CN113811608A - 毛螺菌科细菌nd2006 cas12a突变型基因和由其编码的多肽 - Google Patents
毛螺菌科细菌nd2006 cas12a突变型基因和由其编码的多肽 Download PDFInfo
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Abstract
本发明涉及用于CRISPR/Cas12a核酸内切酶系统的突变型毛螺菌科(Lachnospiraceae)细菌ND2006(Lb)Cas12a核酸和蛋白质，以及它们的使用方法。特别地，本发明涉及一种分离的突变型LbCas12a蛋白，其中该分离的突变型LbCas12a蛋白在CRISPR/Cas12a核酸内切酶系统中具有活性。本发明还包括分离的编码突变型LbCas12a蛋白的核酸，具有突变型LbCas12a蛋白的核糖核蛋白复合物和CRISPR/Cas12a核酸内切酶系统。
Description
相关申请的交叉引用
本申请要求于2019年2月22日提交的美国临时申请第62/808,984号的权益，所述临时申请的公开内容特此通过引用整体并入。
技术领域
本发明涉及基于毛螺菌科(Lachnospiraceae)细菌Cas12a的CRISPR基因，由其编码的多肽，稳定表达Cas12的哺乳动物细胞系，crRNA以及这些材料在CRISPR-Cas12a系统和方法的组合物中的用途。
背景技术
Cas12a(以前称为Cpf1)是2类/V型CRISPR RNA引导的核酸内切酶。(Zetsche,B等人,(2015)Cas12a是2类CRISPR-Cas系统的单一RNA引导的核酸内切酶(Cas12a is asingle RNA-guided endonuclease of a class 2CRISPR-Cas system).Cell 163:1-13)。Cas12a是一种用于基因组编辑的有效核酸酶，并且是Cas9酶的替代物。Cas12a是一种～1300个氨基酸的蛋白质，并且比来自化脓性链球菌(S.pyogenes)的Cas9略小。Cas12系统不利用单独的tracrRNA，而是只需要长度为40-45个核苷酸的单个短crRNA，所述crRNA指定目标DNA序列并同时引导RNA与Cas12a核酸酶的结合。(Hur,J.K.等人,(2016)通过Cas12a核糖核蛋白的电穿孔在小鼠中进行靶向诱变(Targeted mutagenesis in mice byelectroporation of Cas12a ribonucleoproteins).Nature Biotechnology,34:807-808)。Cas12a的PAM识别序列是TTTV，它允许扩大覆盖Cas9无法访问的基因组中富含腺嘌呤和胸苷的区域。
Cas12a的切割导致DNA中出现一个具有4-5个核苷酸突出端的交错双链断裂，从而在PAM位点远端留下交错末端(Gao,P.等人,(2016)V型CRISPR-Cas Cas12a核酸内切酶采用独特的机制进行crRNA介导的靶DNA识别(Type V CRISPR-Cas Cas12a endonucleaseemploys a unique mechanism for crRNA-mediated target DNA recognition).CellResearch 26:901-913)。然后可以经由非同源末端连接(NHEJ)修复这些双链断裂，这通常会导致在切割位点或位点处的突变或插入/缺失或者可以产生精确的编辑事件的同源定向修复(HDR)。此外，当Cas12a切割时，它比Cas9离PAM更远，离目标位点也更远。因此，原间隔子，尤其是原间隔子的种子序列，不太可能被编辑，因此如果第一次没有发生所需的修复事件，则有可能进行第二轮切割。
LbCas12a是来自毛螺菌科细菌ND2006(Lb)物种的成簇规则间隔短回文重复序列(CRISPR)适应性免疫系统的RNA引导的核酸内切酶。Cas12a核酸酶被归类为2类V型CRISPR系统，当与CRISPR RNA(crRNA)形成复合物时，所述系统提供具有5个核苷酸的5'-突出端的交错DNA双链断裂[1]。LbCas12a:crRNA复合物被称为CRISPR核糖核蛋白(RNP)复合物。
LbCas12a以及AsCas12a(氨基酸球菌属(Acidaminococcus)菌种BV3L6)于2015年首次被表征[1]，此后已成功用于真核细胞的基因组编辑[1-8]。两种Cas12a变体As和Lb共有34％的序列同一性，并且都由Nureki小组进行结晶[9-10]。两种Cas12a变体的RuvC和Nuc结构域在结构上相似，并通过相似的机制切割目标DNA[9-10]。两种变体都将TTTV识别为典型PAM，并且已经显示可以容许CTTV、TCTV和TTCV作为非典型PAM[6,10]。
Zhang及其同事报道了工程化的Cas12a蛋白，其显示出改变的PAM特异性[11]。他们的主要目标是在AsCas12a上进行结构引导诱变筛选，接着在LbCas12a中进行镜像突变。这产生了两种突变型变体，AsCas12a-S542R/K607R和AsCas12a-S542R/K548V/N552R，它们分别识别PAM TYCV和TATV。这些突变体保留了这些CRISPR蛋白的高特异性，并且将这些突变引入LbCas12a(分别为G532R/K595R和G532R/K538V/T542R)导致类似的PAM改变特异性[11]。
发明内容
本发明涉及Cas12a CRISPR基因和突变体、由其编码的多肽、稳定表达Cas12a的哺乳动物细胞系和它们在CIRSPR-Cas12a系统和方法的组合物中的用途。显示了使用来自毛螺菌科细菌ND2006(Lb)的Cpf1系统的实例，但这并不旨在限制范围，本发明的范围扩展到从其他物种分离的Cas12a同源物或直系同源物。
此外，本发明涉及利用CRISPR/LbCas12a核酸酶系统在精确位置切割活生物体的双链DNA的能力。此外，本发明描述了LbCas12a的单氨基酸取代，与野生型Cas12a变体即LbCas12a和AsCas12a相比，其增强了基因组编辑效率，并且被称为LbCas12a-E795L。本发明还包括LbCas12a的六个其他突变体，N527R、D559P、N527R/D559P、N527R/E795L、D559P/E795L和N527R/D559P/E795L，它们显示出与野生型LbCas12a相似的基因组编辑。
在第一个实施方式中，提供了分离的突变型Cas12a蛋白。所述分离的突变型Cas12a蛋白在成簇规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白核酸内切酶系统(“CRISPR/Cas12a核酸内切酶系统”)中具有活性。相对于野生型CRISPR/Cas12a核酸内切酶系统，所述CRISPR/Cas12a核酸内切酶系统保持了在靶编辑(on-target editing)活性。在另一方面，Cas12a蛋白是从毛螺菌科细菌ND2006(Lb)中分离出来的。
在第二个实施方式中，提供了分离的核糖核蛋白(RNP)复合物。所述RNP复合物包括突变型Cas12a蛋白和crRNA。分离的核糖核蛋白复合物作为CRISPR/Cas12a核酸内切酶系统具有活性，其中所得CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
在第三个实施方式中，提供了分离的编码突变型Cas12a蛋白的核酸。所述突变型Cas12a蛋白在CRISPR/Cas12a核酸内切酶系统中具有活性，其中所述CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
在第四个实施方式中，提供了CRISPR/Cas12a核酸内切酶系统。所述CRISPR/Cas12a核酸内切酶系统包括突变型Cas12a蛋白和crRNA。所述CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
在第五个实施方式中，提供了一种进行基因编辑的方法，所述方法具有保持的在靶编辑活性。所述方法包括使候选编辑DNA目标位点基因座与活性CRISPR/Cas12a核酸内切酶系统接触的步骤，所述系统具有与合适的crRNA形成复合物的突变型Cas12a蛋白。所述相互作用可以在任何情况下发生，例如在活体动物中、活细胞中或体外分离的DNA中。
在另一个实施方式中，本发明的CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas核酸内切酶系统显示出保持的在靶编辑活性，并且当与野生型CRISPR/Cas核酸内切酶系统相比时，可以显示出降低的脱靶编辑活性。在另一方面，本发明的CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性，并且可以显示出与野生型CRISPR/Cas12a核酸内切酶系统相比降低的脱靶编辑活性。
在另一个实施方式中，本发明的CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas核酸内切酶系统显示出保持的在靶编辑活性，并且当与野生型CRISPR/Cas核酸内切酶系统相比时，可以显示出降低的脱靶编辑活性。在另一方面，本发明的CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性，并且可以显示出与野生型CRISPR/Cas12a核酸内切酶系统相比降低的脱靶编辑活性。
附图说明
图1A至图1D显示了AsCas12a(顶部)和LbCas12a(底部)的一级和二级结构比对。α螺旋用波浪线表示，β链用箭头表示，并且转角用“TT”表示。相同的残基用实心红色框出，并且相似的残基用蓝色轮廓框出。
图2显示了与野生型LbCas12a和AsCas12a-M537R/F870L突变体相比，LbCas12a突变体在HEK 293人类细胞中48小时后的编辑效率。
图3显示了在IDT
电穿孔增强剂的存在下，与野生型LbCas12a相比，LbCas12a突变体在HEK293人类细胞中48小时后的编辑效率。
图4显示了在不存在IDT
电穿孔增强剂的情况下，与野生型LbCas12a相比，LbCas2a突变体在HEK293人类细胞中48小时后的编辑效率。
图5显示了在IDT
电穿孔增强剂的存在下，与野生型LBCas12a和AsCas12a-M537R/F870L突变体相比，LbCas12a突变体在HEK293人类细胞中48小时后的编辑效率。
图6A显示了在IDT
电穿孔增强剂的存在下，与AsCas12a野生型和AsCas12a-M537R/F870L突变体相比，以1.0μM剂量的RNP递送的LbCas12a野生型和E795L突变型LbCas12a在HEK293细胞中48小时后测量的编辑效率。
图6B显示了在IDT
电穿孔增强剂的存在下，与AsCas12a野生型和AsCas12a-M537R/F870L突变体相比，以0.22μM剂量的RNP递送的LbCas12a野生型和E795L突变型LbCas12a在HEK293细胞中48小时后测量的编辑效率。
图6C显示了在IDT
电穿孔增强剂的存在下，与AsCas12a野生型和AsCas12a-M537R/F870L突变体相比，以0.05μM剂量的RNP递送的LbCas12a野生型和E795L突变型LbCas12a在HEK293细胞中48小时后测量的编辑效率。
图6D显示了在IDT
电穿孔增强剂的存在下，与AsCas12a野生型和AsCas12a-M537R/F870L突变体相比，以0.01μM剂量的RNP递送的LbCas12a野生型和E795L突变型LbCas12a在HEK293细胞中48小时后测量的编辑效率。
具体实施方式
本文描述的本发明的方法和组合物提供了用于CRISPR/Cas12a系统的突变型LbCas12a核酸和多肽。本发明描述了新型Cas12a突变体，即使当作为RNP复合物递送时，其将脱靶编辑活性降低至低水平，同时保持相对于野生型蛋白的高在靶编辑活性。本发明的这些和其他优点以及额外的发明特征将从本文提供的本发明的描述中显而易见。
Cas12a通过将可靶向的PAM序列范围从基因组中富含GC的区域(Cas9)扩展到富含AT的区域(Cas12a)而对Cas9提供了有用的补充，从而扩大了可以使用CRISPR基因组工程方法修饰的序列范围。除了具有富含T的PAM位点外，Cas12a系统与Cas9相比的另一个优势是使用单个短RNA分子。
在第一实施方式中，提供了分离的突变型Cas12a蛋白。所述分离的突变型Cas12a蛋白在成簇规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白核酸内切酶系统(“CRISPR/Cas12a核酸内切酶系统”)中具有活性。所述CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas核酸内切酶系统显示出保持的在靶编辑活性。在另一方面，Cas12a蛋白是从毛螺菌科细菌ND2006(Lb)中分离出来的。优选的单突变型Cas12a蛋白包括WT-LbCas12a中在以下位置引入的取代突变：N527、D559和E795。示例性的单突变型Cas12a蛋白包括以下引入WT-LbCas12a中的特定突变：N527R、D559P和E795L。示例性的单突变型Cas12a蛋白包括选自SEQ ID NO.3-9的至少一个成员。额外的取代突变可以包括在单突变型Cas12a蛋白氨基酸序列的氨基酸背景中，前提是所得Cas12a蛋白作为CRISPR/Cas12a核酸内切酶系统具有活性，其中所得CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
优选的多取代突变型Cas12a蛋白包括在WT-LbCas12a中引入以下位置中的至少两者的突变：N527/D559、D559/E795、N527/E795和N527/D559/E795。示例性的多取代突变型Cas12a蛋白包括WT-LbCas12a中选自以下氨基酸突变的突变：N527R/D559P、D559P/E795L、N527R/E795L和N527R/D559P/E795L。示例性的多取代突变型Cas12a蛋白包括选自SEQ IDNO:3-9的至少一个成员。额外的取代突变可以包括在单突变型Cas12a蛋白氨基酸序列的氨基酸背景中，前提是所得Cas12a蛋白作为CRISPR/Cas12a核酸内切酶系统具有活性，其中所得CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
在第二实施方式中，提供了分离的核糖核蛋白复合物。RNP包括突变型Cas12a蛋白和crRNA复合物。在一个方面，crRNA包括针对给定基因座的特定编辑目标位点的
crRNA(Integrated DNA Technologies,Inc.(Coralville,IA,(US))。优选的突变型Cas9蛋白包括如上所述的那些。
在另一个实施方式中，提供了分离的编码突变型LbCas12a蛋白的核酸。优选的分离的核酸编码如上所述的突变型LbCas12a蛋白。可以使用重组DNA程序或化学合成方法从编码野生型LbCas12a蛋白的核酸容易地产生编码突变型LbCas12a蛋白的示例性分离核酸。用于此目的的优选核酸包括优化用于在细菌(例如大肠杆菌)或哺乳动物(例如人类)细胞中表达LbCas12a蛋白的那些。用于在大肠杆菌和人类细胞中表达WT-LbCas12a的示例性密码子优化核酸包括SEQ ID NO.1。此外，本发明考虑了WT-Cas12a和突变型LbCas12a的融合蛋白，其中WT-Cas12a和突变型LbCas12a的编码序列与编码融合蛋白在真核细胞中的核定位(“NLS”)的氨基酸序列或促进蛋白质的纯化的氨基酸序列融合。
在另一个实施方式中，分离的核酸包括编码上述突变型LbCas12a蛋白之一的mRNA。在第二方面，分离的核酸包括编码上述突变型LbCas12a蛋白之一的基因的DNA。优选的DNA包括对编码突变型LbCas12a蛋白的基因进行编码的载体。此类递送方法包括本领域技术人员熟知的质粒和各种病毒递送载体。还可以使用合适的表达载体将突变型LbCas12a蛋白稳定转化到细胞中，以产生组成型或诱导性表达突变型LbCas12a的细胞系。上述方法也可以应用于胚胎以产生组成型或诱导性表达突变型LbCas12a的后代动物。
在另一个实施方式中，提供了CRISPR/Cas12a核酸内切酶系统。所述CRISPR/Cas12a核酸内切酶系统包括突变型LbCas12a蛋白。优选的突变型LbCas12a蛋白包括上述那些。在一个方面，CRISPR/Cas12a核酸内切酶系统由DNA表达载体编码。在一个实施方式中，DNA表达载体选自细菌表达载体或真核表达载体。在另一方面，CRISPR/Cas12a核酸内切酶系统包含含有突变型LbCas12a蛋白和crRNA的核糖核蛋白复合物。
在另一个实施方式中，提供了一种进行基因编辑的方法，所述方法具有增加的在靶编辑活性。所述方法包括将候选编辑目标位点基因座与具有突变型LbCas12a蛋白的活性CRISPR/Cas12a核酸内切酶系统接触的步骤。在一个方面，所述方法包括在WT-LbCas12a中在以下位置之一引入突变的单突变型LbCas12a蛋白：N527、D559和E795。示例性的单突变型LbCas12a蛋白包括以下引入WT-LbCas12a中的特定突变：N527R、D559P和E795L。示例性的单突变型LbCas12a蛋白包括选自SEQ ID NO.3-9的至少一个成员。额外的取代突变可以包括在单突变型LbCas12a蛋白氨基酸序列的氨基酸背景中，前提是所得LbCas12a蛋白作为CRISPR/Cas12a核酸内切酶系统具有活性，其中所得CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
在另一个实施方式中，所述方法包括多取代突变型LbCas12a蛋白，所述多取代突变型LbCas12a蛋白包括在WT-LbCas12a中引入以下位置中的至少两者的突变：N527/D559、D559/E795、N527/E795和N527/D559/E795。示例性的多取代突变型Cas12a蛋白包括WT-LbCas12a中选自以下氨基酸突变的突变：N527R/D559P、D559P/E795L、N527R/E795L和N527R/D559P/E795L。示例性的多取代突变型LbCas12a蛋白包括选自SEQ ID NO:3-9的至少一个成员。额外的取代突变可以包括在单突变型LbCas12a蛋白氨基酸序列的氨基酸背景中，前提是所得LbCas12a蛋白作为CRISPR/Cas12a核酸内切酶系统具有活性，其中所得CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
基于Cas12a和LbCas12a的工具的应用多种多样。所述应用包括但不限于：植物基因编辑、酵母基因编辑、哺乳动物基因编辑、活体动物器官中的细胞编辑、胚胎编辑、快速生成敲除/敲入动物系、生成动物疾病状态模型、纠正疾病状态、插入报告基因和全基因组功能筛选。
实施例1
野生型和突变型LbCas12a蛋白和AsCas12a蛋白的DNA和氨基酸序列。
下面的列表显示了本发明中描述的不同野生型(WT)和突变型Cas12a核酸酶。本领域技术人员应理解，许多不同的DNA序列可以编码/表达相同的氨基酸(AA)序列，因为在许多情况下，超过一个密码子可以编码相同的氨基酸。下面显示的DNA序列仅用作示例，并且设想了编码相同蛋白质(例如，相同氨基酸序列)的其他DNA序列。还应理解，可以向所述序列添加额外特征、元件或标签，例如NLS结构域等。显示WT LbCas12a(Cpf1)、WT AsCas12a和突变型N527R LbCas12a、突变型D559P LbCas12a、突变型E759L LbCas12a、双突变型N527R/D559P LbCas12a、双突变型N527R/E795L LbCas12a、双突变型D559P/E795L LbCas12a、三突变型N527R/D559P/E795L LbCas12a和双突变型M537R/F870L AsCas12a的实例。对于LbCas12a和AsCas12a突变体，仅提供了氨基酸和DNA序列，但设想了可以添加NLS结构域和His标签结构域以促进用于产生用于哺乳动物细胞的重组蛋白。
SEQ ID NO:1
WT毛螺菌科细菌ND2006(Lb)Cas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGAACCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGGACAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATGAACTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:2
WT LbCas12氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:3
突变型N527R LbCas12a氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQRPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:4
突变型D559P LbCas12a氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMPKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:5
突变型E795L LbCas12a氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYLLHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:6
突变型N527R/D559P LbCas12a氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQRPQFMGGWDKDKETDYRATILRYGSKYYLAIMPKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:7
突变型527R/E795L LbCas12a氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQRPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYLLHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:8
突变型D559P/E795L LbCas12a氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMPKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYLLHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:9
突变型N527R/D559P/E795L LbCas12a氨基酸序列
MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQRPQFMGGWDKDKETDYRATILRYGSKYYLAIMPKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYLLHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKH
SEQ ID NO:10
突变型N527R LbCas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGCGTCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGGACAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATGAACTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:11
突变型D559P LbCas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGAACCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGCCGAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATGAACTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:12.
突变型E795L LbCas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGAACCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGGACAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATCTGCTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:13
突变型N527R/D559P LbCas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGCGTCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGCCGAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATGAACTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:14
突变型N527R/E795L LbCas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGCGTCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGGACAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATCTGCTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:15
突变型D559P/E795L LbCas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGAACCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGCCGAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATCTGCTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:16
突变型N527R/D559P/E795L LbCas12a DNA序列
ATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGATCTTCGATAAACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCAGCAAAGATATTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTACTTTCAGCGTCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGCCGAAAAAATACGCAAAATGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATCTGCTGCATATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTTTGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACAT
SEQ ID NO:17
WT AsCas12a DNA序列
ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAAC
SEQ ID NO:18
WT AsCas12a氨基酸序列
MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN
SEQ ID NO:19
突变型M537R/F870L AsCas12a DNA序列
ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGATTATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAATCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCAAATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGCGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCCTGTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTAACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAAC
SEQ ID NO:20
突变型M537R/F870L AsCas12a氨基酸序列
MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN
实施例2
LbCas12a突变体在大肠杆菌细胞中的过表达和纯化
本实施例展示了七种Cas12a突变体N527R、D559P、E795L、N527R/D559P、D559P/E795L、N527R/E795L和N527R/D559P/E795L的过表达和纯化。LbCas12a突变体是使用标准PCR条件和引物(表1)通过定点诱变引入的。转化到大肠杆菌BL21(DE3)细胞中后，使用具有适当菌株的菌落接种具有卡那霉素(0.05mg/mL)的TB培养基，并在37℃生长至达到约0.9的OD，然后将烧瓶冷却至18℃持续30分钟。使用1M IPTG(500μL)的添加来诱导蛋白质表达，接着在18℃生长19小时。收获细胞并且将细胞沉淀重悬并在15-20kpsi下预冷却至4℃的Avestin Emulsiflex C3上裂解三个回合。裂解液在4℃以16,000×g离心20分钟以去除细胞碎片。
将澄清的裂解液置于HisTrap HP柱上。程序包括用His结合缓冲液(20mM NaPO4pH 6.8、0.5M NaCl、10mM咪唑、5％甘油)平衡树脂，接着上样。样品用His结合缓冲液洗涤，接着进行额外的标准洗涤和由10％His洗脱缓冲液(10mM NaPO4 pH 6.8、250mM NaCl、150mM咪唑、5％甘油)组成的10％“B”洗涤液洗涤。最后，使用His洗脱缓冲液洗脱样品。然后将LbCas12a突变体置于HiTrap肝素HP柱上。程序包括用肝素结合缓冲液(20mM NaPO4 pH6.8、250mM NaCl、10％甘油)平衡树脂，接着上样。然后用肝素结合缓冲液洗涤样品，接着用由5％肝素洗脱缓冲液(10mM NaPO4 pH 6.8、1M NaCl、10％甘油)组成的5％“B”洗涤液洗涤。最后，使用肝素洗脱缓冲液(10mM NaPO4，pH 6.8，1M NaCl，10％甘油)洗脱纯化的蛋白质。最后，使用肝素洗脱纯化的蛋白质。
将纯化的LbCas12a突变体浓缩至约10mg/mL并在-20℃储存于储存缓冲液(25mMTris-HCl pH 7.4、0.3M NaCl、1mM EDTA、1mM DTT、50％甘油)中。
表1.用于定点诱变的引物的序列，其中氨基酸密码子加下划线。
所有引物以DNA寡核苷酸定购自Intergrated DNA Technologies。
实施例3
新型LbCas12a取代突变体在经由核糖核蛋白复合物递送到人类细胞中时增强了基于人类细胞系的活性测定中的切割活性
以下实施例展示了LbCas12a突变体在作为RNP复合物递送时提高基因组编辑效率的能力。该实施例证明了LbCas12a突变体的能力，其在以高剂量递送时显示出相当的基因组编辑效率，以及当利用电穿孔转染通过核糖核蛋白(RNP)复合物以低剂量递送到人类细胞中时提高的基因组编辑效率。
RNP复合物是通过将纯化的LbCas12a和sgRNA(表2，条目3和9)在PBS缓冲液中以1:1.2的比率在室温下孵育10分钟形成的。使用Lonza 4D-NucleofectorTM和
96孔穿梭装置在存在
Cpf1电穿孔增强剂(3μM，Integrated DNA Technologies)下将RNP复合物(5μM最终剂量)转染到HEK293永生化人类细胞中。实验以一式三份生物重复物进行，并且在37℃下48小时后，用QuickExtractTMDNA提取溶液(50μL)裂解粘附细胞。
表2.在HEK293细胞的基因组编辑中使用的Cas12a(Cpf1)sgRNA的序列。所有sgRNA以RNA寡核苷酸定购自Intergrated DNA Technologies，其中在5'端和3'端均具有
端修饰。
表2.续
粗裂解物在65℃下孵育15分钟，接着在98℃下加热灭活3分钟。粗基因组DNA在TE缓冲液中稀释5倍并用作PCR模板。使用
DNA聚合酶(New England Biolabs)和以下参数，使用PCR(表3中列出的引物)扩增HPRT基因座的1.2kbp片段：98℃持续30秒，接着重复以下循环24次：98℃持续10秒、65℃持续15秒和72℃持续1分钟，接着在72℃下最终延伸2分钟。通过加入NEBuffer 2并最初加热到95℃持续10分钟和缓慢冷却至室温来形成异源双链体。然后将异源双链体在37℃下用2U的T7核酸内切酶I(New England Biolabs)切割1小时。通过毛细管电泳(Fragment Analyzer,Advanced Analytical)分析切割产物。
表3.在通过T7EI分析之前用于编辑基因组HEK293 DNA的扩增的引物的序列。所有引物以DNA寡核苷酸定购自Intergrated DNA Technologies。
HEK293人类细胞中野生型和突变型LbCas12a的核酸内切酶活性描述于图2和表4中。LbCas12a突变体D559P、E795L和D559P/E795L的RNP递送导致与野生型LbCas12a和AsCas12a-M537R/F870L类似的活性(～80％切割)。由于此初始筛选是为了确定最高剂量(5μM)下的活性，因此进行了较低浓度(2、1和0.05μM)RNP的剂量反应，以确定这些突变体是否可以带来增强的活性。
表4a.与野生型LbCas12a和AsCas12a-M537R/F870L相比LbCas12a突变体在HEK293人类细胞中48小时后在HPRT-38115处的核酸内切酶活性。数值计算为切割百分比。
表4b.与野生型LbCas12a和AsCas12a-M537R/F870L相比LbCas12a突变体在HEK293人类细胞中48小时后在HPRT-38330处的核酸内切酶活性。数值计算为切割百分比。
如上所述实现了剂量反应，将RNP的量减少了2倍(存在和不存在
电穿孔增强剂)，并从2μM剂量开始。结果示于图3-4和表5-6中。LbCas12a的RNP递送需要添加
Cpf1电穿孔增强剂以实现最大切割效率。在这些剂量下，LbCas12a突变体与野生型相比显示出活性相似或略有增加；因此，需要研究新的位点和更低的剂量以显示活性差异。
表5a.在
电穿孔增强剂的存在下与野生型LbCas12a相比LbCas12a突变体在HEK293人类细胞中48小时后在HPRT-38115处的核酸内切酶活性。数值计算为切割百分比。
表5b.在
电穿孔增强剂的存在下与野生型LbCas12a相比LbCas12a突变体在HEK293人类细胞中48小时后在HPRT-38330处的核酸内切酶活性。数值计算为切割百分比。
表6a.在不存在
电穿孔增强剂的情况下与野生型LbCas12a相比LbCas12a突变体在HEK293人类细胞中48小时后在HPRT-38115处的核酸内切酶活性。数值计算为切割百分比。
表6b.在不存在
电穿孔增强剂的情况下与野生型LbCas12a相比LbCas12a突变体在HEK293人类细胞中48小时后在存在HPRT-38330处的核酸内切酶活性。数值计算为切割百分比。
本实施例中的最终剂量反应如上所述进行设置，从2μM剂量开始，将RNP的量进一步降低5倍。对于LbCas12a RNP使用sgRNA 2和8并且对于AsCas12a RNP使用sgRNA 14和20来形成RNP(表1)。结果示于图5和表7中。与野生型LbCas12a(～22％切割)和AsCas12a-M537R/F870L(～19％切割)相比，LbCas12a-E795L的RNP递送在低至0.4μM下在HPRT基因座的38228位点处显示出增加的活性(～90％切割)，并且在38104位点处保留了高切割活性(～90％切割)，如由野生型LbCas12a所见。LbCas12a核酸酶的单个E795L突变体在最低剂量(0.4μM)下将基因组编辑活性提高至多4.5倍。
表7a.与野生型LbCas12a和AsCas12a-M537R/F870L相比LbCas12a突变体在HEK293人类细胞中48小时后在HPRT-38104处的核酸内切酶活性。数值计算为切割百分比。
表7b.与野生型LbCas12a和AsCas12a-M537R/F870L相比LbCas12a突变体在HEK293人类细胞中48小时后在HPRT-38228处的核酸内切酶活性。数值计算为切割百分比。
实施例4
当以低剂量经由核糖核蛋白复合物递送到人类细胞中时，单个LbCas12a取代突变体增强了基于人类细胞系的活性测定中的切割活性。
以下实施例展示了突变体E795L LbCas12a在利用电穿孔转染通过RNP复合物以低剂量递送到人类细胞中时显示出增加的基因组编辑效率的能力。当野生型或突变型Cas12a作为RNP复合物递送到人类细胞中时，本发明提高了基因组编辑效率。
通过在室温下将纯化的Cas12a和sgRNA(表2，LbCas12a RNP的条目1-12和AsCas12a RNP的条目13-24)以1:1.2的比率在PBS缓冲液中孵育10分钟来形成RNP复合物。使用Lonza 4D-NucleofectorTM和
96孔穿梭装置和
Cpf1电穿孔增强剂(3μM，Integrated DNA Technologies)将所述RNP复合物(1、0.22、0.05和0.01μM最终剂量)转染到HEK293永生化人类细胞中。实验以一式两份生物学重复进行，并且在37℃下48小时后，用QuickExtractTMDNA提取溶液(50μL)裂解粘附细胞。
粗裂解物在65℃下孵育15分钟，接着在98℃下加热灭活3分钟。粗基因组DNA在TE缓冲液中稀释15倍并用作PCR模板。使用
DNA聚合酶(New England Biolabs)和以下参数，使用PCR(表3中列出的引物)扩增HPRT基因座的1.2kbp片段：98℃持续30秒，接着重复以下循环24次：98℃持续10秒、65℃持续15秒和72℃持续1分钟，接着在72℃下最终延伸2分钟。通过加入NEBuffer 2并最初加热到95℃持续10分钟和缓慢冷却至室温来形成异源双链体。然后将所述异源双链体在37℃下用2U的T7核酸内切酶I(New England Biolabs)切割1小时。通过毛细管电泳(Fragment Analyzer,Advanced Analytical)分析切割产物。
野生型和突变型LbCas12a与野生型和突变型AsCas12a在HEK293人类细胞中的核酸内切酶活性描述于图6A、图6b、图6C、图6D和表8中。总体而言，与野生型LbCas12a和AsCas12a相比LbCas12a-E795L的RNP递送显示出提高的活性。0.05μM剂量显示即使在低剂量下酶的活性也增加(图6c)。在此剂量下，LbCas12a-E795L在HPRT 38146位点处的活性超过野生型LbCas12a的活性高达23倍(表8，条目51和55)，而在HPRT 38186位点处超过野生型AsCas12a的活性高达3倍(表8，条目103和107)。在LbCas12a变体的最高可区分剂量(0.22μM，图6b)下，相比于野生型形式E795L突变体在HPRT38146位点处相比于野生型形式的活性增加高达11倍(表8，条目50和54)，而在相同位点处相对于野生型AsCas12a为几乎2倍(表8，条目54和58)。
表8.与野生型LbCas12a和AsCas12a和AsCas12a-M537R/F870L相比LbCas12a-E795L在HEK293人类细胞中48小时后在HPRT-38228处的核酸内切酶活性。数值计算为切割百分比。
表8.续
表8.续
表8.续
本文引用的所有参考文献，包括出版物、专利申请和专利，都特此通过引用并入，其程度就如同每个参考文献被单独地和具体地指示为通过引用并入并以其整体在本文阐述一样。
在描述本发明的上下文中(尤其是在权利要求书的上下文中)，术语“一个”和“一种”和“所述”以及类似指代的使用应被解释为涵盖单数和复数，除非本文另有说明或与上下文明显矛盾。除非另有说明，否则术语“包含”、“具有”、“包括”和“含有”应被解释为开放式术语(即，意指“包括但不限于”)。除非本文另有说明，否则对本文数值范围的叙述仅旨在用作单独提及落在所述范围内的每个单独值的简写方法，并且每个单独的值都并入本说明书中，就如同它在本文中单独叙述一样。除非本文另有说明或以其他方式与上下文明显矛盾，否则本文中描述的所有方法可以以任何合适的顺序执行。本文提供的任何和所有实例或示例性语言(例如，“诸如”)的使用仅旨在更好地阐明本发明而不对本发明的范围构成限制，除非另有声明。说明书中的任何语言都不应被解释为指出任何未要求保护的要素对于本发明的实践是必不可少的。
本发明的优选实施方式描述于本文中，包括本发明人已知的用于实施本发明的最佳模式。在阅读上述描述后，那些优选实施方式的变化可为本领域普通技术人员所显而易见。本发明人期望技术人员适当地采用此类变化，并且本发明人打算以不同于本文中具体描述的方式来实践本发明。因此，本发明包括在适用法律允许的情况下权利要求书中叙述的主题的所有修改和等效物。此外，除非本文另有说明或以其他方式与上下文明显矛盾，否则本发明涵盖上述要素的所有可能变化形式的任何组合。
术语“野生型LbCas12a”(“野生型Lb酶”或“WT-LbCas12a”)包括具有与天然存在的毛螺菌科细菌ND2006 Cas12a(例如，SEQ ID NO:2)相同的氨基酸序列并且当与合适的crRNA组合以形成并激活CRISPR/Cas12a核酸内切酶系统时具有生化和生物学活性的蛋白质。术语“野生型AsCas12a”(“野生型As酶”或“WT-AsCas12a”)包括具有与天然存在的氨基酸球菌属菌种BV3L6 Cas12a(例如，SEQ ID NO:18)相同的氨基酸序列并且当与合适的crRNA组合以形成并激活CRISPR/Cas12a核酸内切酶系统时具有生化和生物学活性的蛋白质。
术语“突变型LbCas12a蛋白”包括具有与野生型毛螺菌科细菌ND2006 Cas12a不同的氨基酸序列并且当与合适的crRNA组合以形成活性CRISPR-Cas12a核酸内切酶系统时具有生化和生物学活性的蛋白质形式。这包括具有与野生型毛螺菌科细菌ND2006 Cas12a不同的氨基酸序列的直系同源物和Cas12a变体。
术语“多肽”是指包含多于一个氨基酸的任何线性或分支的肽。多肽包括蛋白质或其片段或其融合物，条件是此类蛋白质、片段或融合物保留有用的生化或生物学活性。
融合蛋白通常包括额外的氨基酸信息，这些信息对于额外氨基酸信息共价连接的蛋白质来说不是天然的。这种额外的氨基酸信息可以包括能够纯化或鉴定融合蛋白的标签。这种额外的氨基酸信息可以包括使融合蛋白能够被转运到细胞中和/或被转运到细胞内的特定位置的肽。用于这些目的的标签的实例包括以下：AviTag，它是一种允许通过酶BirA进行生物素化因此可以通过链霉亲和素分离蛋白质的肽(GLNDIFEAQKIEWHE)；钙调蛋白标签，它是一种由蛋白钙调蛋白结合的肽(KRRWKKNFIAVSAANRFKKKISSSGAL)；聚谷氨酸标签，它是一种与阴离子交换树脂如Mono-Q有效结合的肽(EEEEEE)；E标签，它是一种被抗体识别的肽(GAVPVYPDPLEPR)；FLAG标签，它是一种被抗体识别的肽(DYKDDDDK)；HA标签，它是一种来自血凝素的被抗体识别的肽(YPYDVPDYA)；His标签，它是一种由镍或钴螯合物结合的通常5-10个组氨酸(HHHHHH)；Myc标签，它是一种衍生自c-myc的被抗体识别的肽(EQKLISEEDL)；NE标签，它是一种由单克隆IgG1抗体识别的新型18个氨基酸合成肽(TKENPRSNQEESYDDNES)，其可用于广泛的应用，包括蛋白质印迹、ELISA、流式细胞术、免疫细胞化学、免疫沉淀和重组蛋白亲和纯化；S标签，它是一种源自核糖核酸酶A的肽(KETAAAKFERQHMDS)；SBP标签，它是一种与链霉亲和素结合的肽(MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP)；Softag 1，它旨在用于哺乳动物表达(SLAELLNAGLGGS)；Softag 3，它旨在用于原核表达(TQDPSRVG)；Strep标签，它是一种与链霉亲和素或称为streptactin的修饰链霉亲和素结合的肽(Strep标签II：WSHPQFEK)；TC标签，它是一种被FlAsH和ReAsH双砷化合物识别的四半胱氨酸标签(CCPGCC)；V5标签，这是一种被抗体识别的肽(GKPIPNPLLGLDST)；VSV标签，一种被抗体识别的肽(YTDIEMNRLGK)；Xpress标签(DLYDDDDK)；Isopeptag，它是一种与菌毛蛋白-C蛋白共价结合的肽(TDKDMTITFTNKKDAE)；SpyTag，它是一种与SpyCatcher蛋白共价结合的肽(AHIVMVDAYKPTK)；SnoopTag，一种与SnoopCatcher蛋白共价结合的肽(KLGDIEFIKVNK)；BCCP(生物素羧基载体蛋白)，它是一个被BirA生物素化以便能够被链霉亲和素识别的蛋白质结构域；谷胱甘肽-S-转移酶标签，它是一种与固定化谷胱甘肽结合的蛋白质；绿色荧光蛋白标签，它是一种自发地发荧光并且可以被抗体结合的蛋白质；HaloTag，它是一种突变型细菌卤代烷烃脱卤酶，它与反应性卤代烷烃底物共价连接以允许与多种底物连接；麦芽糖结合蛋白标签，一种与直链淀粉琼脂糖结合的蛋白质；Nustag；硫氧还蛋白标签；和Fc标签，源自免疫球蛋白Fc结构域，它允许二聚化和增溶并且可用于在Protein-A Sepharose上进行纯化。核定位信号(NLS)，例如从SV40获得的核定位信号(NLS)，允许蛋白质在进入细胞后立即转运至细胞核。鉴于天然Cas9蛋白是细菌来源的并且因此不天然地包含NLS基序，当用于目标基因组DNA底物位于细胞核中的真核细胞时，向重组Cas9蛋白添加一个或多个NLS基序预期显示改进的基因组编辑活性。本领域技术人员应理解这些不同的融合标签技术，以及如何制造和使用包含它们的融合蛋白。
参考文献
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序列表
<110> 合成DNA技术公司（Integrated DNA Technologies, Inc. ）
<120> 毛螺菌科细菌ND2006 CAS12A突变型基因和由其编码的多肽
<130> PA2020-5
<150> US62808984
<151> 2019-02-22
<160> 20
<170> PatentIn 3.5版
<210> 1
<211> 3684
<212> DNA
<213> 合成核酸
<400> 1
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagaa cccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catggacaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atgaactgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 2
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 2
Met Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp
20 25 30
Asn Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys
35 40 45
Gly Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp
50 55 60
Val Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu
65 70 75 80
Phe Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn
85 90 95
Leu Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn
100 105 110
Glu Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu
115 120 125
Pro Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe
130 135 140
Asn Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn
145 150 155 160
Met Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile
165 170 175
Asn Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys
180 185 190
Val Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys
195 200 205
Ile Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe
210 215 220
Phe Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile
225 230 235 240
Ile Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn
245 250 255
Glu Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys
260 265 270
Phe Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser
275 280 285
Phe Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe
290 295 300
Arg Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys
305 310 315 320
Leu Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile
325 330 335
Phe Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe
340 345 350
Gly Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp
355 360 365
Ile His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp
370 375 380
Arg Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu
385 390 395 400
Gln Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu
405 410 415
Ile Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser
420 425 430
Glu Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys
435 440 445
Asn Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys
450 455 460
Ser Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr
465 470 475 480
Asn Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile
485 490 495
Leu Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr
500 505 510
Gln Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro
515 520 525
Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala
530 535 540
Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 3
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 3
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 Arg Pro
515 520 525
Gln Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala
530 535 540
Thr Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 4
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 4
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 Pro Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 5
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 5
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 Leu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 6
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 6
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 Arg 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 Pro Lys
545 550 555 560
Lys Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly
565 570 575
Asn Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met
580 585 590
Leu Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro
595 600 605
Ser Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly
610 615 620
Asp Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys
625 630 635 640
Asp Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn
645 650 655
Phe Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu
660 665 670
Val Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys
675 680 685
Glu Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His
705 710 715 720
Thr Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile
725 730 735
Arg Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys
740 745 750
Lys Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys
755 760 765
Asn Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr
770 775 780
Lys Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 7
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 7
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 Arg 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 Leu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 8
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 8
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 Pro 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 Leu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 9
<211> 1228
<212> PRT
<213> 合成氨基酸
<400> 9
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 Arg 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 Pro 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 Leu Leu His Ile Pro Ile
785 790 795 800
Ala Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val
805 810 815
Arg Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp
820 825 830
Arg Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly
835 840 845
Asn Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn
850 855 860
Gly Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu
865 870 875 880
Lys Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile
885 890 895
Lys Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys
900 905 910
Glu Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn
915 920 925
Ser Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln
930 935 940
Lys Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys
945 950 955 960
Lys Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile
965 970 975
Thr Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe
980 985 990
Ile Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr
995 1000 1005
Gly Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp
1010 1015 1020
Ser Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro
1025 1030 1035
Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser
1040 1045 1050
Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr
1055 1060 1065
Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val
1070 1075 1080
Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu
1085 1090 1095
Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala
1100 1105 1110
Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1115 1120 1125
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly
1130 1135 1140
Arg Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp
1145 1150 1155
Gly Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala
1160 1165 1170
Ile Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala
1175 1180 1185
Arg Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp
1190 1195 1200
Glu Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp
1205 1210 1215
Leu Glu Tyr Ala Gln Thr Ser Val Lys His
1220 1225
<210> 10
<211> 3684
<212> DNA
<213> 合成核酸
<400> 10
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagcg tccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catggacaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atgaactgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 11
<211> 3684
<212> DNA
<213> 合成核酸
<400> 11
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagaa cccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catgccgaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atgaactgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 12
<211> 3684
<212> DNA
<213> 合成核酸
<400> 12
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagaa cccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catggacaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atctgctgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 13
<211> 3684
<212> DNA
<213> 合成核酸
<400> 13
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagcg tccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catgccgaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atgaactgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 14
<211> 3684
<212> DNA
<213> 合成核酸
<400> 14
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagcg tccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catggacaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atctgctgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 15
<211> 3684
<212> DNA
<213> 合成核酸
<400> 15
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagaa cccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catgccgaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atctgctgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 16
<211> 3684
<212> DNA
<213> 合成核酸
<400> 16
atgagcaaac tggaaaagtt caccaactgt tatagcctga gcaaaaccct gcgttttaaa 60
gcaattccgg ttggtaaaac ccaagagaac attgataata aacgcctgct ggtcgaagat 120
gaaaaacgcg ctgaagatta taaaggcgtg aaaaaactgc tggatcgcta ttatctgagc 180
ttcattaacg atgtgctgca cagcattaaa ctgaagaacc tgaacaacta tatcagcctg 240
tttcgtaaaa aaacccgcac cgaaaaagaa aacaaagagc tggaaaacct ggaaatcaat 300
ctgcgtaaag aaatcgccaa agcgtttaaa ggtaacgagg gttataaaag cctgttcaag 360
aaagacatca tcgaaaccat tctgccggaa tttctggatg ataaagatga aattgccctg 420
gtgaatagct ttaatggctt taccaccgca tttaccggct tttttgataa tcgcgaaaac 480
atgttcagcg aagaagcaaa aagcaccagc attgcatttc gctgcattaa tgaaaatctg 540
acccgctaca ttagcaacat ggatatcttt gaaaaagtgg acgcgatctt cgataaacac 600
gaagtgcaag agatcaaaga gaaaatcctg aacagcgatt atgacgtcga agattttttt 660
gaaggcgagt tctttaactt cgttctgacc caagaaggta tcgacgttta taacgcaatt 720
attggtggtt ttgttaccga aagcggtgag aaaatcaaag gcctgaatga atatatcaac 780
ctgtataacc agaaaaccaa acagaaactg ccgaaattca aaccgctgta taaacaggtt 840
ctgagcgatc gtgaaagcct gagcttttat ggtgaaggtt ataccagtga tgaagaggtt 900
ctggaagttt ttcgtaacac cctgaataaa aacagcgaga tctttagcag catcaaaaag 960
cttgagaaac tgttcaaaaa ctttgatgag tatagcagcg caggcatctt tgttaaaaat 1020
ggtccggcaa ttagcaccat cagcaaagat atttttggcg aatggaatgt gatccgcgat 1080
aaatggaatg ccgaatatga tgatatccac ctgaaaaaaa aggccgtggt gaccgagaaa 1140
tatgaagatg atcgtcgtaa aagcttcaag aaaattggta gctttagcct ggaacagctg 1200
caagaatatg cagatgcaga tctgagcgtt gtggaaaaac tgaaagaaat catcattcag 1260
aaggtggacg agatctataa agtttatggt agcagcgaaa aactgttcga tgcagatttt 1320
gttctggaaa aaagcctgaa aaagaatgat gccgttgtgg ccattatgaa agatctgctg 1380
gatagcgtta agagcttcga gaattacatc aaagcctttt ttggtgaggg caaagaaacc 1440
aatcgtgatg aaagtttcta tggcgatttt gtgctggcct atgatattct gctgaaagtg 1500
gaccatattt atgatgccat tcgcaattat gttacccaga aaccgtatag caaagacaag 1560
ttcaaactgt actttcagcg tccgcagttt atgggtggtt gggataaaga taaagaaacc 1620
gattatcgtg ccaccatcct gcgttatggt agtaaatact atctggccat catgccgaaa 1680
aaatacgcaa aatgcctgca gaaaatcgac aaagatgatg tgaatggcaa ctatgaaaaa 1740
atcaactaca aactgctgcc tggtccgaat aaaatgctgc cgaaagtgtt ctttagcaag 1800
aaatggatgg cctattataa cccgagcgag gatattcaaa agatctacaa aaatggcacc 1860
tttaaaaagg gcgacatgtt caatctgaac gattgccaca aactgatcga tttcttcaaa 1920
gattcaattt cgcgttatcc gaaatggtcc aatgcctatg attttaactt tagcgaaacc 1980
gaaaaataca aagacattgc cggtttttat cgcgaagtgg aagaacaggg ctataaagtg 2040
agctttgaaa gcgcaagcaa aaaagaggtt gataagctgg ttgaagaggg caaactgtat 2100
atgttccaga tttacaacaa agattttagc gacaaaagcc atggcacccc gaatctgcat 2160
accatgtact ttaaactgct gttcgacgaa aataaccatg gtcagattcg tctgagcggt 2220
ggtgccgaac tgtttatgcg tcgtgcaagt ctgaaaaaag aagaactggt tgttcatccg 2280
gcaaatagcc cgattgcaaa caaaaatccg gacaatccga aaaaaaccac gacactgagc 2340
tatgatgtgt ataaagacaa acgttttagc gaggatcagt atctgctgca tatcccgatt 2400
gccatcaata aatgcccgaa aaacatcttt aagatcaaca ccgaagttcg cgtgctgctg 2460
aaacatgatg ataatccgta tgtgattggc attgatcgtg gtgaacgtaa cctgctgtat 2520
attgttgttg ttgatggtaa aggcaacatc gtggaacagt atagtctgaa cgaaattatc 2580
aacaacttta acggcatccg catcaaaacc gactatcata gcctgctgga caagaaagaa 2640
aaagaacgtt ttgaagcacg tcagaactgg accagtattg aaaacatcaa agaactgaaa 2700
gccggttata ttagccaggt ggttcataaa atctgtgagc tggtagaaaa atacgatgca 2760
gttattgcac tggaagatct gaatagcggt ttcaaaaata gccgtgtgaa agtcgaaaaa 2820
caggtgtatc agaaattcga gaaaatgctg atcgacaaac tgaactacat ggtcgacaaa 2880
aaaagcaatc cgtgtgcaac cggtggtgca ctgaaaggtt atcagattac caacaaattt 2940
gaaagcttta aaagcatgag cacccagaac ggctttatct tctatattcc ggcatggctg 3000
accagcaaaa ttgatccgag caccggtttt gtgaacctgc tgaaaacaaa atatacctcc 3060
attgccgaca gcaagaagtt tattagcagc tttgatcgca ttatgtatgt tccggaagag 3120
gacctgtttg aattcgcact ggattacaaa aatttcagcc gtaccgatgc cgactacatc 3180
aaaaaatgga aactgtacag ctatggtaac cgcattcgca tttttcgcaa cccgaagaaa 3240
aacaatgtgt tcgattggga agaagtttgt ctgaccagcg catataaaga acttttcaac 3300
aaatacggca tcaactatca gcagggtgat attcgtgcac tgctgtgtga acagagcgat 3360
aaagcgtttt atagcagttt tatggcactg atgagcctga tgctgcagat gcgtaatagc 3420
attaccggtc gcaccgatgt ggattttctg attagtccgg tgaaaaattc cgatggcatc 3480
ttttatgata gccgcaatta cgaagcacaa gaaaatgcaa ttctgccgaa aaacgcagat 3540
gcaaatggtg catataacat tgcacgtaaa gttctgtggg caattggcca gtttaagaaa 3600
gcagaagatg agaagctgga caaagtgaaa attgcgatca gcaataaaga gtggctggaa 3660
tacgcacaga ccagcgttaa acat 3684
<210> 17
<211> 3921
<212> DNA
<213> 合成核酸
<400> 17
atgacccagt ttgaaggttt caccaatctg tatcaggtta gcaaaaccct gcgttttgaa 60
ctgattccgc agggtaaaac cctgaaacat attcaagaac agggcttcat cgaagaggat 120
aaagcacgta acgatcacta caaagaactg aaaccgatta tcgaccgcat ctataaaacc 180
tatgcagatc agtgtctgca gctggttcag ctggattggg aaaatctgag cgcagcaatt 240
gatagttatc gcaaagaaaa aaccgaagaa acccgtaatg cactgattga agaacaggca 300
acctatcgta atgccatcca tgattatttc attggtcgta ccgataatct gaccgatgca 360
attaacaaac gtcacgccga aatctataaa ggcctgttta aagccgaact gtttaatggc 420
aaagttctga aacagctggg caccgttacc accaccgaac atgaaaatgc actgctgcgt 480
agctttgata aattcaccac ctatttcagc ggcttttatg agaatcgcaa aaacgtgttt 540
agcgcagaag atattagcac cgcaattccg catcgtattg tgcaggataa tttcccgaaa 600
ttcaaagaga actgccacat ttttacccgt ctgattaccg cagttccgag cctgcgtgaa 660
cattttgaaa acgttaaaaa agccatcggc atctttgtta gcaccagcat tgaagaagtt 720
tttagcttcc cgttttacaa tcagctgctg acccagaccc agattgatct gtataaccaa 780
ctgctgggtg gtattagccg tgaagcaggc accgaaaaaa tcaaaggtct gaatgaagtg 840
ctgaatctgg ccattcagaa aaatgatgaa accgcacata ttattgcaag cctgccgcat 900
cgttttattc cgctgttcaa acaaattctg agcgatcgta ataccctgag ctttattctg 960
gaagaattca aatccgatga agaggtgatt cagagctttt gcaaatacaa aacgctgctg 1020
cgcaatgaaa atgttctgga aactgccgaa gcactgttta acgaactgaa tagcattgat 1080
ctgacccaca tctttatcag ccacaaaaaa ctggaaacca tttcaagcgc actgtgtgat 1140
cattgggata ccctgcgtaa tgccctgtat gaacgtcgta ttagcgaact gaccggtaaa 1200
attaccaaaa gcgcgaaaga aaaagttcag cgcagtctga aacatgagga tattaatctg 1260
caagagatta ttagcgcagc cggtaaagaa ctgtcagaag catttaaaca gaaaaccagc 1320
gaaattctgt cacatgcaca tgcagcactg gatcagccgc tgccgaccac cctgaaaaaa 1380
caagaagaaa aagaaatcct gaaaagccag ctggatagcc tgctgggtct gtatcatctg 1440
ctggactggt ttgcagttga tgaaagcaat gaagttgatc cggaatttag cgcacgtctg 1500
accggcatta aactggaaat ggaaccgagc ctgagctttt ataacaaagc ccgtaattat 1560
gccaccaaaa aaccgtatag cgtcgaaaaa ttcaaactga actttcagat gccgaccctg 1620
gcaagcggtt gggatgttaa taaagaaaaa aacaacggtg ccatcctgtt cgtgaaaaat 1680
ggcctgtatt atctgggtat tatgccgaaa cagaaaggtc gttataaagc gctgagcttt 1740
gaaccgacgg aaaaaaccag tgaaggtttt gataaaatgt actacgacta ttttccggat 1800
gcagccaaaa tgattccgaa atgtagcacc cagctgaaag cagttaccgc acattttcag 1860
acccatacca ccccgattct gctgagcaat aactttattg aaccgctgga aatcaccaaa 1920
gagatctacg atctgaataa cccggaaaaa gagccgaaaa aattccagac cgcatatgca 1980
aaaaaaaccg gtgatcagaa aggttatcgt gaagcgctgt gtaaatggat tgatttcacc 2040
cgtgattttc tgagcaaata caccaaaacc accagtatcg atctgagcag cctgcgtccg 2100
agcagccagt ataaagatct gggcgaatat tatgcagaac tgaatccgct gctgtatcat 2160
attagctttc agcgtattgc cgagaaagaa atcatggacg cagttgaaac cggtaaactg 2220
tacctgttcc agatctacaa taaagatttt gccaaaggcc atcatggcaa accgaatctg 2280
cataccctgt attggaccgg tctgtttagc cctgaaaatc tggcaaaaac ctcgattaaa 2340
ctgaatggtc aggcggaact gttttatcgt ccgaaaagcc gtatgaaacg tatggcacat 2400
cgtctgggtg aaaaaatgct gaacaaaaaa ctgaaagacc agaaaacccc gatcccggat 2460
acactgtatc aagaactgta tgattatgtg aaccatcgtc tgagccatga tctgagtgat 2520
gaagcacgtg ccctgctgcc gaatgttatt accaaagaag ttagccacga gatcattaaa 2580
gatcgtcgtt ttaccagcga caaattcttt tttcatgtgc cgattaccct gaattatcag 2640
gcagcaaata gcccgagcaa atttaaccag cgtgttaatg catatctgaa agaacatcca 2700
gaaacgccga ttattggtat tgatcgtggt gaacgtaacc tgatttatat caccgttatt 2760
gatagcaccg gcaaaatcct ggaacagcgt agcctgaata ccattcagca gtttgattac 2820
cagaaaaaac tggataatcg cgagaaagaa cgtgttgcag cacgtcaggc atggtcagtt 2880
gttggtacaa ttaaagacct gaaacagggt tatctgagcc aggttattca tgaaattgtg 2940
gatctgatga ttcactatca ggccgttgtt gtgctggaaa acctgaattt tggctttaaa 3000
agcaaacgta ccggcattgc agaaaaagca gtttatcagc agttcgagaa aatgctgatt 3060
gacaaactga attgcctggt gctgaaagat tatccggctg aaaaagttgg tggtgttctg 3120
aatccgtatc agctgaccga tcagtttacc agctttgcaa aaatgggcac ccagagcgga 3180
tttctgtttt atgttccggc accgtatacg agcaaaattg atccgctgac cggttttgtt 3240
gatccgtttg tttggaaaac catcaaaaac catgaaagcc gcaaacattt tctggaaggt 3300
ttcgattttc tgcattacga cgttaaaacg ggtgatttca tcctgcactt taaaatgaat 3360
cgcaatctga gttttcagcg tggcctgcct ggttttatgc ctgcatggga tattgtgttt 3420
gagaaaaacg aaacacagtt cgatgcaaaa ggcaccccgt ttattgcagg taaacgtatt 3480
gttccggtga ttgaaaatca tcgtttcacc ggtcgttatc gcgatctgta tccggcaaat 3540
gaactgatcg cactgctgga agagaaaggt attgtttttc gtgatggctc aaacattctg 3600
ccgaaactgc tggaaaatga tgatagccat gcaattgata ccatggttgc actgattcgt 3660
agcgttctgc agatgcgtaa tagcaatgca gcaaccggtg aagattacat taatagtccg 3720
gttcgtgatc tgaatggtgt ttgttttgat agccgttttc agaatccgga atggccgatg 3780
gatgcagatg caaatggtgc atatcatatt gcactgaaag gacagctgct gctgaaccac 3840
ctgaaagaaa gcaaagatct gaaactgcaa aacggcatta gcaatcagga ttggctggca 3900
tatatccaag aactgcgtaa c 3921
<210> 18
<211> 1307
<212> PRT
<213> 合成氨基酸
<400> 18
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> 19
<211> 3921
<212> DNA
<213> 合成核酸
<400> 19
atgacccagt ttgaaggttt caccaatctg tatcaggtta gcaaaaccct gcgttttgaa 60
ctgattccgc agggtaaaac cctgaaacat attcaagaac agggcttcat cgaagaggat 120
aaagcacgta acgatcacta caaagaactg aaaccgatta tcgaccgcat ctataaaacc 180
tatgcagatc agtgtctgca gctggttcag ctggattggg aaaatctgag cgcagcaatt 240
gatagttatc gcaaagaaaa aaccgaagaa acccgtaatg cactgattga agaacaggca 300
acctatcgta atgccatcca tgattatttc attggtcgta ccgataatct gaccgatgca 360
attaacaaac gtcacgccga aatctataaa ggcctgttta aagccgaact gtttaatggc 420
aaagttctga aacagctggg caccgttacc accaccgaac atgaaaatgc actgctgcgt 480
agctttgata aattcaccac ctatttcagc ggcttttatg agaatcgcaa aaacgtgttt 540
agcgcagaag atattagcac cgcaattccg catcgtattg tgcaggataa tttcccgaaa 600
ttcaaagaga actgccacat ttttacccgt ctgattaccg cagttccgag cctgcgtgaa 660
cattttgaaa acgttaaaaa agccatcggc atctttgtta gcaccagcat tgaagaagtt 720
tttagcttcc cgttttacaa tcagctgctg acccagaccc agattgatct gtataaccaa 780
ctgctgggtg gtattagccg tgaagcaggc accgaaaaaa tcaaaggtct gaatgaagtg 840
ctgaatctgg ccattcagaa aaatgatgaa accgcacata ttattgcaag cctgccgcat 900
cgttttattc cgctgttcaa acaaattctg agcgatcgta ataccctgag ctttattctg 960
gaagaattca aatccgatga agaggtgatt cagagctttt gcaaatacaa aacgctgctg 1020
cgcaatgaaa atgttctgga aactgccgaa gcactgttta acgaactgaa tagcattgat 1080
ctgacccaca tctttatcag ccacaaaaaa ctggaaacca tttcaagcgc actgtgtgat 1140
cattgggata ccctgcgtaa tgccctgtat gaacgtcgta ttagcgaact gaccggtaaa 1200
attaccaaaa gcgcgaaaga aaaagttcag cgcagtctga aacatgagga tattaatctg 1260
caagagatta ttagcgcagc cggtaaagaa ctgtcagaag catttaaaca gaaaaccagc 1320
gaaattctgt cacatgcaca tgcagcactg gatcagccgc tgccgaccac cctgaaaaaa 1380
caagaagaaa aagaaatcct gaaaagccag ctggatagcc tgctgggtct gtatcatctg 1440
ctggactggt ttgcagttga tgaaagcaat gaagttgatc cggaatttag cgcacgtctg 1500
accggcatta aactggaaat ggaaccgagc ctgagctttt ataacaaagc ccgtaattat 1560
gccaccaaaa aaccgtatag cgtcgaaaaa ttcaaactga actttcagcg tccgaccctg 1620
gcaagcggtt gggatgttaa taaagaaaaa aacaacggtg ccatcctgtt cgtgaaaaat 1680
ggcctgtatt atctgggtat tatgccgaaa cagaaaggtc gttataaagc gctgagcttt 1740
gaaccgacgg aaaaaaccag tgaaggtttt gataaaatgt actacgacta ttttccggat 1800
gcagccaaaa tgattccgaa atgtagcacc cagctgaaag cagttaccgc acattttcag 1860
acccatacca ccccgattct gctgagcaat aactttattg aaccgctgga aatcaccaaa 1920
gagatctacg atctgaataa cccggaaaaa gagccgaaaa aattccagac cgcatatgca 1980
aaaaaaaccg gtgatcagaa aggttatcgt gaagcgctgt gtaaatggat tgatttcacc 2040
cgtgattttc tgagcaaata caccaaaacc accagtatcg atctgagcag cctgcgtccg 2100
agcagccagt ataaagatct gggcgaatat tatgcagaac tgaatccgct gctgtatcat 2160
attagctttc agcgtattgc cgagaaagaa atcatggacg cagttgaaac cggtaaactg 2220
tacctgttcc agatctacaa taaagatttt gccaaaggcc atcatggcaa accgaatctg 2280
cataccctgt attggaccgg tctgtttagc cctgaaaatc tggcaaaaac ctcgattaaa 2340
ctgaatggtc aggcggaact gttttatcgt ccgaaaagcc gtatgaaacg tatggcacat 2400
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gaaacgccga ttattggtat tgatcgtggt gaacgtaacc tgatttatat caccgttatt 2760
gatagcaccg gcaaaatcct ggaacagcgt agcctgaata ccattcagca gtttgattac 2820
cagaaaaaac tggataatcg cgagaaagaa cgtgttgcag cacgtcaggc atggtcagtt 2880
gttggtacaa ttaaagacct gaaacagggt tatctgagcc aggttattca tgaaattgtg 2940
gatctgatga ttcactatca ggccgttgtt gtgctggaaa acctgaattt tggctttaaa 3000
agcaaacgta ccggcattgc agaaaaagca gtttatcagc agttcgagaa aatgctgatt 3060
gacaaactga attgcctggt gctgaaagat tatccggctg aaaaagttgg tggtgttctg 3120
aatccgtatc agctgaccga tcagtttacc agctttgcaa aaatgggcac ccagagcgga 3180
tttctgtttt atgttccggc accgtatacg agcaaaattg atccgctgac cggttttgtt 3240
gatccgtttg tttggaaaac catcaaaaac catgaaagcc gcaaacattt tctggaaggt 3300
ttcgattttc tgcattacga cgttaaaacg ggtgatttca tcctgcactt taaaatgaat 3360
cgcaatctga gttttcagcg tggcctgcct ggttttatgc ctgcatggga tattgtgttt 3420
gagaaaaacg aaacacagtt cgatgcaaaa ggcaccccgt ttattgcagg taaacgtatt 3480
gttccggtga ttgaaaatca tcgtttcacc ggtcgttatc gcgatctgta tccggcaaat 3540
gaactgatcg cactgctgga agagaaaggt attgtttttc gtgatggctc aaacattctg 3600
ccgaaactgc tggaaaatga tgatagccat gcaattgata ccatggttgc actgattcgt 3660
agcgttctgc agatgcgtaa tagcaatgca gcaaccggtg aagattacat taatagtccg 3720
gttcgtgatc tgaatggtgt ttgttttgat agccgttttc agaatccgga atggccgatg 3780
gatgcagatg caaatggtgc atatcatatt gcactgaaag gacagctgct gctgaaccac 3840
ctgaaagaaa gcaaagatct gaaactgcaa aacggcatta gcaatcagga ttggctggca 3900
tatatccaag aactgcgtaa c 3921
<210> 20
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<213> 合成氨基酸
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Claims (12)
1.一种分离的突变型LbCas12a，所述分离的突变型LbCas12a包含选自以下的取代突变：
a)引入野生型LbCas12a蛋白中选自以下位置的单一取代突变：N527、D559和E795：或
b)引入野生型LbCas12a蛋白中选自以下位置中的至少两者的多个取代突变：N527、D559和E795。
2.根据权利要求1所述的分离的突变型LbCas12a蛋白，其中所述分离的突变型LbCas12a蛋白选自SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5。
3.根据权利要求1所述的分离的突变型LbCas12a蛋白，其中所述分离的突变型LbCas12a蛋白选自SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8和SEQ ID NO.9。
4.一种分离的核糖核蛋白复合物，所述分离的核糖核蛋白复合物包含：
a)根据权利要求1所述的突变型LbCas12a蛋白；和
b)gRNA复合物，
其中所述分离的核糖核蛋白复合物作为CRISPR/Cas12a核酸内切酶系统具有活性，其中所得CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
5.根据权利要求4所述的分离的核糖核蛋白复合物，其中所述突变型LbCas12a蛋白选自SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8和SEQ ID NO.9。
6.一种包含突变型LbCas12a蛋白和gRNA的CRISPR/Cas12a核酸内切酶系统，其中所述CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
7.根据权利要求6所述的CRISPR/Cas12a核酸内切酶系统，其中所述CRISPR/Cas12a核酸内切酶系统由DNA表达载体编码。
8.根据权利要求7所述的CRISPR/Cas12a核酸内切酶系统，其中所述DNA表达载体包括质粒携带载体。
9.根据权利要求8所述的CRISPR/Cas12a核酸内切酶系统，其中所述DNA表达载体选自细菌表达载体和真核表达载体。
10.一种分离的编码突变型LbCas12a蛋白的核酸，其中所述突变型LbCas12a蛋白在CRISPR/Cas12a核酸内切酶系统中具有活性，其中所述CRISPR/Cas12a核酸内切酶系统相对于野生型CRISPR/Cas12a核酸内切酶系统显示出保持的在靶编辑活性。
11.根据权利要求10所述的分离的编码突变型LbCas12a蛋白的核酸，其中所述突变型LbCas12a蛋白包含选自以下的取代突变：
a)引入野生型Cas12a蛋白中选自以下位置的单一取代突变：N527、D559和E795；或
b)引入野生型Cas12a蛋白中选自以下位置中的至少两者的多个取代突变：N527、D559和E795。
12.根据权利要求10所述的分离的编码突变型Cas12a蛋白的核酸，其中所述突变型Cas12a蛋白选自SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.14、SEQ IDNO.14、SEQ ID NO.15和SEQ ID NO.16。
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| WO2023049926A2 (en) | 2021-09-27 | 2023-03-30 | Vor Biopharma Inc. | Fusion polypeptides for genetic editing and methods of use thereof |
| WO2023247753A1 (en) | 2022-06-23 | 2023-12-28 | BASF Agricultural Solutions Seed US LLC | Diversifying base editing |
| WO2024073751A1 (en) | 2022-09-29 | 2024-04-04 | Vor Biopharma Inc. | Methods and compositions for gene modification and enrichment |
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