Leu Glu Pro Lys Ala Val Ala Gly Val Ala Gly Ile Thr Val Lys Ser
165 170 175
Gly Ile Glu Asp Phe Ser Ile Ile Pro Phe Glu Gln Glu Ala Gly Ile
180 185 190
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
195 200 205
Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp Ile Tyr Thr
210 215 220
Phe Lys Lys Gln Phe Ser Asn Asn Lys Lys Ser Val Ser His Arg Cys
225 230 235 240
Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg Ala Cys Phe
245 250 255
Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu Arg Gly Ile
260 265 270
His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr Leu Arg Asp
275 280 285
Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp Ser Pro Cys
290 295 300
Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln Glu Leu Arg
305 310 315 320
Gly Asn Gly His Thr Leu Lys Ile Trp Val Cys Lys Leu Tyr Tyr Glu
325 330 335
Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg Asp Asn Gly
340 345 350
Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys Cys Arg Lys
355 360 365
Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn Arg Trp Leu
370 375 380
Glu Lys Thr Leu Lys Arg Ala Glu Lys Arg Arg Ser Glu Leu Ser Ile
385 390 395 400
Met Phe Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro Ala Val Ser
405 410 415
Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser
420 425 430
Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp
435 440 445
Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp
450 455 460
Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val
465 470 475 480
Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala
485 490 495
Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg
500 505 510
Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu
515 520 525
Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe
530 535 540
His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu
545 550 555 560
Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu
565 570 575
Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr
580 585 590
Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile
595 600 605
Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn
610 615 620
Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln
625 630 635 640
Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala
645 650 655
Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile
660 665 670
Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile
675 680 685
Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu
690 695 700
Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp
705 710 715 720
Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe
725 730 735
Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu
740 745 750
Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile
755 760 765
Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu
770 775 780
Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln
785 790 795 800
Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu
805 810 815
Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr
820 825 830
Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln
835 840 845
Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu
850 855 860
Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys
865 870 875 880
Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr
885 890 895
Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr
900 905 910
Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val
915 920 925
Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe
930 935 940
Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu
945 950 955 960
Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val
965 970 975
Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys
980 985 990
Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys
995 1000 1005
Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
1010 1015 1020
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
1025 1030 1035
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu
1040 1045 1050
Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr
1055 1060 1065
Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys
1070 1075 1080
Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
1085 1090 1095
Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile
1100 1105 1110
Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
1115 1120 1125
Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
1130 1135 1140
His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln
1145 1150 1155
Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu
1160 1165 1170
Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys
1175 1180 1185
Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu
1190 1195 1200
Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys
1205 1210 1215
Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly
1220 1225 1230
Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu
1235 1240 1245
Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
1250 1255 1260
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
1265 1270 1275
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu
1280 1285 1290
Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys
1295 1300 1305
Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
1310 1315 1320
Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile
1325 1330 1335
Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly
1340 1345 1350
Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val
1355 1360 1365
Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
1370 1375 1380
Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu
1385 1390 1395
Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg
1400 1405 1410
Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His
1415 1420 1425
His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu
1430 1435 1440
Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp
1445 1450 1455
Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln
1460 1465 1470
Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile
1475 1480 1485
Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
1490 1495 1500
Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1505 1510 1515
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu
1520 1525 1530
Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr
1535 1540 1545
Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp
1550 1555 1560
Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly
1565 1570 1575
Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala
1580 1585 1590
Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu
1595 1600 1605
Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
1610 1615 1620
Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys
1625 1630 1635
Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu
1640 1645 1650
Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys
1655 1660 1665
Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr
1670 1675 1680
Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn
1685 1690 1695
Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp
1700 1705 1710
Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu
1715 1720 1725
Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
1730 1735 1740
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1745 1750 1755
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe
1760 1765 1770
Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val
1775 1780 1785
Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu
1790 1795 1800
Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser
1805 1810 1815
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys Lys Lys Arg
1820 1825 1830
Lys Val
1835
<210> 100
<211> 213
<212> PRT
<213> 噬菌体Mu
<400> 100
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ala Lys Pro Ala Lys Arg Ile Lys Ser Ala Ala Ala Ala
20 25 30
Tyr Val Pro Gln Asn Arg Asp Ala Val Ile Thr Asp Ile Lys Arg Ile
35 40 45
Gly Asp Leu Gln Arg Glu Ala Ser Arg Leu Glu Thr Glu Met Asn Asp
50 55 60
Ala Ile Ala Glu Ile Thr Glu Lys Phe Ala Ala Arg Ile Ala Pro Ile
65 70 75 80
Lys Thr Asp Ile Glu Thr Leu Ser Lys Gly Val Gln Gly Trp Cys Glu
85 90 95
Ala Asn Arg Asp Glu Leu Thr Asn Gly Gly Lys Val Lys Thr Ala Asn
100 105 110
Leu Val Thr Gly Asp Val Ser Trp Arg Val Arg Pro Pro Ser Val Ser
115 120 125
Ile Arg Gly Met Asp Ala Val Met Glu Thr Leu Glu Arg Leu Gly Leu
130 135 140
Gln Arg Phe Ile Arg Thr Lys Gln Glu Ile Asn Lys Glu Ala Ile Leu
145 150 155 160
Leu Glu Pro Lys Ala Val Ala Gly Val Ala Gly Ile Thr Val Lys Ser
165 170 175
Gly Ile Glu Asp Phe Ser Ile Ile Pro Phe Glu Gln Glu Ala Gly Ile
180 185 190
Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Pro Lys
195 200 205
Lys Lys Arg Lys Val
210
<210> 101
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp10间隔区
<400> 101
gagtccgagc agaagaagaa 20
<210> 102
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp11间隔区
<400> 102
ggaatccctt ctgcagcacc 20
<210> 103
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp12间隔区
<400> 103
gaacacaaag catagactgc 20
<210> 104
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp13间隔区
<400> 104
ggcccagact gagcacgtga 20
<210> 105
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp14间隔区
<400> 105
ggcactgcgg ctggaggtgg 20
<210> 106
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp15间隔区
<400> 106
gtcatcttag tcattacctg 20
<210> 107
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp88间隔区
<400> 107
gcacaaccag tggaggcaag 20
<210> 108
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp89间隔区
<400> 108
gctccagagc cgtgcgaatg 20
<210> 109
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp90间隔区
<400> 109
gcactaccta cgtcagcacc 20
<210> 110
<211> 20
<212> DNA
<213> 人工的
<220>
<223> PWsp91间隔区
<400> 110
gtgttccagt ttcctttaca 20
<210> 111
<211> 1479
<212> PRT
<213> 人工的
<220>
<223> APOBEC3A-dLbCas12a
<400> 111
Met Ala Gly Ser Lys Lys Arg Arg Ile Lys Gln Asp Glu Ala Ser Pro
1 5 10 15
Ala Ser Gly Pro Arg His Leu Met Asp Pro His Ile Phe Thr Ser Asn
20 25 30
Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr Leu Cys Tyr Glu Val
35 40 45
Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met Asp Gln His Arg Gly
50 55 60
Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg
65 70 75 80
His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp
85 90 95
Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys
100 105 110
Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn
115 120 125
Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg Ile Tyr Asp Tyr Asp
130 135 140
Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln
145 150 155 160
Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His Cys Trp Asp Thr Phe
165 170 175
Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp Asp Gly Leu Asp Glu
180 185 190
His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln
195 200 205
Gly Asn Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro
210 215 220
Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly
225 230 235 240
Gly Ser Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys
245 250 255
Thr Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile
260 265 270
Asp Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr
275 280 285
Lys Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn
290 295 300
Asp Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser
305 310 315 320
Leu Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu
325 330 335
Asn Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly
340 345 350
Asn Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile
355 360 365
Leu Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser
370 375 380
Phe Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu
385 390 395 400
Asn Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys
405 410 415
Ile Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu
420 425 430
Lys Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu
435 440 445
Lys Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu
450 455 460
Phe Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala
465 470 475 480
Ile Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu
485 490 495
Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro
500 505 510
Lys Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu
515 520 525
Ser Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val
530 535 540
Phe Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys
545 550 555 560
Lys Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly
565 570 575
Ile Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile
580 585 590
Phe Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp
595 600 605
Asp Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp
610 615 620
Asp Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln
625 630 635 640
Leu Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys
645 650 655
Glu Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser
660 665 670
Ser Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys
675 680 685
Lys Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val
690 695 700
Lys Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu
705 710 715 720
Thr Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp
725 730 735
Ile Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val
740 745 750
Thr Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn
755 760 765
Pro Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg
770 775 780
Ala Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp
785 790 795 800
Lys Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn
805 810 815
Gly Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys
820 825 830
Met Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn
835 840 845
Pro Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys
850 855 860
Gly Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe
865 870 875 880
Lys Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe
885 890 895
Asn Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg
900 905 910
Glu Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys
915 920 925
Lys Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln
930 935 940
Ile Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu
945 950 955 960
His Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln
965 970 975
Ile Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu
980 985 990
Lys Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn
995 1000 1005
Lys Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp
1010 1015 1020
Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His
1025 1030 1035
Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile
1040 1045 1050
Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr
1055 1060 1065
Val Ile Gly Ile Ala Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val
1070 1075 1080
Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu Asn
1085 1090 1095
Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr
1100 1105 1110
His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg
1115 1120 1125
Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly
1130 1135 1140
Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys
1145 1150 1155
Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys
1160 1165 1170
Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu
1175 1180 1185
Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys Ser
1190 1195 1200
Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr
1205 1210 1215
Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
1220 1225 1230
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser
1235 1240 1245
Thr Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala
1250 1255 1260
Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val
1265 1270 1275
Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe
1280 1285 1290
Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser
1295 1300 1305
Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn
1310 1315 1320
Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu
1325 1330 1335
Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg
1340 1345 1350
Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe
1355 1360 1365
Met Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr
1370 1375 1380
Gly Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser
1385 1390 1395
Asp Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn
1400 1405 1410
Ala Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile
1415 1420 1425
Ala Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu
1430 1435 1440
Asp Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu
1445 1450 1455
Trp Leu Glu Tyr Ala Gln Thr Ser Val Lys His Gly Ser Lys Lys
1460 1465 1470
Arg Arg Ile Lys Gln Asp
1475
<210> 112
<211> 23
<212> DNA
<213> Glycine max
<400> 112
gtaagaagct cttcaccgtt cca 23
<210> 113
<211> 194
<212> PRT
<213> 噬菌体Mu
<400> 113
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Ala Lys Pro Ala Lys Arg Ile Lys Ser Ala Ala Ala Ala
20 25 30
Tyr Val Pro Gln Asn Arg Asp Ala Val Ile Thr Asp Ile Lys Arg Ile
35 40 45
Gly Asp Leu Gln Arg Glu Ala Ser Arg Leu Glu Thr Glu Met Asn Asp
50 55 60
Ala Ile Ala Glu Ile Thr Glu Lys Phe Ala Ala Arg Ile Ala Pro Ile
65 70 75 80
Lys Thr Asp Ile Glu Thr Leu Ser Lys Gly Val Gln Gly Trp Cys Glu
85 90 95
Ala Asn Arg Asp Glu Leu Thr Asn Gly Gly Lys Val Lys Thr Ala Asn
100 105 110
Leu Val Thr Gly Asp Val Ser Trp Arg Val Arg Pro Pro Ser Val Ser
115 120 125
Ile Arg Gly Met Asp Ala Val Met Glu Thr Leu Glu Arg Leu Gly Leu
130 135 140
Gln Arg Phe Ile Arg Thr Lys Gln Glu Ile Asn Lys Glu Ala Ile Leu
145 150 155 160
Leu Glu Pro Lys Ala Val Ala Gly Val Ala Gly Ile Thr Val Lys Ser
165 170 175
Gly Ile Glu Asp Phe Ser Ile Ile Pro Phe Glu Gln Glu Ala Gly Ile
180 185 190
Ser Gly

Claims (94)

1.一种修饰靶核酸的方法，所述方法包括：
使靶核酸接触：
CRISPR-Cas效应蛋白(例如，CRISPR酶)，
指导核酸(例如，指导RNA)，
胞嘧啶脱氨酶，和
腺嘌呤脱氨酶，
其中胞嘧啶脱氨酶和腺嘌呤脱氨酶并行地和/或同时地修饰靶核酸，任选地在包含CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶的试剂的单次递送中进行修饰，和/或
其中CRISPR-Cas效应蛋白与胞嘧啶脱氨酶和/或腺嘌呤脱氨酶形成复合物或包含在复合物中，从而修饰靶核酸。
2.权利要求1所述的方法，其中指导核酸包含RNA募集基序，任选地其中RNA募集基序是MS2发夹。
3.权利要求1或2所述的方法，其中CRISPR-Cas效应蛋白包含指导核酸或复合物进一步包含指导核酸。
4.前述权利要求中任一项所述的方法，其中将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
5.前述权利要求中任一项所述的方法，其中将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶不融合至Cas9。
6.前述权利要求中任一项所述的方法，其中将胞嘧啶脱氨酶与腺嘌呤脱氨酶融合在一起以提供融合蛋白，任选地其中融合蛋白包含SEQ ID NO：79-83中任一项的序列的全部或部分。
7.前述权利要求中任一项所述的方法，其中胞嘧啶脱氨酶和/或腺嘌呤脱氨酶包含MS2加帽蛋白(MCP)或其部分，任选地其中将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶与MCP或其部分融合在一起。
8.权利要求7所述的方法，其中MCP蛋白或其部分结合RNA募集基序(例如，MS2发夹)。
9.权利要求2-8中任一项所述的方法，其中使用MS2发夹将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶募集至靶核酸。
10.前述权利要求中任一项所述的方法，其中使靶核酸接触进一步包括使靶核酸接触CRISPR-Cas效应蛋白、指导核酸、胞嘧啶脱氨酶、腺嘌呤脱氨酶和糖基化酶抑制剂(例如，尿嘧啶糖基化酶抑制剂(UGI))。
11.权利要求10所述的方法，其中将糖基化酶抑制剂融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
12.前述权利要求中任一项所述的方法，其中CRISPR-Cas效应蛋白包含肽标签(例如，SunTag)，任选地其中肽标签包含一个或多个(例如，1、2、3、4或更多个)GCN4表位。
13.权利要求12所述的方法，其中腺嘌呤脱氨酶和/或胞嘧啶脱氨酶包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将腺嘌呤脱氨酶和/或胞嘧啶脱氨酶与亲和多肽融合一起。
14.权利要求13所述的方法，其中使用亲和多肽将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶募集至靶核酸。
15.权利要求12-14中任一项所述的方法，其中通过融合至MCP或其部分将肽标签(例如，Sun Tag)募集至RNA募集基序(例如，MS2发夹)并且使用亲和多肽将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶募集至肽标签。
16.权利要求1-11中任一项所述的方法，其中腺嘌呤脱氨酶和/或胞嘧啶脱氨酶包含肽标签(例如SunTag)，任选地其中肽标签包含一个或多个(例如1、2、3、4或更多个)GCN4表位。
17.权利要求16所述的方法，其中CRISPR-Cas效应蛋白包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将CRISPR-Cas效应蛋白与亲和多肽融合在一起。
18.权利要求17所述的方法，其中使用亲和多肽将CRISPR-Cas效应蛋白募集至靶核酸。
19.权利要求16-18中任一项所述的方法，其中将肽标签(例如，Sun Tag)募集至RNA募集基序(例如，MS2发夹)并且使用亲和多肽将CRISPR-Cas效应蛋白募集至肽标签。
20.前述权利要求中任一项所述的方法，其中CRISPR-Cas效应蛋白是V型CRISPR-Cas效应蛋白。
21.前述权利要求中任一项所述的方法，其中靶核酸在基因的非编码区(例如，启动子区)中或其中靶核酸在基因的编码区中。
22.前述权利要求中任一项所述的方法，进一步包括使靶核酸接触目标多肽(例如，包含SEQ ID NO：100或SEQ ID NO：113的全部或部分的多肽)，任选地其中将目标多肽融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
23.一种碱基编辑组合物或系统，包括：
CRISPR-Cas效应蛋白(例如，CRISPR酶)，
指导核酸(例如，指导RNA)，
胞嘧啶脱氨酶，和
腺嘌呤脱氨酶，
其中CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和腺嘌呤脱氨酶形成复合物或包含在复合物中。
24.权利要求23所述的碱基编辑组合物或系统，其中指导核酸包含RNA募集基序，任选地其中RNA募集基序是MS2发夹。
25.权利要求23或24所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包含指导核酸和/或复合物进一步包含指导核酸。
26.权利要求23-25中任一项所述的碱基编辑组合物或系统，其中将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
27.权利要求23-26中任一项所述的碱基编辑组合物或系统，其中将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶不融合至Cas9。
28.权利要求23-27中任一项所述的碱基编辑组合物或系统，其中将胞嘧啶脱氨酶与腺嘌呤脱氨酶融合在一起以提供融合蛋白，任选地其中融合蛋白包含SEQ ID NO：79-83中任一项的序列的全部或部分。
29.权利要求23-28中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶和/或腺嘌呤脱氨酶包含MS2加帽蛋白(MCP)或其部分，任选地其中将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶与MCP或其部分融合在一起。
30.权利要求29所述的碱基编辑组合物或系统，其中MCP蛋白或其部分结合至RNA募集基序(例如，MS2发夹)。
31.权利要求23-30中任一项所述的碱基编辑组合物或系统，还包含糖基化酶抑制剂(例如，尿嘧啶糖基化酶抑制剂(UGI))和/或目标多肽(例如，包含SEQ ID NO：100或SEQ IDNO：113的全部或部分的多肽)，任选地其中将糖基化酶抑制剂和/或目标多肽融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
32.权利要求31所述的碱基编辑组合物或系统，其中将糖基化酶抑制剂融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
33.权利要求23-32中任一项所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包含肽标签(例如，SunTag)，任选地其中肽标签包含一个或多个(例如，1、2、3、4或更多个)GCN4表位。
34.权利要求33所述的碱基编辑组合物或系统，其中腺嘌呤脱氨酶和/或胞嘧啶脱氨酶包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将腺嘌呤脱氨酶和/或胞嘧啶脱氨酶与亲和多肽融合在一起。
35.权利要求34所述的碱基编辑组合物或系统，其中使用亲和多肽将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶募集至靶核酸。
36.权利要求33-35中任一项所述的碱基编辑组合物或系统，其中通过融合至MCP或其部分将肽标签(例如Sun Tag)募集至RNA募集基序(例如MS2发夹)并且使用亲和多肽将胞嘧啶脱氨酶和/或腺嘌呤脱氨酶募集至肽标签。
37.权利要求23-32中任一项所述的碱基编辑组合物或系统，其中腺嘌呤脱氨酶和/或胞嘧啶脱氨酶包含肽标签(例如SunTag)，任选地其中肽标签包含一个或多个(例如1、2、3、4或更多个)GCN4表位。
38.权利要求37所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将CRISPR-Cas效应蛋白与亲和多肽融合一起。
39.权利要求38所述的碱基编辑组合物或系统，其中使用亲和多肽将CRISPR-Cas效应蛋白募集至靶核酸。
40.权利要求37-39中任一项所述的碱基编辑组合物或系统，其中将肽标签(例如，SunTag)募集至RNA募集基序(例如，MS2发夹)并且使用亲和多肽将CRISPR-Cas效应蛋白募集至肽标签。
41.权利要求23-40中任一项所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白是V型CRISPR-Cas效应蛋白。
42.一种修饰靶核酸的方法，所述方法包括：
使靶核酸接触：
CRISPR-Cas效应蛋白(例如，CRISPR酶)，
指导核酸(例如，指导RNA)，和
胞嘧啶脱氨酶，
其中所述方法将靶核酸的胞嘧啶(C)修饰为腺嘌呤(A)、鸟嘌呤(G)或胸腺嘧啶(T)，从而修饰靶核酸。
43.权利要求42所述的方法，其中胞嘧啶脱氨酶是载脂蛋白B mRNA-编辑复合物(APOBEC)脱氨酶(例如APOBECl脱氨酶、APOBEC2脱氨酶、APOBEC3A脱氨酶、APOBEC3B脱氨酶、APOBEC3C脱氨酶、APOBEC3D脱氨酶、APOBEC3F脱氨酶、APOBEC3G脱氨酶、APOBEC3H脱氨酶、APOBEC4脱氨酶和/或rAPOBECl脱氨酶)、人活化诱导脱氨酶(hAID)、FERNY脱氨酶和/或CDA1脱氨酶(例如，pmCDA1、atCDA1(例如，At2g19570))和/或其进化形式，任选地其中胞嘧啶脱氨酶包含SEQ ID NO：40-49中任一项的氨基酸序列的全部或部分。
44.权利要求42或43所述的方法，其中使靶核酸接触进一步包括使靶核酸接触CRISPR-Cas效应蛋白、指导核酸、胞嘧啶脱氨酶和尿嘧啶N-糖基化酶(UNG)。
45.权利要求42-44中任一项所述的方法，其中所述方法包括调节CRISPR-Cas效应蛋白的DNA结合亲和力。
46.权利要求42-45中任一项所述的方法，其中指导核酸与靶核酸具有不完全的互补性。
47.权利要求42-46中任一项所述的方法，其中CRISPR-Cas效应蛋白包含Cas9，任选地其中Cas9与靶核酸具有减弱的相互作用。
48.权利要求42-47中任一项所述的方法，其中所述方法包括调节(例如，增加或减少)CRISPR-Cas效应蛋白在靶核酸处的停留时间。
49.权利要求42-48中任一项所述的方法，其中所述方法包括产生用作DNA聚合酶的模板的脱碱基位点。
50.权利要求42-49中任一项所述的方法，其中使靶核酸接触进一步包括使靶核酸接触CRISPR-Cas效应蛋白、指导核酸、胞嘧啶脱氨酶和AP核酸内切酶I(APE1)抑制剂，任选地其中APE1抑制剂是有机化合物和/或核酸(例如，siRNA)。
51.权利要求42-50中任一项所述的方法，其中所述方法包括抑制APE1。
52.权利要求42-51中任一项所述的方法，其中所述方法包括抑制或减少插入缺失形成，任选地与不存在APE1抑制剂时的插入缺失形成的量相比。
53.权利要求42-52中任一项所述的方法，其中使靶核酸接触进一步包括使靶核酸接触CRISPR-Cas效应蛋白、指导核酸、胞嘧啶脱氨酶和DNA连接酶IV抑制剂。
54.权利要求42-53中任一项所述的方法，其中使靶核酸接触进一步包括使靶核酸接触CRISPR-Cas效应蛋白、指导核酸、胞嘧啶脱氨酶和DNA-PKcs抑制剂。
55.权利要求42-54中任一项所述的方法，其中所述方法包括减少非同源末端连接(NHEJ)。
56.权利要求42-55中任一项所述的方法，其中使靶核酸接触进一步包括使靶核酸接触CRISPR-Cas效应蛋白、指导核酸、胞嘧啶脱氨酶和外源聚合酶，任选地其中通过CRISPR-Cas效应蛋白将外源聚合酶募集至靶核酸。
57.权利要求56所述的方法，其中将外源聚合酶融合至V型CRISPR-Cas效应蛋白，任选地其中将外源聚合酶融合至Cas9(例如dCas9或nCas9)。
58.权利要求56或57所述的方法，其中外源聚合酶是密码子优化的。
59.权利要求30-47中任一项所述的方法，其中CRISPR-Cas效应蛋白包含肽标签(例如，SunTag)，任选地其中肽标签包含一个或多个(例如，1、2、3、4或更多个)GCN4表位。
60.权利要求59所述的方法，其中胞嘧啶脱氨酶包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将胞嘧啶脱氨酶与亲和多肽融合在一起。
61.权利要求60所述的方法，其中使用亲和多肽将胞嘧啶脱氨酶募集至靶核酸。
62.权利要求42-58中任一项所述的方法，其中胞嘧啶脱氨酶包含肽标签(例如SunTag)，任选地其中肽标签包含一个或多个(例如1、2、3、4或更多个)GCN4表位。
63.权利要求62所述的方法，其中CRISPR-Cas效应蛋白包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将CRISPR-Cas效应蛋白与亲和多肽融合在一起。
64.权利要求63所述的方法，其中使用亲和多肽将CRISPR-Cas效应蛋白募集至靶核酸。
65.权利要求62-64中任一项所述的方法，其中将肽标签(例如，Sun Tag)募集至RNA募集基序(例如，MS2发夹)并且使用亲和多肽将CRISPR-Cas效应蛋白募集至肽标签。
66.权利要求42-65中任一项所述的方法，其中所述方法提供大于0.1％、0.5％、1％、2％、5％、10％、20％、30％、40％、50％、60％、70％或80％的碱基置换频率，任选其中C至非T编辑(例如C至G编辑和/或C至A编辑)的碱基置换频率大于约0.1％、0.5％、1％、2％、5％、10％、20％、30％、40％、50％、60％、70％或80％，和/或其中所述方法提供对于C→G∶C→T变化高至约1∶1或1.5∶1的比率。
67.权利要求42-66中任一项所述的方法，其中靶核酸存在于植物细胞或真核细胞(例如哺乳动物细胞)中。
68.权利要求42-67中任一项所述的方法，其中胞嘧啶脱氨酶包含MS2加帽蛋白(MCP)或其部分，任选地其中将MCP或其部分融合至胞嘧啶脱氨酶氨基酸的N末端。
69.权利要求42-68中任一项所述的方法，其中胞嘧啶脱氨酶包含Cas9结构域，任选地其中将胞嘧啶脱氨酶融合至Cas9结构域。
70.权利要求42-69中任一项所述的方法，其中胞嘧啶脱氨酶包含失活的LbCpf1(dLbCpf1)，任选地其中将胞嘧啶脱氨酶融合至dLbCpf1。
71.权利要求42-70中任一项所述的方法，其中胞嘧啶脱氨酶是密码子优化的。
72.权利要求71所述的方法，其中针对单子叶植物表达或针对子叶植物表达对胞嘧啶脱氨酶进行密码子优化。
73.权利要求42-72中任一项所述的方法，进一步包括使靶核酸与目标多肽(例如，包含SEQ ID NO：100或SEQ ID NO：113的全部或部分的多肽)接触，任选地其中将目标多肽融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
74.一种碱基编辑组合物或系统，包括：
CRISPR-Cas效应蛋白(例如，CRISPR酶)，
指导核酸(例如，指导RNA)，和
胞嘧啶脱氨酶，
其中组合物或系统缺乏糖基化酶抑制剂(例如，尿嘧啶糖基化酶抑制剂(UGI))。
75.权利要求74所述的碱基编辑组合物或系统，其中指导核酸包含RNA募集基序，任选地其中RNA募集基序是MS2发夹。
76.权利要求74或75所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包含指导核酸和/或复合物进一步包含指导核酸。
77.权利要求74-76中任一项所述的碱基编辑组合物或系统，其中指导核酸与靶核酸具有不完全的互补性。
78.权利要求74-77中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶是载脂蛋白B mRNA-编辑复合物(APOBEC)脱氨酶(例如APOBEC1脱氨酶、APOBEC2脱氨酶、APOBEC3A脱氨酶、APOBEC3B脱氨酶、APOBEC3C脱氨酶、APOBEC3D脱氨酶、APOBEC3F脱氨酶、APOBEC3G脱氨酶、APOBEC3H脱氨酶、APOBEC4脱氨酶和/或rAPOBEC1脱氨酶)、人活化诱导脱氨酶(hAID)、FERNY脱氨酶和/或CDA1脱氨酶(例如pmCDA1、atCDA1(例如At2g19570))和/或其进化形式，任选地其中胞嘧啶脱氨酶包含SEQ ID NO：40-49中任一项的氨基酸序列的全部或部分。
79.权利要求74-78中任一项所述的碱基编辑组合物或系统，其中将CRISPR-Cas效应蛋白融合至胞嘧啶脱氨酶。
80.权利要求74-79中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶包含MS2加帽蛋白(MCP)或其部分，任选地其中将胞嘧啶脱氨酶与MCP或其部分融合在一起。
81.权利要求80所述的碱基编辑组合物或系统，其中MCP蛋白或其部分结合至RNA募集基序(例如，MS2发夹)。
82.权利要求74-81中任一项所述的碱基编辑组合物或系统，还包含糖基化酶结构域(例如，UNG结构域)。
83.权利要求74-82中任一项所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包括V型CRISPR-Cas效应蛋白。
84.权利要求74-83中任一项所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包含Cas9，任选地与靶核酸具有减弱的相互作用的Cas9。
85.权利要求74-84中任一项所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包含(例如，融合至)外源聚合酶，任选地其中外源聚合酶任选地是密码子优化的。
86.权利要求74-85中任一项所述的碱基编辑组合物或系统，其中CRISPR-Cas效应蛋白包含肽标签(例如，SunTag)并且胞嘧啶脱氨酶包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将胞嘧啶脱氨酶与亲和多肽融合在一起。
87.权利要求74-86中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶包含肽标签(例如，SunTag)并且CRISPR-Cas效应蛋白包含能够结合肽标签的亲和多肽(例如，scFv)，任选地其中将CRISPR-Cas效应蛋白与亲和多肽融合在一起。
88.权利要求74-87中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶包含MCP或其部分，任选地其中将MCP或其部分融合至胞嘧啶脱氨酶氨基酸序列的N末端。
89.权利要求74-88中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶包含Cas9、Cas12、Cas13或Cas14结构域。
90.权利要求89所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶包含Cas9结构域，任选地其中将胞嘧啶脱氨酶融合至Cas9结构域。
91.权利要求74-88中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶包含失活的LbCpf1(dLbCpf1)，任选地其中将胞嘧啶脱氨酶融合至dLbCpf1。
92.权利要求74-91中任一项所述的碱基编辑组合物或系统，其中胞嘧啶脱氨酶是密码子优化的，任选地针对单子叶植物表达和/或双子叶植物表达进行密码子优化。
93.权利要求74-91中任一项所述的碱基编辑组合物或系统，进一步包含目标多肽(例如，包含SEQ ID NO：100或SEQ ID NO：113的全部或部分的多肽)，任选地其中将目标多肽融合至CRISPR-Cas效应蛋白、胞嘧啶脱氨酶和/或腺嘌呤脱氨酶。
94.权利要求1-22中任一项所述的方法，其中靶核酸存在于植物细胞或真核细胞(例如哺乳动物细胞)中。
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