CN117043324A - 用于治疗先天性肌营养不良的治疗性lama2载荷 - Google Patents

Abstract

本发明涉及一种组合物，所述组合物包含a)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；b)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由转座酶组成；以及c)包含编码层粘连蛋白‑α2蛋白或其功能变体或片段的转基因的核酸构建体。本发明还涉及所述组合物的治疗用途，用于将LAMA2转基因整合到细胞基因组内的特定位点，特别是用于治疗先天性肌营养不良。

Description

用于治疗先天性肌营养不良的治疗性LAMA2载荷

技术领域

本发明涉及一种组合物及其治疗用途，特别是用于治疗先天性肌营养不良的用途，所述组合物包含含有与转座酶融合的位点特异性DNA结合蛋白的融合蛋白和LAMA2转基因，以将LAMA2转基因整合到细胞基因组内的特定位点。

背景技术

先天性肌营养不良(CMD)是一类严重的早发型肌营养不良，其影响骨骼肌/心肌以及中枢神经系统。

在CMD中，层粘连蛋白α2链缺陷型先天性肌营养不良(laminin-α2chain-deficient co-genital muscular dystrophy，LAMA2 MD)，也称为分区蛋白缺陷型先天性营养不良1A型(merosin-deficient congenital dystrophy type 1A，MDC1A)，其特征在于严重的张力减退、肌无力、骨骼畸形、不能移动和呼吸功能不全。该疾病有一些较晚发作的较轻微形式，其具有相似的症状但具有较广泛的表型变异性。

目前对于MDC1A没有可用的治愈方法。目前临床策略集中在管理上(补充进食，针对对呼吸功能不全的非侵入性通气支持，以及针对关节挛缩、脊柱缺损和其它问题的物理治疗)(Nguyen等人,2019.Appl Clin Genet.12:113–130)。

MDC1A是由两个拷贝的LAMA2基因中均存在的功能丧失性隐性突变引起的，所述LAMA2基因编码层粘连蛋白α2链，这是层粘连蛋白-211的一个亚基。层粘连蛋白-211是一种细胞外基质蛋白，功能是在收缩期间稳定基底膜和肌肉纤维。层粘连蛋白α2链蛋白的长度为3122个氨基酸残基，因此其DNA编码序列(CDS)的长度为约9.3kb。与许多其它肌营养不良一样，MDC1A因此是由非常大的基因中的突变引起的，该基因大小超过了AAV载体或慢病毒载体的载货(cargo)容量。此外，迄今为止，在整个LAMA2基因中已描述了几十种不同的突变，从单点错义、剪接位点或框内突变到数千碱基区域缺失(Oliveira等人,2018.HumMutat.39(10):1314-1337)，如此多以致于有疗效的基因治疗方法面临挑战，而基因置换策略迄今为止尚不可用。

已经对MDC1A测试了其它基于基因纠正的策略。使用了外显子跳跃法(exon-skipping approach)，其通常指使用合成的反义寡核苷酸来抑制剪接增强子位点并防止特定外显子参与剪接(Aoki等人,2013.Biomed Res Int.2013:402369)。然而，这种外显子跳跃法不是通用的，并且仅可以用于一些具有缺失的患者，在此阅读框可以通过跳过与缺失相邻的额外外显子而恢复(Shieh,2018.Neurotherapeutics.15(4):840-848)。外显子跳跃恢复了截短但具有部分功能的蛋白的翻译，但不能重现全长层粘连蛋白α2蛋白的益处。

CRISPR/Cas9也用于纠正LAMA2突变，特别是切除含有剪接位点突变的LAMA2内含子2区域，并通过非同源末端连接(NHEJ)产生功能性供体剪接位点，从而导致LAMA2转录物的外显子2的纳入以及全长层粘连蛋白α2链蛋白的恢复(Kemaladewi等人,2017.NatMed.23(8):984-989)。作为外显子跳跃，这种策略仅能用于一些患者，而不是通用的。

然而，NHEJ不适合于将大的转基因整合到基因组中，并且尽管依赖于同源指导的修复(HDR)途径的基因修饰在理论上可以纠正大多数致病性点突变，但发现其是“极其低效的”，如Kemaladewi&Cohn(2019.Emerg Top Life Sci.3(1):11-18)所述。总之，Kemaladewi&Cohn呼唤开发用以纠正引起肌营养不良的突变的替代策略。

最后，CRISPR/Cas9也用于诱导基因表达，其使用与转录激活结构域(例如VP64，其针对LAMA1基因的启动子)融合的催化性失活的Cas9(SadCas9)，从而允许结构类似的层粘连蛋白α1链蛋白的表达增加(Kemaladewi等人,2019.Nature.572(7767):125-130)。然而，这种方法需要转基因的连续表达。

因此，仍然需要开发用于治疗MDC1A的永久且有效的策略，所述策略是通用的，且不仅限于受某些突变影响的某些患者。

发明内容

LAMA2基因的基因替换的治疗价值颇具前景，但却由于载货大小的限制而迄今为止一直面临挑战。本发明人使用了包含与转座酶(例如高活性PiggyBac转座酶)融合的位点特异性DNA结合蛋白(例如Cas9)的融合蛋白，将LAMA2基因的健康全长拷贝整合至细胞基因组内的特定位点中。因此，本发明人能够提供具有大的LAMA2表达盒(9.3kb的CDS)的治疗性载荷(payload)，用于离体和体内递送，且与不同的基因递送技术相容。

因此，本发明涉及一种组合物，所述组合物包含a)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合并切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；b)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由转座酶组成；和c)包含编码层粘连蛋白-α2蛋白或其功能变体或片段的转基因的核酸构建体，所述层粘连蛋白-α2蛋白优选是SEQ ID NO：74的层粘连蛋白-α2蛋白。

在一个实施方案中，a)和b)的第一和第二蛋白融合在一起，任选地通过接头融合在一起。

在一个实施方案中，c)的核酸构建体包含选自以下的启动子：SEQ ID NO：76的CMV启动子、SEQ ID NO：77的CAG启动子、SEQ ID NO：78的EF1-α启动子、SEQ ID NO：79的SV-40启动子和SEQ ID NO：80的EalbAAT启动子。在一个实施方案中，c)的核酸构建体包含SEQ IDNO：81的剪接受体(splice acceptor)。在一个实施方案中，c)的核酸构建体包含poly(A)信号序列(优选选自SEQ ID NO：83-85)和/或隔绝元件(insulator element)(优选选自SEQID NO：86-87)。在一个实施方案中，c)的核酸构建体侧翼为反向末端重复序列(ITR)，优选SEQ ID NO:88和89的5’-ITR和3’-ITR。

在一个实施方案中，c)的核酸构建体包含在选自以下的载体中：质粒载体、微环载体、犬骨DNA供体载体(doggy bone DNA donor vector)、慢病毒载体和逆转录病毒载体。

在一个实施方案中，所述位点特异性DNA结合蛋白是包含Cas蛋白的RNA引导的核酸酶(RNA-guided nuclease)，并且其中所述组合物还包含引导RNA(guide RNA)，所述引导RNA包含靶核酸序列的互补序列，用于将所述LAMA2转基因整合在细胞基因组的特定位点中，优选地，所述Cas蛋白是酿脓链球菌(S.pyogenes)Cas 9蛋白。

在一个实施方案中，引导RNA包含SEQ ID NO：90-97中任一者。

在一个实施方案中，所述转座酶是修饰的高活性Piggybac转座酶或睡美人(Sleepy Beauty或Sleeping Beauty)转座酶，优选地，修饰的高活性PiggyBac转座酶与未修饰的高活性Piggybac相比包含一个或多个增加切除活性的氨基酸突变，以及与未修饰的高活性Piggybac相比包含一个或多个降低DNA结合活性的氨基酸突变。

在一个实施方案中，所述高活性PiggyBac转座酶是修饰的高活性PiggyBac转座酶，其包含选自以下的氨基酸的至少一个突变：V34、T43、Y177、M194、R202、S230、R245、R275、R277、G325、S351、N347、R372、K375、R376、E377、E380、A411、D450、T560、S564、S573、M589、S592和F594，优选地包含氨基酸Y177、R202、S230、R245、R275、R277、G325、N347、S351、E377、D450、R372和K375、E377、T560、S564、S573、M589、S592、F594的突变，所述位置编号对应于SEQ ID NO：9的未修饰的高活性Piggybac的氨基酸编号。在一个实施方案中，所述高活性PiggyBac转座酶是修饰的高活性PiggyBac转座酶，其包含选自以下的氨基酸的至少一个突变：M194、R245、R275、R277、G325、R372、K375、R376、E377、E380、D450和S573，优选地包含氨基酸D450、R372和R375的突变，所述位置编号对应于SEQ ID NO：9的未修饰的高活性Piggybac的氨基酸编号。

在一个实施方案中，所述转座酶通过接头在N-末端与所述位点特异性DNA结合蛋白融合，所述接头优选是包括GGS、XTEN或FOKI的肽接头，更优选是SEQ ID NO：53的XTEN。

在一个实施方案中，所述组合物被包装在纳米颗粒中。

本发明还涉及用于将LAMA2转基因整合到细胞基因组内的靶核酸序列中的体外方法，所述方法包括将本发明的组合物引入细胞。

本发明还涉及可通过本发明的体外方法获得的工程化细胞，其中所述工程化细胞包含整合在其基因组内的核酸，所述核酸包含编码层粘连蛋白-α2蛋白的转基因，其侧翼为用于整合酶和/或转座酶介导的基因插入的操作序列(operational sequence)。

在一个实施方案中，转基因侧翼的操作序列包含SEQ ID NO：88和89或由SEQ IDNO：88和89组成。

在一个实施方案中，inter-ITR大小为至少300bp。

在一个实施方案中，转基因编码全长层粘连蛋白-α2蛋白。

本发明还涉及包含本发明组合物或本发明工程化细胞的药物组合物，任选地与一种或多种药学上可接受的赋形剂组合。

本发明还涉及本发明的组合物、本发明的工程化细胞或本发明的药物组合物，用于治疗，特别是用于治疗有此需要的受试者的分区蛋白缺陷型先天性肌营养不良1型(MDC1A)。

定义

如本文所用，单数形式“一”、“该”和“所述”包括单数和复数形式，除非上下文另有明确指示。因此，例如，提到“剂”时包括单个剂和多个这样的剂。

术语“核酸序列”和“核苷酸序列”可互换使用，是指由单体核苷酸组成或包含单体核苷酸的任何分子。核酸可以是寡核苷酸或多核苷酸。核苷酸序列可以是DNA、RNA或其混合物。核苷酸序列可以是化学修饰的或人工的。核苷酸序列包括肽核酸(PNA)、吗啉代和锁核酸(LNA)以及二元醇核酸(GNA)和苏糖核酸(TNA)。这些序列中的每一个通过分子主链的改变而区别于天然存在的DNA或RNA。也可使用硫代磷酸核苷酸。其它脱氧核苷酸类似物包括但不限于甲基膦酸酯、氨基磷酸酯、二硫代磷酸酯、N3'P5'-氨基磷酸酯和低聚核糖核苷酸硫代磷酸酯及其2'-O-烯丙基类似物和2'-O-甲基核糖核苷酸甲基膦酸酯，它们可以用于本公开的核苷酸中。

术语“转基因”是指外源核酸序列，特别是编码基因产物的外源DNA或cDNA。基因产物可以是RNA、肽或蛋白。除了基因产物的编码区(CDS)外，转基因还可以包括一个或多个操作序列或与一个或多个操作序列相结合以促进或增强表达，例如启动子、增强子、响应元件、报告元件、隔绝元件、聚腺苷酸化信号和/或其它功能元件。除非另有说明，否则本公开的实施方案可以利用任何已知的合适启动子、增强子、响应元件、报告元件、隔绝元件、聚腺苷酸化信号和/或其它功能元件。合适的元件和序列是本领域技术人员公知的。

术语“核酸构建体”指通过使用重组DNA技术产生的人造核酸分子。核酸构建体是单链或双链的核酸分子，其已经被修饰以含有以自然界不存在的方式组合和并置的核酸序列区段。核酸构建体通常是“载体”，即用于将外源产生的DNA递送到宿主细胞中的核酸分子。

术语“载体”或“表达载体”可以指自主复制载体，即作为染色体外实体存在的载体，其复制不依赖于染色体复制，例如质粒、染色体外元件、微型染色体(mini-chromosome)或人工染色体，其可以包含任何确保自我复制的手段(means)；或整合载体，即当引入宿主细胞时整合到其基因组中并与其整合进的染色体一起复制的载体。

术语“序列同一性”或“同一性”是指根据两个多肽或多核苷酸序列的比对在位置中的匹配(相同的氨基酸残基或核苷酸)的数目(％)。通过在比对时比较序列以使重叠最大化的同时使序列空位最小化，来确定序列同一性。特别地，可以使用多种数学全局或局部比对算法中的任一种来确定序列同一性，这取决于两个序列的长度。相似长度的序列优选使用全局比对算法(例如Needleman和Wunsch算法[Needleman&Wunsch,1970.J Mol Biol.48(3):443-53])进行比对，该算法在整个长度上最佳地比对序列，而长度显著不同的序列优选使用局部比对算法(例如Smith和Waterman算法[Smith&Waterman,1981.J Mol Biol.147(1):195-197]或Altschul算法[Altschul等人,1997.Nucleic Acids Res.25(17):3389-3402；Altschul等人,2005.FEBS J.272(20):5101-9])进行比对。用于确定氨基酸序列同一性百分比的比对可以以本领域技术范围内的各种方式实现，例如，使用可获自因特网网站如http://blast.ncbi.nlm.nih.gov/或http://www.ebi.ac.uk/Tools/emboss/上的公众可用的计算机软件。本领域技术人员可以确定用于测量比对的适当参数，包括在被比较的序列的全长上实现最大比对所需的任何算法。为了本文的目的，％核苷酸或氨基酸序列同一性数值是指使用成对序列比对程序EMBOSS Needle产生的值，所述程序使用Needleman-Wunsch算法产生两个序列的最佳全局比对，其中所有检索参数均被设定为默认值，即，评分矩阵＝BLOSUM62，空位开放＝10，空位延伸＝0.5，末端空位罚分＝假(false)，末端空位开放＝10，末端空位延伸＝0.5。

术语“融合物”是指其中的两个或更多个亚单位分子连接在一起的分子。在一些实施方案中，两个亚单位之间的连接是共价的；或者，两个亚单位之间的连接可以是非共价的，并且依赖于例如分子间相互作用。亚单位分子可以是相同化学类型的分子，或者可以是不同化学类型的分子。

术语“融合蛋白”指包含来自至少两种不同蛋白的蛋白结构域的杂合多肽。例如，一个蛋白结构域可位于融合蛋白的氨基末端(N-末端)部分或羧基末端(C-末端)蛋白，从而分别形成“氨基末端融合蛋白”或“羧基末端融合蛋白”。在优选的实施方案中，融合蛋白是单链多肽，其可以完全由核酸序列编码，并且包括至少两个蛋白结构域，所述至少两个蛋白结构域通过肽结合直接共价连接，或任选地通过肽接头共价连接。

术语“接头”是指连接两个相邻分子或部分的化学基团或分子。

术语“结合蛋白”指能够非共价结合另一分子的蛋白。结合蛋白可以结合例如DNA分子(DNA结合蛋白)、RNA分子(RNA结合蛋白)和/或蛋白分子(蛋白结合蛋白)。在蛋白结合蛋白的情况下，它可以结合相同蛋白的一个或多个分子以形成同型二聚体、同型三聚体等；和/或它可以结合至一种或多种不同蛋白的一个或多个分子。结合蛋白可以具有多于一种类型的结合活性。例如，锌指蛋白具有DNA结合活性、RNA结合活性和蛋白结合活性。

术语“Cas9”或“Cas9核酸酶”是指RNA引导的核酸酶，其包含Cas9蛋白或其片段(例如，包含Cas9的活性或无活性DNA切割结构域和/或Cas9的gRNA结合结构域的蛋白)。Cas9核酸酶有时也称为casn1核酸酶或CRISPR(成簇规律间隔短回文重复序列)相关核酸酶。CRISPR是适应性免疫系统，它提供了针对可移动基因元件(病毒、可转座元件和接合质粒)的保护。CRISPR簇含有间隔区、与先前的移动元件互补的序列，和靶入侵核酸。CRISPR簇被转录并加工成CRISPR RNA(crRNA)。在II型CRISPR系统中，pre-crRNA的正确加工需要反式编码的小RNA(tracrRNA)、内源核糖核酸酶3(rnc)和Cas9蛋白。tracrRNA用作核糖核酸酶3辅助的pre-crRNA加工的指导。随后，Cas9/crRNA/tracrRNA通过内切核酸酶切割与间隔区互补的线性或环状dsDNA靶。不与crRNA互补的靶链首先通过核酸内切酶切割，然后通过核酸外切酶以3'-5'修剪。在自然界，DNA结合和切割通常需要蛋白和两种RNA。然而，可以对单引导RNA(“sgRNA”或简单地为“gRNA”)进行工程改造，以将crRNA和tracrRNA两者的方面并入单RNA物质中。

Cas9识别CRISPR重复序列中的短基序(PAM或原型间隔区邻近基序)以帮助区分自我和非我。Cas9核酸酶序列和结构是本领域技术人员公知的。Cas9直向同源物已在各种物种中均有描述，包括但不限于酿脓链球菌(S.pyogenes)和嗜热链球菌(S.thermophilus)。基于本公开内容，其它合适的Cas9核酸酶和序列对本领域技术人员是显而易见的，并且这样的Cas9核酸酶和序列包括来自Chylinski等人,2013.(RNA Biol.10(5):726-37)中公开的生物体和基因座的Cas9序列，该文献的全部内容通过引用并入本文。

在一些实施方案中，Cas9核酸酶具有无活性的(例如失活的)DNA切割结构域。核酸酶失活的Cas9蛋白可以互换地称为“dCas9”蛋白(核酸酶“死亡”的Cas9)。产生具有无活性的DNA切割结构域的Cas9蛋白(或其片段)的方法是本领域已知的(参见，例如，Jinek等人,2012.Science.337(6096):816-821；Qi等人,2013.Cell.152(5):1173-83，其全部内容通过引用并入本文)。

术语“锌指蛋白”指通过一个或多个锌指以序列特异性方式结合DNA的蛋白或较大蛋白内的结构域，所述锌指是锌指蛋白的结合结构域内的氨基酸序列的区域，其结构通过锌离子配位而稳定。术语“锌指蛋白”通常缩写为“ZFP”。

术语“锌指核酸酶”指通过将锌指DNA结合结构域融合至DNA切割结构域而产生的人工限制性酶。可以对锌指结构域进行工程改造以靶向特定的目标DNA序列，并且这使得锌指核酸酶能够靶向复杂基因组内的独特序列。“锌指核酸酶”通常缩写为“ZFN”或“ZNP”。

术语“切割”指DNA分子共价骨架的断裂。切割可以通过多种方法引发，包括但不限于磷酸二酯键的酶促水解或化学水解。单链切割和双链切割都是可能的，双链切割可以作为两个不同的单链切割事件的结果而发生。DNA切割可导致产生平端(blunt end)或交错末端(staggered end)。在某些实施方案中，融合多肽用于所靶向的双链DNA切割。

术语“特异性”是指选择性结合与所选序列具有一定程度序列同一性的序列的能力。

术语“插入”和“整合”是指将核酸序列添加至第二核酸序列或添加至基因组或其部分中。与插入或整合有关的术语“特异性”、“位点特异性”、“靶向的”和“中靶的(on-targeted)”在本文中可互换使用，是指将核酸插入第二核酸的特定位点或插入基因组或其部分的特定位点。相反，术语“随机”、“非靶向都”和“脱靶的(off-targeted)”是指核酸非特异性和无意地插入到不想要的位点。术语“总”或“全部”指插入的总数。

术语“突变”是指序列例如核酸或氨基酸序列内的残基被另一残基取代；和/或核酸或氨基酸序列中一个或多个残基的缺失或插入。在本文中，突变通常通过指明原始残基，然后指明残基在序列内的位置，然后指明新取代残基来描述。本文提供的进行氨基酸取代(突变)的各种方法都是本领域公知的，并且由例如Green&Sambrook,2012(Molecularcloning:a laboratory manual(第4版).Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.)提供。在优选的实施方案中，术语蛋白中的突变是指氨基酸取代。

术语“转座酶”是指与转座子末端结合并通过切割-和-粘贴(cut-and-paste)机制或复制转座机制催化其移动到基因组的另一部分的酶。

术语“修饰的”是指与相应的未修饰的蛋白或核酸序列不同的蛋白或核酸序列。

术语“接头”是指连接两个相邻分子或部分的化学基团或分子。

具体实施方式

本发明人使用了包含与转座酶融合的位点特异性DNA结合蛋白的融合蛋白，以将LAMA2转基因整合到细胞基因组内的特定位点。

因此，本发明涉及一种组合物，其包含以下或由以下组成：

a)第一蛋白或编码所述第一蛋白的核酸构建体，优选cDNA或mRNA，所述第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；

b)第二蛋白或编码所述第二蛋白的核酸构建体，优选cDNA或mRNA，所述第二蛋白包含转座酶或由转座酶组成；以及

c)核酸构建体，其包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因组成。

在一个实施方案中，所述组合物包含以下或由以下组成：

a)融合蛋白或编码所述融合蛋白的核酸构建体，优选cDNA或mRNA，所述融合蛋白包含以下或由以下组成：(i)第一蛋白，所述第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成，和(ii)第二蛋白，所述第二蛋白包含转座酶或由转座酶组成；以及

b)核酸构建体，其包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因组成。

根据本发明，第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白，或由其组成。

目前的基因组工程化工具，包括工程化的锌指蛋白(ZFP)、转录激活子样效应物核酸酶(TALEN)和最近的RNA引导的DNA核酸酶如Cas9，均影响基因组中的序列特异性DNA切割。这种可编程切割可导致DNA在切割位点处通过非同源末端连接(NHEJ)而突变，或通过同源指导的修复(HDR)导致切割位点周围DNA的置换。

在一个实施方案中，位点特异性DNA结合蛋白选自包含以下或由以下组成的组：RNA引导的DNA核酸酶、锌指蛋白和转录激活子样效应物核酸酶。

在一个实施方案中，位点特异性DNA结合蛋白选自包含以下或由以下组成的组：RNA引导的核酸酶和锌指蛋白。

在一个实施方案中，位点特异性DNA结合蛋白是RNA引导的核酸酶。

在一个实施方案中，位点特异性DNA结合蛋白是Cas9蛋白(例如但不限于酿脓链球菌Cas9(SpCas9)、金黄色葡萄球菌(Staphylococcus aureus)Cas9(SaCas9)或空肠弯曲杆菌(Campylobacter jejuni)Cas9(CjCas9)；一些其它合适的实例将在下面描述)，或其变体(例如切口酶Cas9(nCas9)或死Cas9(dCas9))、Cas12a蛋白、Cas12b蛋白、Cpf1蛋白或Casx蛋白，包括其变体和功能片段。

在一个实施方案中，位点特异性DNA结合蛋白是Cas9蛋白，包括其变体和功能片段。

CRISPR-Cas9系统是通过序列特异性双链断裂(DSB)使基因失活或修饰基因的高效工具。这些DSB被细胞DNA损伤响应机制所识别，并可通过内源性DSB修复途径修复。主要的修复途径是非同源末端连接(NHEJ)，其经常导致小的插入和/或缺失，这可以产生移码突变并破坏基因的功能。该途径可用于产生基因敲除突变。或者，在修复模板(例如，包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由所述转基因组成的核酸构建体)存在时，损伤可通过同源指导的修复(HDR)无缝修复。然而，尽管取得了显著的进展，但是引入准确基因修饰的HDR介导的基因组编辑的效率远低于NHEJ介导的基因破坏。此外，通过HDR途径的大的数kb的置换面临挑战，并且需要选择和/或大群体细胞分选。因此，HDR途径的主要应用目前限于基因内关键区域的局部置换，而不是大的全长基因的置换。如上所述，本发明弥补了这种缺陷。

在一个实施方案中，Cas9蛋白包含(i)活性DNA切割结构域和(ii)引导RNA结合结构域。

在已知的Cas9蛋白中，酿脓链球菌Cas9蛋白已被广泛用作基因组工程化的工具。所述Cas9蛋白是含有两个不同核酸酶结构域的大的多结构域蛋白。

在一个实施方案中，Cas9蛋白选自包含以下或由以下组成的组：具有SEQ ID NO：19的来自溃疡棒杆菌(Corynebacterium ulcerans)的Cas9蛋白(NCBI Refs:NC_015683.1,NC_017317.1)；具有SEQ ID NO：20的来自白喉棒杆菌(Corynebacterium diphtheria)的Cas9蛋白(NCBI Refs:NC_016782.1,NC_016786.1)；具有SEQ ID NO：21的来自梅毒螺原体(Spiroplasma syrphidicola)的Cas9蛋白(NCBI Ref:NC_021284.1)；具有SEQ ID NO：22的来自中间普雷沃氏菌(Prevotella intermedia)的Cas9蛋白(NCBI Ref:NC_017861.1)；具有SEQ ID NO：23的来自台湾螺原体(Spiroplasma taiwanense)的Cas9蛋白(NCBI Ref:NC_021846.1)；具有SEQ ID NO：24的来自海豚链球菌(Streptococcus iniae)的Cas9蛋白(NCBI Ref:NC_021314.1)；具有SEQ ID NO：25的来自Belliella baltica的Cas9蛋白(NCBIRef:NC_018010.1)；具有SEQ ID NO：26的来自热带冷弯菌(Psychroflexus torquisi)的Cas9蛋白(NCBI Ref:NC_018721.1)；具有SEQ ID NO：27的来自嗜热链球菌(Streptococcusthermophilus)的Cas9蛋白(NCBI Ref:YP_820832.1)；具有SEQ ID NO：28的来自无害利斯特氏菌(Listeria innocua)的Cas9蛋白(NCBI Ref:NP_472073.1)；具有SEQ ID NO：29的来自空肠弯曲杆菌(Campylobacter jejuni)的Cas9蛋白(CjCas9)(NCBI Ref:YP_002344900.1)(由SEQ ID NO：63编码)；具有SEQ ID NO：30的来自脑膜炎奈瑟氏球菌(Neisseria meningitidis)的Cas9蛋白(NCBI Ref:YP_002342100.1)；具有SEQ ID NO：68的来自金黄色葡萄球菌(Staphylococcus aureus)的Cas9蛋白(SaCas9)(由SEQ ID NO：60编码)；和具有SEQ ID NO：31的来自酿脓链球菌的Cas9蛋白(SpCas9)(NCBI Ref:NC_017053.1)。

在一个实施方案中，当本文提及野生型Cas9蛋白时，除非另有说明，否则所述野生型Cas9蛋白对应于具有SEQ ID NO：31的来自酿脓链球菌的Cas9(spCas9)。

在一个实施方案中，Cas9蛋白可以是“Cas9变体”。如本文所用，“Cas9变体”是与如本文所述的Cas9蛋白具有同源性的蛋白，并且包括其片段。

在一个实施方案中，Cas9变体可以与具有SEQ ID NO:31的野生型Cas9蛋白或与具有SEQ ID NO:19-30或68的任何其他Cas9蛋白至少约70％同一、至少约80％同一、至少约90％同一、至少约95％同一、至少约96％同一、至少约97％同一、至少约98％同一、至少约99％同一、至少约99.5％同一或至少约99.9％同一。

在一个实施方案中，Cas9变体包含具有一个或几个氨基酸取代的Cas9蛋白的氨基酸序列。例如，已知Cas9的DNA切割结构域包括两个亚结构域，即HNH核酸酶亚结构域和RuvC1亚结构域。HNH亚结构域切割与gRNA互补的链，而RuvC1亚结构域切割非互补链。

这些亚结构域内的突变可使Cas9的核酸酶活性沉默。例如，已知取代D10A和H841A使具有SEQ ID NO：31的酿脓链球菌Cas9蛋白的核酸酶活性完全失活，导致死Cas9(dCas9)，其仍保留其以sgRNA被编程的方式结合DNA的能力。原则上，当与另一蛋白或结构域融合时，dCas9可以简单地通过与合适的sgRNA共表达而将所述蛋白靶向几乎任何DNA序列。在一个实施方案中，dCas9蛋白由与SEQ ID NO：59具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，dCas9蛋白包含与SEQ ID NO：67具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列，或由其组成。

至于Cas9切口酶(nCas9)，它是Cas9核酸酶的变体，区别在于在RuvC核酸酶结构域中的点突变(D10A)，这使其能够对DNA产生切口但不切割DNA。在一个实施方案中，nCas9蛋白由与SEQ ID NO：57具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，nCas9蛋白包含与SEQ ID NO：65具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列，或由其组成。在一些实施方案中，SaCas9切口酶(SanCas9)由与SEQ ID NO：58具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、约99％序列或约100％同一性的核酸序列编码。在一个实施方案中，SaCas9切口酶(SanCas9)包含与SEQID NO：66具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。

在一个实施方案中，Cas9变体包含Cas9的片段，使得所述片段与具有SEQ ID NO:31的野生型Cas9蛋白或具有SEQ ID NO:19-30或68的任何其他Cas9蛋白的对应片段至少约70％同一、至少约80％同一、至少约90％同一、至少约95％同一、至少约96％同一、至少约97％同一、至少约98％同一、至少约99％同一、至少约99.5％同一或至少约99.9％同一。

在一个实施方案中，Cas9变体仅包含DNA切割结构域或引导RNA结合结构域中的一者。

在一个实施方案中，示例性Cas9变体是人源化Cas9(hCas9)或其变体或功能片段。如本文所用，术语“人源化Cas9”或“hCas9”是指针对人类细胞的序列优化的Cas9蛋白。

在一个实施方案中，hCas9蛋白由与SEQ ID NO：56具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，hCas9蛋白包含与SEQ ID NO：64具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。

在一个实施方案中，位点特异性DNA结合蛋白是cpf1蛋白。在一个实施方案中，cpf1蛋白由与SEQ ID NO：61具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，cpf1蛋白包含与SEQ ID NO：69具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。

在一个实施方案中，位点特异性DNA结合蛋白是CasX蛋白。在一个实施方案中，CasX由与SEQ ID NO：62具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，CasX蛋白包含与SEQ ID NO：70具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。

如下文将进一步详述地，本公开的某些方面还涉及包含核酸构建体的载体或质粒(例如表达载体、包装载体等)，所述核酸构建体编码位点特异性DNA结合蛋白，特别是RNA引导的核酸酶，特别是本文所述的Cas9蛋白中的任一种；所述载体或质粒优选适于在宿主细胞中表达，所述宿主细胞例如是哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞。

在一个实施方案中，位点特异性DNA结合蛋白是锌指蛋白(ZFP)。

锌指蛋白是可以以序列特异性方式结合DNA的蛋白。ZFP在真核生物中分布不均匀。ZFP已经被鉴定参与DNA识别、RNA结合和蛋白结合。考虑到折叠结构域中的蛋白骨架的整体形状，锌指蛋白的某些分类基于“折叠组(fold group)”。锌指最常见的“折叠组”是C2H2或Cys2His2样(“经典锌指”)、高音谱号锌指(treble clef)和锌带锌指(zincribbon)。表征这些蛋白的代表性基序公布于Li&Liu,2020(Int J Mol Sci.21(4):1361)的表1，该表通过引用并入本文。

ZFP可以是可结合基因组中特定基因组DNA序列的任何ZFP、其变体或功能片段。ZFP的非限制性实例包括包含选自以下的折叠组或锌指基序的ZFP：C2H2锌指、塞结状锌指(gag knuckle)、高音谱号锌指、锌带锌指、Zn2/Cys6样锌指或TAZ2结构域样锌指或其任何组合。在一个实施方案中，ZFP是C2H2锌指蛋白。

在一个实施方案中，ZFP是工程化的ZFP。工程化的锌指阵列可与DNA切割结构域(通常是FokI的切割结构域)融合以产生锌指核酸酶。此类锌指-FokI融合物已经成为用于操控基因组的有用试剂。

ZFP可以包含2、3、4、5、6、7、8、9、10、11、12个或更多个锌指结构域。ZFP可以包含2至12个、2至10个、2至8个、3至8个、4至8个或5至8个锌指结构域。在一个实施方案中，ZFP包含6个锌指结构域。

常见的模块组装方法包括组合各自可识别3个碱基对的DNA序列的单独锌指，以产生识别长度为9个碱基对至18个碱基对的靶位点的3-指、4-指、5-指或6-指阵列。另一种方法使用2-指模块来生成具有多达六个单独锌指的锌指阵列。

在一个实施方案中，可将ZFP的结合结构域工程化以结合目标序列。与天然存在的ZFP相比，工程化的锌指结合结构域可具有提高的结合特异性。

在一个实施方案中，编码ZFP的示例性核酸序列包含SEQ ID NO：32、SEQ ID NO：34、SEQ ID NO：36或SEQ ID NO：38，或由其组成。在一个实施方案中，由这些序列编码的示例性氨基酸序列包含SEQ ID NO：33、SEQ ID NO：35、SEQ ID NO：37或SEQ ID NO：39，或由其组成。

在一个实施方案中，ZFP包含与SEQ ID NO：33、35、37或39中任一者具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。

在一个实施方案中，ZFP不具有Gal4 DNA结合结构域。Gal4结合CGG-N₁₁-CCG，其中N可以是任何碱基。该蛋白是半乳糖诱导的基因如GAL1、GAL2、GAL7、GAL10和MEL1的基因表达的正调节物，MEL1编码用于将半乳糖转化为葡萄糖的酶。它识别这些基因的上游激活序列(UAS-G)中的17个碱基对的序列。因此，Gal4识别基因组中的短且非常频繁的序列，因此不是位点特异性的。在一个实施方案中，ZFP具有经工程改造为位点特异性的Gal4DNA结合结构域。

如下文将进一步详述地，本公开的某些方面涉及包含编码位点特异性DNA结合蛋白，特别是本文所述的ZFP的核酸构建体的载体或质粒(例如，表达载体、包装载体等)；所述载体或质粒优选适于在宿主细胞中表达，所述宿主细胞例如是哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞。

根据本发明，第二蛋白包含转座酶或由转座酶组成。

转座子是可以进行转座的染色体片段，例如，在宿主DNA中不存在互补序列时可以作为整体易位的DNA。转座子可用于在人细胞中进行长程(long-range)DNA工程化。用于哺乳动物细胞的常见转座子系统包括但不限于，睡美人(SB，其从失活的转座子重构)和PiggyBac(PB，其从Trichoplusia蛾分离)。PiggyBac具有比SB更高的转座活性，并且可被无痕切除。

天然DNA转座子通常含有编码转座酶蛋白的单个基因，其侧翼为携带转座酶结合位点的反向末端重复序列(ITR)。在它们的转座过程中，转座酶蛋白识别这些ITR以催化所述元件切除和随后以随机方式在别处再整合。此外，这些转座子中的一些可经改造用于基因治疗方案，将它们作为双组分系统使用，其中质粒含有表达盒，其中置于转座子ITR之间的目标DNA序列(例如LAMA2转基因)可在共转染质粒的指导下被引入宿主基因组中，所述共转染质粒含有编码转座酶的序列或其体外合成的mRNA。根据本公开内容，使用基于转座子的系统来有效介导LAMA2转基因在细胞中的稳定整合和持续表达。

在一个实施方案中，用于本发明的转座酶或修饰的转座酶可以是能将LAMA2转基因插入基因组特定位点的任何转座酶。

转座酶的非限制性实例包括Frog Prince、睡美人、高活性睡美人、PiggyBac和高活性PiggyBac。

在一个实施方案中，转座酶是高活性PiggyBac转座酶(hyPB)。

野生型高活性PiggyBac转座酶具有包含SEQ ID NO：9或由SEQ ID NO：9组成的氨基酸序列。编码该蛋白的示例性核酸序列如SEQ ID NO：71所示。

在一个实施方案中，转座酶是修饰的高活性PiggyBac转座酶。

如本文所用，“修饰的高活性PiggyBac转座酶”是指与具有SEQ ID NO：9的野生型高活性PiggyBac转座酶相比，包含一个或多个氨基酸取代，通常不超过1、2、3、4、5、6、7、8、9或10个氨基酸取代的转座酶。更具体而言，修饰的高活性PiggyBac与野生型高活性PiggyBac转座酶相比包含(i)增加切除活性的一个或多个氨基酸取代，和/或(ii)降低DNA结合活性的一个或多个氨基酸取代。在一个实施方案中，修饰的高活性PiggyBac转座酶包含与SEQ ID NO：9中所示序列至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一的氨基酸序列。

在一些实施方案中，与野生型高活性PiggyBac转座酶相比，修饰的高活性PiggyBac转座酶包含增加切除活性的一个或多个氨基酸取代。

在一个实施方案中，修饰的高活性PiggyBac转座酶在由氨基酸位置编号[194-200]、[214-222]、[434-442]和/或[446-456]限定的区域内，例如在氨基酸位置D198、D201、R202、M212和/或S213处，包含增加切除活性的一个或多个氨基酸取代；编号基于具有SEQID NO:9的野生型的高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶在选自位置450、560、564、573、589、592和/或594的氨基酸位置处包含增加切除活性的一个或多个氨基酸取代；编号基于具有SEQ ID NO:9的野生型的高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶在选自位置M194和D450的氨基酸位置处包含增加切除活性的一个或多个氨基酸取代；编号基于具有SEQ ID NO:9的野生型高活性PiggyBac转座酶。在一个实施方案中，所述一个或多个氨基酸取代选自M194V和D450N，编号基于具有SEQ ID NO:9的野生型高活性PiggyBac转座酶。

在一个实施方案中，与野生型高活性PiggyBac转座酶相比，修饰的高活性PiggyBac转座酶包含降低DNA结合活性的一个或多个氨基酸取代。

在一个实施方案中，修饰的高活性PiggyBac转座酶在选自位置254、275、277、347、372、375和/或465的氨基酸位置处包含降低DNA结合活性的一个或多个氨基酸取代；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶在选自位置R275、N347、R372、K375、R376、E377和/或E380的氨基酸位置处包含降低DNA结合活性的一个或多个氨基酸取代；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶在选自位置R372、K375、R376、E377和/或E380的氨基酸位置处包含降低DNA结合活性的一个或多个氨基酸取代；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。在一个实施方案中，所述一个或多个氨基酸取代选自R372A、K375A、R376A、E377A和/或E380A；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶在选自位置N347、R372和K375的氨基酸位置处包含降低DNA结合活性的一个或多个氨基酸取代；编号基于具有SEQ IDNO：9的野生型高活性PiggyBac转座酶。在一个实施方案中，所述一个或多个氨基酸取代选自N347S、N347A、R372A和/或K375A；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。在一个实施方案中，所述一个或多个氨基酸取代选自N347S或N347A；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含增加切除活性的一个或多个氨基酸取代，如上文所定义；和降低DNA结合活性的一个或多个氨基酸取代，如上文所定义。

在一个实施方案中，修饰的高活性PiggyBac转座酶在位置D450处包含至少一个增加切除活性的取代，且在位置N347、R372和K375处包含至少两个降低DNA结合活性的取代；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶；优选地，所述修饰的高活性PiggyBac转座酶包含双突变N347S和D450N或三重取代D450N、R372A和K375A；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。在一个实施方案中，修饰的高活性PiggyBac转座酶包含双突变N347S和D450N，所述位置编号对应于SEQ ID NO：9的未修饰的高活性Piggybac的氨基酸编号。

在一些实施方案中，修饰的高活性PiggyBac转座酶不包含三重突变D450N、R372A和K375A，所述位置编号对应于SEQ ID NO：9的未修饰的高活性PiggyBac的氨基酸编号。

在一个实施方案中，如先前实施方案中所公开，修饰的高活性PiggyBac转座酶在由氨基酸位置编号[158-169]定义的区域中还包含至少一个取代，例如A166S；和/或在位置Y527、R518、K525和/或N463处的至少一个取代；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

通常，所述修饰的高活性PiggyBac转座酶包含与SEQ ID NO：1的未修饰的高活性PiggyBac转座酶具有至少85％、至少90％、至少95％同一性的氨基酸序列。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶在选自以下的位置处还包含下列氨基酸取代的一个或多个：34、43、117、202、230、245、268、275、277、287、290、315、325、341、346、347、350、351、356、357、388、409、412、432、447、460、461、465、517、560、564、571、573、576、586、587、589、592和594；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶包含下列氨基酸取代或取代组合的其中一个：V34M、T43I、Y177H、R202K、S230N、R245A、D268N、R275A、R277A、K287A、K290A、K287A/K290A、R315A、G325A、R341A、D346N、N347A、N347S、T350A、S351E、S351P、S351A、K356E、N357A、R388A、K409A、A411T、K412A、K432A、D447A、D447N、D450N、R460A、K461A、W465A、S517A、T560A、S564P、S571N、S573A、K576A、H586A、I587A、M589V、S592G、F594L、D450N/R372A/K375A、R275A/R277A、K409A/K412A、R460A/K461A、R275A/R277A/N347S/K375A/T560A/S573A/M589V/S592G和R245A/R275A/R277A/R372A/W465A；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶包含下列氨基酸取代或氨基酸取代组合的其中一个：

-R372A/K375A/D450N,

-R372A/K375A/R376A/D450N,

-K375A/R376A/E377A/E380A/D450N,

-R372A/K375A/R376A/E377A/E380A/D450N,

-M194V,

-M194V/R372A/K375A,

-S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L,

-R245A/R275A/R277A/R372A/W465A/M589V,

-R275A/325A/R372A/T560A,

-N347A/D450N,

-N347S/D450N/T560A/S573A/F594L,

-R202K/R275A/N347S/R372A/D450N/T560A/F594L,

-R275A/N347S/K375A/D450N/S592G,

-R275A/N347S/R372A/D450N/T560A/F594L，

-R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L

-R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G，

-R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L，

-V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P，

-G325A/N347S/K375A/D450N/S573A/M589V/S592G,

-S230N/R277A/N347S/K375A/D450N，

-T43I/R372A/K375A/A411T/D450N，

-G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G，或

-Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G；

编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶包含下列氨基酸取代或氨基酸取代组合的其中一个：

-K375A/R376A/E377A/E380A/D450N,

-R372A/K375A/R376A/E377A/E380A/D450N,

-M194V,

-M194V/R372A/K375A,

-R245A/R275A/R277A/R372A/W465A/M589V,

-R275A/325A/R372A/T560A,

-N347A/D450N,

-N347S/D450N/T560A/S573A/F594L,

-R202K/R275A/N347S/R372A/D450N/T560A/F594L,

-R275A/N347S/K375A/D450N/S592G,

-R275A/N347S/R372A/D450N/T560A/F594L，

-R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L

-R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G，

-R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L，

-G325A/N347S/K375A/D450N/S573A/M589V/S592G,

-S230N/R277A/N347S/K375A/D450N，

-G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G，或

-Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G；

编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在优选实施方案中，所述修饰的高活性PiggyBac转座酶包含下列氨基酸取代或氨基酸取代组合的其中一个：

-R372A/K375A/D450N,

-S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L,

-R245A/R275A/R277A/R372A/W465A/M589V,

-N347A/D450N,

-N347S/D450N/T560A/S573A/F594L,

-R202K/R275A/N347S/R372A/D450N/T560A/F594L,

-R275A/N347S/K375A/D450N/S592G,

-R275A/N347S/R372A/D450N/T560A/F594L,

-R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L，

-R275A/325A/R372A/T560A,

-R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G，

-R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L，

-G325A/N347S/K375A/D450N/S573A/M589V/S592G,

-S230N/R277A/N347S/K375A/D450N，

-G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G，或

-Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G；

编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶具有选自SEQ ID NO：1-8、10-18、108-113和122-130中任一项的氨基酸序列。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶具有选自SEQ ID NO：1-8和10-18中任一项的氨基酸序列。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶具有选自SEQ ID NO：108-113中任一项的氨基酸序列。

在一个实施方案中，所述修饰的高活性PiggyBac转座酶具有选自SEQ ID NO：122-130中任一项的氨基酸序列。

在一个实施方案中，相对于野生型高活性PiggyBac转座酶，修饰的高活性PiggyBac转座酶可包含在保守催化三联体中涉及的一个或多个取代，例如在氨基酸268和/或346处(例如D268N和/或D346N)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ ID NO：11的修饰的高活性PiggyBac转座酶。

在一个实施方案中，相对于野生型高活性PiggyBac转座酶，修饰的高活性PiggyBac转座酶可包含对切除至关重要的一个或多个取代，例如在氨基酸287、287/290和/或460/461处(例如K287A、K287A/K290A和/或R460A/K461A)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ ID NO：12的修饰的高活性PiggyBac转座酶。

在一个实施方案中，相对于野生型高活性PiggyBac转座酶，修饰的高活性PiggyBac转座酶可包含参与靶连接(target joining)的一个或多个取代，例如在氨基酸351、356和/或379处(例如S351E、S351P、S351A和/或K356E)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ ID NO：13的修饰的高活性PiggyBac转座酶。

在一个实施方案中，相对于野生型高活性PiggyBac转座酶，修饰的高活性PiggyBac转座酶可包含对整合至关重要的一个或多个取代，例如在氨基酸560、564、571、573、589、592和/或594处(例如T560A、S564P、S571N、S573A、M589V、S592G和/或F594L)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ ID NO：14的修饰的高活性PiggyBac转座酶。

在一个实施方案中，相对于hyPB，修饰的高活性PiggyBac转座酶可以包含参与比对(alignment)的一个或多个取代，例如，在氨基酸325、347、350、357和/或465处(例如，G325A、N347A、N347S、T350A和/或W465A)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ ID NO：15的修饰的高活性PiggyBac转座酶。

在一个实施方案中，相对于野生型高活性PiggyBac转座酶，修饰的高活性PiggyBac转座酶可包含高度保守的一个或多个取代，例如在氨基酸576和/或587处(例如K576A和/或I587A)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ ID NO：16的修饰的高活性PiggyBac转座酶。

在一个实施方案中，相对于野生型高活性PiggyBac转座酶，修饰的高活性PiggyBac转座酶可包含参与Zn²⁺结合的一个或多个取代，例如586(例如H586A)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ ID NO：17的修饰的高活性PiggyBac转座酶。

在一个实施方案中，相对于野生型高活性PiggyBac转座酶，可编程转座酶可包含参与整合的一个或多个取代，例如315、341、372和/或375(例如R315A、R341A、R372A和/或K375A)；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶或基于具有SEQ IDNO：18的修饰的高活性PiggyBac转座酶。

在一个实施方案中，选择的修饰的高活性PiggyBac转座酶与野生型高活性PiggyBac转座酶相比具有将DNA整合到基因组中的高特异性。在一个实施方案中，修饰的高活性PiggyBac转座酶包含这样的氨基酸序列，其相对于SEQ ID NO：9-18和108-113中任一项具有一个或多个本文公开的修饰，并且与SEQ ID NO：1-18和108-113所示的序列分别保持至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的同一性。

如实施例中所示，新开发的高活性PiggyBac转座酶取代文库已用于鉴定实施特异性靶向转座的修饰的高活性PiggyBac。使用这样的文库鉴定具有阳性靶向转座的修饰的高活性PiggyBac。

在一个实施方案中，修饰的高活性PiggyBac转座酶可以包含选自以下氨基酸的一个或多个氨基酸的取代：245、275、275、277、325、347、351、372、375、388、450、465、560、564、573、589、592、594；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含一个或多个选自以下的氨基酸取代：R245A、R275A、R275A、R277A、R275A/R277A、G325A、N347A、N347S、S351E、S351P、S351A、R372A、K375A、R388A、D450N、W465A、T560A、S564P、S573A、M589V、S592G或F594L；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸取代D450N；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸修饰N347A；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。在另一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸取代N347S；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含双氨基酸取代D450N和N347A；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。在另一个实施方案中，修饰的高活性PiggyBac转座酶包含双氨基酸取代D450N和N347S；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸取代R372A、K375A和D450；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸取代R245A和D450；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸取代R245A、G325A和S573P；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸修饰R245A、G325A、D450和S573P；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸取代N347S和D450N；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含氨基酸取代N347A和D450N；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。在一个实施方案中，所述修饰的高活性PiggyBac转座酶包含SEQ ID NO：110的氨基酸序列。

在一个实施方案中，修饰的高活性PiggyBac转座酶包含一个或几个选自L25F、R36A、I42K、G59D、I212K、N245S、K252A和/或Q271L的氨基酸取代；编号基于具有SEQ ID NO：9的野生型高活性PiggyBac转座酶。

本文提供的修饰的高活性PiggyBac转座酶可与本文公开的其它元件融合，例如能够结合和切割靶核酸序列的位点特异性DNA结合蛋白；或者可以单独使用，或者与这些其它元件组合使用。在一个实施方案中，本文公开的修饰的高活性PiggyBac转座酶包含SEQ IDNO：9的氨基酸序列，其中：

位置34处的氨基酸是V或M，

位置43处的氨基酸是T或I，

位置177处的氨基酸是Y或H，

位置202处的氨基酸是R或K，

位置230处的氨基酸是S或N，

位置245处的氨基酸是A，位置268处的氨基酸是D或N，

位置275处的氨基酸是R或A，

位置277处的氨基酸是R或A，

位置325处的氨基酸是A或G，

位置347处的氨基酸是N、S或A，

位置351处的氨基酸是E、P或A，

位置372处的氨基酸是R或A，

位置375处的氨基酸是A或K，

位置388处的氨基酸是R或A，

位置409处的氨基酸是K或A，

位置411处的氨基酸是A或T，

位置412处的氨基酸是K或A，

位置450处的氨基酸是D或N，

位置460处的氨基酸是R或A，

位置465处的氨基酸是W或A，

位置517处的氨基酸是S或A，

位置560处的氨基酸是T或A，

位置564处的氨基酸是P或S，

位置571处的氨基酸是S或N，

位置573处的氨基酸是S或A，

位置576处的氨基酸是K或A，

位置586处的氨基酸是H或A，

位置587处的氨基酸是I或A，

位置589处的氨基酸是M或V，

位置592处的氨基酸是G或S，和/或

位置594处的氨基酸是L或F。

在一个实施方案中，所述转座酶是睡美人转座酶。

在一个实施方案中，所述睡美人转座酶包含与SEQ ID NO：73具有至少85％、90％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列，或由其组成。编码该蛋白的示例性核酸序列如SEQ ID NO：72所示。

在一个实施方案中，所述转座酶是修饰的睡美人转座酶。在一个实施方案中，所述修饰的睡美人转座酶与野生型睡美人转座酶相比包含一个或多个取代。

在一个实施方案中，所述睡美人转座酶中的所述一个或多个取代选自L25F、R36A、I42K、G59D、I212K、N245S、K252A和/或Q271L；编号基于具有SEQ ID NO：73的野生型睡美人转座酶。

在一个实施方案中，转座酶不是Himar1C9突变体。

如下文将进一步详述地，本公开的某些方面还涉及包含编码转座酶，特别是本文所述的任何高活性PiggyBac转座酶或修饰的高活性PiggyBac转座酶的核酸构建体的载体或质粒(例如，表达载体、包装载体等)；所述载体或质粒优选适于在宿主细胞中表达，所述宿主细胞例如是哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞。

在一个实施方案中，将包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成的第一蛋白(如上所述)，和包含转座酶或由转座酶组成的第二蛋白(如上所述)，直接地或通过接头间接地融合在一起，以形成融合蛋白。

一方面涉及位点特异性DNA结合蛋白而另一方面涉及转座酶的任何实施方案，经必要的变更都适用于本文所述的融合蛋白的情况。

因此，在一个实施方案中，融合蛋白包含以下或由以下组成：

(i)第一蛋白，所述第一蛋白包含如上所述的RNA引导的DNA核酸酶、锌指蛋白或转录激活子样效应物核酸酶或由其组成，和

(ii)第二蛋白，所述第二蛋白包含如上所述的转座酶或由其组成。

在一个实施方案中，融合蛋白包含以下或由以下组成：

(i)第一蛋白，所述第一蛋白包含如上所述的RNA引导的DNA核酸酶或锌指蛋白或由其组成，和

(ii)第二蛋白，所述第二蛋白包含如上所述的转座酶或由其组成。

在一个实施方案中，融合蛋白包含以下或由以下组成：

(i)第一蛋白，所述第一蛋白包含如上所述的RNA引导的DNA核酸酶或由其组成，和

(ii)第二蛋白，所述第二蛋白包含如上所述的转座酶或由其组成。

在一个实施方案中，融合蛋白包含以下或由以下组成：

(i)第一蛋白，所述第一蛋白包含如上所述的Cas9蛋白或其变体或由如上所述的Cas9蛋白或其变体组成，和

(ii)第二蛋白，所述第二蛋白包含如上文所述的高活性PiggyBac转座酶或修饰的高活性PiggyBac转座酶，特别是如上所述的修饰的高活性PiggyBac转座酶，或由其组成。

在一个实施方案中，第一蛋白和第二蛋白可以以任一顺序在融合蛋白中定向。

在一个实施方案中，融合蛋白包含直接或通过接头间接地融合在第二蛋白C-末端的第一蛋白，或由其组成。换句话说，融合蛋白从N-末端到C-末端包含以下或由以下组成：(i)第二蛋白(即转座酶)；(ii)任选地，接头；和(iii)第一蛋白(即位点特异性DNA结合蛋白，优选是RNA引导的DNA核酸酶；更优选是Cas9蛋白或其变体)。

在一个实施方案中，融合蛋白包含直接或通过接头间接地融合在第二蛋白N-末端的第一蛋白，或由其组成。换句话说，融合蛋白从N-末端到C-末端包含以下或由以下组成：(i)第一蛋白(即位点特异性DNA结合蛋白，优选是RNA引导的DNA核酸酶；更优选是Cas9蛋白或其变体)；(ii)任选地，接头；和(iii)第二蛋白(即转座酶)。

在一个实施方案中，融合蛋白包含接头。

接头的合适实例包括在第一蛋白和第二蛋白之间(以任何顺序)的肽接头。

在一个实施方案中，肽接头选自包含以下或由以下组成的组：(GGS)_n、具有SEQ IDNO：114的(GGGGS)_n、(G)_n、具有SEQ ID NO：115的(EAAAK)_n、XTEN接头和(XP)_n基序以及任何这些的组合，其中n独立地为1至50之间的整数。

在一个实施方案中，接头的长度为12至24个氨基酸，或由长度为36至72个核苷酸的核酸序列编码。

在一个实施方案中，接头是XTEN接头或(GGS)_n接头。

在一个实施方案中，所述接头选自表1中所示的接头。

表1：接头

在一个实施方案中，所述接头包含选自包含以下或由以下组成的组的氨基酸序列：SEQ ID NO：41、SEQ ID NO：43、SEQ ID NO：45、SEQ ID NO：47、SEQ ID NO：49、SEQ IDNO：51、SEQ ID NO：53、SEQ ID NO：55或其任意组合；分别由SEQ ID NO：40、SEQ ID NO：42、SEQ ID NO：44、SEQ ID NO：46、SEQ ID NO：48、SEQ ID NO：50、SEQ ID NO：52、SEQ ID NO：54的示例性核酸序列编码。

在一个实施方案中，所述接头包含SEQ ID NO：41的氨基酸序列或由其组成；由SEQID NO：40的示例性核酸序列编码。

本文还提供了从本公开中提供的任何核酸构建体的表达获得的融合蛋白。

在一个实施方案中，融合蛋白是三重融合蛋白。

这样的三重融合蛋白可以包含以下或由以下组成：

-一个第一蛋白(即一个位点特异性DNA结合蛋白)和两个第二蛋白(即两个转座酶)；或

-两个第一蛋白(即两个位点特异性DNA结合蛋白)和一个第二蛋白(即一个转座酶)。

在一个实施方案中，三重融合物包含一个第一蛋白(即一个位点特异性DNA结合蛋白)和两个第二蛋白(即两个转座酶)，或由其组成，并且所述三重融合物从N-末端至C-末端包含：

-(i)位点特异性DNA结合蛋白，(ii)第一转移酶；(iii)第二转座酶；或

-(i)第一转移酶；(ii)位点特异性DNA结合蛋白，(iii)第二转座酶；或

-(i)第一转移酶；(ii)第二转座酶，(iii)位点特异性DNA结合蛋白。

在一个实施方案中，第一和第二转座酶是相同的。在一个实施方案中，第一和第二转座酶是不同的。例如，第一转座酶可以是高活性PiggyBac转座酶，第二转座酶可以是修饰的高活性PiggyBac转座酶，其选自本文所述的任何修饰的高活性PiggyBac转座酶。或者，第一和第二转座酶可以都是修饰的高活性PiggyBac转座酶，但各自具有不同的取代或不同的取代组合，如本文所述。

在一个实施方案中，第一和第二转座酶能够形成功能性二聚体。

在一个实施方案中，三重融合物包含两个第一蛋白(即两个位点特异性DNA结合蛋白)和一个第二蛋白(即一个转座酶)或由其组成，并且所述三重融合物从N-末端到C-末端包含：

-(i)第一位点特异性DNA结合蛋白，(ii)第二位点特异性DNA结合蛋白；(iii)转座酶；或

-(i)第一位点特异性DNA结合蛋白；(ii)转座酶，(iii)第二位点特异性DNA结合蛋白；或

-(i)转座酶；(ii)第一位点特异性DNA结合蛋白，(iii)第二位点特异性DNA结合蛋白。

在一个实施方案中，第一和第二位点特异性DNA结合蛋白是相同的。在一个实施方案中，第一和第二位点特异性DNA结合蛋白是不同的。例如，第一位点特异性DNA结合蛋白可以是Cas9蛋白，第二位点特异性DNA结合蛋白可以是Cas9蛋白的变体，其选自本文所述的Cas9蛋白变体中的任一种。或者，第一和第二位点特异性DNA结合蛋白可以都是Cas9蛋白变体，但各自是不同的变体。

在一个实施方案中，三重融合蛋白任选地在其蛋白中的两个之间或在所述三个蛋白之间包含接头。

本文还公开了一种融合蛋白，其包含：

(i)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由转座酶组成，如上所述，和

(ii)RNA结合蛋白或编码所述RNA结合蛋白的核酸构建体，所述RNA结合蛋白能够结合至少一个特定RNA序列。

在一个实施方案中，融合蛋白包含如上所述的接头。

在一个实施方案中，第二蛋白包含转座酶或由转座酶组成，所述转座酶为具有SEQID NO：9的高活性PiggyBac。在一个实施方案中，第二蛋白包含转座酶或由转座酶组成，所述转座酶为与具有SEQ ID NO：9的高活性PiggyBac相比包含一个或多个氨基酸突变的修饰的高活性PiggyBac。特别地，所述修饰的高活性PiggyBac可以是本文公开的那些中的任一个。

在一个实施方案中，转座酶/RNA结合蛋白融合物可以进一步与包含位点特异性DNA结合蛋白或由其组成的第一蛋白融合，如上所述。

在一些实施方案中，所述RNA结合蛋白是MS2噬菌体衣壳蛋白(MCP)或其片段。

在一些实施方案中，MCP与SEQ ID NO：132具有至少75％、80％、85％、90％、95％、96％、97％、98％、99％或100％的同一性(例如由具有SEQ ID NO：131的核酸序列编码)。

在一些实施方案中，所述RNA结合蛋白能够结合至少一个特定RNA序列，所述RNA序列包含四环。术语“四环”与术语“茎环”和“发夹环”可互换使用。

在一些实施方案中，所述至少一个四环是MS2 RNA四环结合序列。

在一些实施方案中，所述四环包含在引导RNA(gRNA)内。在某些实施方案中，gRNA与Cas9蛋白形成复合物，如上所述。

在一些实施方案中，gRNA包含至少一个MS2 RNA四环结合序列。在一些实施方案中，gRNA包含多于一个MS2 RNA四环结合序列。

在一些实施方案中，包含至少一个MS2 RNA四环结合序列的gRNA与SEQ ID NO：134具有至少75％、80％、85％、90％、95％、96％、97％、98％、99％或100％的同一性(例如由具有SEQ ID NO：133的DNA序列编码)。

在一些实施方案中，融合蛋白中的MCP非共价结合包含在gRNA本身中的至少一个MS2 RNA四环结合序列，gRNA本身非共价结合Cas9蛋白；具体而言，融合蛋白与Cas9/gRNA复合物的结合将修饰的高活性PiggyBac转座酶的切除活性指导向由Cas9/gRNA复合物特异性识别的位点。

如下文将进一步详述地，本公开的某些方面还涉及包含编码本文所述融合蛋白的核酸构建体的载体或质粒(例如，表达载体、包装载体等)；所述载体或质粒优选适于在宿主细胞中表达，所述宿主细胞例如是哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞。

根据本发明，所述组合物可以包含第一蛋白和/或第二蛋白(或包含两者的融合蛋白)，或是如上所述地作为蛋白；或是作为编码这些蛋白的核酸构建体。

因此，在一个实施方案中，本发明的组合物包含以下或由以下组成：

a)编码上述第一蛋白的核酸构建体，所述第一蛋白包含能够结合并切割上述靶核酸序列的位点特异性DNA结合蛋白或由其组成；

b)编码上述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由转座酶组成；以及

c)包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因组成的核酸构建体。

在另一个实施方案中，本发明的组合物包含以下或由以下组成：

a)编码上述融合蛋白的核酸构建体，所述融合蛋白包含以下或由以下组成：(i)包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成的第一蛋白，和(ii)包含转座酶或由转座酶组成的第二蛋白；以及

b)包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因组成的核酸构建体。

在一个实施方案中，编码融合蛋白的核酸构建体还包含编码如上所述的第一和第二蛋白之间的接头的核酸序列；或者在三重融合蛋白的情况下，包含编码在其蛋白中的两个之间或者在这三个蛋白之间的接头的核酸序列。

根据本公开，第一和第二蛋白，或包含所述第一和第二蛋白或由所述第一和第二蛋白组成的融合蛋白，实现和/或促进转基因，特别是编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因位点特异性插入基因组中。

一些实施方案涉及质粒或载体(例如，表达载体)，其包含以下之一：

-编码第一蛋白的核酸构建体；或

-编码第二蛋白的核酸构建体；或

-编码第一蛋白的核酸构建体和编码第二蛋白的核酸构建体；或

-编码融合蛋白或三重融合蛋白的核酸构建体。

在一些实施方案中，质粒是包装质粒。在一些实施方案中，质粒还包含编码衣壳蛋白例如gag和pol的多核苷酸。在一些实施方案中，质粒与包含编码病毒包膜蛋白的多核苷酸的第二质粒(包膜质粒)和包含含有LAMA2转基因的核酸构建体的第三质粒组合，其中当将所述组合引入到生产细胞系(例如真核细胞、原核细胞和/或细胞系)时，产生了包含编码LAMA2转基因的核酸构建体和编码第一蛋白、第二蛋白、第一和第二蛋白两者或融合蛋白的核酸构建体的病毒颗粒。

在一些实施方案中，将质粒与包含编码衣壳蛋白例如gag和pol的多核苷酸的第二质粒(包装质粒，其中包装质粒缺乏功能性整合酶)、包含编码病毒包膜蛋白的多核苷酸的第三质粒(包膜质粒)和包含含有LAMA2转基因的核酸构建体的第四质粒组合，其中当将所述组合引入生产细胞系(例如真核和原核细胞和/或细胞系)时，产生包含含有LAMA2转基因的核酸构建体和编码第一蛋白、第二蛋白、第一和第二蛋白两者或融合蛋白的核酸构建体的病毒颗粒。

在一个实施方案中，使用慢病毒颗粒将第一蛋白、第二蛋白、第一和第二蛋白两者或融合蛋白、和/或LAMA2转基因递送至细胞。

在一个实施方案中，核酸构建体包含：编码第一蛋白的第一多核苷酸序列，所述第一蛋白包含经工程改造以结合靶核酸序列的位点特异性DNA结合蛋白或由其组成；编码第二蛋白的第二多核苷酸序列，所述第二蛋白包含转座酶或由转座酶组成，所述转座酶使得LAMA2转基因能够插入到基因组；和任选地，第三多核苷酸序列，所述第三多核苷酸序列包含编码第一和第二多核苷酸之间的接头的核酸序列。在一些实施方案中，第一蛋白是如上所述的锌指蛋白或Cas9蛋白或其变体；和/或所述第二蛋白是如上所述的高活性PiggyBac转座酶或修饰的高活性PiggyBac转座酶。

产生融合蛋白的合适接头的实例已在上文描述。

在一些实施方案中，不需要接头，因为第一蛋白从与第二蛋白不同的质粒表达。

在一个实施方案中，不使用接头，第一和/或第二多核苷酸序列分别包含编码第一和第二蛋白的核酸，并且在其至少一个末端还包含产生接头功能的额外核苷酸。

在一个实施方案中，核酸构建体是DNA或RNA形式。

本文还提供了包含本公开中提供的任何核酸构建体的载体。特别地，所述载体适于在哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞中表达。本文还提供了包含本公开中提供的任何核酸构建体或载体的宿主细胞。

根据本发明，所述组合物进一步包含核酸构建体，所述核酸构建体包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因组成，该转基因在本文中也称为“LAMA2转基因”。

在一个实施方案中，LAMA2转基因可以是编码层粘连蛋白-α2蛋白，特别是野生型的哺乳动物的、优选人的层粘连蛋白-α2蛋白，其功能变体或片段的任何核酸序列。

在一个实施方案中，LAMA2转基因可包含LAMA2野生型基因(或其功能变体或片段)、LAMA2 cDNA(或其功能变体或片段)或LAMA2小基因(或其功能变体或片段)。

在一个实施方案中，LAMA2转基因包含全长LAMA2基因。

层粘连蛋白-α2链蛋白由LAMA2基因(基因ID：3908，在2020年11月24日更新)编码，其被转录并翻译成390kDa的蛋白。层粘连蛋白-α2是称为层粘连蛋白-211(或分区蛋白)的异源三聚交叉形分子的组分，所述层粘连蛋白-211由通过二硫键彼此结合的三个亚基(α、β和γ)组成。

本领域已知有许多不同的哺乳动物层粘连蛋白-α2蛋白的编码序列，包括但不限于来自人、猪、黑猩猩、狗、牛、小鼠、兔或大鼠的层粘连蛋白-α2蛋白，并且可以容易地在序列数据库中找到。或者，本领域技术人员可以基于多肽序列容易地确定编码序列。

具体地，LAMA2转基因可编码人层粘连蛋白亚基α2同种型a前体(NCBI参考：NP_000417，在2020年10月22日提交)或层粘连蛋白亚基α2同种型b前体(NCBI参考：NP_001073291.2，在2020年10月24日提交)。

在一个实施方案中，LAMA2转基因编码人层粘连蛋白亚基α2同种型前体，优选具有SEQ ID NO：74。

翻译后，层粘连蛋白-α2链通常被切割成彼此非共价结合的N-末端片段和C-末端片段。

在一个实施方案中，LAMA2转基因可编码层粘连蛋白-α2蛋白的不同人类成熟同种型，优选选自包含以下或由以下组成的组：层粘连蛋白亚基α2同种型X1(NCBI参考：XP_005267038.1，具有SEQ ID NO：116)、层粘连蛋白亚基α2同种型X2(NCBI参考：XP_011534122.1，具有SEQ ID NO：117)、层粘连蛋白亚基α2同种型X3(NCBI参考：XP_005267039.1，具有SEQ ID NO：118)、层粘连蛋白亚基α2同种型X4(NCBI参考：XP_016866340.1，具有SEQ ID NO：119)、层粘连蛋白亚基α2同种型X5(NCBI参考：XP_01686634.1，具有SEQ ID NO：120)和层粘连蛋白亚基α2同种型X6(NCBI参考：XP_005267038.1，具有SEQ ID NO：121)。

如本文所用，当提及层粘连蛋白-α2蛋白或编码其的核酸时，术语“片段”或“功能片段”是指衍生自如上所述的层粘连蛋白-α2蛋白的蛋白、多肽或核酸，其仍然保留全长层粘连蛋白-α2蛋白的活性，但其序列与全长蛋白、多肽或核酸不是100％相同。功能片段可以具有比相应的天然分子更多、更少或相同数量的残基，和/或可以含有一个或多个氨基酸或核苷酸取代。优选地，功能片段是指层粘连蛋白-α2蛋白的成熟形式，其在蛋白的N-末端不包含信号肽。技术人员可以容易地确定信号肽对应于层粘连蛋白-α2蛋白的哪一部分，例如基于Uniprot数据库上公开可用的信息。

在一个实施方案中，LAMA2转基因包含具有SEQ ID NO：75的序列。在一个实施方案中，LAMA2转基因包含人LAMA2小基因(human LAMA2minigene)，其包含具有SEQ ID NO：82的合成内含子。在一个实施方案中，所述合成内含子包含在具有SEQ ID NO：75的编码序列的3925和3926位核碱基之间。

在一个实施方案中，LAMA2转基因可以是编码保留全长层粘连蛋白-α2蛋白活性的功能性层粘连蛋白-α2蛋白变体的任何核酸序列。特别地，层粘连蛋白-α2蛋白变体保留通过二硫键结合层粘连蛋白-211β和γ亚基以形成功能性交叉形层粘连蛋白-211或层粘连蛋白-221的能力。层粘连蛋白形成独立的网络，并通过巢蛋白(entactin)、纤连蛋白和串珠蛋白聚糖(perlecan)与IV型胶原网络相关连。它们还通过整联蛋白受体和其它质膜分子如肌营养不良聚糖糖蛋白复合物和卢瑟伦血型(lutheran blood group)糖蛋白结合至细胞膜。通过这些相互作用，层粘连蛋白关键性地促成了细胞附着和分化、细胞形状和移动、组织表型的维持和组织存活的促进。

优选地，如本文所用，术语“变体”或“功能变体”，在提到层粘连蛋白-α2蛋白时，是指具有与野生型层粘连蛋白-α2蛋白序列具有至少70、75、80、85、90、95或99％序列同一性的氨基酸或核苷酸序列的多肽。

更优选地，术语“变体”或“功能变体”指具有与野生型层粘连蛋白-α2蛋白序列的差异在于小于30、25、20、15、10或5个取代、插入和/或缺失的氨基酸序列的多肽。在一个实施方案中，变体与野生型层粘连蛋白-α2蛋白序列的差异之处在于一个或多个保守取代，优选小于15、10或5个保守取代。保守取代的实例在碱性氨基酸(精氨酸、赖氨酸和组氨酸)、酸性氨基酸(谷氨酸和天冬氨酸)、极性氨基酸(谷氨酰胺和天冬酰胺)、疏水性氨基酸(甲硫氨酸、亮氨酸、异亮氨酸和缬氨酸)、芳香族氨基酸(苯丙氨酸、色氨酸和酪氨酸)和小氨基酸(甘氨酸、丙氨酸、丝氨酸和苏氨酸)的组内。

在一个实施方案中，LAMA2转基因因此包含与SEQ ID NO：75具有至少70、75、80、85、90、95或99％序列同一性的序列。

在一个实施方案中，LAMA2转基因可包括编码层粘连蛋白-α2蛋白、其变体或片段的优化序列。

术语“优化的”，在核酸序列的上下文中，是指密码子优化，并且意指通常表现偏向于人的密码子被改变为不表现偏向于人的同义密码子(即，编码相同氨基酸残基的密码子)。因此，密码子的改变不会导致所编码蛋白中的任何氨基酸改变。

根据本发明，LAMA2转基因包含在核酸构建体中。

在一个实施方案中，核酸构建体包含LAMA2转基因，其与指导所述转基因在细胞中表达的一个或多个控制序列有效连接。

启动子含有在引入宿主细胞后介导LAMA2转基因表达的转录控制序列。启动子可以是在细胞中显示转录活性的任何多核苷酸，包括突变的、截短的和杂合的启动子。启动子可以是组成型或诱导型启动子，优选组成型启动子，更优选强组成型启动子。

合适的启动子的实例包括但不限于CMV启动子，优选具有SEQ ID NO：76；CAG启动子，优选具有SEQ ID NO：77；EF1-α启动子，具有优选SEQ ID NO：78；SV-40启动子，优选具有SEQ ID NO：79；和杂合白蛋白增强子-α1-抗胰蛋白酶(EalbAAT)肝特异性启动子，优选具有SEQ ID NO：80。

在另一个实施方案中，LAMA2转基因也可以插入细胞基因组中，其插入方式使得其表达由整合位点处或整合位点附近的内源启动子驱动。特别地，包含LAMA2转基因的核酸构建体可含有剪接受体，以在转基因被整合到主动表达的基因如内源LAMA2或白蛋白基因的内含子中时确保基因表达，从而具有对表达的内源控制。

剪接受体位点提供信号，以靶向待表达的剪接受体位点之后的序列(Padgett等人,1988.Annu Rev Biochem.55:1119-1150)。剪接受体位点是通常参与RNA剪接以去除内含子RNA序列的核苷酸序列。剪接受体位点通常参与内含子的切除，在此期间，其被称为剪接体(spliceosome)的RNA-蛋白复合物结合、被切割且然后与已被切割的剪接供体位点连接。剪接受体序列是本领域公知的，并且可以容易地从外显子和内含子之间的位置处的基因获得，在所述位置它们介导剪接。或者，剪接受体位点可以被化学或酶促合成。剪接受体(SA)位点通常以高度保守的AG二核苷酸结束。所述序列的其余核苷酸主要是胞苷和/或胸苷。在一个实施方案中，剪接受体具有SEQ ID NO：81的序列。

控制序列还可以包括适当的转录起始、终止和增强子序列；有效的RNA加工信号，如剪接和聚腺苷酸化信号；稳定细胞质mRNA的序列；增强翻译效率的序列(即Kozak共有序列)；和/或增强蛋白稳定性的序列。大量表达控制序列，例如天然的、组成型的、诱导型的和/或组织特异性的序列，是本领域已知的，并且可用于驱动LAMA2转基因的表达。通常，LAMA2转基因与转录启动子和转录终止子有效连接。

ploy(A)信号通常由以下组成：A)共有序列AAUAAA，已经显示其是前信使RNA(pre-mRNA)的3'末端切割和聚腺苷酸化以及促进下游转录终止所必需的，和b)在AAUAAA序列上游和下游的额外元件，其控制AAUAAA作为ploy(A)信号的利用效率。这些基序在哺乳动物基因中有相当大的变异性。

在一个实施方案中，核酸构建体中的聚腺苷酸化信号序列是哺乳动物基因或病毒基因的聚腺苷酸化信号序列。合适的聚腺苷酸化信号包括但不限于SV40早期聚腺苷酸化信号、SV40晚期聚腺苷酸化信号、HSV胸苷激酶聚腺苷酸化信号、鱼精蛋白(protamine)基因聚腺苷酸化信号、腺病毒5EIb聚腺苷酸化信号、生长激素聚腺苷酸化信号、PBGD聚腺苷酸化信号以及计算机设计的聚腺苷酸化信号(合成的)等。在一个实施方案中，聚腺苷酸化信号序列是具有选自包含SEQ ID NO：83、84和85或由SEQ ID NO：83、84和85组成的组的序列的聚腺苷酸。

在一个实施方案中，包含LAMA2转基因的核酸构建体还包含隔绝元件，以增加转基因表达水平、避免沉默和降低表达变异性。

隔绝物是一类复杂的顺式作用调节序列，其防止异染色质的扩散和基因的沉默(屏障活性)并具有增强子阻断活性。隔绝物的长度通常为300bp至2000bp。隔绝物的示例有许多，包括CTCF隔绝物、gypsy隔绝物和β-球蛋白基因座。

在一个实施方案中，隔绝元件序列在核酸构建体两个末端的侧翼。

在一个实施方案中，包含LAMA2转基因的核酸构建体包含具有SEQ ID NO：86和87的隔绝元件。

在一个实施方案中，本文公开的转座酶识别携带转座酶结合位点的反向末端重复(ITR)元件，以催化ITR之间的元件的切除和随后的再整合。

因此，在一个具体实施方案中，所述包含LAMA2转基因的核酸构建体侧翼是携带转座酶结合位点的反向末端重复序列(ITR)，优选SEQ ID NO：88和89的5'-ITR和3'-ITR。

在一个实施方案中，ITR之间的大小增加会正调节LAMA2转基因向基因组DNA的转移。

在一个实施方案中，ITR之间的大小为至少105bp。在一个实施方案中，ITR之间的大小为至少200bp。在一个实施方案中，ITR之间的大小为至少300bp。

如本文所用，术语“至少300bp”指300bp、400bp、500bp、600bp、700bp、800bp、900bp、1kb、2kb或更大。

在一个实施方案中，包含LAMA2转基因的核酸构建体包含在表达载体中。

合适载体的实例包括但不限于重组的整合或非整合的病毒载体和衍生自重组噬菌体DNA、质粒DNA或粘粒DNA的载体。在一个实施方案中，所述载体是质粒载体、微环载体或犬骨DNA供体载体。优选地，载体是重组的整合的或非整合的病毒载体。重组病毒载体的实例包括但不限于衍生自疱疹病毒、逆转录病毒、慢病毒、牛痘病毒、腺病毒、腺相关病毒或牛乳头瘤病毒的载体。

在一个实施方案中，所述病毒载体是慢病毒载体或逆转录病毒载体。因此，本发明还涉及包含含有LAMA2转基因的核酸构建体或包含含有所述核酸构建体的表达载体的病毒颗粒，如上所述。特别地，核酸构建体或表达载体可以包装到病毒衣壳中以产生“病毒颗粒”，也称为“病毒载体颗粒”。

在一个实施方案中，核酸构建体或表达载体被包装到慢病毒的或AAV衍生的衣壳中，以产生“慢病毒颗粒”或“AAV颗粒”。

根据本发明，第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白，或由其组成。

如本文所用，“靶序列”或“靶核酸序列”或“靶位点”是定义核酸的一部分的序列，所述核酸例如是在基因组中的核酸，只要存在足够的结合条件，结合分子将结合至所述部分。

所述第一蛋白可被工程化以结合细胞基因组内的任何选择的序列，称为“靶核酸序列”。例如，序列5'-GAATTC-3'是EcoRI限制性核酸内切酶的靶位点。

在一个实施方案中，靶核酸序列在细胞基因组中的安全港基因座内。在一个实施方案中，靶核酸序列在细胞基因组中的内源LAMA2基因内。特别地，第一和/或第二蛋白可以被工程化以结合和/或整合编码层粘连蛋白-α2蛋白的转基因到安全港基因座内。

“安全港基因座(safe harbor locus)”是指细胞基因组的区域，在此整合的物质可充分表达而不干扰内源基因结构或功能。安全港基因座包括，但不限于，AAVSl(PPP1R12C的内含子1)、HPRT、HI 1、hRosa26、白蛋白和F-A区。安全港基因座可以是普遍表达的基因和/或具有组织(例如肌肉)特异性表达的基因的外显子或内含子。安全港基因座可选自：PPP1R12C的外显子1、内含子1或外显子2，HPRT的外显子1、内含子1或外显子2，以及hRosa26的外显子1、内含子1或外显子2，白蛋白基因的内含子1。安全港基因座还可包括缺乏内源基因并具有开放染色质的基因组区域，其允许插入的转基因的表达而不干扰基因组结构或功能。

在一个实施方案中，第一和/或第二蛋白被工程化以结合LAMA2转基因并将其整合到细胞基因组内的内源LAMA2基因中，特别是整合到内源LAMA2基因的内含子1中。

在一个实施方案中，第一蛋白，任选地与引导RNA组合，结合选自SEQ ID NO：90-97任一项的靶核酸序列。

在一个实施方案中，本发明的组合物还包含引导RNA。

如本文所用，“引导RNA”、“gRNA”或“单引导RNA”是指促进gRNA/Cas复合物特异性靶向或归巢于靶核酸的核酸。

特别地，gRNA是指包含反式激活crRNA(tracrRNA)和crRNA的RNA分子。优选地，所述引导RNA对应于可以单独使用或融合在一起使用的crRNA和tracrRNA。与靶序列配对的互补序列募集Cas蛋白以结合并切割靶序列处的DNA。

在一个实施方案中，引导RNA被工程化以包含与靶序列的一部分互补的序列，所述靶序列优选选自SEQ ID NO：90-97的任一项。

如本文所用，术语“互补序列”是指在标准低严谨条件下可与多核苷酸的另一部分杂交的多核苷酸序列(例如crRNA或tracRNA的一部分)。优选地，依赖于链之间的Watson-Crick碱基配对，即腺嘌呤和胸腺嘧啶(A-T)核苷酸以及鸟嘌呤和胞嘧啶(G-C)核苷酸之间的内在碱基配对，序列根据两条核酸链之间的互补性彼此互补。

本公开还涉及将LAMA2转基因整合到细胞基因组内的靶核酸序列中的方法。所述方法包括将上述组合物引入细胞的步骤，使得第一和第二蛋白(单独地或作为上述融合蛋白的一部分)切割靶核酸序列并将LAMA2转基因整合在所述靶核酸序列中。

所述方法包括在细胞中引入：第一和第二蛋白(单独地或作为上述融合蛋白的一部分)或编码它们的一个或几个核酸构建体；引导RNA；以及包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由该转基因组成的核酸构建体。

这些元件可以在细胞中原位合成，这是将如上所述的编码所述元件的一个或多个核酸构建体或表达载体引入细胞的结果。或者，所述元件可以在细胞外产生，然后被引入其中。

在一个实施方案中，将如上所述的包含LAMA2转基因的核酸构建体、表达载体或病毒颗粒引入细胞。

在一个实施方案中，编码第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的一个或几个多核苷酸可以以mRNA形式被转染，其被直接引入细胞中，例如通过电穿孔或脂质纳米颗粒。

在一个实施方案中，引导RNA也可在细胞外产生，然后被引入细胞中，例如通过电穿孔或脂质纳米颗粒。

在一个实施方案中，第一和第二蛋白(单独地或作为上述融合蛋白的一部分)可以单独地或与引导RNA预先复合而引入细胞中。

在一个实施方案中，引导RNA和/或第一和第二蛋白(单独地或作为上述融合蛋白的一部分)由核酸构建体或表达载体编码。

本发明的产品(蛋白和核酸)可以通过脂质体递送方式、聚合物载体、化学载体、lipoplex、polyplex、树枝状聚合物(dendrimer)、纳米颗粒、乳液、天然胞吞作用或吞噬途径(作为非限制性实例)以及物理方法(例如电穿孔)递送到细胞或亚细胞区室内部。

所述核酸构建体或表达载体可以通过本领域已知的任何方法引入细胞，并且作为非限制性实例包括将核酸构建体或表达载体整合到细胞基因组中的稳定转化方法、不将核酸构建体或表达载体整合到细胞基因组中的瞬时转化方法以及病毒介导的方法。例如，瞬时转化方法包括例如显微注射、电穿孔或粒子轰击。

本发明的核酸分子或核酸构建体或表达载体可以使用任何已知的技术转移到细胞中，所述技术包括但不限于磷酸钙转染、DEAE-葡聚糖转染、电穿孔、显微注射、生物射弹(biolistic)、病毒感染或脂质体介导的转染。

在一个实施方案中，RNA，优选编码第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的引导RNA或mRNA，可以在体外产生，例如通过体外转录。然后可以通过电穿孔将RNA引入细胞(例如描述于等人,2011.Cytotherapy.13(5):629-640；Rabinovich等人,2009.Hum Gene Ther.20(1):51-61；和Beatty等人,2013.CancerImmunol Res.2(2):112-20)。

或者，RNA可以通过其它方式引入，例如通过脂质体或阳离子分子等。

在一个实施方案中，引入细胞的一个或多个核酸构建体或载体可以以游离方式表达或可以整合到细胞的基因组中。

在一个实施方案中，编码第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的多核苷酸如引导RNA和mRNA，和/或包含LAMA2转基因或由LAMA2转基因组成的核酸构建体，可通过纳米颗粒，优选脂质纳米颗粒(LNP)递送至细胞或患者。本领域已知的任何脂质或脂质组合都可以用于产生LNP。用于产生LNP的脂质的实例是：DOTMA、DOSPA、DOTAP、DMRIE、DC-胆固醇、DOTAP-胆固醇、GAP-DMORIE-DPyPE和GL67A-DOPE-DMPE-聚乙二醇(PEG)。阳离子脂质的实例是：98N12-5、C12-200、DLin-KC2-DMA(KC2)、DLin-MC3-DMA(MC3)、XTC、MD1和7C1。中性脂质的实例是：DPSC、DPPC、POPC、DOPE和SM。PEG修饰的脂质的实例是：PEG-DMG、PEG-CerC14和PEG-CerC20。

在一个实施方案中，所述方法是体外或离体方法。体外方法在细胞培养物上进行。

在另一个方面，本公开涉及通过上述方法可获得或获得的分离的工程化细胞。所述工程化细胞至少包含如上所述的编码层粘连蛋白-α2蛋白的转基因。

在一个实施方案中，分离的工程化细胞包含至少一个编码层粘连蛋白-α2的转基因，其包括指导所述转基因在细胞中表达的任何一个或几个控制序列，如上所述。

在一些实施方案中，分离的工程化细胞包含至少一个编码层粘连蛋白-α2的转基因，其包含如上所述的侧翼ITR。

在一个实施方案中，inter-ITR大小为至少105bp、至少200bp、至少300bp或更大。

本公开的工程化细胞可以用于离体基因治疗目的。在这些实施方案中，将如上所述的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)，引导RNA和LAMA2转基因或一个或多个核酸构建体、表达载体或病毒颗粒引入细胞。所述分离的细胞随后可以移植到患者或受试者。所述植入步骤可以使用本领域已知的任何植入方法来完成。例如，遗传修饰的细胞可以直接注射到患者的血液中或注射到目标肌肉中，以其他方式施用于患者。移植的细胞可以具有自体的、同种异体的或异源的来源。特别地，所述细胞可以是从供体或患者骨骼肌、平滑肌或心肌分离的肌细胞，或是来自骨髓细胞或外周血的间充质干细胞。对于临床应用，细胞分离通常将在良好的生产实践(GMP)条件下进行。

合适的细胞包括但不限于真核和原核细胞和/或细胞系。优选地，所述细胞是真核细胞，例如哺乳动物细胞，这些包括但不限于人，非人灵长类动物，例如猿、黑猩猩、猴子和红毛猩猩(orangutan)，驯养动物，包括狗和猫，以及家畜如马、牛、猪、绵羊和山羊，或其它哺乳动物物种，包括但不限于小鼠、大鼠、豚鼠、兔、仓鼠等。本领域技术人员将根据待移植的患者或受试者选择更合适的细胞。

所述工程化细胞也可以是肌肉细胞。如本文所用，术语“肌肉”是指心肌(即心脏)和骨骼肌。如本文所用，术语“肌肉细胞”指肌细胞、肌管、成肌细胞和/或卫星细胞。

所述工程化细胞可以是具有自我更新和多能性特性的细胞，例如干细胞或诱导多能干细胞。合适的干细胞还包括例如胚胎干细胞、诱导多能干细胞(iPSC)、造血干细胞、神经元干细胞和间充质干细胞。干细胞优选是间充质干细胞。间充质干细胞(MSC)能够分化成成骨细胞、软骨细胞、脂肪细胞或肌细胞中的至少一种，并且可以从任何类型的组织分离。通常，MSC从骨髓、脂肪组织、脐带或外周血中分离。获得它们的方法是本领域技术人员公知的。诱导多能干细胞(也称为iPS细胞或iPSC)是一类可直接从成年细胞产生的多能干细胞。Yamanaka等人通过将Oct3/4、Sox2、Klf4和c-Myc基因转移到小鼠和人成纤维细胞中，并迫使细胞表达这些基因来诱导iPS细胞(WO 2007/069666)。Thomson等人随后使用Nanog和Lin28代替Klf4和c-Myc来生产人iPS细胞(WO 2008/118820)。在某些实施方案中，所述细胞是成肌细胞。成肌细胞可以来源于干细胞，例如iPSC，包括来源于患有肌肉障碍如肌营养不良的患者的iPSC。

本公开的组合物优选以药物组合物的形式使用，所述药物组合物包含治疗有效量的本发明的产品，例如编码所述LAMA2转基因的核酸构建体、编码第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的核酸构建体、包含引导RNA的多核苷酸或核酸构建体、编码所述产品的表达载体或病毒颗粒。

在本公开的上下文中，治疗有效量是指足以逆转、减轻或抑制所述术语所应用的病症或病况的进展，或逆转、减轻或抑制所述术语所应用的病症或病况的一种或多种症状的进展的剂量。

有效剂量的确定和调整取决于多种因素，例如所用的组合物，施用途径，所考虑的个体的身体特征如性别、年龄和体重，同时给药以及医学领域技术人员将认识到的其它因素。

在本公开的各种实施方案中，药物组合物包含药学上可接受的载体和/或媒介物。

“药学上可接受的载体”是指当适当地施用于哺乳动物，尤其是人时，不产生不利的、过敏的或其它不良反应的媒介物。药学上可接受的载体或赋形剂是指任何类型的无毒固体、半固体或液体填料，稀释剂，包封材料或制剂助剂。

优选地，药物组合物含有对于能够被注射的制剂是药学上可接受的媒介物。这些可以特别是等渗的、无菌的盐水溶液(磷酸二氢钠或磷酸氢二钠、氯化钠、氯化钾、氯化钙或氯化镁等或这些盐的混合物)，或干燥的、特别是冷冻干燥的组合物，在根据情况向其添加无菌水或生理盐水后，允许配制可注射溶液。

适于注射使用的药物形式包括无菌水溶液或悬浮液。溶液或悬浮液可以包含与细胞相容的添加剂。溶液或悬浮液可以包含与非病毒载体、病毒载体和纳米颗粒相容并且不阻止组分进入靶细胞的添加剂。在所有情况下，所述形式必须是无菌的，并且必须是流体，达到能够容易注射的程度。它在生产和储存条件下必须是稳定的，并且必须在保存下抵抗微生物如细菌和真菌的污染作用。合适的溶液的实例有缓冲液，例如磷酸盐缓冲盐水(PBS)或林格乳酸盐(Ringer lactate)。

本公开的组合物用于治疗，尤其是用于治疗LAMA2相关的肌营养不良。

LAMA2相关的肌营养不良是引起用于运动的肌肉(骨骼肌)虚弱和消耗(萎缩)的疾病。这种病症的严重程度不同，从严重的早发型到较温和的迟发型。

早发型的LAMA2相关的肌营养不良显现于出生时或在生命的最初几个月内。它被认为是称为先天性肌营养不良的一类肌肉疾病的一部分，有时也被称为先天性肌营养不良1A型。受影响的婴儿可能具有严重的肌无力、肌张力缺乏(张力减退)、自发运动少和关节畸形(挛缩)。面部和咽喉肌肉的虚弱会导致进食困难，并且不能以预期的速度生长和增重。当胸腔中的肌肉变弱时发生的呼吸功能不全引起哭泣弱并产生呼吸问题，这可导致频繁的、潜在地威胁生命的肺部感染。

随着患病儿童的成长，他们经常发展为异常的、逐渐恶化的脊柱左右弯曲(脊柱侧凸)和背部向内弯曲(脊柱前凸)。患有早发型的LAMA2相关的肌营养不良的儿童通常无法建立行走能力。言语困难可能是由于面部肌肉的虚弱和舌头变大。癫痫发作发生在约三分之一的患有早发型的LAMA2相关的肌营养不良的个体中；在极少情况下，在这种形式的病症中发生心脏并发症。

迟发型的LAMA2相关的肌营养不良的症状在儿童期或成年期后期变得明显，并且与被分类为肢带型肌营养不良(limb-girdle muscular dystrophy)的一组肌肉病症的症状相似。在晚发型的LAMA2相关的肌营养不良中，最受影响的肌肉是那些最接近身体的肌肉(近端肌肉)，特别是肩部、上臂、骨盆区和大腿的肌肉。患有迟发型的LAMA2相关的肌营养不良的儿童有时具有运动技能如行走的延迟发育，但通常能达到在没有帮助下行走的能力。随着时间的推移，他们可能出现背部僵硬、关节挛缩、脊柱侧凸和呼吸问题。然而，大多数受影响的个体保有步行和爬楼梯的能力。

本公开还提供了本公开的治疗LAMA2相关的肌营养不良的方法，包括：向患者施用治疗有效量的如上所述的组合物、工程化细胞或药物组合物。

“治疗有效量”是指这样的量，其在获得期望治疗结果所需的剂量和时间段内是有效的，并且预防、延迟或逆转LAMA2相关的肌营养不良的至少一种或多种体征或症状，如骨骼肌无力和萎缩。本公开的产品或包含它的药物组合物的治疗有效量可以根据多种因素而变化，例如个体的疾病状态、年龄、性别和体重，以及产品或药物组合物在个体中引起期望的响应的能力。可以调整剂量方案以提供最佳治疗响应。治疗有效量通常也是产品或药物组合物的任何毒性或有害作用被治疗有益效果所超过的量。

如本文所用，术语“患者”或“个体”表示哺乳动物。优选地，本公开的患者或个体是人。

在本公开的上下文中，如本文所用的术语“治疗”意指逆转、减轻或抑制LAMA2相关的肌营养不良或所述术语所应用的病症的进展，或逆转、减轻或抑制所述术语所应用的病症或病况的一种或多种症状的进展，尤其是减少骨骼肌的虚弱和萎缩和/或改善骨骼肌功能。

本公开的药物组合物通常根据已知程序以在患者中有效诱导治疗效果的剂量和时间段施用。

施用可以是全身的或局部的。全身施用优选胃肠外施用，如皮下(SC)、肌内(IM)、血管内如静脉内(IV)或动脉内、腹膜内(IP)、皮内(ID)、经间质(interstitial)或其他。施用可以是例如通过注射或灌注。在一些优选的实施方案中，施用是胃肠外施用，优选血管内如静脉内(IV)或动脉内。除非另有说明，否则本公开的实施将采用本领域技术人员所熟知的常规技术。这些技术在文献中有充分的解释。

本公开还提供了用于实施所公开的方法的试剂盒，如本文所述。试剂盒可含有第一和第二蛋白(单独地或作为上述融合蛋白的一部分)或编码它们的核酸构建体，以及包含如本文所述的LAMA2转基因的核酸构建体。在一些方面，试剂盒可包含慢病毒颗粒，所述慢病毒颗粒包含编码第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的核酸构建体以及包含本文所述的LAMA2转基因或由本文所述的LAMA2转基因组成的核酸构建体。

本发明的试剂盒还可包括使用试剂盒的组分来实施本发明方法的说明书。用于实施本发明方法的说明书通常记录在合适的记录介质上。例如，说明书可以印刷在诸如纸或塑料等的基材上。因此，说明书可以作为包装插页存在于试剂盒中，存在于试剂盒或其组分的容器的标签中(即，与包装或子包装相连)，等等。在其它实施方案中，说明书作为存在于合适的计算机可读存储介质例如CD-ROM、磁盘等上的电子存储数据文件而存在。在其它实施方案中，实际的说明书不存在于试剂盒中，但提供了从远程来源，例如通过因特网获得说明书的手段。该实施方案的一个实例是试剂盒包括网址，在所述网址上可以查看说明书和/或可以下载说明书。与说明书一样，这种获得说明书的手段也记录在合适的基材上。

本公开通常涉及组合物或试剂盒，其包含：

第一组合物，所述第一组合物包含：

(i)如本文定义的第一蛋白，或编码所述第一蛋白的核酸，

(ii)如本文所定义的第二蛋白，或编码所述第二蛋白的核酸，和

(iii)包含LAMA2转基因或由LAMA2转基因组成的核酸构建体。

本公开还涉及组合物或试剂盒，其包含：

第一组合物，所述第一组合物包含：

(i)如本文定义的融合蛋白，或编码所述融合蛋白的核酸，

(ii)包含LAMA2转基因或由LAMA2转基因组成的核酸构建体。

在一个实施方案中，组合物或试剂盒包含在微环、质粒或病毒载体中，特别是在非整合病毒载体中，例如在非整合慢病毒载体中的外源核酸。在一个实施方案中，本文公开的组合物或试剂盒包含在纳米颗粒中。

在一个实施方案中，编码第一和/或第二蛋白(单独地或作为上述融合蛋白的一部分)的核酸构建体是RNA、DNA或蛋白的形式，编码LAMA2转基因的多核苷酸序列是RNA或DNA的形式，这取决于递送方法。特别地，编码外源核酸的多核苷酸序列是RNA形式。

在一个实施方案中，所述组合物或试剂盒是无病毒的，并且所述包装载体是纳米颗粒，例如聚合物的或脂质的纳米颗粒。包装载体也可以是与组合物的元件结合的载体。在一些实施方案中，组合物包含在病毒载体中，特别是慢病毒颗粒中。

在一个实施方案中，组合物或试剂盒包含(a)RNA形式的编码第一和第二蛋白的核酸构建体，所述第一和第二蛋白单独地或作为上述融合蛋白的一部分(例如包含Cas9和转座酶)，(b)如果需要的话，引导RNA(例如作为单独的线性单链RNA分子)，和(c)DNA形式的包含用于插入的LAMA2转基因或由其组成的多核苷酸(例如，在载体中)，其包含在包装载体中或与包装载体结合。

在一个实施方案中，所述组合物包含(a)蛋白形式的本文所述的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)(例如包含Cas9和转座酶)，(b)如果需要的话，引导RNA(例如作为单独的线性单链RNA分子)，其中融合蛋白和引导RNA形成核糖核酸蛋白复合物(RNP)，和(c)DNA形式的包含用于插入的LAMA2转基因或由其组成的多核苷酸(例如，在载体中)，其包含在包装载体中或与包装载体结合。

在一个实施方案中，所述组合物包含(a)DNA形式的编码第一和第二蛋白的核酸构建体，所述第一和第二蛋白单独地或作为上述融合蛋白的一部分(例如，包含Cas9和转座酶)，(b)如果需要的话，引导RNA(例如作为单独的线性RNA分子或作为载体中的DNA)，和(c)DNA形式的包含用于插入的LAMA2转基因或由其组成的多核苷酸(例如，在载体中)，其包含在包装载体中或与包装载体结合。

在一个实施方案中，所述组合物包含(a)蛋白形式的第一和第二蛋白，单独地或作为上述融合蛋白的一部分(例如，包含Cas9和整合酶)，(b)如果需要的话，引导RNA(例如作为与融合蛋白复合的单独的RNA分子)，和(c)包含用于插入的LAMA2转基因或由其组成的多核苷酸，其包含在包装载体中或与包装载体结合。在一个具体实施方案中，包装载体是慢病毒颗粒。在一些实施方案中，第一和第二蛋白，单独或作为上述融合蛋白的一部分，通过gag-pol或VPR(病毒蛋白R)与慢病毒衣壳结合。在一些实施方案中，包含LAMA2转基因或由其组成的多核苷酸为RNA形式，作为整合酶的载荷。

在一个实施方案中，当第一蛋白是ZFP时，可以不需要引导RNA。

本公开通常涉及试剂盒，其包含：

第一组合物，所述第一组合物包含：

(i)第一和第二蛋白，单独地或作为上述融合蛋白的一部分，或编码它们的核酸，其中所述第一和第二蛋白包含第一引导RNA切口酶Cas9(通常为具有SEQ ID NO：65的SpCas9切口酶)和修饰的高活性PiggyBac的氨基酸序列，和

(ii)第一引导RNA核酸，

第二组合物，所述第二组合物包含：

(iii)第一和第二蛋白，单独地或作为上述融合蛋白的一部分，或编码它们的核酸，其中所述第一和第二蛋白包含第二引导RNA切口酶Cas9(通常为具有SEQ ID NO：66的SaCas9切口酶)和修饰的高活性Piggybac的氨基酸序列，

(iv)第二引导RNA核酸，

以及包含LAMA2转基因的核酸构建体；

其中第一和第二蛋白(在第一和第二组合物中的每一者中)各自能够异二聚化，并由第一和第二引导RNA确定在基因组DNA区域的相邻位点处产生双切割，并且任选地在所述相邻位点之间插入所述核酸。

下文公开了一些另外的实施方案。

E1：一种组合物，其包含：a)包含第一蛋白和第二蛋白的融合蛋白，所述第一蛋白由能够结合靶核酸序列的位点特异性DNA结合蛋白组成，所述靶核酸序列优选地包含在白蛋白的内含子1、Lama2或Rosa 26基因座的内含子1内，所述第二蛋白由转座酶组成；或核酸构建体，优选编码所述融合蛋白的mRNA，b)包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因的核酸构建体。

E2：所述转基因编码SEQ ID NO：74的层粘连蛋白-α2蛋白。

E3：b)的核酸构建体包含选自以下的启动子：SEQ ID NO：76的CMV启动子、SEQ IDNO：77的CAG启动子、SEQ ID NO：78的EF1-α启动子、SEQ ID NO：79的SV-40启动子和SEQ IDNO：80的EalbAAT启动子或SEQ ID NO：81的剪接受体。

E4：b)的核酸构建体包含ploy(A)信号序列，优选选自SEQ ID NO：83-85。

E5：b)的核酸构建体还可以包含隔绝元件，优选选自SEQ ID NO：86-87。

E6：b)的核酸构建体侧翼为反向末端重复序列(ITR)，优选SEQ ID NO：88和89的5'-ITR和3'-ITR。

E7：b)的核酸构建体可以包含在选自以下的载体中：质粒载体、微环载体、犬骨DNA供体载体、慢病毒载体和逆转录病毒载体。

E8：所述位点特异性DNA结合蛋白是包含Cas蛋白的RNA引导的核酸酶，并且所述组合物还包含引导RNA，所述引导RNA包含靶核酸序列的互补序列，用于将所述LAMA2转基因整合在细胞基因组的特定位点中，优选地，所述Cas蛋白是酿脓链球菌Cas 9蛋白。

E9：所述引导RNA包含选自SEQ ID NO：90-97的序列的互补序列。

E10：所述转座酶是修饰的高活性Piggybac转座酶或睡美人转座酶，优选地，修饰的高活性PiggyBac转座酶与未修饰的高活性Piggybac相比包含一个或多个增加切除活性的氨基酸突变，并且与未修饰的高活性Piggybac相比包含一个或多个降低DNA结合活性的氨基酸突变。

E11：所述高活性PiggyBac转座酶是修饰的高活性PiggyBac转座酶，其包含选自以下的氨基酸的至少一个突变：M194、R245、G325、R372、K375、R376、E377、E380、D450和S573，优选包含氨基酸D450、R372和K375的突变，所述位置编号对应于SEQ ID NO：9的未修饰的高活性Piggybac的氨基酸编号。

E12：优选地，转座酶通过接头在N-末端与所述位点特异性DNA结合蛋白融合，所述接头优选为包括GGS、XTEN或FOKI的肽接头，更优选地为SEQ ID NO：53的XTEN。

E13：所述组合物被包装在纳米颗粒中。

E14：将LAMA2转基因整合到细胞基因组内的靶核酸序列的体外方法，包括将上述组合物引入细胞，以及可通过所述方法获得的工程化细胞，其包含整合到其基因组中的LAMA2转基因。

E15：药物组合物，其包含如上所述的组合物或工程化细胞，任选地与一种或多种药学上可接受的赋形剂组合。

E16：所述组合物、工程化细胞或药物组合物用于治疗，特别是用于在有此需要的受试者中治疗分区蛋白缺陷型先天性肌营养不良1A型(MDC1A)。

现在将参照附图，用下列非限制性的实施例来举例说明本发明。

附图说明

图1：已经产生的用于在MCD1A的Lama2动物模型中进行治疗性基因置换的不同Lama2载荷的代表图。

图2：用装载GFP或Lama2的慢病毒感染的Hek293T细胞上的病毒拷贝数。基于拷贝数的qPCR估计来计算滴度。由于Lama2而增加的载货大小将病毒生产效率降低了2个数量级。

图3：循环白细胞(A)和浸润的肌细胞(B)的FACS分析；在移植后4周从小鼠分离后。显示了空的慢病毒转导和装载GFP的病毒的比较。

图4：用于离体慢病毒介导的Lama2基因转移至骨髓衍生细胞以治疗MCD1A模型的实验设计和工作流程。

图5：用表达Lama2的骨髓衍生细胞移植6周和12周后，经治疗和未经治疗的MCD1A模型相比于野生型动物的相对力量(A)。在相同动物中治疗后的肌肉力量增加(B)。在两种情况下的结果显示了握力测定的分析。

图6：经治疗和未经治疗的MCD1A模型相比于野生型动物的随时间的Kaplan Meier存活曲线。

图7：用于体内的无病毒的转座酶介导的Lama2基因转移到肝细胞中以治疗MCD1A模型的实验设计和工作流程。

图8：转基因拷贝数(A；C)和转基因表达(B；D)；在肝细胞中处理后4周分别通过qPCR和RT-qPCR测量。显示了RFP基因报告子的数据，其通过高活性PiggyBac稳定整合(A；B)；和Lama2基因的数据，其通过与工程化PiggyBac融合的可编程转座酶Cas9而稳定整合(C；D)。

图9：在用Lama2处理MDC1A模型1周和4周后，用Elisa测试循环血清中层粘连蛋白的存在，其中Lama2与或不与用以稳定表达的插入机制(insertion machinery)共转染。

图10：(A)单独的RFP转座子(游离体)或与hyPB或FICAT R372A_K375A_D450N mRNA一起，使用体内JetPEI试剂递送，用于靶向小鼠基因组中的Rosa26安全港。通过半定量qPCR测量肝脏中RFP转基因的相对拷贝数，并相对于Tfrc基因(二倍体基因组)的相对双拷贝进行归一化。(B)微环荧光素酶转座子的肝整合。通过流体动力学注射递送微环荧光素酶转座子、靶向Rosa26基因座的sgRNA和FiCAT(Cas9-hyPB R372A_K375A_D450N)mRNA，并监测荧光素酶信号。(C)肝脏基因组DNA中转座子3'ITR与Rosa26基因座之间的连接PCR。向小鼠流体动力学注射FiCAT R372A_K375A_D450N质粒DNA或mRNA、靶向Rosa26基因座的gRNA和微环转座子GFP载荷，并在注射后5周处死。进行PCR以扩增基因组+链整合。n＝2-3只动物/条件，数字对应于不同的个体。66％的用FiCAT mRNA或pDNA处理的小鼠显示靶向插入。在FICAT中检测到的条带的大小对应于扩增插入的预期大小。在视为背景的游离体样品中检测到大小较大的条带。

图11：(a)C2C12细胞用单独的RFP转座子(游离体)转导，或用与FiCAT R372A_K375A_D450N和靶向Lama2基因的gRNA(间隔区271.1)组合的RFP转座子转导，转导后监测RFP阳性细胞2周。对n＝2的技术重复的平均值±SD作图。n＝3的代表图像。(b)Karyoplot显示在c2c12基因组中检测的插入。(c)以+链(1，3)和-链(2，4)载荷插入显示了3'ITR和Lama2基因座之间的连接PCR(下图)，比较了FiCAT(3，4)和游离体(1，2)处理的富集群体。n＝3的代表图像。(d)在中靶连接(Lama2位点)处的覆盖。

图12：相对于具有300bp inter-ITR大小的插入，在报告细胞系中的AAVS1位点中的1/2GFP转座子的靶向插入。用与高活性PiggyBac(hyPB)R372A_K375A_D450N(Cas9-PBx3)或hyPB R372A_K375A_D450N的二聚体(Cas9-PBx3-PBx3)融合的Cas9，测试105bp、200bp和300bp的inter-ITR大小的1/2GFP的插入。

图13：可以用不同的Cas变体来工程改造可编程转座酶，所述Cas变体例如Casx、CjCas9 Cpf1或SaCas9，其中一些在靶位点处的可编程插入方面获得与SpCas9类似的结果。用3个独立的gRNA将每种测试的Cas变体靶向分裂GFP报告细胞系的特定靶区域。

图14：FiCAT R372A-K375A-D450N的可编程插入活性，使用四种不同的核酸酶蛋白。SpCas9用作对照，用于仅用gRNA-TRAC-1进行的可编程插入(左)。每种核酸酶均与三个独立的gRNA(1-3)一起用于在1/2GFP报告细胞系中的靶向插入。

图15：(a)通过Casx(左)和Cpf1(中)的编辑活性。(b)通过SaCas9(左)、CjCas9(中)的编辑活性。对于两个技术重复显示了具有插入缺失的读数的平均值％+/-SD，N＝3个生物学重复的代表性图像。靶向TRAC-1位点的SpCas9用于参照(右)。

图16：可编程转座酶可以作为两个hyPB结构域和Cas9核酸酶的二聚体多肽而工程化，导致与Cas9-hyPB相比更好的可编程插入。将分裂GFP报告细胞系用于将分裂GFP转座子可编程插入靶位点。hyPB R372A-K375A-D450N的突变体已用于与Cas9融合的单体或二聚体。条件：1-仅用hyPB作为插入机制的阴性对照；2：Cas9-hyPB R372A-K375A-D450N于pcDNA表达载体中的阳性对照；3：Cas9-hyPB R372A-K375A-D450N于慢病毒表达载体中的阳性对照；4：在C-末端与两个hyPB R372A-K375A-D450N单元融合的Cas9核酸酶；5：与hyPB R372A-K375A-D450N的两个单元融合的Cas9核酸酶，一个在C-末端融合，另一个在N-末端融合。

图17：发生了可编程转座的细胞的几轮选择能够从文库中选择最佳突变体组合。我们鉴定了几种突变体，当与Cas9融合时，它们与Cas9-hyPB R372A-K375A-D450N相比具有更好的富集和可编程插入的能力。

图18：中靶效率随选择轮数增加。将从每轮中选择的多个变体与靶向AAVS1的gRNA和1/2GFP转座子共转染到报告细胞系中。质粒的量通过PB拷贝数校正，以针对克隆效率归一化。

图19：(A)所选择的几个最佳候选物的中靶效率。基于从最后一轮选择的96个随机克隆中的最高中靶活性，选择了六个候选物个体。将个体的中靶活性与Cas9-hyPB R372A-K375A-D450N进行比较。(B)Logo显示了在几个最佳中靶活性变体中的主要PB残基。

图20：通过Cas9和单gRNA(gRNA-TCR1或AAVS1-3)或通过切口酶Cas9和靶向附近位置的两个gRNA(gRNA-TCR1和AAVS1-3)，以及与修饰的hyPB(突变体R372A-K375A-D405N)融合的可编程DNA结合结构域(ZnF)产生的双链断裂导致了靶向插入。为了有效的中靶插入，需要双链断裂和PB在插入位点上的共定位。这可以通过核酸酶Cas9或切口酶Cas9的双切割来实现。

图21：与SpCas9或SaCas9融合的二聚的hyPB R372A-K375A-D450N用于1/2GFP报告细胞系中靶向插入的可编程插入活性。

图22：中靶效率随选择轮数而增加。(A)将从每轮选择的多个变体与靶向AAVS1的gRNA和1/2GFP转座子共转染到报告细胞系中。质粒的量通过PB拷贝数校正，以针对克隆效率归一化。(B)产生表达每轮的多个变体的慢病毒，并用于感染报告细胞系。

图23：在用gRNA tcr1和1/2GFP MC转座子共转染的cas9_PB文库4和5轮富集之后，从多个变体分离的单个突变体相对于FiCAT(hyPB R372A-K375A-D450N)的特异性靶整合。

图24：靶向插入1/2GFP报告细胞系的可编程插入活性的相对比较。(A)与SpCas9蛋白融合的hyPB R372A-K375A-D450N(左)和与MCP蛋白融合的hyPB R372A-K375A-D450N且单独添加SpCas9(右)之间的比较。(B)单独添加SpCas9的与MCP蛋白融合的3个hyPB突变体(R372A-K375A-D450N、R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)之间的比较。

图25：靶向插入1/2GFP报告蛋白细胞系的可编程插入活性的比较。(A)hyPBR372A-K375A-D450N与SpCas9蛋白的共表达(左)和包含hyPB R372A-K375A-D450N和SpCas9蛋白的融合蛋白(右)之间的比较。(B)与SpCas9共表达的3个hyPB突变体(R372A-K375A-D450N、R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)之间的相对比较。

图26：包含SpCas和hyPB R372A-K375A-D450N的第一融合蛋白以及包含MCP蛋白和hyPB突变体(R372A-K375A-D450N、R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)的第二融合蛋白的共表达的靶向插入到1/2GFP报告细胞系中的可编程插入活性的相对比较。

图27：包含SpCas和hyPB R372A-K375A-D450N的融合蛋白和3个hyPB突变体(R372A-K375A-D450N、R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)的共表达的靶向插入1/2GFP报告细胞系的可编程插入活性的相对比较。

图28：融合至hyPB R272A-K275A-D450N的二聚体的SpCas9(左)与融合至第一hyPBR272A-K275A-D450N且融合至第二hyPB突变体的SpCas9(右)之间的靶向插入1/2GFP报告细胞系的可编程插入活性的比较。

实施例

1.LAMA2载荷结构

可以使用LAMA2编码序列，其包含用于输出的前导序列，用于小家鼠(Musmusculus)(SEQ ID NO：94)或智人(Homo sapiens)(SEQ ID NO：75)。LAMA2编码序列可以含有用于载荷特异性检测和基因转移后追踪的部分序列进行再编码(recoding)的序列，以将其与我们进行基因编辑的生物体中存在的内源基因区分开来。

将该LAMA2基因克隆到组成型启动子(SEQ ID NO：76-79)或组织特异性启动子(SEQ ID NO：80)或剪接受体(SEQ ID NO：81)的下游。具有剪接受体的形式可以整合在主动表达的基因的第一内含子上以具有对表达的內源控制。

所述编码序列还可以具有用于载荷的小基因形式的合成内含子(SEQ ID NO：82)。表达盒可含有poly-A信号(SEQ ID NO：83-85)和隔绝元件(SEQ ID NO：86-87)。

所述载荷可以是以下形式：

1.质粒载体，含有细菌复制起点和抗生素抗性。

2.微环载体，其除了质粒载体组分外还含有32个SceI限制酶靶位点和在Lama2表达盒侧翼的2个AttB和AttP重组位点。

3.犬骨DNA供体载体，其除了质粒载体组分外还含有在Lama2表达盒侧翼的TelN靶位点，用于供体DNA的载货末端的线性化和保护。

4.慢病毒或γ逆转录病毒载荷载体，除了慢病毒/逆转录病毒操作元件之外，其具有在Lama2表达盒侧翼的长末端重复，所述慢病毒/逆转录病毒操作元件可被工程化以减小大小，从而允许用于治疗性载货的额外空间。

在所有这些情况下，Lama2表达盒的侧翼可以是用于转座酶识别(Piggybac和睡美人)的反向末端重复(ITR)。

图1显示了含有Lama2转基因的不同载货的图示。

发明人进一步测试了载荷的ITR之间的大小是否会影响融合蛋白的结合能力，并因此影响其随后转移到基因组中。尝试了三种inter-ITR大小用于载荷效率的靶向插入：大约300bp、200bp和105bp，在具有FiCAT(Cas9+在残基R372A_K375A_D450N上突变的高活性PiggyBac转座酶)和FiCAT二聚体(Cas9+在残基R372A_K375A_D450N上突变的高活性PiggyBac转座酶的二聚体)和靶向报告细胞系中AAVS1位点的gRNA的1/2GFP转座子的微环(MC)载荷中(图12)。最佳的inter-ITR大小是300bp和200bp，而105bp的靶向整合效率较低。

除了基于慢病毒和逆转录病毒的递送之外，不同的Lama2载荷可使用polyplex和脂质纳米颗粒递送，或通过电穿孔或显微注射作为裸核酸转导到靶细胞中。

2.Lama2载荷的离体转导

使用用于第二代慢病毒生产的标准方案，生产含有处于CMV启动子控制下的小家鼠(mus musculus)Lama2编码序列的慢病毒颗粒。考虑到该载荷的尺寸大，将颗粒浓缩，并通过感染Hek293T细胞后的病毒拷贝数qPCR估计来计算病毒滴度(图2)。

尽管与报告基因GFP相比，生产效率低两倍，但该滴度足以用于离体转导细胞，该细胞随后可用于Lama2小鼠模型中的细胞治疗。采用两种不同的方法。将分离的肌肉干细胞转导并移植到受体模型的肌肉中。或者，用含有Lama2表达盒的慢病毒转导衍生自骨髓细胞的造血祖细胞，然后可通过静脉内注射将这些祖细胞移植到条件化模型(conditionedmodel)中。

作为离体治疗的概念验证，本发明人分离了骨髓来源的造血祖细胞，将它们首先用表达GFP的慢病毒转导，然后移植到条件化小鼠(conditioned mice)中。然后，本发明人通过观察循环血液中白细胞的GFP表达来检查处理后4周的移植，并发现阳性结果(图3A)。此外，还检查了循环细胞向发炎肌肉的浸润，并发现阳性结果(图3B)。

鉴于离体转导的造血祖细胞的阳性浸润和载荷表达，本发明人然后用表达Lama2的慢病毒颗粒重复所述实验(图4)。小鼠在治疗后12周恢复肌肉力量，通过握力测定法测量(图5A和5B)。此外，还观察了所治疗的Lama2动物模型的存活(图6)，表明离体细胞转导和移植对治疗MDC1A患者的治疗价值。

在另一系列实验中，本发明人进行了RFP报告子至C2C12鼠成肌细胞系中的LAMA2靶向插入，效率为～20％(图11)。使用连接PCR和(STAT)-PCR来测量中靶和脱靶效率。这种提高的效率是使用包含Cas9和在残基R372A_K375A_D450N上突变的高活性PiggyBac转座酶的融合蛋白(称为FiCAT)的结果。

3.Lama2载荷的体内转导

鉴于肝脏作为体内蛋白生产的生物反应器的能力，本发明人研究了使用基于转座酶的无病毒基因递送方法，用Lama2表达载体转导动物模型的体内肝细胞的可能性。

将用体内JetPei(PolyPlus)配制的高活性PiggyBac转座酶或经工程改造的高活性PiggyBac与可编程核酸酶蛋白(SpCas9)的融合转座酶作为质粒载体或mRNA分子和转座子Lama2质粒载体(图7)静脉内注射到动物模型中，3至4周后收集组织，用于获得肝脏中的载荷拷贝数(RFP数据作为概念验证)(图8A、8C)和肝脏中的表达分析(RFP，图8C；Lama2，图8D)以及Lama2的循环血液(图9)。注射1周后采集100μL血液，并在终点采集全血。

处理4周后肝细胞中转基因拷贝数显示阳性结果，并进一步与RFP报告基因比较，证实了不同大小的载荷具有相似的转导效率(图8A，8B)。当没有共同递送转座酶时，利用游离体递送为参考。RFP报告子和Lama2基因的转基因表达也在肝细胞中得到证实(图8B，8D)，并且在递送后1周和4周的血液中检测Lama2表达(图9)。

4.用于体内基因递送的改进工具

为了改进用于体内基因递送的可编程转座酶，本发明人针对FiCAT(Cas9-hyPBR372A_K375A_D450N)使用了质粒DNA(pDNA)和mRNA两者并将其以质粒或MC形式递送至小鼠肝，靶向Rosa26基因组安全港(编码RFP或荧光素酶的转座子，用于概念验证)。与内源基因TFRC相比，观察到高拷贝数的转基因(图10A)，以及转基因表达随时间保持(图10B)。使用3'ITR和基因组基因座之间的连接的PCR，来测量新形成的中靶插入(图10C)。

4.1.与hyPB融合的Cas变体的结果

为了进一步表征工程化的hyPB进行可编程转座的能力，我们将测试的可编程转座酶的SpCas9模块替换为来自不同生物体的具有核酸酶活性的Cas蛋白(即SEQ ID NO：72的SaCas9、SEQ ID NO：74的cpf1、SEQ ID NO：75的Casx和SEQ ID NO：29的CjCas9)。设计并克隆靶向分裂GFP报告子上游区域的特异性gRNA，用于Hershey细胞系转染。通过GFP表达测定靶向转座(图13)。

这些结果在另一系列实验中得到证实：我们获得了CjCas9和LbCpf1的良好的可编程插入活性，而Casx在我们的测定中没有实现任何可编程整合。值得注意的是，SaCas9在测试的Cas蛋白中具有最高水平的可编程插入，其水平与融合至修饰的hyPB的SpCas9相似(图14)。通过Ilumina NGS测定了所用的不同Cas蛋白和为每种蛋白设计的三种不同gRNA的插入缺失(图15)，以归一化显示。

这些积极的结果证实了用于可编程转座的工程化的hyPB对于任何序列特异性核酸酶模块都是有用的。

4.2.与PB突变体的二聚体融合的Cas9的进一步结果

鉴于PB在进行转座时以二聚体发挥作用的性质，我们尝试产生了Cas9和hyPBR372A-K375A-D450N突变体的融合蛋白。我们比较了这些融合物与单独的Cas9-PB突变体的中靶活性。我们观察到Cas9-PB-PB构型具有更好的性能；而PB-Cas9-PB构型并不优于Cas9-PB单体融合物(图16)。对于与Cas9的二聚融合物，我们使用记录版本的hyPB R372A-K375A-D450N突变体来促进克隆和表达。

有趣的是，如果融合至二聚hyPB R372A-K375A-D450N的Cas9是SaCas9而不是SpCas9，则活性进一步增加(图21)。在二聚体hyPB的情况下，SaCas9的性能与SpCas9相比增加与在单体hyPB情况下获得的结果一致(图13和14)。

4.3.ZNF-PB突变体恢复的结果

我们想进一步探索由促进由SpCas9切口酶变体(D10A)进行的单链切割的两个gRNA在靠近(4个核苷酸)靶位点诱导的双链断裂(DSB)活性在促进靶向整合，同时通过非诱导性DSB降低在脱靶位点中的脱靶活性中的作用。通过与D450N突变体和R372A-K375A-D450N突变体融合，我们使用锌指-PB融合物来指导定位转座子，并使用通过独立的切口酶Cas9产生的两个在位(on-site)单链断裂或通过Cas9核酸酶产生的单个DSB对其进行补充。

Znf-PB融合物没有表现出靶向插入活性或表现出非常低的靶向插入活性，当与使用单或双gRNA引导的-Cas9(核酸酶或切口酶)在Znf结合位点附近引入DSB组合时，该靶向插入活性得以恢复(图20)。

4.4Cas9-hyPB突变变体的结果

为了进一步研究能够以更好的效率进行可编程转座的突变组合，进行了数轮细胞选择，其中通过分裂GFP报告系统的可编程插入来重构GFP。有趣的是，我们观察到了有几种组合表现优于Cas9-hyPB R372A-K375A-D450N(图17)。特别值得提及的是，在HyPB上在AA：A351-A372-A375-A388-N450-A465-A573-V589-G592-L594处突变的融合至Cas9的hyPB变体(也鉴定为SEQ ID NO：2)，与R372A-K375A-D450N(SEQ ID NO：1)相比，其在阳性细胞群中富集了数倍；以及程度较低的A245-A275-A277-A372-A465-V589(SEQ ID NO：3)和A275-A325-A372-A560(SEQ ID NO：4)。

在另一系列实验中，PiggyBac DNA文库由Twist Bioscience产生，与Cas9融合克隆至慢病毒载体中，并转化至stb4感受态细胞中，确保x100变体复杂度(variantcomplexity)。通过maxiprep纯化质粒，并与慢病毒包装质粒共转染到Hek293T细胞中。慢病毒用于感染1/2GFP报告细胞系。感染的细胞用1/2GFP转座子和靶向AAVS1序列的gRNA转染。通过流式细胞术分选选择GFP阳性细胞，并提取基因组DNA。从提取的gDNA扩增PB，再克隆到慢病毒载体中以重新开始新的一轮。选择表现最佳的可编程转座酶变体，并分别用AAVS1gRNA和MC1/2GFP转染。

首先，随机选择96个变体，并单独筛选表现最佳的变体(图18)。对高中靶插入的最佳PB氨基酸变体的总结证实了突变D450N、R372A和K375A的重要性；但也凸显了有助于提高靶向效率的其它重要残基(图19B)。选择具有最佳中靶效率的六种PB变体(图19A)。与FiCAT(Cas9-hyPB R372A-K375A-D450N)相比，使用以下变体的单独中靶活性显著提高：N347A-D450N、N347S-D450N-T560A-S573A-F594L、R202K-R275A-N347S-R372A-D450N-T560A-F594L、R275A-N347S-K375A-D450N-S592G、R275A-N347S-R372A-D450N-T560A-F594L和R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L(双向t检验)。

重复该实验并进行验证(图22A)。我们还生产了表达每轮的多个变体的慢病毒和感染的报告细胞系，通过PB变体CN校正了其滴度，从而证明中靶效率随轮数的类似增加(图22B)。在4轮和5轮的Cas9_PB文库富集之后，从多个变体分离单个突变体。通过用FiCAT突变体、gRNA tcr1和1/2GFP MC转座子转染中靶报告细胞系，单独对突变体进行了测试。显示了与FiCAT R372A_K375A_D450N相比的最佳FiCAT突变体(图23)。与FiCAT(Cas9-hyPB R372A-K375A-D450N)相比，使用以下变体显著提高了单独的中靶活性：R202K-R275A-N347S-R372A-D450N-T560A-F594L、R245A-N347S-R372A-D450N-T560A-S564P-S573A-S592G、R275A-N347S-R372A-D450N-T560A-F594L、N347A-D450N、R277A-G325A-N347A-R375A-D450N-T560A-S564P-S573A-S592G-F594L、N347S-D450N-T560A-S573A-F594L、V34M-R275A-G325A-N347S-S351A-R372A-K375A-D450N-T560A-S564P、G325A-N347S-K375A-D450N-S573A-M589V-S592G、S230N-R277A-N347S-K375A-D450N、T43I-R372A-K375A-A411T-D450N、G325A-N347S-S351A-K375A-D450N-S573A-M589V-S592G、Y177H-R275A-G325A-K375A-D450N-T560A-S564P-S592G。

在包含SpCas9和两个hyPB的三重融合蛋白中进一步证实了突变体R202K-R275A-N347S-R372A-D450N-T560A-F594L、R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L和R275A-N347S-R372A-D450N-T560A-F594L与突变体R372A-K375A-D450N相比的优越性(图28)。

4.5.Cas9和hyPB非共价连接的结果

除了通过接头将Cas9与hyPB R372A-R375A-D450N共价结合之外，我们使用MS2-MCP系统，通过含有结合MCP蛋白的MS2序列的四环的修饰的gRNA，将Cas9和由MCP蛋白和hyPB R372A-R375A-D450N组成的融合蛋白连接。

与Cas9-hyPB R372A-R375A-D450N融合蛋白相比，MCP-hyPB R372A-R375A-D450N融合蛋白与Cas9的组合具有增加的可编程插入活性(图24A)。此外，我们将MCP蛋白与hyPB的其他突变体融合，以与SpCas9组合进行可编程转座。所用的两种变体(R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)均优于R372A-R375A-D450N(图24B)。

4.6.用于可编程转座的Cas9和hyPB解偶联的结果

我们还尝试了在没有接头也不使用MS2-MCP系统的情况下的hyPB R372A-R375A-D450N和SpCas9的性能。我们在同一细胞中共表达SpCas9和hyPB R372A-R375A-D450N，并且记录了与Cas9-hyPB R372A-R375A-D450N融合蛋白相比的可编程插入活性增加(图25A)。我们扩展了测试的hYPB突变变体的数目，所述hYPB突变变体不与Cas9融合，但同时表达，并且一起作用以实现可编程转座的活性(图25B)。

4.7.Cas9-hyPB和MCP-hyPB融合蛋白的共表达的结果

我们共转染融合至MCP蛋白的hyPB R372A-R375A-D450N突变体和融合至SpCas9的hyPB突变体，以便获得其中一个单体非共价连接的融合物的二聚体版本。比较了融合至SpCas9的几种hyPB突变体的特异性靶整合(图26)。

4.8.Cas9-hyPB和hyPB变体的共表达的结果

以类似的方式，我们独立地共转染了SpCas9 hyPB R372A-R375A-D450N融合蛋白和hyPB突变体，以获得其中一个单体未连接的融合物的二聚体版本(图27)。

材料和方法

慢病毒的生产、浓缩和滴定

为了生产病毒，使用pSICO(GFP)或pLV-Lama2(Lama2)和pmd2.g(VSVG＝包膜)和pax2(含有包装蛋白，包括IN)，并且有时使用仅含有wt-整合酶以使非感染性整合酶恢复的质粒，来共转染细胞。首先将6×10⁵个HEK392T细胞(第8轮传代)接种在6孔组织平板的孔中并孵育过夜。在开始病毒生产前5小时，将细胞培养基更换成每孔为含1:1000CD(二磷酸氯喹；储备液＝25mM)的1.7mL培养基。以1.6：1.32：0.72：3.32(pSICO：pax2：VSVG：wtIN-恢复)的摩尔比感染质粒。PEI(聚乙烯亚胺；储备液＝1mg/mL)用作转染试剂，而针对1μg用于转染的总DNA使用3μL PEI。将DNA稀释于83μL Opti-MEM(Thermo Scientific；#31985062)中，并将PEI稀释于另一83μL中。将两种溶液混合后，在室温下温育15-20分钟。将每种转染混合物逐滴添加到CD培养基中的细胞中。将细胞孵育过夜。第二天更换培养基，并添加2.5mL新鲜培养基。第二天，将细胞上清液以1000rpm离心5分钟，并通过45μm过滤器。在4℃下以19,500rpm超速离心90分钟，并以1:100的原始培养基体积重悬过夜。将上清液贮存于-80℃。

为了测定病毒滴度，用产生的病毒感染HEK293T细胞，并计数GFP阳性细胞的量，因为GFP在病毒包装序列上编码。因此，在6孔板的每孔中接种75000个HEK393T细胞。用含有1:1000聚凝胺的1mL培养基和500μL先前生产的病毒上清液(1:3)的混合物感染细胞。次日更换培养基。再次日，吸出培养基，并使用200μL胰蛋白酶分离细胞。加入800μL正常培养基终止反应，并用细胞计量术分析。在非荧光Lama2载荷的情况下，从细胞提取gDNA并进行qPCR以确定相对于基因组的载荷拷贝数。

体内Lama2递送

动物实验程序得到巴塞罗那生物医药研究中心(Barcelona BiomedicalResearch Park)的动物实验伦理委员会的批准。C57BL/6J或Lama2模型dj2y/dj2y，8-10周龄，用于所述研究。所述动物购自Jackson Laboratories，使用雄性和雌性动物而不作区分。

在8周龄处死供体小鼠，从后肢骨收集骨髓细胞；使用小鼠谱系细胞耗减试剂盒(Mouse Lineage Cell Depletion Kit，Miltenyi Biotec)按照制造商说明书进行谱系阴性耗减；并在具有适当刺激因子的干细胞培养基(Stem Cell Technologies)中用慢病毒转导细胞过夜。然后收获这些细胞并通过眼眶后注射移植到受体小鼠中(在通过辐射进行条件化后)。

移植后4周，从经治疗的小鼠收集血液和胫骨肌。在FACS分析之前，根据制造商的说明书，使用裂解缓冲液(Thermo Fisher Scientific)处理血液样品。在FACS分析之前，将胫骨肌切碎，进一步用librase/分散酶(dispase)消化处理，以分离浸润的细胞。

治疗后6周和12周，根据制造商的说明书，使用Bioseb装置进行握力测定。

用RiboMAX大规模RNA生产系统-T7(Promega)，按照制造商的说明生产hyPB mRNA。Rosa26 gRNA(25)购自IDT。以1:2.5:2.5的比例通过眼眶后注射hyPB mRNA、靶向Rosa26的gRNA和PB512-B或Lama2转座子。将总共55μg核酸与In vivo-JetPEI(Polyplus转染)以NP比率7进行复合。注射后10天将动物安乐死，分离肝脏并匀浆。从肝脏样品中提取基因组DNA和RNA。分别通过qPCR、RT-qPCR或Elisa试验获得相对于Tfrc内源基因的转座子相对拷贝数和转基因相对表达。

使用IVIS光谱成像系统(Caliper Life Sciences)，在施用FiCAT-gRNA-转座子或转座子对照后的不同时间点进行荧光素酶表达的成像。根据制造商的说明书，在腹膜内注射D-荧光素钾盐(Gold Biotechnology)后5分钟进行成像。

从肝脏中收获gDNA和RNA

根据Blood&Tissue Kit Protocol进行基因组DNA提取。将肝组织在PBS(磷酸盐缓冲盐水)中匀浆。将20μL蛋白酶K(由试剂盒提供)与200μL缓冲液AL一起添加。涡旋后，将样品在56℃下温育10分钟。添加200μL乙醇(96-100％)并短暂涡旋后，将混合物转移至置于3mL收集管中的Dneasy Mini离心柱中，并以8000rpm离心1分钟。将离心柱移至新的2mL收集管中，并添加500μL缓冲液AW1。将管在8000rpm下离心1分钟。使用缓冲液AW2重复所述洗涤步骤(离心3分钟)。然后，将离心液转移至新的1.5mL微量离心管中，将200μL缓冲液AE添加到离心柱膜的中心，通过使管静置1分钟来洗脱DNA，然后以8000rpm离心1分钟。用NanoDrop(ThermoFisher Scientific)测量浓度。

qPCR和RT-qPCR

通过qPCR分析基因组DNA样品的25ng/μL和10ng/μL稀释液。将5μL的每种稀释液与4.4μL PowerUP SYBR Green MasterMix(Fisher Scientific)和0.3μL正向和反向寡聚物混合。使用靶向Lama2、RFP和TfrC内源基因的寡聚物(SEQ ID NO：100-105)。

使用高容量RNA至cDNA试剂盒(High-Capacity RNA-to-cDNA kit，ThermoFisherScientific)进行mRNA向cDNA的逆转录。在20μL反应中，将1μg mRNA样品与10μL缓冲液和1μL酶混合物混合。将反应在37℃下温育2小时，并在80℃下使失活10分钟。将RT产物稀释10倍，并将5μL样品与4.4μL PowerUP SYBR Green MasterMix(Fisher Scientific)以及0.3μL正向和反向寡聚物混合以进行qPCR。使用靶向Lama2、RFP和GAPDH内源基因的寡聚物(SEQID NO：102-107)。

酶联免疫吸附测定(ELISA)

在台式离心机中，将全血在4℃以1200xg离心10分钟。分离血浆并在80℃下储存直至进行ELISA测定。使用人层粘连蛋白亚基α-2(LAMA-2)ELISA试剂盒(Human Lamininsubunit alpha-2(LAMA-2)ELISA kit，Cusabio)。用80μL样品缓冲液稀释20μL血浆。将稀释的样品置于板中并孵育2小时。对孔进行吸出，但不洗涤，添加100μL生物素抗体。将板温育1小时，用洗涤缓冲液洗涤3次。将100μL HRP-抗生物素蛋白添加至每个孔中，并在37℃下温育1h。小心进行5次洗涤的洗涤步骤。添加90μL TMB底物，并将板在37℃下温育20分钟。添加50μL终止溶液，轻柔敲击平板，在5分钟内在平板读数仪中测定450nm处的光密度。

荧光激活细胞术分析(FACS)

分离肌肉中的循环白血细胞和浸润的细胞，然后测量emGFP表达(BD LSRFortessa；BD Biosciences，具有530/30滤波器的蓝色488nm激光和具有610/20滤波器的黄绿色561nm激光)。

序列表

<110> 庞培法布拉大学（Universitat Pompeu Fabra）

<120> 用于治疗先天性肌营养不良的治疗性LAMA2载荷

<130> IBIO-2200/PCT

<150> EP21209721.6

<151> 2021-11-22

<150> EP20214691.6

<151> 2020-12-16

<160> 134

<170> BiSSAP 1.3.6

<210> 1

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

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<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<400> 2

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<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<400> 3

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Ala Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Ser

580 585 590

Cys Phe

<210> 4

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<400> 4

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 5

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<400> 5

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ala Ile

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Arg

580 585 590

Cys Leu

<210> 6

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<400> 6

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Ser

580 585 590

Cys Leu

<210> 7

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<400> 7

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Ala Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 8

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<400> 8

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Arg

580 585 590

Cys Leu

<210> 9

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 高活性PiggyBac aa序列

<400> 9

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 10

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

<220>

<221> SITE

<222> 245..245

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 268..268

<223> 其中氨基酸可以是Asp, Asn

<220>

<221> SITE

<222> 275..275

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 277..277

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 287..287

<223> 其中氨基酸可以是Ala, Lys

<220>

<221> SITE

<222> 290..290

<223> 其中氨基酸可以是Ala, Lys

<220>

<221> SITE

<222> 315..315

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 325..325

<223> 其中氨基酸可以是Gly, Ala

<220>

<221> SITE

<222> 341..341

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 346..346

<223> 其中氨基酸可以是Asp, Asn

<220>

<221> SITE

<222> 347..347

<223> 其中氨基酸可以是Asn, Ala, Ser

<220>

<221> SITE

<222> 350..350

<223> 其中氨基酸可以是Thr, Ala

<220>

<221> SITE

<222> 351..351

<223> Xaa可以是Ser, Glu, Pro, Ala

<220>

<221> SITE

<222> 356..356

<223> 其中氨基酸可以是Lys, Glu

<220>

<221> SITE

<222> 357..357

<223> 其中氨基酸可以是Asn, Ala

<220>

<221> SITE

<222> 372..372

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 375..375

<223> 其中氨基酸可以是Lys, Ala

<220>

<221> SITE

<222> 388..388

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 409..409

<223> 其中氨基酸可以是Lys, Ala

<220>

<221> SITE

<222> 412..412

<223> 其中氨基酸可以是Lys, Ala

<220>

<221> SITE

<222> 432..432

<223> 其中氨基酸可以是Lys, Ala

<220>

<221> SITE

<222> 460..460

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 461..461

<223> 其中氨基酸可以是Ala, Lys

<220>

<221> SITE

<222> 465..465

<223> 其中氨基酸可以是Trp, Ala

<220>

<221> SITE

<222> 560..560

<223> 其中氨基酸可以是Thr, Ala

<220>

<221> SITE

<222> 564..564

<223> 其中氨基酸可以是Ser, Pro

<220>

<221> SITE

<222> 571..571

<223> 其中氨基酸可以是Asn, Ser

<220>

<221> SITE

<222> 573..573

<223> 其中氨基酸可以是Ser, Ala

<220>

<221> SITE

<222> 576..576

<223> 其中氨基酸可以是Lys, Ala

<220>

<221> SITE

<222> 586..586

<223> 其中氨基酸可以是His, 任何天然存在的氨基酸

<220>

<221> SITE

<222> 587..587

<223> 其中氨基酸可以是Ile, Ala

<220>

<221> SITE

<222> 589..589

<223> 其中氨基酸可以是Met, Val

<220>

<221> SITE

<222> 592..592

<223> 其中氨基酸可以是Ser, Gly

<220>

<221> SITE

<222> 594..594

<223> 其中氨基酸可以是Phe, Leu

<400> 10

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Xaa Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Xaa Glu Gln Leu Leu

260 265 270

Gly Phe Xaa Gly Xaa Cys Pro Phe Arg Val Tyr Ile Pro Asn Xaa Pro

275 280 285

Ser Xaa Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Xaa Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Xaa Asn Ile Thr Cys Xaa Xaa Trp Phe Xaa Xaa Ile

340 345 350

Pro Leu Ala Xaa Xaa Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

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

370 375 380

Ser Arg Ser Xaa Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Xaa Pro Ala Xaa Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Xaa

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Xaa Xaa Thr Asn Arg

450 455 460

Xaa Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Xaa

545 550 555 560

Tyr Cys Pro Xaa Lys Ile Arg Arg Lys Ala Xaa Ala Xaa Cys Lys Xaa

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Xaa Xaa Asp Xaa Cys Gln Xaa

580 585 590

Cys Xaa

<210> 11

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其具有在催化三联体中的突变

<220>

<221> SITE

<222> 268..268

<223> Xaa可以是Asp, Asn

<220>

<221> SITE

<222> 346..346

<223> Xaa可以是Asp, Asn

<400> 11

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Xaa Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Xaa Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 12

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其在对切除至关重要的氨基酸中具有突变

<220>

<221> SITE

<222> 287..287

<223> Xaa可以是Lys, Ala

<220>

<221> SITE

<222> 290..290

<223> Xaa可以是Lys, Ala

<220>

<221> SITE

<222> 460..460

<223> Xaa可以是Arg, Ala

<220>

<221> SITE

<222> 461..461

<223> Xaa可以是Lys, Ala

<400> 12

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Xaa Pro

275 280 285

Ser Xaa Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Xaa Xaa Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 13

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其具有参与靶连接的突变

<220>

<221> SITE

<222> 351..351

<223> Xaa可以是Ser, Glu, Pro, Ala

<220>

<221> SITE

<222> 356..356

<223> Xaa可以是Lys, Glu

<400> 13

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Xaa Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 14

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其具有对于整合至关重要的突变

<220>

<221> SITE

<222> 560..560

<223> 其中氨基酸可以是Thr, Ala,

<220>

<221> SITE

<222> 564..564

<223> 其中氨基酸可以是Ser, Pro

<220>

<221> SITE

<222> 571..571

<223> 其中氨基酸可以是Asn, Ser

<220>

<221> SITE

<222> 573..573

<223> 其中氨基酸可以是Ser, Ala

<220>

<221> SITE

<222> 589..589

<223> 其中氨基酸可以是Met, Val

<220>

<221> SITE

<222> 592..592

<223> 其中氨基酸可以是Ser, Gly

<220>

<221> SITE

<222> 594..594

<223> 其中氨基酸可以是Phe, Leu

<400> 14

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Xaa

545 550 555 560

Tyr Cys Pro Xaa Lys Ile Arg Arg Lys Ala Xaa Ala Xaa Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Xaa Cys Gln Xaa

580 585 590

Cys Xaa

<210> 15

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其具有参与比对的突变

<220>

<221> SITE

<222> 325..325

<223> Xaa可以是Gly, Ala

<220>

<221> SITE

<222> 347..347

<223> Xaa可以是Asn, Ala, Ser

<220>

<221> SITE

<222> 350..350

<223> Xaa可以是Thr, Ala

<220>

<221> SITE

<222> 465..465

<223> Xaa可以是Trp, Ala

<400> 15

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Xaa Trp Phe Xaa Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Xaa Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 16

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其具有高度保守氨基酸处的突变

<220>

<221> SITE

<222> 576..576

<223> Xaa可以是lys, Ala

<220>

<221> SITE

<222> 587..587

<223> Xaa可以是Ile, Ala

<400> 16

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Xaa

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Xaa Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 17

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其具有参与Zn2+结合的突变

<220>

<221> SITE

<222> 586..586

<223> 其中氨基酸可以是His, 任何天然存在的氨基酸

<400> 17

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Xaa Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 18

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac，其具有参与整合的突变

<220>

<221> SITE

<222> 315..315

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 341..341

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 372..372

<223> 其中氨基酸可以是Arg, Ala

<220>

<221> SITE

<222> 375..375

<223> 其中氨基酸可以是Lys, Ala

<400> 18

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

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

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

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

130 135 140

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

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

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

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

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

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

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

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Xaa Gly Thr Gln Thr Asn

305 310 315 320

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

325 330 335

His Gly Ser Cys Xaa Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

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

355 360 365

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

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

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

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

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

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 19

<211> 897

<212> PRT

<213> 人工序列

<220>

<223> 来自溃疡棒杆菌（Corynebacterium ulcerans）的Cas9

<400> 19

Met Thr Asn Ala Val Ala Asn His His Val Leu Trp Ala Lys Phe Asp

1 5 10 15

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

20 25 30

Thr Ala Ala Thr Cys Leu Phe Asn His Trp Leu Arg Lys Gly Leu Arg

35 40 45

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

50 55 60

Phe Val Ala Gly Ile His Asp Leu Gly Lys Ala Asn Pro Tyr Phe Gln

65 70 75 80

Ala Gln Arg Arg Asn Lys Lys Glu Glu Trp Ile Thr Leu Arg Asp Ala

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Leu Ser Asp Glu Asp Asp Leu Glu Asp Ile Glu Asp Ile Phe Asp Asp

165 170 175

Asn Gly Trp Ser Pro Thr His Glu Leu Leu Val Ser Ser Leu Leu Gln

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Glu Leu Phe Phe His Gln Pro Glu Lys Trp Ile Ala Asn Arg Lys Ala

245 250 255

Phe Cys Arg Glu Ile Ile Glu Asn Thr Val Gly Thr Tyr His Pro Trp

260 265 270

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

275 280 285

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

290 295 300

Val Met Glu Thr Thr Gly Ala Gly Lys Thr Glu Ala Ala Leu Leu Arg

305 310 315 320

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

325 330 335

Thr Thr Asn Ala Ile Met Asp Arg Ile Gly Lys Ile Phe Asp Gly Thr

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Ala Leu Ala Asn Ala His Val Val Val Asp Glu Val His Thr Met Asp

435 440 445

Gln Tyr Gln Ser Glu Leu Met Ser Gly Leu Leu Glu Trp Trp Ser Ala

450 455 460

Thr Asp Thr Pro Val Thr Leu Leu Thr Ala Thr Met Pro Ala Trp Gln

465 470 475 480

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

485 490 495

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

500 505 510

Glu Thr Ser Gln Glu Asn Ile Pro Thr Glu Ala Phe Thr Ile Pro Ile

515 520 525

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

530 535 540

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

545 550 555 560

Ile Cys Asn Thr Val Gly Arg Ala Gln Ser Ile Ala Glu Ala Leu Ala

565 570 575

His Glu Ser Pro Ile Val Leu His Ser Arg Met Thr Ala Gly His Arg

580 585 590

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

595 600 605

Ala Asn Ala Thr Leu Val Ile Gly Thr Gln Ala Ile Glu Ala Ser Leu

610 615 620

Asp Ile Asp Leu Asp Leu Leu Arg Thr Glu Leu Cys Pro Ala Pro Ser

625 630 635 640

Leu Ile Gln Arg Ala Gly Arg Leu Trp Arg Arg Leu Asp Pro Gln Arg

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Pro Ala Asp Ile Gln Asp Phe Ile Asp Ala Thr Thr Pro Gly Leu Gln

705 710 715 720

Glu Leu Phe Gln Lys Val Ser Leu Pro Glu Asp Cys Gly Ser Ala Glu

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Phe Leu Gln Ile Thr Ser Ala Asn Asn Leu Glu Glu Ser Ala Thr Arg

785 790 795 800

Leu Ile Asp Tyr Pro Ser Ile Ser Ala Ile Leu Cys Asp Pro Thr Gly

805 810 815

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

820 825 830

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

835 840 845

Ser Ile Pro His Ser Lys Lys Phe Leu Pro Ile Thr Ser Arg Glu Ile

850 855 860

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

865 870 875 880

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

885 890 895

Lys

<210> 20

<211> 876

<212> PRT

<213> 人工序列

<220>

<223> 来自白喉棒杆菌（Corynebacterium diphtheria）的Cas9

<400> 20

Met Asn Pro His Glu Glu Leu Trp Ala Lys Gln Lys Gly Leu Ala Lys

1 5 10 15

Pro Tyr Pro Leu Leu Ala His Leu Leu Asp Ser Ala Ala Val Ala Gly

20 25 30

Ala Leu Trp Asp His Trp Leu Arg Gln Asp Leu Arg Gln Met Phe Ile

35 40 45

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

50 55 60

Ser His Asp Ile Gly Lys Ala Thr Pro Leu Phe Gln Tyr Gln Lys Ala

65 70 75 80

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

85 90 95

Gly Arg Tyr Gln Lys Pro Leu Pro Ser Ser Tyr Leu Val Lys Lys Thr

100 105 110

Ser Gly Gly Pro Asn Arg His Glu Gln Trp Ser Ser Phe Ala Ser Lys

115 120 125

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

130 135 140

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

145 150 155 160

Arg His Gln Arg Lys Ala Ala Glu Asp Leu Ala Lys Ser Gly Trp Ser

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Thr Ile Ala Leu Gln Asn Gln Val Gly Leu Leu Asn Leu Met Ala Pro

290 295 300

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

305 310 315 320

Glu Asn Asp Arg Leu Ile Phe Leu Leu Pro Thr Gln Ala Thr Ser Asn

325 330 335

Ala Ile Met Arg Arg Val Gln Gly Ile Tyr Ser Asp Thr Pro Asn Ala

340 345 350

Ala Ala Leu Ala His Ser Leu Ala Ser Val Glu Asp Phe Tyr Gln Thr

355 360 365

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

370 375 380

Glu Ser Ser Met Ser Gly Gly Leu Tyr Pro Ser Ser Phe Val Cys Ser

385 390 395 400

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

405 410 415

Ala Leu Ala Thr Ala Leu Pro Gly Lys Trp Ile His Leu Arg Ile Leu

420 425 430

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

435 440 445

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

450 455 460

Leu Lys Thr Lys Ile Thr Phe Leu Thr Ala Thr Met Pro Ser Trp Gln

465 470 475 480

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

485 490 495

Pro Thr Val Phe Pro Ala Ala Glu Thr Val Leu Pro Gly Gln Phe Asn

500 505 510

Arg Thr Leu Ile Asp Ser Asp Ser Thr Thr Ile Asp Phe Thr Met Glu

515 520 525

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

530 535 540

Thr Thr Arg Leu Asn Ala Pro His Ala Arg Ile Gly Leu Ile Cys Asn

545 550 555 560

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

565 570 575

Asp Arg Ile Val Leu Leu His Ser Arg Met Thr Thr Glu His Arg Arg

580 585 590

Arg Ser Ala Glu Leu Leu Glu Ser Leu Leu Gly Pro Asn Gly Asn Arg

595 600 605

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

610 615 620

Ile Asp Leu Asp Ile Leu Arg Thr Glu Leu Cys Pro Ala Pro Ser Leu

625 630 635 640

Val Gln Arg Ala Gly Arg Val Trp Arg Arg Asn Asp Pro Tyr Arg Ser

645 650 655

Ser Arg Ile Thr Ala Asp His Lys Pro Ile Ser Val Val Phe Ile Ala

660 665 670

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

675 680 685

Arg Thr Gln Arg Trp Leu Glu Lys His Asn Gln Met Phe Leu Pro Gln

690 695 700

Met Ala Gln Glu Phe Ile Asp Ala Ala Thr Val Asp Leu Asp Thr Ala

705 710 715 720

Thr Ser Glu Met Asp Leu Asp Ala Leu Ala Leu Met Gly Ile His Leu

725 730 735

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

740 745 750

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

755 760 765

Asp Glu Ala Gln Thr Arg Leu Ile Glu Glu Gly Thr His Leu Arg Ile

770 775 780

Ile Leu Gly Asp Glu Asn Glu Ser Ile Pro Gly Gly Trp Lys His Gly

785 790 795 800

Leu Ser Ser Leu Leu Lys Leu Lys Ala Ser Asp Arg Glu Ser Leu Arg

805 810 815

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

820 825 830

Gln Leu Leu Tyr Gln His Asn Leu Val Pro Leu Ser Ser Ser Lys Thr

835 840 845

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

850 855 860

Lys Asn Leu Gly Phe Ile Trp Pro Glu Glu Lys Asp

865 870 875

<210> 21

<211> 773

<212> PRT

<213> 人工序列

<220>

<223> 来自梅毒螺原体（Spiroplasma syrphidicola）的Cas9

<400> 21

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

1 5 10 15

Cys Gly Trp Ala Val Thr Gly Gln Met Glu Asp Gly Asn Trp Val Leu

20 25 30

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

35 40 45

Gly Thr Thr Asn Ala Ala Ala Arg Arg Leu Lys Arg Gly Ala Arg Arg

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Val Asp Leu Leu Leu Ala Lys Gln Ala Lys Phe Tyr Pro Glu Leu Thr

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Arg Cys Thr Phe Leu Arg Glu Leu Ser Val Gly Tyr Lys Ser Ser Ile

290 295 300

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

305 310 315 320

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

325 330 335

Leu Tyr Asn Glu Ala Gly Lys Lys Gly Lys Thr Leu Leu Lys Glu Ile

340 345 350

Leu Lys Lys His His Ile Asn Asp Asp Ile Phe Asp Thr Asn Ala Tyr

355 360 365

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

370 375 380

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

385 390 395 400

Asn Ile Tyr His Gln Leu Gly Phe Ile Ile His Thr Asn Ile Thr Pro

405 410 415

Glu Arg Lys Glu Lys Ala Ile Asn Gln Trp Leu Leu Glu Asn Asn Ile

420 425 430

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

435 440 445

Ile Ser Thr Thr Val Lys Thr Ser Phe Lys Trp Met Ser Ile Ala Ile

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Ile Asp Pro Asp Leu Trp Arg Asn Pro Ile Val Phe Arg Ala Ile Asn

515 520 525

Gln Ala Arg Lys Val Ile Lys Lys Leu Phe Glu Lys Tyr Thr Phe Ile

530 535 540

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

545 550 555 560

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

565 570 575

Ala Lys Ala Lys Glu Phe Leu Met Ala Asn Gly Ile Ile Val Asn Asp

580 585 590

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

595 600 605

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

610 615 620

Val Leu Gln Ile Asp His Ile Ile Pro Tyr Ser Lys Leu Ala Asp Asp

625 630 635 640

Ser Phe Asn Asn Lys Val Leu Val Phe Ser Lys Glu Asn Gln Glu Lys

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Ile Thr Ser Arg Phe Arg Arg Thr Trp Leu Arg Asn Ser Pro Trp Gly

755 760 765

Leu Glu Lys Lys Ser

770

<210> 22

<211> 1380

<212> PRT

<213> 人工序列

<220>

<223> 来自中间普雷沃氏菌（Prevotella intermedia）的Cas9

<400> 22

Met Lys Arg Ile Leu Gly Leu Asp Leu Gly Thr Thr Ser Ile Gly Trp

1 5 10 15

Ala Leu Val Asn Glu Ala Glu Asn Asn Asn Glu Ala Ser Ser Ile Val

20 25 30

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

35 40 45

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

50 55 60

His Gly Ala Arg Ile Asn Leu Gln Arg Tyr Lys Leu Arg Arg Gln Asn

65 70 75 80

Leu His Asp Cys Leu Gln Lys Gln Gly Trp Leu Gly Thr Glu Ala Met

85 90 95

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

100 105 110

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

115 120 125

Phe Met Leu Asn Lys Lys Arg Gly Tyr Lys Ser Asn Arg Lys Ala Asn

130 135 140

Asn Lys Glu Asp Gly Gln Leu Phe Asp Gly Met Thr Ile Ala Lys Lys

145 150 155 160

Leu Tyr Glu Glu His Leu Thr Pro Ala Glu Tyr Ser Leu Gln Leu Leu

165 170 175

Asn Lys Gly Lys Lys Phe Thr Gln Gly Tyr Tyr Arg Ser Asp Leu Asn

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Tyr Leu Trp Ala Ser Leu Glu Glu Asn Pro His Ile Ser Ile Lys Asn

305 310 315 320

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

325 330 335

Glu Thr Gln Ala Ala Phe His Lys Gln Leu Thr Pro Glu Leu Lys Gln

340 345 350

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

355 360 365

Lys Ser Leu Ile Ser Val Cys Glu Leu Glu Gln Arg Lys Val Lys Ala

370 375 380

Thr Ile Asp Gly Lys Glu Lys Glu Ile Thr Ile Gly Pro Lys Val Ala

385 390 395 400

Pro Lys Ser Ser Pro Val Phe Gln Glu Phe Arg Ile Trp Gln Asn Leu

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ile Leu Phe Asp Cys Tyr Asn Arg Ile Ile Thr Leu Thr Gly His Glu

485 490 495

Glu Cys Asp Phe Lys Lys Ile Lys Ala Ser Glu Ile Arg His Tyr Val

500 505 510

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

515 520 525

Asp Pro Ser Leu Lys Lys His Glu Leu Glu Lys Gln Pro Met Tyr Gln

530 535 540

Leu Trp His Leu Leu Tyr Ser Tyr Glu Ser Asp Asn Ser Arg Thr Gly

545 550 555 560

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

565 570 575

Glu Tyr Ala Thr Val Leu Cys Asp Val Val Phe Glu Glu Asp Tyr Gly

580 585 590

Asn Leu Ser Val Lys Ala Met Arg Glu Ile Leu Pro Tyr Leu Gln Ala

595 600 605

Gly Asn Asp Tyr Ser Gln Ala Cys Ala Tyr Ala Gly Tyr Asn His Ser

610 615 620

Arg His Ser Leu Thr Lys Glu Glu Leu Asp Gln Lys Val Tyr Lys Glu

625 630 635 640

Arg Leu Glu Leu Leu Pro Lys Asn Ser Leu Arg Asn Pro Val Val Glu

645 650 655

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

660 665 670

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

675 680 685

Ser Ser Ala Ala Asp Arg Lys Lys Thr Thr His Ala Ile Ser Gln Gly

690 695 700

Asn Ala Glu Asn Gln Arg Ile Arg Glu Ile Leu Glu Lys Glu Phe Ser

705 710 715 720

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

725 730 735

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

740 745 750

Thr Lys Asp Lys Leu Phe Ser Lys Asp Phe Asp Ile Glu His Ile Ile

755 760 765

Pro Lys Ala Arg Leu Phe Asp Asp Ser Phe Ser Asn Lys Thr Leu Glu

770 775 780

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

785 790 795 800

Ile Lys Glu Lys Tyr Gly Glu Asp Gly Ala Glu Ala Tyr Lys Lys Lys

805 810 815

Leu Asp Met Leu Leu Glu Asn Asp Ala Ile Ser Arg Pro Lys Tyr Asn

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

Arg Leu Arg Glu Asp Trp Gln Leu Val Asp Val Met Lys Glu Leu Asn

885 890 895

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

900 905 910

Asp Gly Arg Lys Ile Lys Arg Ile Lys Asp Trp Thr Lys Arg Asn Asp

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

Lys Leu Arg Phe Asn Ala Pro Ile Pro Val Asn Glu Phe Arg Ala Glu

980 985 990

Ala Lys Arg His Leu Ser Ala Ile Leu Val Ser Ile Lys Ala Lys Asn

995 1000 1005

Lys Val Met Thr Gln Asn Val Asn Lys Ile Lys Thr Lys His Gly Ile

1010 1015 1020

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

1025 1030 1035 1040

Ile Tyr Gly Thr Lys Met Arg Pro Ile Ile Lys Met Val Lys Val Gly

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

Val Ser Ile Glu Gly Val Leu Ser Ala Ile Pro Leu His Thr Leu Lys

1185 1190 1195 1200

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

1205 1210 1215

Gly Asn Tyr Val Gln Thr Ser Asn Asn His His Ile Ala Phe Tyr Tyr

1220 1225 1230

Asp Glu Asp Gly Asn Leu Gln Asp Asn Ala Val Ser Phe Phe Glu Ala

1235 1240 1245

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

1250 1255 1260

Arg Asp Lys Gly Trp Arg Phe Leu Phe Thr Met Lys Gln Asn Glu Tyr

1265 1270 1275 1280

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

1285 1290 1295

Leu Thr Asp Glu Ala Asn Tyr Gly Ile Ile Ser Pro Asn Leu Tyr Arg

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

Asp Ile Ile Lys Val Arg Ile Asn Ser Thr Gly Lys Ile Val Ala Val

1365 1370 1375

Gly Glu Tyr Asp

1380

<210> 23

<211> 535

<212> PRT

<213> 人工序列

<220>

<223> 来自台湾螺原体（Spiroplasma taiwanense）的Cas9

<400> 23

Met Trp Ser Arg Lys Ile Leu Lys Ala Gly Ser Arg Leu Phe Asp Glu

1 5 10 15

Ala Asn Leu Ser Asp Lys Ile Ala Ser Lys Arg Arg Glu Gln Arg Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Tyr Tyr Asp Gly Pro Gly Pro Lys Asp Lys Asn Leu Leu Asn Pro Ser

195 200 205

Lys Tyr Gly Trp Lys Asn Gln Glu Glu Phe Phe Asp Arg Phe Ala Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Asn Leu Thr Leu Ile Asn Gln Lys Glu Lys Ala Glu Asn Ile Thr Leu

275 280 285

Lys Lys Ile Ala Lys Tyr Leu Lys Ile Asn Glu Lys Asn Ile Thr Gly

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Glu Leu Asn Phe Asp Phe Phe Asp Lys Glu Thr Cys Glu Lys Leu Ala

370 375 380

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

385 390 395 400

Lys Leu Ile Glu Glu Met Phe His Asp Asn Leu Asn His Met Glu Ala

405 410 415

Leu Ala Lys Leu Lys Ile Lys Pro Asp Tyr Lys Leu Lys Val Asp Leu

420 425 430

Thr Asn Phe Lys Thr Ile Pro Ile Leu Arg Glu Lys Ile Asn Glu Met

435 440 445

Tyr Ile Ser Pro Val Val Lys Arg Ala Leu Ile Glu Ser Leu Lys Ile

450 455 460

Ile Lys Glu Leu Glu Arg His Phe Lys Asp Phe Glu Ile Lys Asp Ile

465 470 475 480

Val Ile Glu Met Ala Lys Lys Asn Ser Ala Glu Lys Lys Gln Phe Ile

485 490 495

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

500 505 510

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

515 520 525

Phe Leu Leu Leu Ser Glu Gln

530 535

<210> 24

<211> 1281

<212> PRT

<213> 人工序列

<220>

<223> 来自海豚链球菌（Streptococcus iniae）的Cas9

<400> 24

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

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Met

20 25 30

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

35 40 45

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

50 55 60

Lys Arg Thr Thr Arg Arg Arg Tyr Thr Arg Arg Lys Tyr Arg Ile Lys

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Glu Asn Thr Asp Val His Val Leu Phe Leu Asn Leu Val Asn Ile Tyr

180 185 190

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

195 200 205

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

210 215 220

Leu Ile Ala Glu Ile Pro Asn Gln Lys Arg Asn Met Leu Phe Gly Asn

225 230 235 240

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

245 250 255

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

260 265 270

Glu Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Ile Ala Ala Lys Lys Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Ile Thr Val Lys Gly Ala Ser Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asn Glu Leu Lys Ala Ile Ile Arg Arg Gln Glu Lys Phe Tyr Pro Phe

420 425 430

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

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Asn Gly Gln Ser Ser Phe Ala Trp

450 455 460

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

465 470 475 480

Val Val Asp Gln Glu Ala Ser Ala Arg Ala Phe Ile Glu Arg Met Thr

485 490 495

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

500 505 510

Pro Leu Tyr Glu Met Phe Met Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

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

530 535 540

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

545 550 555 560

Val Lys Gln Leu Lys Glu Glu Tyr Phe Ser Lys Met Lys Cys Phe His

565 570 575

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

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Phe Lys Asp Lys Ala Phe Leu Asp

595 600 605

Asp Glu Ala Asn Gln Asp Ile Leu Glu Glu Ile Val Trp Thr Leu Thr

610 615 620

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

625 630 635 640

Asp Val Phe Glu Lys Ser Val Leu Lys Lys Leu Lys Lys Arg His Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Gln Lys Leu Ile Asn Gly Ile Lys Asp

660 665 670

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

675 680 685

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

690 695 700

Ala Lys Ile Ile Lys Asn Glu Gln Glu Lys Thr Ile Lys Asn Glu Ser

705 710 715 720

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

725 730 735

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

740 745 750

Gly Gln Asn Pro Asp Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

Gly Lys Ser Asp Asn Val Pro Asn Ile Glu Thr Val Asn Lys Met Lys

865 870 875 880

Ser Phe Trp Tyr Lys Gln Leu Lys Ser Gly Ala Ile Ser Gln Arg Lys

885 890 895

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

900 905 910

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

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Phe Asn Ser Asn Leu Thr

930 935 940

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

945 950 955 960

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

965 970 975

Glu Val Asn Asp Tyr His His Ala Gln Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Leu Lys Lys Tyr Pro Lys Leu Glu Ala Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys His Tyr Asp Leu Ala Lys Leu Met Ile Gln

1010 1015 1020

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

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

Val Trp Asp Lys Val Lys Asp Met Gln Thr Ile Arg Lys Val Met Ser

1075 1080 1085

Tyr Pro Gln Val Asn Ile Val Met Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

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

1105 1110 1115 1120

Ala Arg Lys Lys Ser Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Ile Ile Ala Tyr Ser Val Leu Val Val Ala Lys Ile Ala Lys Gly

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

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

1205 1210 1215

Ala Lys Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Asn Lys Tyr

1220 1225 1230

Val Lys Phe Leu Tyr Leu Ala Ser His Tyr Thr Lys Phe Thr Gly Lys

1235 1240 1245

Glu Glu Asp Arg Glu Lys Lys Arg Ser Tyr Val Glu Ser His Leu Tyr

1250 1255 1260

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

1265 1270 1275 1280

Phe

<210> 25

<211> 1352

<212> PRT

<213> 人工序列

<220>

<223> 来自Belliella baltica的Cas9

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Lys Ser Glu Glu Asp Asp Gly Ser Ala Ile Asp Ser Met Gly Ile Ala

145 150 155 160

Lys Glu Leu Tyr Glu Asn Asp Leu Thr Pro Gly Gln Trp Val Tyr Glu

165 170 175

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

180 185 190

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

195 200 205

Pro Asp Ile Phe Asn Ala Ser Phe Val Glu Asp Trp Met Gly Lys Ala

210 215 220

Ser Thr Pro Thr Lys Gln Tyr Phe Asn Lys Lys Gly Val Gln Leu Ala

225 230 235 240

Glu Asn Lys Gly Lys Arg Glu Glu Arg Arg Leu Gln Glu Tyr Lys Trp

245 250 255

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

260 265 270

Ile Leu Ser Gln Ile Asn Ser Gln Ile Ser Asn Ser Ser Gly Tyr Leu

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Lys Val Val Pro Lys Ser His Pro Val Phe Gln Glu Phe Arg Ile Trp

385 390 395 400

Gln Asn Leu Asn Asn Leu Leu Leu Ile Lys Lys Asp Asn Leu Asn Glu

405 410 415

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

420 425 430

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

435 440 445

Leu Lys Pro Asn Glu Trp Glu Phe Asn Phe Thr Lys Ile Glu Gly Asn

450 455 460

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

465 470 475 480

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

485 490 495

Leu Asp Gln Phe Ser Glu Ala Phe Leu Arg Ile Gly Ile Asp Thr Glu

500 505 510

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

515 520 525

Ser Tyr Ile Gln Phe Trp His Leu Leu Tyr Ser Ser Glu Asp Asp Gln

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Asn Gln Met Ile Asn Val Val Asn Ala Ile Leu Lys Asp Pro Glu Leu

645 650 655

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

660 665 670

Asn Ala Glu Gln Arg Lys Asn Met Thr Ser Asn Ile Ala Ser Ala Thr

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

Tyr Ile Ser Lys Lys Ala Lys Glu Ile Leu Phe Glu Ile Ser Arg Arg

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

Lys Ile Lys Asp Trp Ser Lys Arg Asn Asp His Arg His His Ala Met

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

Ser Arg Ile Leu Val Ser Tyr Lys Ala Lys Asn Lys Val Val Thr Ile

980 985 990

Asn Lys Asn Lys Thr Lys Ser Lys Ala Gly Leu Asn Glu Gln Ile Ala

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

Lys Asn Thr Leu Asn Lys Met Pro Ile Tyr Leu Asp Leu Gly Lys Asn

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

Gly Asn Ala Lys Leu Ala Phe Ser Asn Leu Glu Glu Asn Pro Ile Trp

1140 1145 1150

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

1155 1160 1165

Val Ser Asn Val Glu Ser Leu His Val Lys Lys Asp His Phe Gly Glu

1170 1175 1180

Pro Ile Leu Asp Gln Glu Gly Asn Glu Ile Pro Val Asp Phe Val Ser

1185 1190 1195 1200

Thr Gly Asn Asn His His Val Ala Ile Tyr Glu Asp Glu Asn Gly Asn

1205 1210 1215

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

1220 1225 1230

Gln Gly Leu Pro Ile Ile Lys Lys Asn His Thr Leu Gly Trp Lys Phe

1235 1240 1245

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

1250 1255 1260

Phe Val Pro Ala Asp Val Asp Leu Met Asp Glu Gln Asn Tyr His Leu

1265 1270 1275 1280

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

1285 1290 1295

Val Phe Asn Asn His Leu Glu Thr Lys Ala Val Asp Asn Asp Leu Leu

1300 1305 1310

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

1315 1320 1325

Pro Glu His Leu Arg Gly Ile Ile Lys Ile Arg Ile Asn His Leu Gly

1330 1335 1340

Lys Ile Ile Gln Ile Gly Glu Tyr

1345 1350

<210> 26

<211> 1509

<212> PRT

<213> 人工序列

<220>

<223> 来自热带冷弯菌（Psychroflexus torquisI）的Cas9

<400> 26

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

1 5 10 15

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

20 25 30

Leu Gly Val Arg Ile Ile Pro Met Ser Gln Glu Ile Leu Gly Lys Phe

35 40 45

Asp Ala Gly Gln Ser Ile Ser Gln Thr Ala Asp Arg Thr Lys Tyr Arg

50 55 60

Gly Val Arg Arg Leu Tyr Gln Arg Asp Asn Leu Arg Arg Glu Arg Leu

65 70 75 80

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

85 90 95

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

100 105 110

Val Lys Leu Asn Tyr Arg Lys Asn Glu Lys Asn Lys His Glu Phe Val

115 120 125

Phe Met Asn Ser Phe Ile Glu Met Val Ser Glu Phe Lys Asn Ala Gln

130 135 140

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

145 150 155 160

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

165 170 175

Ile Thr Lys Glu Glu Leu Ala Trp Leu Ile Leu Asn Phe Asn Gln Lys

180 185 190

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

195 200 205

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

210 215 220

Lys Val Lys Gly Lys Val Leu Tyr Asn Val Ile Phe Asp Asn Gly Trp

225 230 235 240

Lys Tyr Glu Lys Gln Ile Val Asn Lys Asp Glu Trp Glu Gly Arg Thr

245 250 255

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

260 265 270

Arg Thr Tyr Lys Ala Val Asp Ser Glu Ile Asp Trp Ala Ala Ile Lys

275 280 285

Ala Lys Thr Glu Gln Asp Ile Asn Lys Ala Asn Lys Thr Val Gly Glu

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Ser Leu Tyr Lys Ala Cys Ile Lys Glu Leu Tyr Pro Arg Asn Glu Asn

355 360 365

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

370 375 380

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

385 390 395 400

Ser Gly Cys Gln Phe Glu His Lys Ile Tyr Lys Gln Lys Asn Lys Lys

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Val Thr Thr Gln Leu Leu Pro Asn Asn Glu Ala Tyr Val Thr Leu Phe

465 470 475 480

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

485 490 495

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

500 505 510

Arg Trp Asn Phe Val Glu Asp Lys Lys Tyr Pro Phe Ser Glu Thr Arg

515 520 525

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

530 535 540

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

545 550 555 560

Pro Phe Ile Lys Arg Ile Glu Gln Leu Trp His Ile Ile Tyr Ser Val

565 570 575

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

580 585 590

His Asn Leu Glu Lys Asp Ser Phe Leu Lys Asn Phe Lys Lys Phe Pro

595 600 605

Pro Phe Val Ser Asp Tyr Ala Ser Tyr Ser Lys Lys Ala Ile Ser Lys

610 615 620

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

625 630 635 640

Pro Thr Gln Val Lys Glu Arg Ser Leu Ser Ile Met Glu Arg Val Lys

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Thr Leu Arg Val Val Arg Asp Ile Trp Glu His Tyr Gly Asn Asn Glu

755 760 765

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

770 775 780

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

785 790 795 800

Glu Asn Thr Asn His Arg Ile Arg Glu Val Leu Lys Glu Leu Met Asn

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

Gln Gly Tyr Ile Ser Pro Tyr Thr Gly Lys Ile Ile Pro Leu Thr Lys

885 890 895

Leu Phe Thr His Glu Tyr Gln Ile Glu His Ile Ile Pro Gln Ser Arg

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Glu Ala His Val Asn Lys Tyr Phe Lys Lys Asn Lys Thr Lys Leu Lys

965 970 975

Asn Leu Leu Ser Glu Asp Ile Pro Glu Gly Phe Ile Asn Arg Gln Leu

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

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

1045 1050 1055

Lys Leu Thr Asn Ser Asn Asp Phe Gly Phe Trp Asp Asn Asp Ile Asn

1060 1065 1070

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

1075 1080 1085

Lys Arg Ile Asp His Arg His His Ala Leu Asp Ala Leu Val Val Ala

1090 1095 1100

Cys Thr Ser Arg Asn His Thr His Tyr Leu Ser Ala Leu Asn Ala Glu

1105 1110 1115 1120

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

1125 1130 1135

Gly Asp Tyr Thr Lys Thr Phe Gln Ile Pro Trp Gln Gly Phe Thr Ile

1140 1145 1150

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

1155 1160 1165

Leu Arg Val Ile Asn Lys Thr Asn Asn Lys Phe Trp Ser Tyr Lys Asp

1170 1175 1180

Glu Asn Gly Asn Leu Asn Leu Gly Lys Asp Gly Lys Pro Lys Lys Lys

1185 1190 1195 1200

Leu Arg Lys Gln Thr Lys Gly Tyr Asn Trp Ala Ile Arg Lys Pro Leu

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Pro Asn His Leu Lys His Tyr Leu Asn Asn Lys Gly Glu Ala Asn Phe

1265 1270 1275 1280

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

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

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

1365 1370 1375

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

1380 1385 1390

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

1395 1400 1405

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

1410 1415 1420

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

1425 1430 1435 1440

Cys Asn Phe Ile Pro Phe Gln Val Ser Lys Leu Ile Phe Asn Leu Lys

1445 1450 1455

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

1460 1465 1470

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

1475 1480 1485

Val Met Ile Lys Glu Lys Cys Ile Lys Leu Lys Ile Asp Arg Leu Gly

1490 1495 1500

Asn Ile Ser Lys Ala

1505

<210> 27

<211> 1388

<212> PRT

<213> 人工序列

<220>

<223> 来自嗜热链球菌（Streptococcus thermophilus）的Cas9

<400> 27

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

1 5 10 15

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

20 25 30

Lys Val Leu Gly Asn Thr Ser Lys Lys Tyr Ile Lys Lys Asn Leu Leu

35 40 45

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

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Arg Asn Arg Ile Leu

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Thr Glu Met Ala Thr Leu Asp Asp Ala

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Phe Leu Lys Leu Ile Val Gly Asn Gln Ala Asp Phe Arg Lys Cys Phe

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Met Ile Lys Arg Tyr Asn Glu His Lys Glu Asp Leu Ala Leu Leu Lys

325 330 335

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

340 345 350

Asp Asp Thr Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Lys Thr Asn

355 360 365

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

370 375 380

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

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro Tyr Gln Ile His Leu

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Ser Phe Asp Leu Tyr Leu Pro Glu Glu Lys Val Leu Pro Lys His Ser

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Ser Ser Asn Glu Ala Ile Ile Glu Glu Ile Ile His Thr Leu Thr Ile

610 615 620

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

625 630 635 640

Ile Phe Asp Lys Ser Val Leu Lys Lys Leu Ser Arg Arg His Tyr Thr

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Lys Lys Ile Gln Lys Ala Gln Ile Ile Gly Asp Glu Asp Lys Gly Asn

705 710 715 720

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

725 730 735

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

740 745 750

Gly Gly Arg Lys Pro Glu Ser Ile Val Val Glu Met Ala Arg Glu Asn

755 760 765

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

770 775 780

Leu Glu Lys Ser Leu Lys Glu Leu Gly Ser Lys Ile Leu Lys Glu Asn

785 790 795 800

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

805 810 815

Arg Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Lys Asp Met Tyr Thr Gly

820 825 830

Asp Asp Leu Asp Ile Asp Arg Leu Ser Asn Tyr Asp Ile Asp His Ile

835 840 845

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

850 855 860

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

865 870 875 880

Glu Val Val Lys Lys Arg Lys Thr Phe Trp Tyr Gln Leu Leu Lys Ser

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Lys Phe Asn Asn Lys Lys Asp Glu Asn Asn Arg Ala Val Arg Thr Val

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

Ser Phe Arg Glu Arg Lys Ser Ala Thr Glu Lys Val Tyr Phe Tyr Ser

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

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

1155 1160 1165

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

1170 1175 1180

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

1185 1190 1195 1200

Ile Glu Leu Ile Ile Glu Leu Pro Lys Tyr Ser Leu Phe Glu Leu Ser

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Asn His Arg Lys Tyr Val Glu Asn His Lys Lys Glu Phe Glu Glu Leu

1265 1270 1275 1280

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

1285 1290 1295

Asn Gly Lys Leu Leu Asn Ser Ala Phe Gln Ser Trp Gln Asn His Ser

1300 1305 1310

Ile Asp Glu Leu Cys Ser Ser Phe Ile Gly Pro Thr Gly Ser Glu Arg

1315 1320 1325

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

1330 1335 1340

Phe Leu Gly Val Lys Ile Pro Arg Tyr Arg Asp Tyr Thr Pro Ser Ser

1345 1350 1355 1360

Leu Leu Lys Asp Ala Thr Leu Ile His Gln Ser Val Thr Gly Leu Tyr

1365 1370 1375

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

1380 1385

<210> 28

<211> 1334

<212> PRT

<213> 人工序列

<220>

<223> 来自无害利斯特氏菌（Listeria innocua）的Cas9

<400> 28

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

1 5 10 15

Gly Trp Ala Val Leu Thr Asp Gln Tyr Asp Leu Val Lys Arg Lys Met

20 25 30

Lys Ile Ala Gly Asp Ser Glu Lys Lys Gln Ile Lys Lys Asn Phe Trp

35 40 45

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

50 55 60

Ala Arg Thr Ala Arg Arg Arg Ile Glu Arg Arg Arg Asn Arg Ile Ser

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

His Lys Asn Tyr Pro Thr Ile Tyr His Leu Arg Glu Glu Leu Val Asn

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Asn Gln Val Phe Ala Ser Gly Ile Glu Asp Gly Ser Leu Lys Lys Leu

195 200 205

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

210 215 220

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

225 230 235 240

Ala Gly Met Phe Ala Gln Phe Ile Ser Leu Ile Val Gly Ser Lys Gly

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Ala Val Val Leu Ser Ser Ile Ile Thr Val Ala Glu Thr Glu Thr Asn

305 310 315 320

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

325 330 335

Asp Leu Gly Glu Leu Lys Ala Phe Ile Lys Leu His Leu Pro Lys His

340 345 350

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

355 360 365

Ile Asp Gly Lys Thr Lys Gln Ala Asp Phe Tyr Lys Tyr Met Lys Met

370 375 380

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

385 390 395 400

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

405 410 415

Pro His Gln Leu His Leu Glu Glu Leu Glu Ala Ile Leu His Gln Gln

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Phe Ile Glu Lys Met Thr Asn Lys Asp Thr Tyr Leu Pro Lys Glu Asn

500 505 510

Val Leu Pro Lys His Ser Leu Cys Tyr Gln Lys Tyr Leu Val Tyr Asn

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Ser His Val Glu Ser Pro Thr Ile Glu Gly Leu Glu Asp Ser Phe Asn

580 585 590

Ser Ser Tyr Ser Thr Tyr His Asp Leu Leu Lys Val Gly Ile Lys Gln

595 600 605

Glu Ile Leu Asp Asn Pro Val Asn Thr Glu Met Leu Glu Asn Ile Val

610 615 620

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

625 630 635 640

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

645 650 655

Arg Arg His Tyr Thr Gly Trp Gly Arg Leu Ser Ala Lys Leu Leu Met

660 665 670

Gly Ile Arg Asp Lys Gln Ser His Leu Thr Ile Leu Asp Tyr Leu Met

675 680 685

Asn Asp Asp Gly Leu Asn Arg Asn Leu Met Gln Leu Ile Asn Asp Ser

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Ser Val Met Gly Tyr Pro Pro Gln Thr Ile Val Val Glu Met Ala Arg

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

Tyr Tyr Leu Gln Asn Gly Lys Asp Met Tyr Thr Gly Gln Asp Leu Asp

820 825 830

Ile His Asn Leu Ser Asn Tyr Asp Ile Asp His Ile Val Pro Gln Ser

835 840 845

Phe Ile Thr Asp Asn Ser Ile Asp Asn Leu Val Leu Thr Ser Ser Ala

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Gln Ile Thr Lys Asn Val Ala Asn Ile Leu His Gln Arg Phe Asn Tyr

930 935 940

Glu Lys Asp Asp His Gly Asn Thr Met Lys Gln Val Arg Ile Val Thr

945 950 955 960

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

965 970 975

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

980 985 990

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

995 1000 1005

Pro Glu Phe Val Tyr Gly Asp Tyr His Gln Phe Asp Trp Phe Lys Ala

1010 1015 1020

Asn Lys Ala Thr Ala Lys Lys Gln Phe Tyr Thr Asn Ile Met Leu Phe

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

Thr Asn Trp Asp Pro Met Lys Tyr Gly Gly Leu Asp Ser Pro Asn Met

1105 1110 1115 1120

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

1125 1130 1135

Phe Glu Lys Lys Ile Ile Arg Val Thr Ile Met Glu Arg Lys Ala Phe

1140 1145 1150

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

1155 1160 1165

Lys Val Leu Ala Lys Leu Pro Lys Tyr Thr Leu Tyr Glu Cys Glu Glu

1170 1175 1180

Gly Arg Arg Arg Met Leu Ala Ser Ala Asn Glu Ala Gln Lys Gly Asn

1185 1190 1195 1200

Gln Gln Val Leu Pro Asn His Leu Val Thr Leu Leu His His Ala Ala

1205 1210 1215

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

1220 1225 1230

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

1235 1240 1245

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

1250 1255 1260

Glu Gln Asn Lys Glu Gly Asp Ile Lys Ala Ile Ala Gln Ser Phe Val

1265 1270 1275 1280

Asp Leu Met Ala Phe Asn Ala Met Gly Ala Pro Ala Ser Phe Lys Phe

1285 1290 1295

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

1300 1305 1310

Leu Asn Ser Thr Ile Ile Tyr Gln Ser Ile Thr Gly Leu Tyr Glu Ser

1315 1320 1325

Arg Lys Arg Leu Asp Asp

1330

<210> 29

<211> 984

<212> PRT

<213> 人工序列

<220>

<223> 来自空肠弯曲杆菌（Campylobacter jejuni）的Cas9

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

Arg Leu Ala Arg Ser Ala Arg Lys Arg Leu Ala Arg Arg Lys Ala Arg

50 55 60

Leu Asn His Leu Lys His Leu Ile Ala Asn Glu Phe Lys Leu Asn Tyr

65 70 75 80

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

85 90 95

Ser Leu Ile Ser Pro Tyr Glu Leu Arg Phe Arg Ala Leu Asn Glu Leu

100 105 110

Leu Ser Lys Gln Asp Phe Ala Arg Val Ile Leu His Ile Ala Lys Arg

115 120 125

Arg Gly Tyr Asp Asp Ile Lys Asn Ser Asp Asp Lys Glu Lys Gly Ala

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Lys Gln Arg Glu Phe Gly Phe Ser Phe Ser Lys Lys Phe Glu Glu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Asn Leu Leu Asn Asn Leu Lys Asn Thr Glu Gly Ile Leu Tyr Thr Lys

275 280 285

Asp Asp Leu Asn Ala Leu Leu Asn Glu Val Leu Lys Asn Gly Thr Leu

290 295 300

Thr Tyr Lys Gln Thr Lys Lys Leu Leu Gly Leu Ser Asp Asp Tyr Glu

305 310 315 320

Phe Lys Gly Glu Lys Gly Thr Tyr Phe Ile Glu Phe Lys Lys Tyr Lys

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Leu Lys Leu Val Thr Pro Leu Met Leu Glu Gly Lys Lys Tyr Asp Glu

405 410 415

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

420 425 430

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

435 440 445

Asn Pro Val Val Leu Arg Ala Ile Lys Glu Tyr Arg Lys Val Leu Asn

450 455 460

Ala Leu Leu Lys Lys Tyr Gly Lys Val His Lys Ile Asn Ile Glu Leu

465 470 475 480

Ala Arg Glu Val Gly Lys Asn His Ser Gln Arg Ala Lys Ile Glu Lys

485 490 495

Glu Gln Asn Glu Asn Tyr Lys Ala Lys Lys Asp Ala Glu Leu Glu Cys

500 505 510

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

515 520 525

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

530 535 540

Lys Ile Ser Asp Leu Gln Asp Glu Lys Met Leu Glu Ile Asp His Ile

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Lys Gly Ser Lys Val His Val Glu Ala Lys Ser Gly Met Leu Thr Ser

675 680 685

Ala Leu Arg His Thr Trp Gly Phe Ser Ala Lys Asp Arg Asn Asn His

690 695 700

Leu His His Ala Ile Asp Ala Val Ile Ile Ala Tyr Ala Asn Asn Ser

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

Ile Arg Lys Val Asn Gly Lys Ile Val Lys Asn Gly Asp Met Phe Arg

820 825 830

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

835 840 845

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

850 855 860

Ala Arg Ser Lys Lys Gly Glu Ile Lys Asp Trp Ile Leu Met Asp Glu

865 870 875 880

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

885 890 895

Gln Thr Lys Asp Met Gln Glu Pro Glu Phe Val Tyr Tyr Asn Ala Phe

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

Glu Lys Tyr Ile Val Ser Ala Leu Gly Glu Val Thr Lys Ala Glu Phe

965 970 975

Arg Gln Arg Glu Asp Phe Lys Lys

980

<210> 30

<211> 1082

<212> PRT

<213> 人工序列

<220>

<223> 来自脑膜炎奈瑟氏球菌（Neisseria. meningitidis）的Cas9

<400> 30

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

1 5 10 15

Ile Gly Ile Ala Ser Val Gly Trp Ala Met Val Glu Ile Asp Glu Asp

20 25 30

Glu Asn Pro Ile Cys Leu Ile Asp Leu Gly Val Arg Val Phe Glu Arg

35 40 45

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

50 55 60

Ala Arg Ser Val Arg Arg Leu Thr Arg Arg Arg Ala His Arg Leu Leu

65 70 75 80

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

85 90 95

Phe Asp Glu Asn Gly Leu Ile Lys Ser Leu Pro Asn Thr Pro Trp Gln

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Val Ala Asp Asn Ala His Ala Leu Gln Thr Gly Asp Phe Arg Thr

165 170 175

Pro Ala Glu Leu Ala Leu Asn Lys Phe Glu Lys Glu Ser Gly His Ile

180 185 190

Arg Asn Gln Arg Gly Asp Tyr Ser His Thr Phe Ser Arg Lys Asp Leu

195 200 205

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

210 215 220

Pro His Val Ser Gly Gly Leu Lys Glu Gly Ile Glu Thr Leu Leu Met

225 230 235 240

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

245 250 255

His Cys Thr Phe Glu Pro Ala Glu Pro Lys Ala Ala Lys Asn Thr Tyr

260 265 270

Thr Ala Glu Arg Phe Ile Trp Leu Thr Lys Leu Asn Asn Leu Arg Ile

275 280 285

Leu Glu Gln Gly Ser Glu Arg Pro Leu Thr Asp Thr Glu Arg Ala Thr

290 295 300

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

305 310 315 320

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

325 330 335

Tyr Gly Lys Asp Asn Ala Glu Ala Ser Thr Leu Met Glu Met Lys Ala

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Pro Leu Met Glu Gln Gly Lys Arg Tyr Asp Glu Ala Cys Ala Glu Ile

435 440 445

Tyr Gly Asp His Tyr Gly Lys Lys Asn Thr Glu Glu Lys Ile Tyr Leu

450 455 460

Pro Pro Ile Pro Ala Asp Glu Ile Arg Asn Pro Val Val Leu Arg Ala

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Gln Gln His Gly Lys Cys Leu Tyr Ser Gly Lys Glu Ile Asn Leu Gly

565 570 575

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

580 585 590

Ser Arg Thr Trp Asp Asp Ser Phe Asn Asn Lys Val Leu Val Leu Gly

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

Val Asn Arg Phe Leu Cys Gln Phe Val Ala Asp Arg Met Arg Leu Thr

675 680 685

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

690 695 700

Leu Leu Arg Gly Phe Trp Gly Leu Arg Lys Val Arg Ala Glu Asn Asp

705 710 715 720

Arg His His Ala Leu Asp Ala Val Val Val Ala Cys Ser Thr Val Ala

725 730 735

Met Gln Gln Lys Ile Thr Arg Phe Val Arg Tyr Lys Glu Met Asn Ala

740 745 750

Phe Asp Gly Lys Thr Ile Asp Lys Glu Thr Gly Glu Val Leu His Gln

755 760 765

Lys Thr His Phe Pro Gln Pro Trp Glu Phe Phe Ala Gln Glu Val Met

770 775 780

Ile Arg Val Phe Gly Lys Pro Asp Gly Lys Pro Glu Phe Glu Glu Ala

785 790 795 800

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

805 810 815

Arg Pro Glu Ala Val His Glu Tyr Val Thr Pro Leu Phe Val Ser Arg

820 825 830

Ala Pro Asn Arg Lys Met Ser Gly Gln Gly His Met Glu Thr Val Lys

835 840 845

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

850 855 860

Thr Gln Leu Lys Leu Lys Asp Leu Glu Lys Met Val Asn Arg Glu Arg

865 870 875 880

Glu Pro Lys Leu Tyr Glu Ala Leu Lys Ala Arg Leu Glu Ala His Lys

885 890 895

Asp Asp Pro Ala Lys Ala Phe Ala Glu Pro Phe Tyr Lys Tyr Asp Lys

900 905 910

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

915 920 925

Gln Lys Thr Gly Val Trp Val Arg Asn His Asn Gly Ile Ala Asp Asn

930 935 940

Ala Thr Met Val Arg Val Asp Val Phe Glu Lys Gly Asp Lys Tyr Tyr

945 950 955 960

Leu Val Pro Ile Tyr Ser Trp Gln Val Ala Lys Gly Ile Leu Pro Asp

965 970 975

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

980 985 990

Asp Ser Phe Asn Phe Lys Phe Ser Leu His Pro Asn Asp Leu Val Glu

995 1000 1005

Val Ile Thr Lys Lys Ala Arg Met Phe Gly Tyr Phe Ala Ser Cys His

1010 1015 1020

Arg Gly Thr Gly Asn Ile Asn Ile Arg Ile His Asp Leu Asp His Lys

1025 1030 1035 1040

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

1045 1050 1055

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

1060 1065 1070

Cys Arg Leu Lys Lys Arg Pro Pro Val Arg

1075 1080

<210> 31

<211> 1367

<212> PRT

<213> 人工序列

<220>

<223> 来自酿脓链球菌（Streptococcus pyogenes）的Cas9

<400> 31

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

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

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

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

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

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

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

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

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

260 265 270

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

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

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

580 585 590

Ala Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

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

705 710 715 720

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

725 730 735

Ile Leu Gln Thr Val Lys Ile Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

755 760 765

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

770 775 780

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

785 790 795 800

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

805 810 815

Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu

820 825 830

Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Ile Lys Asp

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

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

945 950 955 960

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

965 970 975

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

980 985 990

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

995 1000 1005

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser

1010 1015 1020

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

1025 1030 1035 1040

Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1045 1050 1055

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

1060 1065 1070

Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met

1075 1080 1085

Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met

1155 1160 1165

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

1170 1175 1180

Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1185 1190 1195 1200

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

1205 1210 1215

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

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1235 1240 1245

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr

1250 1255 1260

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

1265 1270 1275 1280

Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

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

1345 1350 1355 1360

Leu Ser Gln Leu Gly Gly Asp

1365

<210> 32

<211> 528

<212> DNA

<213> 人工序列

<220>

<223> zfp编码序列

<400> 32

atggcccagg ctgctcttga gcccggagag aaaccctaca agtgcccgga gtgcggaaag 60

tccttctctg agcggagtca cctccgagag caccagcgga ctcatacggg cgaaaaacca 120

tacaagtgcc cagaatgtgg taaatctttt tctcgggctg acaacctgac tgaacatcag 180

cgcacgcaca ccggtgaaaa accttacaag tgtccagagt gtggcaagag cttttctagt 240

agaaggacct gtcgagcgca tcagcggact cacaccggcg aaaaacccta taagtgtccg 300

gaatgtggaa agagctttag ccgcaacgac acccttactg aacaccagcg aacacacacg 360

ggagaaaaac catataaatg tccggaatgt ggcaaaagtt ttagtcggag tgataaactt 420

acggagcacc aacggacaca caccggagag aagccatata agtgtcctga atgtggaaag 480

tccttctcac agcttgctca tctgcgagca catcagcgca cacacacc 528

<210> 33

<211> 176

<212> PRT

<213> 人工序列

<220>

<223> ZFP氨基酸序列

<400> 33

Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro

1 5 10 15

Glu Cys Gly Lys Ser Phe Ser Glu Arg Ser His Leu Arg Glu His Gln

20 25 30

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

35 40 45

Ser Phe Ser Arg Ala Asp Asn Leu Thr Glu His Gln Arg Thr His Thr

50 55 60

Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Ser

65 70 75 80

Arg Arg Thr Cys Arg Ala His Gln Arg Thr His Thr Gly Glu Lys Pro

85 90 95

Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Arg Asn Asp Thr Leu

100 105 110

Thr Glu His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro

115 120 125

Glu Cys Gly Lys Ser Phe Ser Arg Ser Asp Lys Leu Thr Glu His Gln

130 135 140

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

145 150 155 160

Ser Phe Ser Gln Leu Ala His Leu Arg Ala His Gln Arg Thr His Thr

165 170 175

<210> 34

<211> 555

<212> DNA

<213> 人工序列

<220>

<223> ZNF-E2C核苷酸

<400> 34

atggcgcagg cggcgctgga accgggcgaa aaaccgtata aatgcccgga atgcggcaaa 60

agctttagcc gcaaagatag cctggtgcgc catcagcgca cccataccgg cgaaaaaccg 120

tataaatgcc cggaatgcgg caaaagcttt agccagagcg gcgatctgcg ccgccatcag 180

cgcacccata ccggcgaaaa accgtataaa tgcccggaat gcggcaaaag ctttagcgat 240

tgccgcgatc tggcgcgcca tcagcgcacc cataccggcg aaaaaccgta taaatgcccg 300

gaatgcggca aaagctttag ccagagcagc catctggtgc gccatcagcg cacccatacc 360

ggcgaaaaac cgtataaatg cccggaatgc ggcaaaagct ttagcgattg ccgcgatctg 420

gcgcgccatc agcgcaccca taccggcgaa aaaccgtata aatgcccgga atgcggcaaa 480

agctttagcc gcagcgataa actggtgcgc catcagcgca cccataccgg caaaaaaacc 540

agcggccagg cgggc 555

<210> 35

<211> 185

<212> PRT

<213> 人工序列

<220>

<223> ZNF-E2C氨基酸

<400> 35

Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro

1 5 10 15

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

20 25 30

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

35 40 45

Ser Phe Ser Gln Ser Gly Asp Leu Arg Arg His Gln Arg Thr His Thr

50 55 60

Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Asp

65 70 75 80

Cys Arg Asp Leu Ala Arg His Gln Arg Thr His Thr Gly Glu Lys Pro

85 90 95

Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu

100 105 110

Val Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro

115 120 125

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

130 135 140

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

145 150 155 160

Ser Phe Ser Arg Ser Asp Lys Leu Val Arg His Gln Arg Thr His Thr

165 170 175

Gly Lys Lys Thr Ser Gly Gln Ala Gly

180 185

<210> 36

<211> 555

<212> DNA

<213> 人工序列

<220>

<223> ZNF-E3核苷酸

<400> 36

atggcgcagg cggcgctgga accgggcgaa aaaccgtata aatgcccgga atgcggcaaa 60

agctttagcg atccgggcgc gctggtgcgc catcagcgca cccataccgg cgaaaaaccg 120

tataaatgcc cggaatgcgg caaaagcttt agccagagca gccatctggt gcgccatcag 180

cgcacccata ccggcgaaaa accgtataaa tgcccggaat gcggcaaaag ctttagcgat 240

tgccgcgatc tggcgcgcca tcagcgcacc cataccggcg aaaaaccgta taaatgcccg 300

gaatgcggca aaagctttag ccagagcagc catctggtgc gccatcagcg cacccatacc 360

ggcgaaaaac cgtataaatg cccggaatgc ggcaaaagct ttagcgattg ccgcgatctg 420

gcgcgccatc agcgcaccca taccggcgaa aaaccgtata aatgcccgga atgcggcaaa 480

agctttagcc agagcagcca tctggtgcgc catcagcgca cccataccgg caaaaaaacc 540

agcggccagg cgggc 555

<210> 37

<211> 185

<212> PRT

<213> 人工序列

<220>

<223> ZNF-E3氨基酸

<400> 37

Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro

1 5 10 15

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

20 25 30

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

35 40 45

Ser Phe Ser Gln Ser Ser His Leu Val Arg His Gln Arg Thr His Thr

50 55 60

Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Asp

65 70 75 80

Cys Arg Asp Leu Ala Arg His Gln Arg Thr His Thr Gly Glu Lys Pro

85 90 95

Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu

100 105 110

Val Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro

115 120 125

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

130 135 140

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

145 150 155 160

Ser Phe Ser Gln Ser Ser His Leu Val Arg His Gln Arg Thr His Thr

165 170 175

Gly Lys Lys Thr Ser Gly Gln Ala Gly

180 185

<210> 38

<211> 528

<212> DNA

<213> 人工序列

<220>

<223> ZNF-TRCa核苷酸

<400> 38

atggcgcagg cggctcttga acccggggag aaaccctata aatgccctga gtgtggcaag 60

agtttttcaa ccacaggaaa cttgacagtc caccaacgga cccacaccgg cgagaaacca 120

tacaagtgtc cggagtgtgg taagtctttc tcaagtcctg ccgaccttac cagacatcaa 180

cgcacacata caggtgaaaa accttacaag tgcccagagt gcggaaaaag tttttcacaa 240

tctggcgacc tccgcaggca ccagcgcact cacaccggtg aaaaaccata caagtgtcct 300

gagtgcggga agagttttag tcaacgagct catctggagc gacaccaaag gactcatact 360

ggggagaaac cgtacaaatg tcccgaatgt gggaagagct tctctaccaa gaattccctt 420

acagagcacc agcgcacgca tacgggagag aagccgtata agtgtccgga atgtggcaag 480

agcttttcca gaagtgacca ccttacaacc caccagagga cgcacacc 528

<210> 39

<211> 176

<212> PRT

<213> 人工序列

<220>

<223> ZNF-TRCa氨基酸

<400> 39

Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro

1 5 10 15

Glu Cys Gly Lys Ser Phe Ser Thr Thr Gly Asn Leu Thr Val His Gln

20 25 30

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

35 40 45

Ser Phe Ser Ser Pro Ala Asp Leu Thr Arg His Gln Arg Thr His Thr

50 55 60

Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln

65 70 75 80

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

85 90 95

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

100 105 110

Glu Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro

115 120 125

Glu Cys Gly Lys Ser Phe Ser Thr Lys Asn Ser Leu Thr Glu His Gln

130 135 140

Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys

145 150 155 160

Ser Phe Ser Arg Ser Asp His Leu Thr Thr His Gln Arg Thr His Thr

165 170 175

<210> 40

<211> 30

<212> DNA

<213> 人工序列

<220>

<223> GGS接头核酸

<400> 40

ggtggatctg gcggtggatc tggtggcggt 30

<210> 41

<211> 10

<212> PRT

<213> 人工序列

<220>

<223> GGS接头氨基酸

<400> 41

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

1 5 10

<210> 42

<211> 36

<212> DNA

<213> 人工序列

<220>

<223> GGS4x接头核酸

<400> 42

ggagggagtg gtgggtccgg tggtagtggc ggatcc 36

<210> 43

<211> 12

<212> PRT

<213> 人工序列

<220>

<223> GGS4x接头氨基酸

<400> 43

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

1 5 10

<210> 44

<211> 45

<212> DNA

<213> 人工序列

<220>

<223> GGS5x接头核酸

<400> 44

ggaggctccg gtgggtctgg tgggagcggt ggtagtggcg gatcc 45

<210> 45

<211> 15

<212> PRT

<213> 人工序列

<220>

<223> GGS5x接头氨基酸

<400> 45

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

1 5 10 15

<210> 46

<211> 54

<212> DNA

<213> 人工序列

<220>

<223> GGS6x接头核酸

<400> 46

ggaggcagtg gtgggagcgg tggttccggg ggtagtggtg gttccggggg atcc 54

<210> 47

<211> 18

<212> PRT

<213> 人工序列

<220>

<223> GGS6x接头氨基酸

<400> 47

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1 5 10 15

Gly Ser

<210> 48

<211> 63

<212> DNA

<213> 人工序列

<220>

<223> GGS7x接头核酸

<400> 48

ggaggttctg gaggctccgg tgggtccggg ggaagtgggg ggtcaggcgg atcaggagga 60

tcc 63

<210> 49

<211> 21

<212> PRT

<213> 人工序列

<220>

<223> GGS7x接头氨基酸

<400> 49

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1 5 10 15

Gly Ser Gly Gly Ser

20

<210> 50

<211> 75

<212> DNA

<213> 人工序列

<220>

<223> GGS8x接头核酸

<400> 50

ggaggtagcg gaggttccgg agggagcggc gggagtgggg gaagcggggg aagtggagga 60

tccgggggag gatcc 75

<210> 51

<211> 21

<212> PRT

<213> 人工序列

<220>

<223> GGS8x接头氨基酸

<400> 51

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1 5 10 15

Gly Ser Gly Gly Ser

20

<210> 52

<211> 48

<212> DNA

<213> 人工序列

<220>

<223>接头 XTEN核酸

<400> 52

tccggtagcg aaacaccggg gacttcagaa tcggccaccc cggagtct 48

<210> 53

<211> 16

<212> PRT

<213> 人工序列

<220>

<223>接头 XTEN氨基酸

<400> 53

Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser

1 5 10 15

<210> 54

<211> 36

<212> DNA

<213> 人工序列

<220>

<223>接头 B核酸

<400> 54

ggaagcgccg gtagtgcggc tgggtctggc gagttc 36

<210> 55

<211> 12

<212> PRT

<213> 人工序列

<220>

<223>接头 B氨基酸

<400> 55

Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe

1 5 10

<210> 56

<211> 4104

<212> DNA

<213> 人工序列

<220>

<223> 人Cas9 (hCas9)核酸

<400> 56

atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720

cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780

gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960

atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620

tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040

gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580

gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080

gacctctctc agctcggtgg agac 4104

<210> 57

<211> 4104

<212> DNA

<213> 人工序列

<220>

<223> 切口酶Cas9 (nCas9)核酸

<400> 57

atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 60

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720

cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780

gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960

atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620

tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040

gattttctta agtccgatgg atttgccaac aggaacttca tgcagttgat ccatgatgac 2100

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580

gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080

gacctctctc agctcggtgg agac 4104

<210> 58

<211> 3213

<212> DNA

<213> 金黄色葡萄球菌（Staphylococcus aureus）

<400> 58

gccaccatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccaag 60

cggaactaca tcctgggcct ggccatcggc atcaccagcg tgggctacgg catcatcgac 120

tacgagacac gggacgtgat cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa 180

aacaacgagg gcaggcggag caagagaggc gccagaaggc tgaagcggcg gaggcggcat 240

agaatccaga gagtgaagaa gctgctgttc gactacaacc tgctgaccga ccacagcgag 300

ctgagcggca tcaaccccta cgaggccaga gtgaagggcc tgagccagaa gctgagcgag 360

gaagagttct ctgccgccct gctgcacctg gccaagagaa gaggcgtgca caacgtgaac 420

gaggtggaag aggacaccgg caacgagctg tccaccaaag agcagatcag ccggaacagc 480

aaggccctgg aagagaaata cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc 540

gaagtgcggg gcagcatcaa cagattcaag accagcgact acgtgaaaga agccaaacag 600

ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga gcttcatcga cacctacatc 660

gacctgctgg aaacccggcg gacctactat gagggacctg gcgagggcag ccccttcggc 720

tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc actgcaccta cttccccgag 780

gaactgcgga gcgtgaagta cgcctacaac gccgacctgt acaacgccct gaacgacctg 840

aacaatctcg tgatcaccag ggacgagaac gagaagctgg aatattacga gaagttccag 900

atcatcgaga acgtgttcaa gcagaagaag aagcccaccc tgaagcagat cgccaaagaa 960

atcctcgtga acgaagagga tattaagggc tacagagtga ccagcaccgg caagcccgag 1020

ttcaccaacc tgaaggtgta ccacgacatc aaggacatta ccgcccggaa agagattatt 1080

gagaacgccg agctgctgga tcagattgcc aagatcctga ccatctacca gagcagcgag 1140

gacatccagg aagaactgac caatctgaac tccgagctga cccaggaaga gatcgagcag 1200

atctctaatc tgaagggcta taccggcacc cacaacctga gcctgaaggc catcaacctg 1260

atcctggacg agctgtggca caccaacgac aaccagatcg ctatcttcaa ccggctgaag 1320

ctggtgccca agaaggtgga cctgtcccag cagaaagaga tccccaccac cctggtggac 1380

gacttcatcc tgagccccgt cgtgaagaga agcttcatcc agagcatcaa agtgatcaac 1440

gccatcatca agaagtacgg cctgcccaac gacatcatta tcgagctggc ccgcgagaag 1500

aactccaagg acgcccagaa aatgatcaac gagatgcaga agcggaaccg gcagaccaac 1560

gagcggatcg aggaaatcat ccggaccacc ggcaaagaga acgccaagta cctgatcgag 1620

aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt acagcctgga agccatccct 1680

ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg accacatcat ccccagaagc 1740

gtgtccttcg acaacagctt caacaacaag gtgctcgtga agcaggaaga aaacagcaag 1800

aagggcaacc ggaccccatt ccagtacctg agcagcagcg acagcaagat cagctacgaa 1860

accttcaaga agcacatcct gaatctggcc aagggcaagg gcagaatcag caagaccaag 1920

aaagagtatc tgctggaaga acgggacatc aacaggttct ccgtgcagaa agacttcatc 1980

aaccggaacc tggtggatac cagatacgcc accagaggcc tgatgaacct gctgcggagc 2040

tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc 2100

tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc 2160

gaggacgccc tgatcattgc caacgccgat ttcatcttca aagagtggaa gaaactggac 2220

aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa agcaggccga gagcatgccc 2280

gagatcgaaa ccgagcagga gtacaaagag atcttcatca ccccccacca gatcaagcac 2340

attaaggact tcaaggacta caagtacagc caccgggtgg acaagaagcc taatagagag 2400

ctgattaacg acaccctgta ctccacccgg aaggacgaca agggcaacac cctgatcgtg 2460

aacaatctga acggcctgta cgacaaggac aatgacaagc tgaaaaagct gatcaacaag 2520

agccccgaaa agctgctgat gtaccaccac gacccccaga cctaccagaa actgaagctg 2580

attatggaac agtacggcga cgagaagaat cccctgtaca agtactacga ggaaaccggg 2640

aactacctga ccaagtactc caaaaaggac aacggccccg tgatcaagaa gattaagtat 2700

tacggcaaca aactgaacgc ccatctggac atcaccgacg actaccccaa cagcagaaac 2760

aaggtcgtga agctgtccct gaagccctac agattcgacg tgtacctgga caatggcgtg 2820

tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg 2880

aatagcaagt gctatgagga agctaagaag ctgaagaaga tcagcaacca ggccgagttt 2940

atcgcctcct tctacaacaa cgatctgatc aagatcaacg gcgagctgta tagagtgatc 3000

ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca tgatcgacat cacctaccgc 3060

gagtacctgg aaaacatgaa cgacaagagg ccccccagga tcattaagac aatcgcctcc 3120

aagacccaga gcattaagaa gtacagcaca gacattctgg gcaacctgta tgaagtgaaa 3180

tctaagaagc accctcagat catcaaaaag ggc 3213

<210> 59

<211> 4104

<212> DNA

<213> 人工序列

<220>

<223> 死Cas9 (dCas9)核酸

<400> 59

atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 60

attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120

cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180

gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240

tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300

ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360

aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420

aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480

atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540

gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600

atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660

cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720

cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780

gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840

cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900

ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960

atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020

cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080

ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140

gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200

aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260

gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320

gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380

agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440

gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500

aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560

tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620

tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680

gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740

agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800

attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860

ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920

catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980

cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040

gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100

tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160

cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220

gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280

atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340

atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400

gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460

gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggctgct 2520

atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580

gataaagcta gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640

aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700

actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760

cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820

accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880

aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940

taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000

tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060

atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120

aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180

ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240

gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300

cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360

gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420

tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480

aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540

tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600

tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660

cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720

cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780

caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840

atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900

cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960

cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020

gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080

gacctctctc agctcggtgg agac 4104

<210> 60

<211> 3207

<212> DNA

<213> 金黄色葡萄球菌（Staphylococcus aureus）

<400> 60

atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc caagcggaac 60

tacatcctgg gcctggacat cggcatcacc agcgtgggct acggcatcat cgactacgag 120

acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag aggccaacgt ggaaaacaac 180

gagggcaggc ggagcaagag aggcgccaga aggctgaagc ggcggaggcg gcatagaatc 240

cagagagtga agaagctgct gttcgactac aacctgctga ccgaccacag cgagctgagc 300

ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc agaagctgag cgaggaagag 360

ttctctgccg ccctgctgca cctggccaag agaagaggcg tgcacaacgt gaacgaggtg 420

gaagaggaca ccggcaacga gctgtccacc aaagagcaga tcagccggaa cagcaaggcc 480

ctggaagaga aatacgtggc cgaactgcag ctggaacggc tgaagaaaga cggcgaagtg 540

cggggcagca tcaacagatt caagaccagc gactacgtga aagaagccaa acagctgctg 600

aaggtgcaga aggcctacca ccagctggac cagagcttca tcgacaccta catcgacctg 660

ctggaaaccc ggcggaccta ctatgaggga cctggcgagg gcagcccctt cggctggaag 720

gacatcaaag aatggtacga gatgctgatg ggccactgca cctacttccc cgaggaactg 780

cggagcgtga agtacgccta caacgccgac ctgtacaacg ccctgaacga cctgaacaat 840

ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt ccagatcatc 900

gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa agaaatcctc 960

gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc cgagttcacc 1020

aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat tattgagaac 1080

gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag cgaggacatc 1140

caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga gcagatctct 1200

aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa cctgatcctg 1260

gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct gaagctggtg 1320

cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt ggacgacttc 1380

atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat caacgccatc 1440

atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga gaagaactcc 1500

aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac caacgagcgg 1560

atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat cgagaagatc 1620

aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat ccctctggaa 1680

gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag aagcgtgtcc 1740

ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaaaacag caagaagggc 1800

aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta cgaaaccttc 1860

aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac caagaaagag 1920

tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt catcaaccgg 1980

aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg gagctacttc 2040

agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac cagctttctg 2100

cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca cgccgaggac 2160

gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc 2220

aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat gcccgagatc 2280

gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa gcacattaag 2340

gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag agagctgatt 2400

aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat cgtgaacaat 2460

ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa caagagcccc 2520

gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa gctgattatg 2580

gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac cgggaactac 2640

ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa gtattacggc 2700

aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag aaacaaggtc 2760

gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg cgtgtacaag 2820

ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga agtgaatagc 2880

aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga gtttatcgcc 2940

tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt gatcggcgtg 3000

aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta ccgcgagtac 3060

ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc ctccaagacc 3120

cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt gaaatctaag 3180

aagcaccctc agatcatcaa aaagggc 3207

<210> 61

<211> 3708

<212> DNA

<213> 人工序列

<220>

<223> cpf1

<400> 61

atggccccaa agaagaagcg gaaggtcagc aagctggaga agtttacaaa ctgctactcc 60

ctgtctaaga ccctgaggtt caaggccatc cctgtgggca agacccagga gaacatcgac 120

aataagcggc tgctggtgga ggacgagaag agagccgagg attataaggg cgtgaagaag 180

ctgctggatc gctactatct gtcttttatc aacgacgtgc tgcacagcat caagctgaag 240

aatctgaaca attacatcag cctgttccgg aagaaaacca gaaccgagaa ggagaataag 300

gagctggaga acctggagat caatctgcgg aaggagatcg ccaaggcctt caagggcaac 360

gagggctaca agtccctgtt taagaaggat atcatcgaga caatcctgcc agagttcctg 420

gacgataagg acgagatcgc cctggtgaac agcttcaatg gctttaccac agccttcacc 480

ggcttctttg ataacagaga gaatatgttt tccgaggagg ccaagagcac atccatcgcc 540

ttcaggtgta tcaacgagaa tctgacccgc tacatctcta atatggacat cttcgagaag 600

gtggacgcca tctttgataa gcacgaggtg caggagatca aggagaagat cctgaacagc 660

gactatgatg tggaggattt ctttgagggc gagttcttta actttgtgct gacacaggag 720

ggcatcgacg tgtataacgc catcatcggc ggcttcgtga ccgagagcgg cgagaagatc 780

aagggcctga acgagtacat caacctgtat aatcagaaaa ccaagcagaa gctgcctaag 840

tttaagccac tgtataagca ggtgctgagc gatcgggagt ctctgagctt ctacggcgag 900

ggctatacat ccgatgagga ggtgctggag gtgtttagaa acaccctgaa caagaacagc 960

gagatcttca gctccatcaa gaagctggag aagctgttca agaattttga cgagtactct 1020

agcgccggca tctttgtgaa gaacggcccc gccatcagca caatctccaa ggatatcttc 1080

ggcgagtgga acgtgatccg ggacaagtgg aatgccgagt atgacgatat ccacctgaag 1140

aagaaggccg tggtgaccga gaagtacgag gacgatcgga gaaagtcctt caagaagatc 1200

ggctcctttt ctctggagca gctgcaggag tacgccgacg ccgatctgtc tgtggtggag 1260

aagctgaagg agatcatcat ccagaaggtg gatgagatct acaaggtgta tggctcctct 1320

gagaagctgt tcgacgccga ttttgtgctg gagaagagcc tgaagaagaa cgacgccgtg 1380

gtggccatca tgaaggacct gctggattct gtgaagagct tcgagaatta catcaaggcc 1440

ttctttggcg agggcaagga gacaaacagg gacgagtcct tctatggcga ttttgtgctg 1500

gcctacgaca tcctgctgaa ggtggaccac atctacgatg ccatccgcaa ttatgtgacc 1560

cagaagccct actctaagga taagttcaag ctgtattttc agaaccctca gttcatgggc 1620

ggctgggaca aggataagga gacagactat cgggccacca tcctgagata cggctccaag 1680

tactatctgg ccatcatgga taagaagtac gccaagtgcc tgcagaagat cgacaaggac 1740

gatgtgaacg gcaattacga gaagatcaac tataagctgc tgcccggccc taataagatg 1800

ctgccaaagg tgttcttttc taagaagtgg atggcctact ataaccccag cgaggacatc 1860

cagaagatct acaagaatgg cacattcaag aagggcgata tgtttaacct gaatgactgt 1920

cacaagctga tcgacttctt taaggatagc atctcccggt atccaaagtg gtccaatgcc 1980

tacgatttca acttttctga gacagagaag tataaggaca tcgccggctt ttacagagag 2040

gtggaggagc agggctataa ggtgagcttc gagtctgcca gcaagaagga ggtggataag 2100

ctggtggagg agggcaagct gtatatgttc cagatctata acaaggactt ttccgataag 2160

tctcacggca cacccaatct gcacaccatg tacttcaagc tgctgtttga cgagaacaat 2220

cacggacaga tcaggctgag cggaggagca gagctgttca tgaggcgcgc ctccctgaag 2280

aaggaggagc tggtggtgca cccagccaac tcccctatcg ccaacaagaa tccagataat 2340

cccaagaaaa ccacaaccct gtcctacgac gtgtataagg ataagaggtt ttctgaggac 2400

cagtacgagc tgcacatccc aatcgccatc aataagtgcc ccaagaacat cttcaagatc 2460

aatacagagg tgcgcgtgct gctgaagcac gacgataacc cctatgtgat cggcatcgat 2520

aggggcgagc gcaatctgct gtatatcgtg gtggtggacg gcaagggcaa catcgtggag 2580

cagtattccc tgaacgagat catcaacaac ttcaacggca tcaggatcaa gacagattac 2640

cactctctgc tggacaagaa ggagaaggag aggttcgagg cccgccagaa ctggacctcc 2700

atcgagaata tcaaggagct gaaggccggc tatatctctc aggtggtgca caagatctgc 2760

gagctggtgg agaagtacga tgccgtgatc gccctggagg acctgaactc tggctttaag 2820

aatagccgcg tgaaggtgga gaagcaggtg tatcagaagt tcgagaagat gctgatcgat 2880

aagctgaact acatggtgga caagaagtct aatccttgtg caacaggcgg cgccctgaag 2940

ggctatcaga tcaccaataa gttcgagagc tttaagtcca tgtctaccca gaacggcttc 3000

atcttttaca tccctgcctg gctgacatcc aagatcgatc catctaccgg ctttgtgaac 3060

ctgctgaaaa ccaagtatac cagcatcgcc gattccaaga agttcatcag ctcctttgac 3120

aggatcatgt acgtgcccga ggaggatctg ttcgagtttg ccctggacta taagaacttc 3180

tctcgcacag acgccgatta catcaagaag tggaagctgt actcctacgg caaccggatc 3240

agaatcttcc ggaatcctaa gaagaacaac gtgttcgact gggaggaggt gtgcctgacc 3300

agcgcctata aggagctgtt caacaagtac ggcatcaatt atcagcaggg cgatatcaga 3360

gccctgctgt gcgagcagtc cgacaaggcc ttctactcta gctttatggc cctgatgagc 3420

ctgatgctgc agatgcggaa cagcatcaca ggccgcaccg acgtggattt tctgatcagc 3480

cctgtgaaga actccgacgg catcttctac gatagccgga actatgaggc ccaggagaat 3540

gccatcctgc caaagaacgc cgacgccaat ggcgcctata acatcgccag aaaggtgctg 3600

tgggccatcg gccagttcaa gaaggccgag gacgagaagc tggataaggt gaagatcgcc 3660

atctctaaca aggagtggct ggagtacgcc cagaccagcg tgaagcac 3708

<210> 62

<211> 2970

<212> DNA

<213> 人工序列

<220>

<223> CasX

<400> 62

atggccccaa agaagaagcg gaaggtcagc atgcaagaga tcaagagaat caacaagatc 60

agaaggagac tggtcaagga cagcaacaca aagaaggccg gcaagacagg ccccatgaaa 120

accctgctcg tcagagtgat gacccctgac ctgagagagc ggctggaaaa cctgagaaag 180

aagcccgaga acatccctca gcctatcagc aacaccagca gggccaacct gaacaagctg 240

ctgaccgact acaccgagat gaagaaagcc atcctgcacg tgtactggga agagttccag 300

aaagaccccg tgggcctgat gagcagagtt gctcagcccg ctcctaagaa catcgaccag 360

agaaagctga tccccgtgaa ggacggcaac gagagactga cctctagcgg ctttgcctgc 420

agccagtgtt gccagcctct gtacgtgtac aagctggaac aagtgaacga caagggcaag 480

ccccacacca actacttcgg cagatgcaac gtgtccgagc acgagaggct gatcctgctg 540

tctcctcaca agcccgaggc caacgatgag ctggtcacat acagcctggg caagttcgga 600

cagagagccc tggacttcta cagcatccac gtgaccaggg agagcaatca ccctgtgaag 660

cccctggaac agatcggcgg caatagctgt gcctctggac ctgtgggaaa agccctgagc 720

gacgcctgta tgggagccgt ggcatccttc ctgaccaagt accaggacat catcctggaa 780

caccagaaag tgatcaagaa gaacgagaaa agactggcca acctcaagga tatcgccagc 840

gctaacggcc tggcctttcc taagatcacc ctgcctccac agcctcacac caaagagggc 900

atcgaggcct acaacaacgt ggtggcccag atcgtgattt gggtcaacct gaatctgtgg 960

cagaagctga agatcggcag ggacgaagcc aagccactgc agagactgaa gggcttccct 1020

agcttccctc tggtggaaag acaggccaat gaagtggatt ggtgggacat ggtctgcaac 1080

gtgaagaagc tgatcaacga gaagaaagag gatggcaagg ttttctggca gaacctggcc 1140

ggctacaaga gacaagaagc cctgctgcct tacctgagca gcgaagagga ccggaagaag 1200

ggcaagaagt tcgccagata ccagttcggc gacctgctgc tgcacctgga aaagaagcac 1260

ggcgaggact ggggcaaagt gtacgatgag gcctgggaga gaatcgacaa gaaggtggaa 1320

ggcctgagca agcacattaa gctggaagag gaaagaagga gcgaggacgc ccaatctaaa 1380

gccgctctga ccgattggct gagagccaag gccagctttg tgatcgaggg cctgaaagag 1440

gccgacaagg acgagttctg cagatgcgag ctgaagctgc agaagtggta cggcgatctg 1500

agaggcaagc ccttcgccat tgaggccgag aacagcatcc tggacatcag cggcttcagc 1560

aagcagtaca actgcgcctt catttggcag aaagacggcg tcaagaaact gaacctgtac 1620

ctgatcatca attacttcaa aggcggcaag ctgcggttca agaagatcaa acccgaggcc 1680

ttcgaggcta acagattcta caccgtgatc aacaaaaagt ccggcgagat cgtgcccatg 1740

gaagtgaact tcaacttcga cgaccccaac ctgattatcc tgcctctggc cttcggcaag 1800

agacagggca gagagttcat ctggaacgat ctgctgagcc tggaaaccgg ctctctgaag 1860

ctggccaatg gcagagtgat cgagaaaacc ctgtacaaca ggagaaccag acaggacgag 1920

cctgctctgt ttgtggccct gaccttcgag agaagagagg tgctggacag cagcaacatc 1980

aagcccatga acctgatcgg catcgaccgg ggcgagaata tccctgctgt gatcgccctg 2040

acagaccctg aaggatgccc actgagcaga ttcaaggact ccctgggcaa ccctacacac 2100

atcctgagaa tcggcgagag ctacaaagag aagcagagga caatccaggc cgccaaagag 2160

gtggaacaga gaagagccgg cggatactct aggaagtacg ccagcaaggc caagaatctg 2220

gccgacgaca tggtccgaaa caccgccaga gatctgctgt actacgccgt gacacaggac 2280

gccatgctga tcttcgagaa tctgagcaga ggcttcggcc ggcagggcaa gagaaccttt 2340

atggccgaga ggcagtacac cagaatggaa gattggctca cagctaaact ggcctacgag 2400

ggactgccca gcaagaccta cctgtccaaa acactggccc agtatacctc caagacctgc 2460

agcaattgcg gcttcaccat caccagcgcc gactacgaca gagtgctgga aaagctcaag 2520

aaaaccgcca ccggctggat gaccaccatc aacggcaaag agctgaaggt tgagggccag 2580

atcacctact acaacaggta caagaggcag aacgtcgtga aggatctgag cgtggaactg 2640

gacagactga gcgaagagag cgtgaacaac gacatcagca gctggacaaa gggcagatca 2700

ggcgaggctc tgagcctgct gaagaagagg tttagccaca gacctgtgca agagaagttc 2760

gtgtgcctga actgcggctt cgagacacac gccgatgaac aggctgccct gaacattgcc 2820

agaagctggc tgttcctgag aagccaagag tacaagaagt accagaccaa caagaccacc 2880

ggcaacaccg acaagagggc ctttgtggaa acctggcaga gcttctacag aaaaaagctg 2940

aaagaagtct ggaagcccgc cgtgactagt 2970

<210> 63

<211> 2976

<212> DNA

<213> 空肠弯曲杆菌（Campylobacter jejuni）

<400> 63

atggccccaa agaagaagcg gaaggtcgcc agaatcctgg ccttcgacat cggcatcagc 60

agcatcggct gggccttcag cgagaacgac gagctgaagg actgcggcgt gcggatcttc 120

accaaggtgg aaaaccccaa gaccggcgag agcctggccc tgcccagaag gctggccaga 180

agcgcccgga agagactggc cagacggaag gcccggctga accacctgaa gcacctgatc 240

gccaacgagt tcaagctgaa ctacgaggac taccagagct tcgacgagtc cctggccaag 300

gcctacaagg gcagcctgat cagcccctac gagctgcggt tccgggccct gaacgagctg 360

ctgagcaagc aggacttcgc cagagtgatc ctgcacattg ccaagcggag aggctacgac 420

gacatcaaga acagcgacga caaagagaag ggcgccatcc tgaaggccat caagcagaac 480

gaggaaaagc tggccaacta ccagtccgtg ggcgagtacc tgtacaaaga gtacttccag 540

aagttcaaag agaacagcaa agaattcacc aacgtgcgga acaagaaaga aagctacgag 600

cggtgtatcg cccagagctt cctgaaggat gagctgaagc tgatcttcaa gaagcagaga 660

gagttcggct tcagcttcag caagaaattc gaggaagagg tgctgagcgt cgccttctac 720

aagagagccc tgaaggactt cagccacctc gtgggcaact gcagcttctt caccgacgag 780

aagagagccc ccaagaacag ccccctggcc ttcatgttcg tggccctgac ccggatcatc 840

aacctgctga acaatctgaa gaacaccgag ggcatcctgt acaccaagga cgacctgaac 900

gccctgctga atgaggtgct gaagaacggc accctgacct acaagcagac caagaagctg 960

ctgggcctga gcgacgacta cgagtttaag ggcgagaagg gcacctactt catcgagttc 1020

aagaagtaca aagagttcat caaggccctg ggcgagcaca acctgagcca ggacgatctg 1080

aatgagatcg ccaaggacat caccctgatc aaggacgaga ttaagctgaa gaaggccctg 1140

gccaaatacg acctgaatca gaaccagatc gacagcctga gcaagctgga attcaaggat 1200

cacctgaaca tcagcttcaa ggctctgaag ctggtcaccc ccctgatgct ggaaggcaag 1260

aagtacgacg aggcctgcaa cgagctgaac ctgaaggtgg ccatcaacga ggacaagaag 1320

gacttcctgc ccgccttcaa cgaaacctac tacaaggacg aagtgaccaa ccccgtggtg 1380

ctgcgggcca tcaaagaata ccggaaggtg ctgaatgccc tgctcaagaa atacggcaag 1440

gtgcacaaga tcaacatcga gctggcccgg gaagtgggca agaaccacag ccagcgggcc 1500

aagatcgaga aagagcagaa cgaaaactac aaggccaaga aggacgctga gctggaatgc 1560

gagaagctgg gactgaagat caacagcaag aacatcctga agctgcggct gttcaaagaa 1620

cagaaagagt tctgcgccta cagcggcgag aagatcaaga tcagcgatct gcaggacgag 1680

aagatgctgg aaatcgacca catctacccc tacagccggt ccttcgacga cagctacatg 1740

aacaaggtgc tggtgttcac caaacagaac caggaaaaac tgaaccagac ccccttcgag 1800

gccttcggca acgacagcgc caagtggcag aaaatcgagg tgctggccaa gaacctgccc 1860

accaagaaac agaagagaat cctggacaag aattacaagg acaaagagca gaagaacttc 1920

aaggaccgga acctgaacga cacccggtat atcgcccggc tggtgctgaa ctacacaaag 1980

gactacctgg atttcctgcc cctgtccgac gacgagaaca ccaagctgaa cgatacccag 2040

aaaggctcca aggtgcacgt ggaagccaag agcggcatgc tgaccagcgc cctgagacac 2100

acctggggct tcagcgccaa ggatcggaac aaccatctgc accacgccat cgacgccgtg 2160

atcattgcct acgccaacaa cagcatcgtg aaggccttct ccgacttcaa gaaagaacag 2220

gaaagcaaca gcgccgagct gtacgccaag aagatctctg agctggacta caagaacaag 2280

cggaagttct tcgagccctt cagcggcttc cggcagaagg tgctggataa gatcgacgag 2340

atcttcgtgt ccaagcccga gcggaagaag ccctctggcg ccctgcacga ggaaaccttc 2400

agaaaagagg aagagttcta ccagtcctac ggcggcaaag aaggcgtgct gaaggccctc 2460

gagctgggca agatcagaaa agtgaacggc aagatcgtga agaacgggga catgttccgg 2520

gtggacatct tcaagcacaa aaagaccaac aagttctacg ccgtgcccat ctacaccatg 2580

gacttcgccc tgaaggtgct gcccaacaag gccgtggccc ggtccaagaa gggcgagatc 2640

aaggactgga ttctgatgga cgagaactac gagttctgct ttagcctgta caaggactcc 2700

ctgatcctga tccagaccaa ggacatgcag gaacccgagt tcgtctacta caacgccttc 2760

accagcagca ccgtgtccct gatcgtgtct aagcacgaca acaagttcga gacactgagc 2820

aagaaccaga agatcctgtt caagaacgcc aacgagaaag aagtgatcgc caagagcatc 2880

ggcatccaga atctgaaggt gttcgagaag tacatcgtgt ccgccctggg agaagtgaca 2940

aaggccgagt tccggcagag agaggacttc aaaaag 2976

<210> 64

<211> 1368

<212> PRT

<213> 人工序列

<220>

<223> 人Cas9 (hCas9)氨基酸

<400> 64

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

1 5 10 15

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

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

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

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

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

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

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

260 265 270

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

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp

1365

<210> 65

<211> 1368

<212> PRT

<213> 人工序列

<220>

<223> 切口酶Cas9 (nCas9)氨基酸

<400> 65

Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp

1365

<210> 66

<211> 1069

<212> PRT

<213> 金黄色葡萄球菌（Staphylococcus aureus）

<400> 66

Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala

1 5 10 15

Ala Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val

20 25 30

Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly

35 40 45

Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg

50 55 60

Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile

65 70 75 80

Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His

85 90 95

Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu

100 105 110

Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu

115 120 125

Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr

130 135 140

Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala

145 150 155 160

Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys

165 170 175

Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr

180 185 190

Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln

195 200 205

Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg

210 215 220

Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys

225 230 235 240

Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe

245 250 255

Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr

260 265 270

Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn

275 280 285

Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe

290 295 300

Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu

305 310 315 320

Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys

325 330 335

Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr

340 345 350

Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala

355 360 365

Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu

370 375 380

Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser

385 390 395 400

Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile

405 410 415

Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala

420 425 430

Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln

435 440 445

Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro

450 455 460

Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile

465 470 475 480

Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg

485 490 495

Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys

500 505 510

Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr

515 520 525

Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp

530 535 540

Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu

545 550 555 560

Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro

565 570 575

Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys

580 585 590

Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu

595 600 605

Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile

610 615 620

Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu

625 630 635 640

Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp

645 650 655

Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu

660 665 670

Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys

675 680 685

Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp

690 695 700

Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp

705 710 715 720

Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys

725 730 735

Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys

740 745 750

Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu

755 760 765

Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp

770 775 780

Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile

785 790 795 800

Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu

805 810 815

Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu

820 825 830

Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His

835 840 845

Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly

850 855 860

Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr

865 870 875 880

Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile

885 890 895

Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp

900 905 910

Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr

915 920 925

Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val

930 935 940

Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser

945 950 955 960

Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala

965 970 975

Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly

980 985 990

Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile

995 1000 1005

Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met

1010 1015 1020

Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr

1025 1030 1035 1040

Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu

1045 1050 1055

Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly

1060 1065

<210> 67

<211> 1368

<212> PRT

<213> 人工序列

<220>

<223> 死Cas9 (dCas9)氨基酸

<400> 67

Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Ala Ala Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Ala Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp

1365

<210> 68

<211> 1069

<212> PRT

<213> 金黄色葡萄球菌（Staphylococcus aureus）

<400> 68

Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala

1 5 10 15

Ala Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val

20 25 30

Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly

35 40 45

Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg

50 55 60

Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile

65 70 75 80

Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His

85 90 95

Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu

100 105 110

Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu

115 120 125

Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr

130 135 140

Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala

145 150 155 160

Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys

165 170 175

Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr

180 185 190

Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln

195 200 205

Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg

210 215 220

Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys

225 230 235 240

Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe

245 250 255

Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr

260 265 270

Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn

275 280 285

Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe

290 295 300

Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu

305 310 315 320

Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys

325 330 335

Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr

340 345 350

Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala

355 360 365

Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu

370 375 380

Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser

385 390 395 400

Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile

405 410 415

Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala

420 425 430

Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln

435 440 445

Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro

450 455 460

Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile

465 470 475 480

Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg

485 490 495

Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys

500 505 510

Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr

515 520 525

Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp

530 535 540

Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu

545 550 555 560

Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro

565 570 575

Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys

580 585 590

Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu

595 600 605

Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile

610 615 620

Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu

625 630 635 640

Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp

645 650 655

Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu

660 665 670

Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys

675 680 685

Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp

690 695 700

Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp

705 710 715 720

Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys

725 730 735

Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys

740 745 750

Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu

755 760 765

Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp

770 775 780

Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile

785 790 795 800

Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu

805 810 815

Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu

820 825 830

Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His

835 840 845

Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly

850 855 860

Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr

865 870 875 880

Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile

885 890 895

Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp

900 905 910

Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr

915 920 925

Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val

930 935 940

Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser

945 950 955 960

Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala

965 970 975

Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly

980 985 990

Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile

995 1000 1005

Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met

1010 1015 1020

Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr

1025 1030 1035 1040

Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu

1045 1050 1055

Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly

1060 1065

<210> 69

<211> 1236

<212> PRT

<213> 人工序列

<220>

<223> Cpf1

<400> 69

Met Ala Pro Lys Lys Lys Arg Lys Val Ser Lys Leu Glu Lys Phe Thr

1 5 10 15

Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro Val

20 25 30

Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu Asp

35 40 45

Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp Arg

50 55 60

Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu Lys

65 70 75 80

Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr Glu

85 90 95

Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys Glu

100 105 110

Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe Lys

115 120 125

Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys Asp

130 135 140

Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe Thr

145 150 155 160

Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys Ser

165 170 175

Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile

180 185 190

Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys His

195 200 205

Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val

210 215 220

Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln Glu

225 230 235 240

Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu Ser

245 250 255

Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln

260 265 270

Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val

275 280 285

Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser

290 295 300

Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn Ser

305 310 315 320

Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe

325 330 335

Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala Ile

340 345 350

Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg Asp

355 360 365

Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Lys Ala Val

370 375 380

Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile

385 390 395 400

Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu

405 410 415

Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp Glu

420 425 430

Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp Phe

435 440 445

Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile Met

450 455 460

Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala

465 470 475 480

Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly

485 490 495

Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile Tyr

500 505 510

Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys

515 520 525

Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp Lys

530 535 540

Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys

545 550 555 560

Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln Lys

565 570 575

Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys

580 585 590

Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser Lys

595 600 605

Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr

610 615 620

Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu Asn Asp Cys

625 630 635 640

His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys

645 650 655

Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys

660 665 670

Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys Val

675 680 685

Ser Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu Val Glu Glu

690 695 700

Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys

705 710 715 720

Ser His Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys Leu Leu Phe

725 730 735

Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu

740 745 750

Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu Val Val His Pro

755 760 765

Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr

770 775 780

Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp

785 790 795 800

Gln Tyr Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn

805 810 815

Ile Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp

820 825 830

Asn Pro Tyr Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr

835 840 845

Ile Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu

850 855 860

Asn Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr

865 870 875 880

His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln

885 890 895

Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile

900 905 910

Ser Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala

915 920 925

Val Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val

930 935 940

Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp

945 950 955 960

Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly

965 970 975

Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys

980 985 990

Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu

995 1000 1005

Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys Thr

1010 1015 1020

Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp

1025 1030 1035 1040

Arg Ile Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp

1045 1050 1055

Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys

1060 1065 1070

Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys

1075 1080 1085

Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys

1090 1095 1100

Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg

1105 1110 1115 1120

Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met

1125 1130 1135

Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg

1140 1145 1150

Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile

1155 1160 1165

Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro

1170 1175 1180

Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val Leu

1185 1190 1195 1200

Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu Asp Lys

1205 1210 1215

Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr

1220 1225 1230

Ser Val Lys His

1235

<210> 70

<211> 990

<212> PRT

<213> 人工序列

<220>

<223> Casx

<400> 70

Met Ala Pro Lys Lys Lys Arg Lys Val Ser Met Gln Glu Ile Lys Arg

1 5 10 15

Ile Asn Lys Ile Arg Arg Arg Leu Val Lys Asp Ser Asn Thr Lys Lys

20 25 30

Ala Gly Lys Thr Gly Pro Met Lys Thr Leu Leu Val Arg Val Met Thr

35 40 45

Pro Asp Leu Arg Glu Arg Leu Glu Asn Leu Arg Lys Lys Pro Glu Asn

50 55 60

Ile Pro Gln Pro Ile Ser Asn Thr Ser Arg Ala Asn Leu Asn Lys Leu

65 70 75 80

Leu Thr Asp Tyr Thr Glu Met Lys Lys Ala Ile Leu His Val Tyr Trp

85 90 95

Glu Glu Phe Gln Lys Asp Pro Val Gly Leu Met Ser Arg Val Ala Gln

100 105 110

Pro Ala Pro Lys Asn Ile Asp Gln Arg Lys Leu Ile Pro Val Lys Asp

115 120 125

Gly Asn Glu Arg Leu Thr Ser Ser Gly Phe Ala Cys Ser Gln Cys Cys

130 135 140

Gln Pro Leu Tyr Val Tyr Lys Leu Glu Gln Val Asn Asp Lys Gly Lys

145 150 155 160

Pro His Thr Asn Tyr Phe Gly Arg Cys Asn Val Ser Glu His Glu Arg

165 170 175

Leu Ile Leu Leu Ser Pro His Lys Pro Glu Ala Asn Asp Glu Leu Val

180 185 190

Thr Tyr Ser Leu Gly Lys Phe Gly Gln Arg Ala Leu Asp Phe Tyr Ser

195 200 205

Ile His Val Thr Arg Glu Ser Asn His Pro Val Lys Pro Leu Glu Gln

210 215 220

Ile Gly Gly Asn Ser Cys Ala Ser Gly Pro Val Gly Lys Ala Leu Ser

225 230 235 240

Asp Ala Cys Met Gly Ala Val Ala Ser Phe Leu Thr Lys Tyr Gln Asp

245 250 255

Ile Ile Leu Glu His Gln Lys Val Ile Lys Lys Asn Glu Lys Arg Leu

260 265 270

Ala Asn Leu Lys Asp Ile Ala Ser Ala Asn Gly Leu Ala Phe Pro Lys

275 280 285

Ile Thr Leu Pro Pro Gln Pro His Thr Lys Glu Gly Ile Glu Ala Tyr

290 295 300

Asn Asn Val Val Ala Gln Ile Val Ile Trp Val Asn Leu Asn Leu Trp

305 310 315 320

Gln Lys Leu Lys Ile Gly Arg Asp Glu Ala Lys Pro Leu Gln Arg Leu

325 330 335

Lys Gly Phe Pro Ser Phe Pro Leu Val Glu Arg Gln Ala Asn Glu Val

340 345 350

Asp Trp Trp Asp Met Val Cys Asn Val Lys Lys Leu Ile Asn Glu Lys

355 360 365

Lys Glu Asp Gly Lys Val Phe Trp Gln Asn Leu Ala Gly Tyr Lys Arg

370 375 380

Gln Glu Ala Leu Leu Pro Tyr Leu Ser Ser Glu Glu Asp Arg Lys Lys

385 390 395 400

Gly Lys Lys Phe Ala Arg Tyr Gln Phe Gly Asp Leu Leu Leu His Leu

405 410 415

Glu Lys Lys His Gly Glu Asp Trp Gly Lys Val Tyr Asp Glu Ala Trp

420 425 430

Glu Arg Ile Asp Lys Lys Val Glu Gly Leu Ser Lys His Ile Lys Leu

435 440 445

Glu Glu Glu Arg Arg Ser Glu Asp Ala Gln Ser Lys Ala Ala Leu Thr

450 455 460

Asp Trp Leu Arg Ala Lys Ala Ser Phe Val Ile Glu Gly Leu Lys Glu

465 470 475 480

Ala Asp Lys Asp Glu Phe Cys Arg Cys Glu Leu Lys Leu Gln Lys Trp

485 490 495

Tyr Gly Asp Leu Arg Gly Lys Pro Phe Ala Ile Glu Ala Glu Asn Ser

500 505 510

Ile Leu Asp Ile Ser Gly Phe Ser Lys Gln Tyr Asn Cys Ala Phe Ile

515 520 525

Trp Gln Lys Asp Gly Val Lys Lys Leu Asn Leu Tyr Leu Ile Ile Asn

530 535 540

Tyr Phe Lys Gly Gly Lys Leu Arg Phe Lys Lys Ile Lys Pro Glu Ala

545 550 555 560

Phe Glu Ala Asn Arg Phe Tyr Thr Val Ile Asn Lys Lys Ser Gly Glu

565 570 575

Ile Val Pro Met Glu Val Asn Phe Asn Phe Asp Asp Pro Asn Leu Ile

580 585 590

Ile Leu Pro Leu Ala Phe Gly Lys Arg Gln Gly Arg Glu Phe Ile Trp

595 600 605

Asn Asp Leu Leu Ser Leu Glu Thr Gly Ser Leu Lys Leu Ala Asn Gly

610 615 620

Arg Val Ile Glu Lys Thr Leu Tyr Asn Arg Arg Thr Arg Gln Asp Glu

625 630 635 640

Pro Ala Leu Phe Val Ala Leu Thr Phe Glu Arg Arg Glu Val Leu Asp

645 650 655

Ser Ser Asn Ile Lys Pro Met Asn Leu Ile Gly Ile Asp Arg Gly Glu

660 665 670

Asn Ile Pro Ala Val Ile Ala Leu Thr Asp Pro Glu Gly Cys Pro Leu

675 680 685

Ser Arg Phe Lys Asp Ser Leu Gly Asn Pro Thr His Ile Leu Arg Ile

690 695 700

Gly Glu Ser Tyr Lys Glu Lys Gln Arg Thr Ile Gln Ala Ala Lys Glu

705 710 715 720

Val Glu Gln Arg Arg Ala Gly Gly Tyr Ser Arg Lys Tyr Ala Ser Lys

725 730 735

Ala Lys Asn Leu Ala Asp Asp Met Val Arg Asn Thr Ala Arg Asp Leu

740 745 750

Leu Tyr Tyr Ala Val Thr Gln Asp Ala Met Leu Ile Phe Glu Asn Leu

755 760 765

Ser Arg Gly Phe Gly Arg Gln Gly Lys Arg Thr Phe Met Ala Glu Arg

770 775 780

Gln Tyr Thr Arg Met Glu Asp Trp Leu Thr Ala Lys Leu Ala Tyr Glu

785 790 795 800

Gly Leu Pro Ser Lys Thr Tyr Leu Ser Lys Thr Leu Ala Gln Tyr Thr

805 810 815

Ser Lys Thr Cys Ser Asn Cys Gly Phe Thr Ile Thr Ser Ala Asp Tyr

820 825 830

Asp Arg Val Leu Glu Lys Leu Lys Lys Thr Ala Thr Gly Trp Met Thr

835 840 845

Thr Ile Asn Gly Lys Glu Leu Lys Val Glu Gly Gln Ile Thr Tyr Tyr

850 855 860

Asn Arg Tyr Lys Arg Gln Asn Val Val Lys Asp Leu Ser Val Glu Leu

865 870 875 880

Asp Arg Leu Ser Glu Glu Ser Val Asn Asn Asp Ile Ser Ser Trp Thr

885 890 895

Lys Gly Arg Ser Gly Glu Ala Leu Ser Leu Leu Lys Lys Arg Phe Ser

900 905 910

His Arg Pro Val Gln Glu Lys Phe Val Cys Leu Asn Cys Gly Phe Glu

915 920 925

Thr His Ala Asp Glu Gln Ala Ala Leu Asn Ile Ala Arg Ser Trp Leu

930 935 940

Phe Leu Arg Ser Gln Glu Tyr Lys Lys Tyr Gln Thr Asn Lys Thr Thr

945 950 955 960

Gly Asn Thr Asp Lys Arg Ala Phe Val Glu Thr Trp Gln Ser Phe Tyr

965 970 975

Arg Lys Lys Leu Lys Glu Val Trp Lys Pro Ala Val Thr Ser

980 985 990

<210> 71

<211> 1782

<212> DNA

<213> 人工序列

<220>

<223> 高活性PiggyBac (PB) 转座酶核酸

<400> 71

atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60

ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120

tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180

tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240

aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300

tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360

cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420

gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480

gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540

ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600

gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660

atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720

ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780

cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840

agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900

agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960

ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020

agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080

gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140

gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200

ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260

gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320

cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380

aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440

agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500

aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560

gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620

cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680

tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740

atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782

<210> 72

<211> 1020

<212> DNA

<213> 人工序列

<220>

<223> 高活性睡美人(SB100) 转座酶核酸

<400> 72

atgggaaaat caaaagaaat cagccaagac ctcagaaaaa gaattgtaga cctccacaag 60

tctggttcat ccttgggagc aatttccaaa cgcctggcgg taccacgttc atctgtacaa 120

acaatagtac gcaagtataa acaccatggg accacgcagc cgtcataccg ctcaggaagg 180

agacgcgttc tgtctcctag agatgaacgt actttggtgc gaaaagtgca aatcaatccc 240

agaacaacag caaaggacct tgtgaagatg ctggaggaaa caggtacaaa agtatctata 300

tccacagtaa aacgagtcct atatcgacat aacctgaaag gccactcagc aaggaagaag 360

ccactgctcc aaaaccgaca taagaaagcc agactacggt ttgcaactgc acatggggac 420

aaagatcgta ctttttggag aaatgtcctc tggtctgatg aaacaaaaat agaactgttt 480

ggccataatg accatcgtta tgtttggagg aagaaggggg aggcttgcaa gccgaagaac 540

accatcccaa ccgtgaagca cgggggtggc agcatcatgt tgtgggggtg ctttgctgca 600

ggagggactg gtgcacttca caaaatagat ggcatcatgg acgccgtgca gtatgtggat 660

atattgaagc aacatctcaa gacatcagtc aggaagttaa agcttggtcg caaatgggtc 720

ttccaacacg acaatgaccc caagcatact tccaaagttg tggcaaaatg gcttaaggac 780

aacaaagtca aggtattgga gtggccatca caaagccctg acctcaatcc tatagaaaat 840

ttgtgggcag aactgaaaaa gcgtgtgcga gcaaggaggc ctacaaacct gactcagtta 900

caccagctct gtcaggagga atgggccaaa attcacccaa attattgtgg gaagcttgtg 960

gaaggctacc cgaaacgttt gacccaagtt aaacaattta aaggcaatgc taccaaatac 1020

<210> 73

<211> 340

<212> PRT

<213> 人工序列

<220>

<223> 高活性睡美人(SB100) 转座酶氨基酸

<400> 73

Met Gly Lys Ser Lys Glu Ile Ser Gln Asp Leu Arg Lys Arg Ile Val

1 5 10 15

Asp Leu His Lys Ser Gly Ser Ser Leu Gly Ala Ile Ser Lys Arg Leu

20 25 30

Ala Val Pro Arg Ser Ser Val Gln Thr Ile Val Arg Lys Tyr Lys His

35 40 45

His Gly Thr Thr Gln Pro Ser Tyr Arg Ser Gly Arg Arg Arg Val Leu

50 55 60

Ser Pro Arg Asp Glu Arg Thr Leu Val Arg Lys Val Gln Ile Asn Pro

65 70 75 80

Arg Thr Thr Ala Lys Asp Leu Val Lys Met Leu Glu Glu Thr Gly Thr

85 90 95

Lys Val Ser Ile Ser Thr Val Lys Arg Val Leu Tyr Arg His Asn Leu

100 105 110

Lys Gly His Ser Ala Arg Lys Lys Pro Leu Leu Gln Asn Arg His Lys

115 120 125

Lys Ala Arg Leu Arg Phe Ala Thr Ala His Gly Asp Lys Asp Arg Thr

130 135 140

Phe Trp Arg Asn Val Leu Trp Ser Asp Glu Thr Lys Ile Glu Leu Phe

145 150 155 160

Gly His Asn Asp His Arg Tyr Val Trp Arg Lys Lys Gly Glu Ala Cys

165 170 175

Lys Pro Lys Asn Thr Ile Pro Thr Val Lys His Gly Gly Gly Ser Ile

180 185 190

Met Leu Trp Gly Cys Phe Ala Ala Gly Gly Thr Gly Ala Leu His Lys

195 200 205

Ile Asp Gly Ile Met Asp Ala Val Gln Tyr Val Asp Ile Leu Lys Gln

210 215 220

His Leu Lys Thr Ser Val Arg Lys Leu Lys Leu Gly Arg Lys Trp Val

225 230 235 240

Phe Gln His Asp Asn Asp Pro Lys His Thr Ser Lys Val Val Ala Lys

245 250 255

Trp Leu Lys Asp Asn Lys Val Lys Val Leu Glu Trp Pro Ser Gln Ser

260 265 270

Pro Asp Leu Asn Pro Ile Glu Asn Leu Trp Ala Glu Leu Lys Lys Arg

275 280 285

Val Arg Ala Arg Arg Pro Thr Asn Leu Thr Gln Leu His Gln Leu Cys

290 295 300

Gln Glu Glu Trp Ala Lys Ile His Pro Asn Tyr Cys Gly Lys Leu Val

305 310 315 320

Glu Gly Tyr Pro Lys Arg Leu Thr Gln Val Lys Gln Phe Lys Gly Asn

325 330 335

Ala Thr Lys Tyr

340

<210> 74

<211> 3123

<212> PRT

<213> 智人（homo sapiens）

<400> 74

Met Pro Gly Ala Ala Gly Val Leu Leu Leu Leu Leu Leu Ser Gly Gly

1 5 10 15

Leu Gly Gly Val Gln Ala Gln Arg Pro Gln Gln Gln Arg Gln Ser Gln

20 25 30

Ala His Gln Gln Arg Gly Leu Phe Pro Ala Val Leu Asn Leu Ala Ser

35 40 45

Asn Ala Leu Ile Thr Thr Asn Ala Thr Cys Gly Glu Lys Gly Pro Glu

50 55 60

Met Tyr Cys Lys Leu Val Glu His Val Pro Gly Gln Pro Val Arg Asn

65 70 75 80

Pro Gln Cys Arg Ile Cys Asn Gln Asn Ser Ser Asn Pro Asn Gln Arg

85 90 95

His Pro Ile Thr Asn Ala Ile Asp Gly Lys Asn Thr Trp Trp Gln Ser

100 105 110

Pro Ser Ile Lys Asn Gly Ile Glu Tyr His Tyr Val Thr Ile Thr Leu

115 120 125

Asp Leu Gln Gln Val Phe Gln Ile Ala Tyr Val Ile Val Lys Ala Ala

130 135 140

Asn Ser Pro Arg Pro Gly Asn Trp Ile Leu Glu Arg Ser Leu Asp Asp

145 150 155 160

Val Glu Tyr Lys Pro Trp Gln Tyr His Ala Val Thr Asp Thr Glu Cys

165 170 175

Leu Thr Leu Tyr Asn Ile Tyr Pro Arg Thr Gly Pro Pro Ser Tyr Ala

180 185 190

Lys Asp Asp Glu Val Ile Cys Thr Ser Phe Tyr Ser Lys Ile His Pro

195 200 205

Leu Glu Asn Gly Glu Ile His Ile Ser Leu Ile Asn Gly Arg Pro Ser

210 215 220

Ala Asp Asp Pro Ser Pro Glu Leu Leu Glu Phe Thr Ser Ala Arg Tyr

225 230 235 240

Ile Arg Leu Arg Phe Gln Arg Ile Arg Thr Leu Asn Ala Asp Leu Met

245 250 255

Met Phe Ala His Lys Asp Pro Arg Glu Ile Asp Pro Ile Val Thr Arg

260 265 270

Arg Tyr Tyr Tyr Ser Val Lys Asp Ile Ser Val Gly Gly Met Cys Ile

275 280 285

Cys Tyr Gly His Ala Arg Ala Cys Pro Leu Asp Pro Ala Thr Asn Lys

290 295 300

Ser Arg Cys Glu Cys Glu His Asn Thr Cys Gly Asp Ser Cys Asp Gln

305 310 315 320

Cys Cys Pro Gly Phe His Gln Lys Pro Trp Arg Ala Gly Thr Phe Leu

325 330 335

Thr Lys Thr Glu Cys Glu Ala Cys Asn Cys His Gly Lys Ala Glu Glu

340 345 350

Cys Tyr Tyr Asp Glu Asn Val Ala Arg Arg Asn Leu Ser Leu Asn Ile

355 360 365

Arg Gly Lys Tyr Ile Gly Gly Gly Val Cys Ile Asn Cys Thr Gln Asn

370 375 380

Thr Ala Gly Ile Asn Cys Glu Thr Cys Thr Asp Gly Phe Phe Arg Pro

385 390 395 400

Lys Gly Val Ser Pro Asn Tyr Pro Arg Pro Cys Gln Pro Cys His Cys

405 410 415

Asp Pro Ile Gly Ser Leu Asn Glu Val Cys Val Lys Asp Glu Lys His

420 425 430

Ala Arg Arg Gly Leu Ala Pro Gly Ser Cys His Cys Lys Thr Gly Phe

435 440 445

Gly Gly Val Ser Cys Asp Arg Cys Ala Arg Gly Tyr Thr Gly Tyr Pro

450 455 460

Asp Cys Lys Ala Cys Asn Cys Ser Gly Leu Gly Ser Lys Asn Glu Asp

465 470 475 480

Pro Cys Phe Gly Pro Cys Ile Cys Lys Glu Asn Val Glu Gly Gly Asp

485 490 495

Cys Ser Arg Cys Lys Ser Gly Phe Phe Asn Leu Gln Glu Asp Asn Trp

500 505 510

Lys Gly Cys Asp Glu Cys Phe Cys Ser Gly Val Ser Asn Arg Cys Gln

515 520 525

Ser Ser Tyr Trp Thr Tyr Gly Lys Ile Gln Asp Met Ser Gly Trp Tyr

530 535 540

Leu Thr Asp Leu Pro Gly Arg Ile Arg Val Ala Pro Gln Gln Asp Asp

545 550 555 560

Leu Asp Ser Pro Gln Gln Ile Ser Ile Ser Asn Ala Glu Ala Arg Gln

565 570 575

Ala Leu Pro His Ser Tyr Tyr Trp Ser Ala Pro Ala Pro Tyr Leu Gly

580 585 590

Asn Lys Leu Pro Ala Val Gly Gly Gln Leu Thr Phe Thr Ile Ser Tyr

595 600 605

Asp Leu Glu Glu Glu Glu Glu Asp Thr Glu Arg Val Leu Gln Leu Met

610 615 620

Ile Ile Leu Glu Gly Asn Asp Leu Ser Ile Ser Thr Ala Gln Asp Glu

625 630 635 640

Val Tyr Leu His Pro Ser Glu Glu His Thr Asn Val Leu Leu Leu Lys

645 650 655

Glu Glu Ser Phe Thr Ile His Gly Thr His Phe Pro Val Arg Arg Lys

660 665 670

Glu Phe Met Thr Val Leu Ala Asn Leu Lys Arg Val Leu Leu Gln Ile

675 680 685

Thr Tyr Ser Phe Gly Met Asp Ala Ile Phe Arg Leu Ser Ser Val Asn

690 695 700

Leu Glu Ser Ala Val Ser Tyr Pro Thr Asp Gly Ser Ile Ala Ala Ala

705 710 715 720

Val Glu Val Cys Gln Cys Pro Pro Gly Tyr Thr Gly Ser Ser Cys Glu

725 730 735

Ser Cys Trp Pro Arg His Arg Arg Val Asn Gly Thr Ile Phe Gly Gly

740 745 750

Ile Cys Glu Pro Cys Gln Cys Phe Gly His Ala Glu Ser Cys Asp Asp

755 760 765

Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro Tyr

770 775 780

Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys Gly Thr

785 790 795 800

Ser Glu Asp Cys Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser Asn

805 810 815

Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile Cys

820 825 830

Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys Ala

835 840 845

Glu Gly Tyr Phe Gly Gln Pro Ser Val Pro Gly Gly Ser Cys Gln Pro

850 855 860

Cys Gln Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser Cys Asp

865 870 875 880

Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly Arg

885 890 895

Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asp Ala

900 905 910

Lys Asn Cys Gln Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe Ser Glu

915 920 925

Val Cys His Ser Gln Thr Gly Gln Cys Glu Cys Arg Ala Asn Val Gln

930 935 940

Gly Gln Arg Cys Asp Lys Cys Lys Ala Gly Thr Phe Gly Leu Gln Ser

945 950 955 960

Ala Arg Gly Cys Val Pro Cys Asn Cys Asn Ser Phe Gly Ser Lys Ser

965 970 975

Phe Asp Cys Glu Glu Ser Gly Gln Cys Trp Cys Gln Pro Gly Val Thr

980 985 990

Gly Lys Lys Cys Asp Arg Cys Ala His Gly Tyr Phe Asn Phe Gln Glu

995 1000 1005

Gly Gly Cys Thr Ala Cys Glu Cys Ser His Leu Gly Asn Asn Cys Asp

1010 1015 1020

Pro Lys Thr Gly Arg Cys Ile Cys Pro Pro Asn Thr Ile Gly Glu Lys

1025 1030 1035 1040

Cys Ser Lys Cys Ala Pro Asn Thr Trp Gly His Ser Ile Thr Thr Gly

1045 1050 1055

Cys Lys Ala Cys Asn Cys Ser Thr Val Gly Ser Leu Asp Phe Gln Cys

1060 1065 1070

Asn Val Asn Thr Gly Gln Cys Asn Cys His Pro Lys Phe Ser Gly Ala

1075 1080 1085

Lys Cys Thr Glu Cys Ser Arg Gly His Trp Asn Tyr Pro Arg Cys Asn

1090 1095 1100

Leu Cys Asp Cys Phe Leu Pro Gly Thr Asp Ala Thr Thr Cys Asp Ser

1105 1110 1115 1120

Glu Thr Lys Lys Cys Ser Cys Ser Asp Gln Thr Gly Gln Cys Thr Cys

1125 1130 1135

Lys Val Asn Val Glu Gly Ile His Cys Asp Arg Cys Arg Pro Gly Lys

1140 1145 1150

Phe Gly Leu Asp Ala Lys Asn Pro Leu Gly Cys Ser Ser Cys Tyr Cys

1155 1160 1165

Phe Gly Thr Thr Thr Gln Cys Ser Glu Ala Lys Gly Leu Ile Arg Thr

1170 1175 1180

Trp Val Thr Leu Lys Ala Glu Gln Thr Ile Leu Pro Leu Val Asp Glu

1185 1190 1195 1200

Ala Leu Gln His Thr Thr Thr Lys Gly Ile Val Phe Gln His Pro Glu

1205 1210 1215

Ile Val Ala His Met Asp Leu Met Arg Glu Asp Leu His Leu Glu Pro

1220 1225 1230

Phe Tyr Trp Lys Leu Pro Glu Gln Phe Glu Gly Lys Lys Leu Met Ala

1235 1240 1245

Tyr Gly Gly Lys Leu Lys Tyr Ala Ile Tyr Phe Glu Ala Arg Glu Glu

1250 1255 1260

Thr Gly Phe Ser Thr Tyr Asn Pro Gln Val Ile Ile Arg Gly Gly Thr

1265 1270 1275 1280

Pro Thr His Ala Arg Ile Ile Val Arg His Met Ala Ala Pro Leu Ile

1285 1290 1295

Gly Gln Leu Thr Arg His Glu Ile Glu Met Thr Glu Lys Glu Trp Lys

1300 1305 1310

Tyr Tyr Gly Asp Asp Pro Arg Val His Arg Thr Val Thr Arg Glu Asp

1315 1320 1325

Phe Leu Asp Ile Leu Tyr Asp Ile His Tyr Ile Leu Ile Lys Ala Thr

1330 1335 1340

Tyr Gly Asn Phe Met Arg Gln Ser Arg Ile Ser Glu Ile Ser Met Glu

1345 1350 1355 1360

Val Ala Glu Gln Gly Arg Gly Thr Thr Met Thr Pro Pro Ala Asp Leu

1365 1370 1375

Ile Glu Lys Cys Asp Cys Pro Leu Gly Tyr Ser Gly Leu Ser Cys Glu

1380 1385 1390

Ala Cys Leu Pro Gly Phe Tyr Arg Leu Arg Ser Gln Pro Gly Gly Arg

1395 1400 1405

Thr Pro Gly Pro Thr Leu Gly Thr Cys Val Pro Cys Gln Cys Asn Gly

1410 1415 1420

His Ser Ser Leu Cys Asp Pro Glu Thr Ser Ile Cys Gln Asn Cys Gln

1425 1430 1435 1440

His His Thr Ala Gly Asp Phe Cys Glu Arg Cys Ala Leu Gly Tyr Tyr

1445 1450 1455

Gly Ile Val Lys Gly Leu Pro Asn Asp Cys Gln Gln Cys Ala Cys Pro

1460 1465 1470

Leu Ile Ser Ser Ser Asn Asn Phe Ser Pro Ser Cys Val Ala Glu Gly

1475 1480 1485

Leu Asp Asp Tyr Arg Cys Thr Ala Cys Pro Arg Gly Tyr Glu Gly Gln

1490 1495 1500

Tyr Cys Glu Arg Cys Ala Pro Gly Tyr Thr Gly Ser Pro Gly Asn Pro

1505 1510 1515 1520

Gly Gly Ser Cys Gln Glu Cys Glu Cys Asp Pro Tyr Gly Ser Leu Pro

1525 1530 1535

Val Pro Cys Asp Pro Val Thr Gly Phe Cys Thr Cys Arg Pro Gly Ala

1540 1545 1550

Thr Gly Arg Lys Cys Asp Gly Cys Lys His Trp His Ala Arg Glu Gly

1555 1560 1565

Trp Glu Cys Val Phe Cys Gly Asp Glu Cys Thr Gly Leu Leu Leu Gly

1570 1575 1580

Asp Leu Ala Arg Leu Glu Gln Met Val Met Ser Ile Asn Leu Thr Gly

1585 1590 1595 1600

Pro Leu Pro Ala Pro Tyr Lys Met Leu Tyr Gly Leu Glu Asn Met Thr

1605 1610 1615

Gln Glu Leu Lys His Leu Leu Ser Pro Gln Arg Ala Pro Glu Arg Leu

1620 1625 1630

Ile Gln Leu Ala Glu Gly Asn Leu Asn Thr Leu Val Thr Glu Met Asn

1635 1640 1645

Glu Leu Leu Thr Arg Ala Thr Lys Val Thr Ala Asp Gly Glu Gln Thr

1650 1655 1660

Gly Gln Asp Ala Glu Arg Thr Asn Thr Arg Ala Lys Ser Leu Gly Glu

1665 1670 1675 1680

Phe Ile Lys Glu Leu Ala Arg Asp Ala Glu Ala Val Asn Glu Lys Ala

1685 1690 1695

Ile Lys Leu Asn Glu Thr Leu Gly Thr Arg Asp Glu Ala Phe Glu Arg

1700 1705 1710

Asn Leu Glu Gly Leu Gln Lys Glu Ile Asp Gln Met Ile Lys Glu Leu

1715 1720 1725

Arg Arg Lys Asn Leu Glu Thr Gln Lys Glu Ile Ala Glu Asp Glu Leu

1730 1735 1740

Val Ala Ala Glu Ala Leu Leu Lys Lys Val Lys Lys Leu Phe Gly Glu

1745 1750 1755 1760

Ser Arg Gly Glu Asn Glu Glu Met Glu Lys Asp Leu Arg Glu Lys Leu

1765 1770 1775

Ala Asp Tyr Lys Asn Lys Val Asp Asp Ala Trp Asp Leu Leu Arg Glu

1780 1785 1790

Ala Thr Asp Lys Ile Arg Glu Ala Asn Arg Leu Phe Ala Val Asn Gln

1795 1800 1805

Lys Asn Met Thr Ala Leu Glu Lys Lys Lys Glu Ala Val Glu Ser Gly

1810 1815 1820

Lys Arg Gln Ile Glu Asn Thr Leu Lys Glu Gly Asn Asp Ile Leu Asp

1825 1830 1835 1840

Glu Ala Asn Arg Leu Ala Asp Glu Ile Asn Ser Ile Ile Asp Tyr Val

1845 1850 1855

Glu Asp Ile Gln Thr Lys Leu Pro Pro Met Ser Glu Glu Leu Asn Asp

1860 1865 1870

Lys Ile Asp Asp Leu Ser Gln Glu Ile Lys Asp Arg Lys Leu Ala Glu

1875 1880 1885

Lys Val Ser Gln Ala Glu Ser His Ala Ala Gln Leu Asn Asp Ser Ser

1890 1895 1900

Ala Val Leu Asp Gly Ile Leu Asp Glu Ala Lys Asn Ile Ser Phe Asn

1905 1910 1915 1920

Ala Thr Ala Ala Phe Lys Ala Tyr Ser Asn Ile Lys Asp Tyr Ile Asp

1925 1930 1935

Glu Ala Glu Lys Val Ala Lys Glu Ala Lys Asp Leu Ala His Glu Ala

1940 1945 1950

Thr Lys Leu Ala Thr Gly Pro Arg Gly Leu Leu Lys Glu Asp Ala Lys

1955 1960 1965

Gly Cys Leu Gln Lys Ser Phe Arg Ile Leu Asn Glu Ala Lys Lys Leu

1970 1975 1980

Ala Asn Asp Val Lys Glu Asn Glu Asp His Leu Asn Gly Leu Lys Thr

1985 1990 1995 2000

Arg Ile Glu Asn Ala Asp Ala Arg Asn Gly Asp Leu Leu Arg Thr Leu

2005 2010 2015

Asn Asp Thr Leu Gly Lys Leu Ser Ala Ile Pro Asn Asp Thr Ala Ala

2020 2025 2030

Lys Leu Gln Ala Val Lys Asp Lys Ala Arg Gln Ala Asn Asp Thr Ala

2035 2040 2045

Lys Asp Val Leu Ala Gln Ile Thr Glu Leu His Gln Asn Leu Asp Gly

2050 2055 2060

Leu Lys Lys Asn Tyr Asn Lys Leu Ala Asp Ser Val Ala Lys Thr Asn

2065 2070 2075 2080

Ala Val Val Lys Asp Pro Ser Lys Asn Lys Ile Ile Ala Asp Ala Asp

2085 2090 2095

Ala Thr Val Lys Asn Leu Glu Gln Glu Ala Asp Arg Leu Ile Asp Lys

2100 2105 2110

Leu Lys Pro Ile Lys Glu Leu Glu Asp Asn Leu Lys Lys Asn Ile Ser

2115 2120 2125

Glu Ile Lys Glu Leu Ile Asn Gln Ala Arg Lys Gln Ala Asn Ser Ile

2130 2135 2140

Lys Val Ser Val Ser Ser Gly Gly Asp Cys Ile Arg Thr Tyr Lys Pro

2145 2150 2155 2160

Glu Ile Lys Lys Gly Ser Tyr Asn Asn Ile Val Val Asn Val Lys Thr

2165 2170 2175

Ala Val Ala Asp Asn Leu Leu Phe Tyr Leu Gly Ser Ala Lys Phe Ile

2180 2185 2190

Asp Phe Leu Ala Ile Glu Met Arg Lys Gly Lys Val Ser Phe Leu Trp

2195 2200 2205

Asp Val Gly Ser Gly Val Gly Arg Val Glu Tyr Pro Asp Leu Thr Ile

2210 2215 2220

Asp Asp Ser Tyr Trp Tyr Arg Ile Val Ala Ser Arg Thr Gly Arg Asn

2225 2230 2235 2240

Gly Thr Ile Ser Val Arg Ala Leu Asp Gly Pro Lys Ala Ser Ile Val

2245 2250 2255

Pro Ser Thr His His Ser Thr Ser Pro Pro Gly Tyr Thr Ile Leu Asp

2260 2265 2270

Val Asp Ala Asn Ala Met Leu Phe Val Gly Gly Leu Thr Gly Lys Leu

2275 2280 2285

Lys Lys Ala Asp Ala Val Arg Val Ile Thr Phe Thr Gly Cys Met Gly

2290 2295 2300

Glu Thr Tyr Phe Asp Asn Lys Pro Ile Gly Leu Trp Asn Phe Arg Glu

2305 2310 2315 2320

Lys Glu Gly Asp Cys Lys Gly Cys Thr Val Ser Pro Gln Val Glu Asp

2325 2330 2335

Ser Glu Gly Thr Ile Gln Phe Asp Gly Glu Gly Tyr Ala Leu Val Ser

2340 2345 2350

Arg Pro Ile Arg Trp Tyr Pro Asn Ile Ser Thr Val Met Phe Lys Phe

2355 2360 2365

Arg Thr Phe Ser Ser Ser Ala Leu Leu Met Tyr Leu Ala Thr Arg Asp

2370 2375 2380

Leu Arg Asp Phe Met Ser Val Glu Leu Thr Asp Gly His Ile Lys Val

2385 2390 2395 2400

Ser Tyr Asp Leu Gly Ser Gly Met Ala Ser Val Val Ser Asn Gln Asn

2405 2410 2415

His Asn Asp Gly Lys Trp Lys Ser Phe Thr Leu Ser Arg Ile Gln Lys

2420 2425 2430

Gln Ala Asn Ile Ser Ile Val Asp Ile Asp Thr Asn Gln Glu Glu Asn

2435 2440 2445

Ile Ala Thr Ser Ser Ser Gly Asn Asn Phe Gly Leu Asp Leu Lys Ala

2450 2455 2460

Asp Asp Lys Ile Tyr Phe Gly Gly Leu Pro Thr Leu Arg Asn Leu Ser

2465 2470 2475 2480

Met Lys Ala Arg Pro Glu Val Asn Leu Lys Lys Tyr Ser Gly Cys Leu

2485 2490 2495

Lys Asp Ile Glu Ile Ser Arg Thr Pro Tyr Asn Ile Leu Ser Ser Pro

2500 2505 2510

Asp Tyr Val Gly Val Thr Lys Gly Cys Ser Leu Glu Asn Val Tyr Thr

2515 2520 2525

Val Ser Phe Pro Lys Pro Gly Phe Val Glu Leu Ser Pro Val Pro Ile

2530 2535 2540

Asp Val Gly Thr Glu Ile Asn Leu Ser Phe Ser Thr Lys Asn Glu Ser

2545 2550 2555 2560

Gly Ile Ile Leu Leu Gly Ser Gly Gly Thr Pro Ala Pro Pro Arg Arg

2565 2570 2575

Lys Arg Arg Gln Thr Gly Gln Ala Tyr Tyr Val Ile Leu Leu Asn Arg

2580 2585 2590

Gly Arg Leu Glu Val His Leu Ser Thr Gly Ala Arg Thr Met Arg Lys

2595 2600 2605

Ile Val Ile Arg Pro Glu Pro Asn Leu Phe His Asp Gly Arg Glu His

2610 2615 2620

Ser Val His Val Glu Arg Thr Arg Gly Ile Phe Thr Val Gln Val Asp

2625 2630 2635 2640

Glu Asn Arg Arg Tyr Met Gln Asn Leu Thr Val Glu Gln Pro Ile Glu

2645 2650 2655

Val Lys Lys Leu Phe Val Gly Gly Ala Pro Pro Glu Phe Gln Pro Ser

2660 2665 2670

Pro Leu Arg Asn Ile Pro Pro Phe Glu Gly Cys Ile Trp Asn Leu Val

2675 2680 2685

Ile Asn Ser Val Pro Met Asp Phe Ala Arg Pro Val Ser Phe Lys Asn

2690 2695 2700

Ala Asp Ile Gly Arg Cys Ala His Gln Lys Leu Arg Glu Asp Glu Asp

2705 2710 2715 2720

Gly Ala Ala Pro Ala Glu Ile Val Ile Gln Pro Glu Pro Val Pro Thr

2725 2730 2735

Pro Ala Phe Pro Thr Pro Thr Pro Val Leu Thr His Gly Pro Cys Ala

2740 2745 2750

Ala Glu Ser Glu Pro Ala Leu Leu Ile Gly Ser Lys Gln Phe Gly Leu

2755 2760 2765

Ser Arg Asn Ser His Ile Ala Ile Ala Phe Asp Asp Thr Lys Val Lys

2770 2775 2780

Asn Arg Leu Thr Ile Glu Leu Glu Val Arg Thr Glu Ala Glu Ser Gly

2785 2790 2795 2800

Leu Leu Phe Tyr Met Ala Arg Ile Asn His Ala Asp Phe Ala Thr Val

2805 2810 2815

Gln Leu Arg Asn Gly Leu Pro Tyr Phe Ser Tyr Asp Leu Gly Ser Gly

2820 2825 2830

Asp Thr His Thr Met Ile Pro Thr Lys Ile Asn Asp Gly Gln Trp His

2835 2840 2845

Lys Ile Lys Ile Met Arg Ser Lys Gln Glu Gly Ile Leu Tyr Val Asp

2850 2855 2860

Gly Ala Ser Asn Arg Thr Ile Ser Pro Lys Lys Ala Asp Ile Leu Asp

2865 2870 2875 2880

Val Val Gly Met Leu Tyr Val Gly Gly Leu Pro Ile Asn Tyr Thr Thr

2885 2890 2895

Arg Arg Ile Gly Pro Val Thr Tyr Ser Ile Asp Gly Cys Val Arg Asn

2900 2905 2910

Leu His Met Ala Glu Ala Pro Ala Asp Leu Glu Gln Pro Thr Ser Ser

2915 2920 2925

Phe His Val Gly Thr Cys Phe Ala Asn Ala Gln Arg Gly Thr Tyr Phe

2930 2935 2940

Asp Gly Thr Gly Phe Ala Lys Ala Val Gly Gly Phe Lys Val Gly Leu

2945 2950 2955 2960

Asp Leu Leu Val Glu Phe Glu Phe Arg Thr Thr Thr Thr Thr Gly Val

2965 2970 2975

Leu Leu Gly Ile Ser Ser Gln Lys Met Asp Gly Met Gly Ile Glu Met

2980 2985 2990

Ile Asp Glu Lys Leu Met Phe His Val Asp Asn Gly Ala Gly Arg Phe

2995 3000 3005

Thr Ala Val Tyr Asp Ala Gly Val Pro Gly His Leu Cys Asp Gly Gln

3010 3015 3020

Trp His Lys Val Thr Ala Asn Lys Ile Lys His Arg Ile Glu Leu Thr

3025 3030 3035 3040

Val Asp Gly Asn Gln Val Glu Ala Gln Ser Pro Asn Pro Ala Ser Thr

3045 3050 3055

Ser Ala Asp Thr Asn Asp Pro Val Phe Val Gly Gly Phe Pro Asp Asp

3060 3065 3070

Leu Lys Gln Phe Gly Leu Thr Thr Ser Ile Pro Phe Arg Gly Cys Ile

3075 3080 3085

Arg Ser Leu Lys Leu Thr Lys Gly Thr Gly Lys Pro Leu Glu Val Asn

3090 3095 3100

Phe Ala Lys Ala Leu Glu Leu Arg Gly Val Gln Pro Val Ser Cys Pro

3105 3110 3115 3120

Ala Asn Ser

<210> 75

<211> 9369

<212> DNA

<213> 智人（homo sapiens）

<400> 75

atgccgggag ccgccggggt cctcctcctt ctgctgctct ccggaggcct cgggggcgta 60

caggcgcaga ggccgcagca gcagcggcag tcacaggcac atcagcaaag aggtttattc 120

cctgctgtcc tgaatcttgc ttctaatgct cttatcacga ccaatgcaac atgtggagaa 180

aaaggacctg aaatgtactg caaattggta gaacatgtcc ctgggcagcc tgtgaggaac 240

ccgcagtgtc gaatctgcaa tcaaaacagc agcaatccaa accagagaca cccgattaca 300

aatgctattg atggaaagaa cacttggtgg cagagtccca gtattaagaa tggaatcgaa 360

taccattatg tgacaattac cctggattta cagcaggtgt tccagatcgc gtatgtgatt 420

gtgaaggcag ctaactcccc ccggcctgga aactggattt tggaacgctc tcttgatgat 480

gttgaataca agccctggca gtatcatgct gtgacagaca cggagtgcct aacgctttac 540

aatatttatc cccgcactgg gccaccgtca tatgccaaag atgatgaggt catctgcact 600

tcattttact ccaagataca ccccttagaa aatggagaga ttcacatctc tttaatcaat 660

gggagaccaa gtgccgatga tccttctcca gaactgctag aatttacctc cgctcgctat 720

attcgcctga gatttcagag gatccgcaca ctgaatgctg acttgatgat gtttgctcac 780

aaagacccaa gagaaattga ccccattgtc accagaagat attactactc ggtcaaggat 840

atttcagttg gagggatgtg catctgctat ggtcatgcca gggcttgtcc acttgatcca 900

gcgacaaata aatctcgctg tgagtgtgag cataacacat gtggcgatag ctgtgatcag 960

tgctgtccag gattccatca gaaaccctgg agagctggaa cttttctaac taaaactgaa 1020

tgtgaagcat gcaattgtca tggaaaagct gaagaatgct attatgatga aaatgttgcc 1080

agaagaaatc tgagtttgaa tatacgtgga aagtacattg gagggggtgt ctgcattaat 1140

tgtacccaaa acactgctgg tataaactgc gagacatgta ctgatggctt cttcagaccc 1200

aaaggggtat ctccaaatta tccaaggcca tgccagccat gtcattgcga tccaattggt 1260

tccttaaatg aagtctgtgt caaggatgag aaacatgctc gacgaggttt ggcacctgga 1320

tcctgtcatt gcaaaactgg ttttggaggt gtgagctgtg atcggtgtgc caggggctac 1380

actggctacc cggactgcaa agcctgtaac tgcagtgggt tagggagcaa aaatgaggat 1440

ccttgttttg gcccctgtat ctgcaaggaa aatgttgaag gaggagactg tagtcgttgc 1500

aaatccggct tcttcaattt gcaagaggat aattggaaag gctgcgatga gtgtttctgt 1560

tcaggggttt caaacagatg tcagagttcc tactggacct atggcaaaat acaagatatg 1620

agtggctggt atctgactga ccttcctggc cgcattcgag tggctcccca gcaggacgac 1680

ttggactcac ctcagcagat cagcatcagt aacgcggagg cccggcaagc cctgccgcac 1740

agctactact ggagcgcgcc ggctccctat ctgggaaaca aactcccagc agtaggagga 1800

cagttgacat ttaccatatc atatgacctt gaagaagagg aagaagatac agaacgtgtt 1860

ctccagctta tgattatctt agagggtaat gacttgagca tcagcacagc ccaagatgag 1920

gtgtacctgc acccatctga agaacatact aatgtattgt tacttaaaga agaatcattt 1980

accatacatg gcacacattt tccagtccgt agaaaggaat ttatgacagt gcttgcgaat 2040

ttgaagagag tcctcctaca aatcacatac agctttggga tggatgccat cttcaggttg 2100

agctctgtta accttgaatc cgctgtctcc tatcctactg atggaagcat tgcagcagct 2160

gtagaagtgt gtcagtgccc accagggtat actggctcct cttgtgaatc ttgttggcct 2220

aggcacaggc gagttaacgg cactattttt ggtggcatct gtgagccatg tcagtgcttt 2280

ggtcatgcgg agtcctgtga tgacgtcact ggagaatgcc tgaactgtaa ggatcacaca 2340

ggtggcccat attgtgataa atgtcttcct ggtttctatg gcgagcctac taaaggaacc 2400

tctgaagact gtcaaccctg tgcctgtcca ctcaatatcc catccaataa ctttagccca 2460

acgtgccatt tagaccggag tcttggattg atctgtgatg gatgccctgt cgggtacaca 2520

ggaccacgct gtgagaggtg tgcagaaggc tattttggac aaccctctgt acctggagga 2580

tcatgtcagc catgccaatg caatgacaac cttgacttct ccatccctgg cagctgtgac 2640

agcttgtctg gctcctgtct gatatgtaaa ccaggtacaa caggccggta ctgtgagctc 2700

tgtgctgatg gatattttgg agatgcagtt gatgcgaaga actgtcagcc ctgtcgctgt 2760

aatgccggtg gctctttctc tgaggtttgc cacagtcaaa ctggacagtg tgagtgcaga 2820

gccaacgttc agggtcagag atgtgacaaa tgcaaggctg ggacctttgg cctacaatca 2880

gcaaggggct gtgttccctg caactgcaat tcttttgggt ctaagtcatt cgactgtgaa 2940

gagagtggac aatgttggtg ccaacctgga gtcacaggga agaaatgtga ccgctgtgcc 3000

cacggctatt tcaacttcca agaaggaggc tgcacagctt gtgaatgttc tcatctgggt 3060

aataattgtg acccaaagac tgggcgatgc atttgccctc ccaataccat tggagagaaa 3120

tgttctaaat gtgcacccaa tacctggggc cacagcatta ccactggttg taaggcttgt 3180

aactgcagca cagtgggatc cttggatttc caatgcaatg taaatacagg ccaatgcaac 3240

tgtcatccaa aattctctgg tgcaaaatgt acagagtgca gtcgaggtca ctggaactac 3300

cctcgctgca atctctgtga ctgcttcctc cctgggacag atgccacaac ctgtgattca 3360

gagactaaaa aatgctcctg tagtgatcaa actgggcagt gcacttgtaa ggtgaatgtg 3420

gaaggcatcc actgtgacag atgccggcct ggcaaattcg gactcgatgc caagaatcca 3480

cttggctgca gcagctgcta ttgcttcggc actactaccc agtgctctga agcaaaagga 3540

ctgatccgga cgtgggtgac tctgaaggct gagcagacca ttctacccct ggtagatgag 3600

gctctgcagc acacgaccac caagggcatt gtttttcaac atccagagat tgttgcccac 3660

atggacctga tgagagaaga tctccatttg gaaccttttt attggaaact tccagaacaa 3720

tttgaaggaa agaagttgat ggcctatggg ggcaaactca agtatgcaat ctatttcgag 3780

gctcgggaag aaacaggttt ctctacatat aatcctcaag tgatcattcg aggtgggaca 3840

cctactcatg ctagaattat cgtcaggcat atggctgctc ctctgattgg ccaattgaca 3900

aggcatgaaa ttgaaatgac agagaaagaa tggaaatatt atggggatga tcctcgagtc 3960

catagaactg tgacccgaga agacttcttg gatatactat atgatattca ttacattctt 4020

atcaaagcta cttatggaaa tttcatgcga caaagcagga tttctgaaat ctcaatggag 4080

gtagctgaac aaggacgtgg aacaacaatg actcctccag ctgacttgat tgaaaaatgt 4140

gattgtcccc tgggctattc tggcctgtcc tgtgaggcat gcttgccggg attttatcga 4200

ctgcgttctc aaccaggtgg ccgcacccct ggaccaaccc tgggcacctg tgttccatgt 4260

caatgtaatg gacacagcag cctgtgtgac cctgaaacat cgatatgcca gaattgtcaa 4320

catcacactg ctggtgactt ctgtgaacga tgtgctcttg gatactatgg aattgtcaag 4380

ggattgccaa atgactgtca gcaatgtgcc tgccctctga tttcttccag taacaatttc 4440

agcccctctt gtgtcgcaga aggacttgac gactaccgct gcacggcttg tccacgggga 4500

tatgaaggcc agtactgtga aaggtgtgcc cctggctata ctggcagtcc aggcaaccct 4560

ggaggctcct gccaagaatg tgagtgtgat ccctatggct cactgcctgt gccctgtgac 4620

cctgtcacag gattctgcac gtgccgacct ggagccacgg gaaggaagtg tgacggctgc 4680

aagcactggc atgcacgcga gggctgggag tgtgtttttt gtggagatga gtgcactggc 4740

cttcttctcg gtgacttggc tcgcctggag cagatggtca tgagcatcaa cctcactggt 4800

ccgctgcctg cgccatataa aatgctgtat ggtcttgaaa atatgactca ggagctaaag 4860

cacttgctgt cacctcagcg ggccccagag aggcttattc agctggcaga gggcaatctg 4920

aatacactcg tgaccgaaat gaacgagctg ctgaccaggg ctaccaaagt gacagcagat 4980

ggcgagcaga ccggacagga tgctgagagg accaacacaa gagcaaagtc cctgggagaa 5040

ttcattaagg agcttgcccg ggatgcagaa gctgtaaatg aaaaagctat aaaactaaat 5100

gaaactctag gaactcgaga tgaggccttt gagagaaatt tggaagggct tcagaaagag 5160

attgaccaga tgattaaaga actgaggagg aaaaatctag agacacaaaa ggaaattgct 5220

gaagatgagt tggtagctgc agaagccctt ctgaaaaaag tgaagaagct gtttggagag 5280

tcccgggggg aaaatgaaga aatggagaag gatctccggg aaaaactggc tgactacaaa 5340

aacaaagttg atgatgcttg ggaccttttg agagaagcca cagataaaat cagagaagct 5400

aatcgcctat ttgcagtaaa tcagaaaaac atgactgcat tggagaaaaa gaaggaggct 5460

gttgaaagcg gcaaacgaca aattgagaac actttaaaag agggcaatga catactcgat 5520

gaagccaacc gtcttgcaga tgaaatcaac tccatcatag actatgttga agacatccaa 5580

actaaattgc cacctatgtc tgaggagctt aatgataaaa tagatgacct ctcccaagaa 5640

ataaaggaca ggaaacttgc tgagaaggtg tcccaggctg agagccacgc agctcagttg 5700

aatgactcat ctgctgtcct tgatggaatc cttgatgagg ctaaaaacat ctccttcaat 5760

gccactgcag ccttcaaagc ctacagcaat attaaggact atattgatga agctgagaaa 5820

gttgccaaag aagccaaaga tcttgcacat gaagctacaa aactggcaac aggtcctcgg 5880

ggtttattaa aggaagatgc caaaggctgt cttcagaaaa gtttcaggat tcttaacgaa 5940

gccaagaagt tagcaaatga tgtaaaagaa aatgaagacc atctaaatgg cttaaaaacc 6000

aggatagaaa atgctgatgc tagaaatggg gatctcttga gaactttgaa tgacactttg 6060

ggaaagttat cagctattcc aaatgataca gctgctaaac tgcaagctgt taaggacaaa 6120

gccagacaag ccaacgacac agctaaagat gtactggcac agattacaga gctccaccag 6180

aacctcgatg gcctgaagaa gaattacaat aaactagcag acagcgtcgc caaaacgaat 6240

gctgtggtta aagatccttc caagaacaaa atcattgccg atgcagatgc cactgtcaaa 6300

aatttagaac aggaagctga ccggctaata gataaactca aacccatcaa ggaacttgag 6360

gataacctaa agaaaaacat ctctgagata aaggaattga taaaccaagc tcggaaacaa 6420

gccaattcta tcaaagtatc tgtgtcttca ggaggtgact gcattcgaac atacaaacca 6480

gaaatcaaga aaggaagtta caataatatt gttgtcaacg taaagacagc tgttgctgat 6540

aacctcctct tttatcttgg aagtgccaaa tttattgact ttctggctat agaaatgcgt 6600

aaaggcaaag tcagcttcct ctgggatgtt ggatctggag ttggacgtgt agagtaccca 6660

gatttgacta ttgatgactc atattggtac cgtatcgtag catcaagaac tgggagaaat 6720

ggaactattt ctgtgagagc cctggatgga cccaaagcca gcattgtgcc cagcacacac 6780

cattcgacgt cccctccagg gtacacgatt ctagatgtgg atgcaaatgc aatgctgttt 6840

gttggtggcc tgactgggaa attaaagaag gctgatgctg tacgtgtgat tacattcact 6900

ggctgcatgg gagaaacata ctttgacaac aaacctatag gtttgtggaa tttccgagaa 6960

aaagaaggtg actgcaaagg atgcactgtc agtcctcagg tggaagatag tgaggggact 7020

attcaatttg atggagaagg ttatgcattg gtcagccgtc ccattcgctg gtaccccaac 7080

atctccactg tcatgttcaa gttcagaaca ttttcttcga gtgcccttct gatgtatctt 7140

gccacacgag acctgagaga tttcatgagt gtggagctca ctgatgggca cataaaagtc 7200

agttacgatc tgggctcagg aatggcttcc gttgtcagca atcaaaacca taatgatggg 7260

aaatggaaat cattcactct gtcaagaatt caaaaacaag ccaatatatc aattgtagat 7320

atagatacta atcaggagga gaatatagca acttcgtctt ctggaaacaa ctttggtctt 7380

gacttgaaag cagatgacaa aatatatttt ggtggcctgc caacgctgag aaacttgagt 7440

atgaaagcaa ggccagaagt aaatctgaag aaatattccg gctgcctcaa agatattgaa 7500

atttcaagaa ctccgtacaa tatactcagt agtcccgatt atgttggtgt taccaaagga 7560

tgttccctgg agaatgttta cacagttagc tttcctaagc ctggttttgt ggagctctcc 7620

cctgtgccaa ttgatgtagg aacagaaatc aacctgtcat tcagcaccaa gaatgagtcc 7680

ggcatcattc ttttgggaag tggagggaca ccagcaccac ctaggagaaa acgaaggcag 7740

actggacagg cctattatgt aatactcctc aacaggggcc gtctggaagt gcatctctcc 7800

acaggggcac gaacaatgag gaaaattgtg atcagaccag agccgaatct gtttcatgat 7860

ggaagagaac attccgttca tgtagagcga actagaggca tctttacagt tcaagtggat 7920

gaaaacagaa gatacatgca aaacctgaca gttgaacagc ctatcgaagt taaaaaactt 7980

ttcgttgggg gtgctccacc tgaatttcaa ccttccccac tcagaaatat tcctcctttt 8040

gaaggctgca tatggaatct tgttattaac tctgtcccca tggactttgc aaggcctgtg 8100

tccttcaaaa atgctgacat tggtcgctgt gcccatcaga aactccgtga agatgaagat 8160

ggagcagctc cagctgaaat agttatccag cctgagccag ttcccacccc agcctttcct 8220

acgcccaccc cagttctgac acatggtcct tgtgctgcag aatcagaacc agctcttttg 8280

atagggagca agcagttcgg gctttcaaga aacagtcaca ttgcaattgc atttgatgac 8340

accaaagtta aaaaccgcct cacaattgag ttggaagtaa gaaccgaagc tgaatccggc 8400

ttgctttttt acatggctcg catcaatcat gctgattttg caacagttca gctgagaaat 8460

ggattgccct acttcagcta tgacttgggg agtggggaca cccacaccat gatccccacc 8520

aaaatcaatg atggccagtg gcacaagatt aagataatga gaagtaagca agaaggaatt 8580

ctttatgtag atggggcttc caacagaacc atcagtccca aaaaagccga catcctggat 8640

gtcgtgggaa tgctgtatgt tggtgggtta cccatcaact acactacccg aagaattggt 8700

ccagtgacct atagcattga tggctgcgtc aggaatctcc acatggcaga ggcccctgcc 8760

gatctggaac aacccacctc cagcttccat gttgggacat gttttgcaaa tgctcagagg 8820

ggaacatatt ttgacggaac cggttttgcc aaagcagttg gtggattcaa agtgggattg 8880

gaccttcttg tagaatttga attccgcaca actacaacga ctggagttct tctggggatc 8940

agtagtcaaa aaatggatgg aatgggtatt gaaatgattg atgaaaagtt gatgtttcat 9000

gtggacaatg gtgcgggcag attcactgct gtctatgatg ctggggttcc agggcatttg 9060

tgtgatggac aatggcataa agtcactgcc aacaagatca aacaccgcat tgagctcaca 9120

gtcgatggga accaggtgga agcccaaagc ccaaacccag catctacatc agctgacaca 9180

aatgaccctg tgtttgttgg aggcttccca gatgacctca agcagtttgg cctaacaacc 9240

agtattccgt tccgaggttg catcagatcc ctgaagctca ccaaaggcac aggcaagcca 9300

ctggaggtta attttgccaa ggccctggaa ctgaggggcg ttcaacctgt atcatgccca 9360

gccaactca 9369

<210> 76

<211> 508

<212> DNA

<213> 人工序列

<220>

<223> CMV启动子

<400> 76

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 360

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 420

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 480

acggtgggag gtctatataa gcagagct 508

<210> 77

<211> 1651

<212> DNA

<213> 人工序列

<220>

<223> CAG启动子

<400> 77

acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 60

tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 120

gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 180

acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 240

accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 300

gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca 360

attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg 420

gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt 480

gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 540

cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg ggagtcgctg cgacgctgcc 600

ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg 660

cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct 720

tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg 780

agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga 840

gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc 900

tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg 960

gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag 1020

ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg 1080

agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc 1140

cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg 1200

agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga 1260

gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc 1320

aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg 1380

cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc 1440

gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc 1500

gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct 1560

ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta 1620

ttgtgctgtc tcatcatttt ggcaaagaat t 1651

<210> 78

<211> 546

<212> DNA

<213> 人工序列

<220>

<223> EF1-alpha启动子

<400> 78

aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60

cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240

acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300

ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360

tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420

cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480

actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540

gcctac 546

<210> 79

<211> 317

<212> DNA

<213> 人工序列

<220>

<223> SV-40启动子

<400> 79

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag 300

gcctaggctt ttgcaaa 317

<210> 80

<211> 725

<212> DNA

<213> 人工序列

<220>

<223> EalbAATp

<400> 80

gacgcgcatg ctcctctaga ctcgacgcgt gttcctagat tacactacac attctgcaag 60

catagcacag agcaatgttc tactttaatt actttcattt tcttgtatcc tcacagccta 120

gaaaataacc tgcgttacag catccactca gtatcccttg agcatgaggt gacactactt 180

aacataggga cgagatggta ctttgtgtct cctgctctgt cagcagggca cagtacttgc 240

tgataccagg gaatgtttgt tcttaaatac catcattccg gacgtgtttg ccttggccag 300

ttttccatgt acatgcagaa agaagtttgg actgatcaat acagtcctct gcctttaaag 360

caataggaaa aggccaactt gtctacgttt agtatgtggc tgtagatctg tacccgccac 420

cccctccacc ttggacacag gacgctgtgg tttctgagcc aggtacaatg actcctttcg 480

gtaagtgcag tggaagctgt acactgccca ggcaaagcgt ccgggcagcg taggcgggcg 540

actcagatcc cagccagtgg acttagcccc tgtttgctcc tccgataact ggggtgacct 600

tggttaatat tcaccagcag cctcccccgt tgcccctctg gatccactgc ttaaatacgg 660

acgaggacag ggccctgtct cctcagcttc aggcaccacc actgacctgg gacagtgaag 720

cggcc 725

<210> 81

<211> 51

<212> DNA

<213> 人工序列

<220>

<223> 剪接受体

<400> 81

gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca g 51

<210> 82

<211> 86

<212> DNA

<213> 人工序列

<220>

<223> 合成的ama2内含子26

<400> 82

gtaagttagt cattgtttgg tgcaaagata ccaatcaatg gttttgcatt cagttttgtc 60

atagtgattt ctcttcttgt tttcag 86

<210> 83

<211> 131

<212> DNA

<213> 人工序列

<220>

<223> SV40-PolyA

<400> 83

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

tatcatgtct g 131

<210> 84

<211> 263

<212> DNA

<213> 人工序列

<220>

<223> BHG-PolyA

<400> 84

gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 60

atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 120

cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 180

ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 240

tggggatgcg gtgggctcta tgg 263

<210> 85

<211> 49

<212> DNA

<213> 人工序列

<220>

<223> HSV-Tk-PolyA

<400> 85

cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttc 49

<210> 86

<211> 232

<212> DNA

<213> 人工序列

<220>

<223> 隔绝物a

<400> 86

gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg 60

gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc 120

ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 180

cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aa 232

<210> 87

<211> 231

<212> DNA

<213> 人工序列

<220>

<223> 隔绝物b

<400> 87

ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 60

aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 120

gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 180

gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc t 231

<210> 88

<211> 63

<212> DNA

<213> 人工序列

<220>

<223> ITR-5'

<400> 88

ccctagaaag ataatcatat tgtgacgtac gttaaagata atcatgcgta aaattgacgc 60

atg 63

<210> 89

<211> 35

<212> DNA

<213> 人工序列

<220>

<223> ITR-3'

<400> 89

catgcgtcaa ttttacgcag actatctttc taggg 35

<210> 90

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-Rosa26-1

<400> 90

agtctttcta gaagatgggc 20

<210> 91

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-Rosa26-2

<400> 91

cagtctttct agaagatggg 20

<210> 92

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-白蛋白-1

<400> 92

caatctttaa atatgttgtg 20

<210> 93

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-Rosa26-4

<400> 93

actccagtct ttctagaaga 20

<210> 94

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-白蛋白-1

<400> 94

caatctttaa atatgttgtg 20

<210> 95

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-白蛋白-2

<400> 95

gttacaggaa aatctgaagg 20

<210> 96

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-白蛋白-3

<400> 96

taactttgag tgtagcagag 20

<210> 97

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> gRNA-白蛋白-4

<400> 97

tatagcatgg tcgagcaggc 20

<210> 98

<211> 3117

<212> PRT

<213> 小家鼠（mus musculus）

<400> 98

Met Pro Ala Ala Thr Ala Gly Ile Leu Leu Leu Leu Leu Leu Gly Thr

1 5 10 15

Leu Glu Gly Ser Gln Thr Gln Arg Arg Gln Ser Gln Ala His Gln Gln

20 25 30

Arg Gly Leu Phe Pro Ala Val Leu Asn Leu Ala Ser Asn Ala Leu Ile

35 40 45

Thr Thr Asn Ala Thr Cys Gly Glu Lys Gly Pro Glu Met Tyr Cys Lys

50 55 60

Leu Val Glu His Val Pro Gly Gln Pro Val Arg Asn Pro Gln Cys Arg

65 70 75 80

Ile Cys Asn Gln Asn Ser Ser Asn Pro Tyr Gln Arg His Pro Ile Thr

85 90 95

Asn Ala Ile Asp Gly Lys Asn Thr Trp Trp Gln Ser Pro Ser Ile Lys

100 105 110

Asn Gly Val Glu Tyr His Tyr Val Thr Ile Thr Leu Asp Leu Gln Gln

115 120 125

Val Phe Gln Ile Ala Tyr Val Ile Val Lys Ala Ala Asn Ser Pro Arg

130 135 140

Pro Gly Asn Trp Ile Leu Glu Arg Ser Leu Asp Asp Val Glu Tyr Lys

145 150 155 160

Pro Trp Gln Tyr His Ala Val Thr Asp Thr Glu Cys Leu Thr Leu Tyr

165 170 175

Asn Ile Tyr Pro Arg Thr Gly Pro Pro Ser Tyr Ala Lys Asp Asp Glu

180 185 190

Val Ile Cys Thr Ser Phe Tyr Ser Lys Ile His Pro Leu Glu Asn Gly

195 200 205

Glu Ile His Ile Ser Leu Ile Asn Gly Arg Pro Ser Ala Asp Asp Pro

210 215 220

Ser Pro Glu Leu Leu Glu Phe Thr Ser Ala Arg Tyr Ile Arg Leu Arg

225 230 235 240

Phe Gln Arg Ile Arg Thr Leu Asn Ala Asp Leu Met Met Phe Ala His

245 250 255

Lys Asp Pro Arg Glu Ile Asp Pro Ile Val Thr Arg Arg Tyr Tyr Tyr

260 265 270

Ser Val Lys Asp Ile Ser Val Gly Gly Met Cys Ile Cys Tyr Gly His

275 280 285

Ala Arg Ala Cys Pro Leu Asp Pro Ala Thr Asn Lys Ser Arg Cys Glu

290 295 300

Cys Glu His Asn Thr Cys Gly Glu Ser Cys Asp Arg Cys Cys Pro Gly

305 310 315 320

Phe His Gln Lys Pro Trp Arg Ala Gly Thr Phe Leu Thr Lys Ser Glu

325 330 335

Cys Glu Ala Cys Asn Cys His Gly Lys Ala Glu Glu Cys Tyr Tyr Asp

340 345 350

Glu Thr Val Ala Ser Arg Asn Leu Ser Leu Asn Ile His Gly Lys Tyr

355 360 365

Ile Gly Gly Gly Val Cys Ile Asn Cys Thr His Asn Thr Ala Gly Ile

370 375 380

Asn Cys Glu Thr Cys Val Asp Gly Phe Phe Arg Pro Lys Gly Val Ser

385 390 395 400

Pro Asn Tyr Pro Arg Pro Cys Gln Pro Cys His Cys Asp Pro Thr Gly

405 410 415

Ser Leu Ser Glu Val Cys Val Lys Asp Glu Lys Tyr Ala Gln Arg Gly

420 425 430

Leu Lys Pro Gly Ser Cys His Cys Lys Thr Gly Phe Gly Gly Val Asn

435 440 445

Cys Asp Arg Cys Val Arg Gly Tyr His Gly Tyr Pro Asp Cys Gln Pro

450 455 460

Cys Asn Cys Ser Gly Leu Gly Ser Thr Asn Glu Asp Pro Cys Val Gly

465 470 475 480

Pro Cys Ser Cys Lys Glu Asn Val Glu Gly Glu Asp Cys Ser Arg Cys

485 490 495

Lys Ser Gly Phe Phe Asn Leu Gln Glu Asp Asn Gln Lys Gly Cys Glu

500 505 510

Glu Cys Phe Cys Ser Gly Val Ser Asn Arg Cys Gln Ser Ser Tyr Trp

515 520 525

Thr Tyr Gly Asn Ile Gln Asp Met Arg Gly Trp Tyr Leu Thr Asp Leu

530 535 540

Ser Gly Arg Ile Arg Met Ala Pro Gln Leu Asp Asn Pro Asp Ser Pro

545 550 555 560

Gln Gln Ile Ser Ile Ser Asn Ser Glu Ala Arg Lys Ser Leu Leu Asp

565 570 575

Gly Tyr Tyr Trp Ser Ala Pro Pro Pro Tyr Leu Gly Asn Arg Leu Pro

580 585 590

Ala Val Gly Gly Gln Leu Ser Phe Thr Ile Ser Tyr Asp Leu Glu Glu

595 600 605

Glu Glu Asp Asp Thr Glu Lys Ile Leu Gln Leu Met Ile Ile Phe Glu

610 615 620

Gly Asn Asp Leu Arg Ile Ser Thr Ala Tyr Lys Glu Val Tyr Leu Glu

625 630 635 640

Pro Ser Glu Glu His Ile Glu Glu Val Ser Leu Lys Glu Glu Ala Phe

645 650 655

Thr Ile His Gly Thr Asn Leu Pro Val Thr Arg Lys Asp Phe Met Ile

660 665 670

Val Leu Thr Asn Leu Glu Arg Val Leu Met Gln Ile Thr Tyr Asn Leu

675 680 685

Gly Met Asp Ala Ile Phe Arg Leu Ser Ser Val Asn Leu Glu Ser Ala

690 695 700

Val Pro Tyr Pro Thr Asp Arg Arg Ile Ala Thr Asp Val Glu Val Cys

705 710 715 720

Gln Cys Pro Pro Gly Tyr Ser Gly Ser Ser Cys Glu Thr Cys Trp Pro

725 730 735

Arg His Arg Arg Val Asn Gly Thr Ile Phe Gly Gly Ile Cys Glu Pro

740 745 750

Cys Gln Cys Phe Ala His Ala Glu Ala Cys Asp Asp Ile Thr Gly Glu

755 760 765

Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro Tyr Cys Asn Glu Cys

770 775 780

Leu Pro Gly Phe Tyr Gly Asp Pro Thr Arg Gly Ser Pro Glu Asp Cys

785 790 795 800

Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser Asn Asn Phe Ser Pro

805 810 815

Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile Cys Asp Glu Cys Pro

820 825 830

Ile Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys Ala Glu Gly Tyr Phe

835 840 845

Gly Gln Pro Ser Ile Pro Gly Gly Ser Cys Gln Pro Cys Gln Cys Asn

850 855 860

Asp Asn Leu Asp Tyr Ser Ile Pro Gly Ser Cys Asp Ser Leu Ser Gly

865 870 875 880

Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly Arg Tyr Cys Glu Leu

885 890 895

Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asn Ala Lys Asn Cys Gln

900 905 910

Pro Cys Arg Cys Asn Ile Asn Gly Ser Phe Ser Glu Ile Cys His Thr

915 920 925

Arg Thr Gly Gln Cys Glu Cys Arg Pro Asn Val Gln Gly Arg His Cys

930 935 940

Asp Glu Cys Lys Pro Glu Thr Phe Gly Leu Gln Leu Gly Arg Gly Cys

945 950 955 960

Leu Pro Cys Asn Cys Asn Ser Phe Gly Ser Lys Ser Phe Asp Cys Glu

965 970 975

Ala Ser Gly Gln Cys Trp Cys Gln Pro Gly Val Ala Gly Lys Lys Cys

980 985 990

Asp Arg Cys Ala His Gly Tyr Phe Asn Phe Gln Glu Gly Gly Cys Ile

995 1000 1005

Ala Cys Asp Cys Ser His Leu Gly Asn Asn Cys Asp Pro Lys Thr Gly

1010 1015 1020

Gln Cys Ile Cys Pro Pro Asn Thr Thr Gly Glu Lys Cys Ser Glu Cys

1025 1030 1035 1040

Leu Pro Asn Thr Trp Gly His Ser Ile Val Thr Gly Cys Lys Val Cys

1045 1050 1055

Asn Cys Ser Thr Val Gly Ser Leu Ala Ser Gln Cys Asn Val Asn Thr

1060 1065 1070

Gly Gln Cys Ser Cys His Pro Lys Phe Ser Gly Met Lys Cys Ser Glu

1075 1080 1085

Cys Ser Arg Gly His Trp Asn Tyr Pro Leu Cys Thr Leu Cys Asp Cys

1090 1095 1100

Phe Leu Pro Gly Thr Asp Ala Thr Thr Cys Asp Leu Glu Thr Arg Lys

1105 1110 1115 1120

Cys Ser Cys Ser Asp Gln Thr Gly Gln Cys Ser Cys Lys Val Asn Val

1125 1130 1135

Glu Gly Val His Cys Asp Arg Cys Arg Pro Gly Lys Phe Gly Leu Asp

1140 1145 1150

Ala Lys Asn Pro Leu Gly Cys Ser Ser Cys Tyr Cys Phe Gly Val Thr

1155 1160 1165

Ser Gln Cys Ser Glu Ala Lys Gly Leu Ile Arg Thr Trp Val Thr Leu

1170 1175 1180

Ser Asp Glu Gln Thr Ile Leu Pro Leu Val Asp Glu Ala Leu Gln His

1185 1190 1195 1200

Thr Thr Thr Lys Gly Ile Ala Phe Gln Lys Pro Glu Ile Val Ala Lys

1205 1210 1215

Met Asp Glu Val Arg Gln Glu Leu His Leu Glu Pro Phe Tyr Trp Lys

1220 1225 1230

Leu Pro Gln Gln Phe Glu Gly Lys Lys Leu Met Ala Tyr Gly Gly Lys

1235 1240 1245

Leu Lys Tyr Ala Ile Tyr Phe Glu Ala Arg Asp Glu Thr Gly Phe Ala

1250 1255 1260

Thr Tyr Lys Pro Gln Val Ile Ile Arg Gly Gly Thr Pro Thr His Ala

1265 1270 1275 1280

Arg Ile Ile Thr Arg His Met Ala Ala Pro Leu Ile Gly Gln Leu Thr

1285 1290 1295

Arg His Glu Ile Glu Met Thr Glu Lys Glu Trp Lys Tyr Tyr Gly Asp

1300 1305 1310

Asp Pro Arg Ile Ser Arg Thr Val Thr Arg Glu Asp Phe Leu Asp Ile

1315 1320 1325

Leu Tyr Asp Ile His Tyr Ile Leu Ile Lys Ala Thr Tyr Gly Asn Val

1330 1335 1340

Val Arg Gln Ser Arg Ile Ser Glu Ile Ser Met Glu Val Ala Glu Pro

1345 1350 1355 1360

Gly His Val Leu Ala Gly Ser Pro Pro Ala His Leu Ile Glu Arg Cys

1365 1370 1375

Asp Cys Pro Pro Gly Tyr Ser Gly Leu Ser Cys Glu Thr Cys Ala Pro

1380 1385 1390

Gly Phe Tyr Arg Leu Arg Ser Glu Pro Gly Gly Arg Thr Pro Gly Pro

1395 1400 1405

Thr Leu Gly Thr Cys Val Pro Cys Gln Cys Asn Gly His Ser Ser Gln

1410 1415 1420

Cys Asp Pro Glu Thr Ser Val Cys Gln Asn Cys Gln His His Thr Ala

1425 1430 1435 1440

Gly Asp Phe Cys Glu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val Arg

1445 1450 1455

Gly Leu Pro Asn Asp Cys Gln Pro Cys Ala Cys Pro Leu Ile Ser Pro

1460 1465 1470

Ser Asn Asn Phe Ser Pro Ser Cys Val Leu Glu Gly Leu Glu Asp Tyr

1475 1480 1485

Arg Cys Thr Ala Cys Pro Arg Gly Tyr Glu Gly Gln Tyr Cys Glu Arg

1490 1495 1500

Cys Ala Pro Gly Tyr Thr Gly Ser Pro Ser Ser Pro Gly Gly Ser Cys

1505 1510 1515 1520

Gln Glu Cys Glu Cys Asp Pro Tyr Gly Ser Leu Pro Val Pro Cys Asp

1525 1530 1535

Arg Val Thr Gly Leu Cys Thr Cys Arg Pro Gly Ala Thr Gly Arg Lys

1540 1545 1550

Cys Asp Gly Cys Glu His Trp His Ala Arg Glu Gly Ala Glu Cys Val

1555 1560 1565

Phe Cys Gly Asp Glu Cys Thr Gly Leu Leu Leu Gly Asp Leu Ala Arg

1570 1575 1580

Leu Glu Gln Met Thr Met Asn Ile Asn Leu Thr Gly Pro Leu Pro Ala

1585 1590 1595 1600

Pro Tyr Lys Ile Leu Tyr Gly Leu Glu Asn Thr Thr Gln Glu Leu Lys

1605 1610 1615

His Leu Leu Ser Pro Gln Arg Ala Pro Glu Arg Leu Ile Gln Leu Ala

1620 1625 1630

Glu Gly Asn Val Asn Thr Leu Val Met Glu Thr Asn Glu Leu Leu Thr

1635 1640 1645

Arg Ala Thr Lys Val Thr Ala Asp Gly Glu Gln Thr Gly Gln Asp Ala

1650 1655 1660

Glu Arg Thr Asn Ser Arg Ala Glu Ser Leu Glu Glu Phe Ile Lys Gly

1665 1670 1675 1680

Leu Val Gln Asp Ala Glu Ala Ile Asn Glu Lys Ala Val Gln Leu Asn

1685 1690 1695

Glu Thr Leu Gly Asn Gln Asp Lys Thr Ala Glu Arg Asn Leu Glu Glu

1700 1705 1710

Leu Gln Lys Glu Ile Asp Arg Met Leu Lys Glu Leu Arg Ser Lys Asp

1715 1720 1725

Leu Gln Thr Gln Lys Glu Val Ala Glu Asp Glu Leu Val Ala Ala Glu

1730 1735 1740

Gly Leu Leu Lys Arg Val Asn Lys Leu Phe Gly Glu Pro Arg Ala Gln

1745 1750 1755 1760

Asn Glu Asp Met Glu Lys Asp Leu Gln Gln Lys Leu Ala Glu Tyr Lys

1765 1770 1775

Asn Lys Leu Asp Asp Ala Trp Asp Leu Leu Arg Glu Ala Thr Asp Lys

1780 1785 1790

Thr Arg Asp Ala Asn Arg Leu Ser Ala Ala Asn Gln Lys Asn Met Thr

1795 1800 1805

Ile Leu Glu Thr Lys Lys Glu Ala Ile Glu Gly Ser Lys Arg Gln Ile

1810 1815 1820

Glu Asn Thr Leu Lys Glu Gly Asn Asp Ile Leu Asp Glu Ala Asn Arg

1825 1830 1835 1840

Leu Leu Gly Glu Ile Asn Ser Val Ile Asp Tyr Val Asp Asp Ile Lys

1845 1850 1855

Thr Lys Leu Pro Pro Met Ser Glu Glu Leu Ser Asp Lys Ile Asp Asp

1860 1865 1870

Leu Ala Gln Glu Ile Lys Asp Arg Arg Leu Ala Glu Lys Val Phe Gln

1875 1880 1885

Ala Glu Ser His Ala Ala Gln Leu Asn Asp Ser Ser Ala Val Leu Asp

1890 1895 1900

Gly Ile Leu Asp Glu Ala Lys Asn Ile Ser Phe Asn Ala Thr Ala Ala

1905 1910 1915 1920

Phe Arg Ala Tyr Ser Asn Ile Lys Asp Tyr Ile Asp Glu Ala Glu Lys

1925 1930 1935

Val Ala Arg Glu Ala Lys Glu Leu Ala Gln Gly Ala Thr Lys Leu Ala

1940 1945 1950

Thr Ser Pro Gln Gly Leu Leu Lys Glu Asp Ala Lys Gly Ser Leu Gln

1955 1960 1965

Lys Ser Phe Arg Ile Leu Asn Glu Ala Lys Lys Leu Ala Asn Asp Val

1970 1975 1980

Lys Gly Asn His Asn Asp Leu Asn Asp Leu Lys Thr Arg Leu Glu Thr

1985 1990 1995 2000

Ala Asp Leu Arg Asn Ser Gly Leu Leu Gly Ala Leu Asn Asp Thr Met

2005 2010 2015

Asp Lys Leu Ser Ala Ile Thr Asn Asp Thr Ala Ala Lys Leu Gln Ala

2020 2025 2030

Val Lys Glu Lys Ala Arg Glu Ala Asn Asp Thr Ala Lys Ala Val Leu

2035 2040 2045

Ala Gln Val Lys Asp Leu His Gln Asn Leu Asp Gly Leu Lys Gln Asn

2050 2055 2060

Tyr Asn Lys Leu Ala Asp Ser Val Ala Lys Thr Asn Ala Val Val Lys

2065 2070 2075 2080

Asp Pro Ser Lys Asn Lys Ile Ile Ala Asp Ala Gly Thr Ser Val Arg

2085 2090 2095

Asn Leu Glu Gln Glu Ala Asp Arg Leu Ile Asp Lys Leu Lys Pro Ile

2100 2105 2110

Lys Glu Leu Glu Asp Asn Leu Lys Lys Asn Ile Ser Glu Ile Lys Glu

2115 2120 2125

Leu Ile Asn Gln Ala Arg Lys Gln Ala Asn Ser Ile Lys Val Ser Val

2130 2135 2140

Ser Ser Gly Gly Asp Cys Val Arg Thr Tyr Arg Pro Glu Ile Lys Lys

2145 2150 2155 2160

Gly Ser Tyr Asn Asn Ile Val Val His Val Lys Thr Ala Val Ala Asp

2165 2170 2175

Asn Leu Leu Phe Tyr Leu Gly Ser Ala Lys Phe Ile Asp Phe Leu Ala

2180 2185 2190

Ile Glu Met Arg Lys Gly Lys Val Ser Phe Leu Trp Asp Val Gly Ser

2195 2200 2205

Gly Val Gly Arg Val Glu Tyr Pro Asp Leu Thr Ile Asp Asp Ser Tyr

2210 2215 2220

Trp Tyr Arg Ile Glu Ala Ser Arg Thr Gly Arg Asn Gly Ser Ile Ser

2225 2230 2235 2240

Val Arg Ala Leu Asp Gly Pro Lys Ala Ser Met Val Pro Ser Thr Tyr

2245 2250 2255

His Ser Val Ser Pro Pro Gly Tyr Thr Ile Leu Asp Val Asp Ala Asn

2260 2265 2270

Ala Met Leu Phe Val Gly Gly Leu Thr Gly Lys Ile Lys Lys Ala Asp

2275 2280 2285

Ala Val Arg Val Ile Thr Phe Thr Gly Cys Met Gly Glu Thr Tyr Phe

2290 2295 2300

Asp Asn Lys Pro Ile Gly Leu Trp Asn Phe Arg Glu Lys Glu Gly Asp

2305 2310 2315 2320

Cys Lys Gly Cys Thr Val Ser Pro Gln Val Glu Asp Ser Glu Gly Thr

2325 2330 2335

Ile Gln Phe Asp Gly Glu Gly Tyr Ala Leu Val Ser Arg Pro Ile Arg

2340 2345 2350

Trp Tyr Pro Asn Ile Ser Thr Val Met Phe Lys Phe Arg Thr Phe Ser

2355 2360 2365

Ser Ser Ala Leu Leu Met Tyr Leu Ala Thr Arg Asp Leu Lys Asp Phe

2370 2375 2380

Met Ser Val Glu Leu Ser Asp Gly His Val Lys Val Ser Tyr Asp Leu

2385 2390 2395 2400

Gly Ser Gly Met Thr Ser Val Val Ser Asn Gln Asn His Asn Asp Gly

2405 2410 2415

Lys Trp Lys Ala Phe Thr Leu Ser Arg Ile Gln Lys Gln Ala Asn Ile

2420 2425 2430

Ser Ile Val Asp Ile Asp Ser Asn Gln Glu Glu Asn Val Ala Thr Ser

2435 2440 2445

Ser Ser Gly Asn Asn Phe Gly Leu Asp Leu Lys Ala Asp Asp Lys Ile

2450 2455 2460

Tyr Phe Gly Gly Leu Pro Thr Leu Arg Asn Leu Ser Met Lys Ala Arg

2465 2470 2475 2480

Pro Glu Val Asn Val Lys Lys Tyr Ser Gly Cys Leu Lys Asp Ile Glu

2485 2490 2495

Ile Ser Arg Thr Pro Tyr Asn Ile Leu Ser Ser Pro Asp Tyr Val Gly

2500 2505 2510

Val Thr Lys Gly Cys Ser Leu Glu Asn Val Tyr Thr Val Ser Phe Pro

2515 2520 2525

Lys Pro Gly Phe Val Glu Leu Ala Ala Val Ser Ile Asp Val Gly Thr

2530 2535 2540

Glu Ile Asn Leu Ser Phe Ser Thr Arg Asn Glu Ser Gly Ile Ile Leu

2545 2550 2555 2560

Leu Gly Ser Gly Gly Thr Leu Thr Pro Pro Arg Arg Lys Arg Arg Gln

2565 2570 2575

Thr Thr Gln Ala Tyr Tyr Ala Ile Phe Leu Asn Lys Gly Arg Leu Glu

2580 2585 2590

Val His Leu Ser Ser Gly Thr Arg Thr Met Arg Lys Ile Val Ile Lys

2595 2600 2605

Pro Glu Pro Asn Leu Phe His Asp Gly Arg Glu His Ser Val His Val

2610 2615 2620

Glu Arg Thr Arg Gly Ile Phe Thr Val Gln Ile Asp Glu Asp Arg Arg

2625 2630 2635 2640

His Met Gln Asn Leu Thr Glu Glu Gln Pro Ile Glu Val Lys Lys Leu

2645 2650 2655

Phe Val Gly Gly Ala Pro Pro Glu Phe Gln Pro Ser Pro Leu Arg Asn

2660 2665 2670

Ile Pro Ala Phe Gln Gly Cys Val Trp Asn Leu Val Ile Asn Ser Ile

2675 2680 2685

Pro Met Asp Phe Ala Gln Pro Ile Ala Phe Lys Asn Ala Asp Ile Gly

2690 2695 2700

Arg Cys Thr Tyr Gln Lys Pro Arg Glu Asp Glu Ser Glu Ala Val Pro

2705 2710 2715 2720

Ala Glu Val Ile Val Gln Pro Gln Pro Val Pro Thr Pro Ala Phe Pro

2725 2730 2735

Phe Pro Ala Pro Thr Met Val His Gly Pro Cys Val Ala Glu Ser Glu

2740 2745 2750

Pro Ala Leu Leu Thr Gly Ser Lys Gln Phe Gly Leu Ser Arg Asn Ser

2755 2760 2765

His Ile Ala Ile Ala Phe Asp Asp Thr Lys Val Lys Asn Arg Leu Thr

2770 2775 2780

Ile Glu Leu Glu Val Arg Thr Glu Ala Glu Ser Gly Leu Leu Phe Tyr

2785 2790 2795 2800

Met Ala Arg Ile Asn His Ala Asp Phe Ala Thr Val Gln Leu Arg Asn

2805 2810 2815

Gly Phe Pro Tyr Phe Ser Tyr Asp Leu Gly Ser Gly Asp Thr Ser Thr

2820 2825 2830

Met Ile Pro Thr Lys Ile Asn Asp Gly Gln Trp His Lys Ile Lys Ile

2835 2840 2845

Val Arg Val Lys Gln Glu Gly Ile Leu Tyr Val Asp Asp Ala Ser Ser

2850 2855 2860

Gln Thr Ile Ser Pro Lys Lys Ala Asp Ile Leu Asp Val Val Gly Ile

2865 2870 2875 2880

Leu Tyr Val Gly Gly Leu Pro Ile Asn Tyr Thr Thr Arg Arg Ile Gly

2885 2890 2895

Pro Val Thr Tyr Ser Leu Asp Gly Cys Val Arg Asn Leu His Met Glu

2900 2905 2910

Gln Ala Pro Val Asp Leu Asp Gln Pro Thr Ser Ser Phe His Val Gly

2915 2920 2925

Thr Cys Phe Ala Asn Ala Glu Ser Gly Thr Tyr Phe Asp Gly Thr Gly

2930 2935 2940

Phe Ala Lys Ala Val Gly Gly Phe Lys Val Gly Leu Asp Leu Leu Val

2945 2950 2955 2960

Glu Phe Glu Phe Arg Thr Thr Arg Pro Thr Gly Val Leu Leu Gly Val

2965 2970 2975

Ser Ser Gln Lys Met Asp Gly Met Gly Ile Glu Met Ile Asp Glu Lys

2980 2985 2990

Leu Met Phe His Val Asp Asn Gly Ala Gly Arg Phe Thr Ala Ile Tyr

2995 3000 3005

Asp Ala Gly Ile Pro Gly His Met Cys Asn Gly Gln Trp His Lys Val

3010 3015 3020

Thr Ala Lys Lys Ile Lys Asn Arg Leu Glu Leu Val Val Asp Gly Asn

3025 3030 3035 3040

Gln Val Asp Ala Gln Ser Pro Asn Ser Ala Ser Thr Ser Ala Asp Thr

3045 3050 3055

Asn Asp Pro Val Phe Val Gly Gly Phe Pro Gly Gly Leu Asn Gln Phe

3060 3065 3070

Gly Leu Thr Thr Asn Ile Arg Phe Arg Gly Cys Ile Arg Ser Leu Lys

3075 3080 3085

Leu Thr Lys Gly Thr Gly Lys Pro Leu Glu Val Asn Phe Ala Lys Ala

3090 3095 3100

Leu Glu Leu Arg Gly Val Gln Pro Val Ser Cys Pro Thr

3105 3110 3115

<210> 99

<211> 9353

<212> DNA

<213> 小家鼠（mus musculus）

<400> 99

atgcctgcgg ccaccgccgg gatcctcttg ctcctgctct tggggacgct cgaaggctcc 60

cagactcagc ggcgacagtc ccaagcgcat caacagagag gtttatttcc tgctgtcctg 120

aatcttgctt cgaatgcact catcacaacc aatgctacat gtggggaaaa aggacccgag 180

atgtactgca agttggtgga acatgtcccc gggcagcctg tgaggaaccc tcagtgccga 240

atctgcaatc agaacagcag caatccatac cagaggcacc cgattacgaa tgctattgat 300

ggcaagaaca catggtggca gagtcccagt atcaagaatg gagtggaata ccattatgtg 360

acaattactc tggatttaca gcaggtgttc cagattgcct acgtaattgt gaaggcagcc 420

aattcccctc ggcctggaaa ctggattttg gaacgttccc tggatgacgt ggagtacaaa 480

ccctggcagt atcatgcggt gacagacacg gagtgcctga ccctctacaa tatctatccc 540

cgcactggac caccatccta cgccaaagat gatgaggtca tctgcacttc attttattcg 600

aagatccacc ctttagaaaa tggagagatt cacatttctt tgatcaatgg gagaccaagt 660

gctgatgacc cctcccctga actcctggaa ttcacctctg ctcgctacat tcgcctgaga 720

tttcagagga tccgcacctt gaatgcagac ttgatgatgt ttgctcacaa agaccccaga 780

gaaatcgatc ccattgtcac acgaagatat tactattctg tcaaggatat ttcagttggc 840

gggatgtgca tctgttatgg tcatgcccgg gcttgtccac ttgaccctgc aacaaataaa 900

tcacgctgtg agtgtgaaca taacacctgt ggggaaagct gtgacaggtg ctgtccagga 960

ttccatcaga agccttggag agctggaacc ttcctcacca agtctgagtg tgaagcatgc 1020

aattgtcacg gaaaagctga ggaatgctat tatgatgaaa ctgttgctag cagaaatcta 1080

agtttaaata tacatgggaa gtacatcgga gggggtgtgt gcatcaactg cacacataac 1140

acggctggga taaattgtga gacatgtgtt gatggattct tcagaccaaa aggggtatca 1200

ccaaattatc caagaccatg ccagccatgt cactgtgatc caactggctc ccttagtgaa 1260

gtctgtgtca aagatgagaa atatgcccag cgagggttga aacctggatc ctgtcactgc 1320

aaaactggct ttggaggcgt gaactgtgat cgctgtgtca ggggttacca tggttaccca 1380

gactgccaac cctgtaactg tagtggcttg gggagcacaa acgaggaccc ttgcgttggg 1440

ccctgtagct gtaaggagaa tgttgaaggt gaagactgta gtcgttgcaa atctggtttc 1500

ttcaacttgc aagaagataa tcagaaaggc tgtgaggagt gtttctgttc aggagtatca 1560

aacagatgtc agagttccta ctggacctat gggaatattc aagacatgcg tggttggtat 1620

ctcacagacc tctctggccg cattcggatg gctccccagc ttgataaccc tgactcacct 1680

cagcagatca gcatcagtaa ctctgaggcc cggaaatccc tgcttgatgg ttactactgg 1740

agtgcaccgc ctccatatct gggaaacaga cttccagctg ttggaggaca gttgtcattt 1800

accatctcat atgacctcga agaagaggaa gacgatacag aaaaaatcct tcagctgatg 1860

attatctttg agggaaatga cttaagaatc agcacagcgt ataaggaggt gtacttagag 1920

ccatctgaag aacacattga ggaggtgtca ctcaaagaag aggcctttac tatacatgga 1980

acaaatttgc cagtcactag aaaagatttc atgattgttc tcacaaattt ggagagagtc 2040

cttatgcaaa tcacatacaa cttagggatg gacgccatct tcaggctgag ttctgtcaat 2100

cttgaatctg ctgtccctta tcctactgat agacgtattg caactgatgt ggaagtttgc 2160

cagtgtccac ctgggtacag tggcagctct tgtgaaacat gttggcctag gcaccgaaga 2220

gttaacggca ccatttttgg tggcatttgt gaaccatgtc agtgctttgc tcatgcagaa 2280

gcctgtgatg acatcacagg agaatgtctg aactgtaagg atcacacagg tgggccgtac 2340

tgcaatgaat gtctccctgg tttctatggt gatcctactc gaggaagccc tgaagactgt 2400

cagccctgtg cctgtccact caatatccca tcaaataact ttagtccaac atgccattta 2460

gaccggagtc tgggattgat ctgtgacgag tgtcctattg ggtacacagg accgcgctgt 2520

gagaggtgtg cagaaggcta ttttggacaa ccttccatac ctggaggatc atgtcagcca 2580

tgccaatgca atgacaacct tgactactcc atccctggca gctgtgacag cctgtctggc 2640

tcctgtctga tttgtaagcc aggtacaaca ggccggtact gtgagctctg tgctgatggg 2700

tattttggag acgcggttaa tgcaaagaac tgtcaaccat gccgttgtaa tatcaatggc 2760

tccttctcag agatttgtca cactagaact gggcaatgtg agtgcagacc caatgtgcag 2820

gggcggcact gtgacgagtg taagcctgaa acctttggcc tgcaactggg aaggggttgt 2880

ctgccctgca actgcaattc ttttgggtct aagtcctttg actgtgaagc aagtgggcag 2940

tgctggtgcc agcctggagt agcagggaag aaatgtgacc gttgtgccca tggctacttc 3000

aacttccaag aaggaggctg catagcttgt gactgttctc atctgggcaa caactgtgac 3060

ccaaaaactg gccaatgcat ttgcccaccc aataccactg gagaaaagtg ttctgagtgt 3120

cttcccaaca cctggggtca cagcattgtc accggctgta aggtttgtaa ctgcagcact 3180

gtggggtcct tggcttctca gtgcaatgta aacacgggcc agtgcagctg tcatccaaaa 3240

ttctctggta tgaaatgctc agagtgcagc cgaggtcact ggaactatcc tctctgcact 3300

ctatgtgact gcttccttcc agggacagat gccacgactt gtgatctgga gactaggaaa 3360

tgctcctgta gtgatcaaac tggacagtgc agctgtaagg tgaatgtgga aggcgtccac 3420

tgtgacaggt gccggcctgg caaatttgga ctagatgcca agaacccact tggctgcagc 3480

agctgctact gctttggagt tactagtcaa tgctctgaag caaaggggct gatccgtacg 3540

tgggtgactt tgagtgatga acagaccatt ctacctctgg tggatgaggc cctgcagcac 3600

acgactacca aaggcattgc tttccagaaa ccagagattg ttgcaaagat ggatgaagtc 3660

aggcaagagc tccatttgga acctttttac tggaaactcc cacaacaatt tgaagggaaa 3720

aagttgatgg cttatggtgg caaactcaag tatgccatct attttgaggc tcgggatgag 3780

acaggctttg ccacatataa acctcaagtt atcattcgag gtggaactcc tactcatgct 3840

agaattatta ccagacacat ggctgcccct ctcattggcc agttgacacg gcatgaaata 3900

gaaatgacag agaaagaatg gaaatattat ggtgatgatc ctcgaatcag tagaactgtg 3960

acccgtgaag acttcttgga tatactatat gatattcact atatccttat caaggctact 4020

tatggaaacg ttgtgagaca aagccgcatt tctgaaatct ccatggaagt agccgaacca 4080

ggacatgtat tagcagggag cccaccagca cacttgatag aaagatgcga ttgccctcct 4140

ggctattctg gcttgtcttg tgagacgtgt gcaccaggat tttatcgact tcgttctgaa 4200

ccaggtggcc gcactcctgg accaacctta ggaacctgtg ttccctgcca atgtaatgga 4260

cacagcagtc agtgtgatcc tgagacctca gtatgccaga attgtcagca tcacactgct 4320

ggtgacttct gtgagcgctg tgcccttggc tactatggaa tcgtcagggg attgccaaat 4380

gactgtcaac catgtgcttg tcctctgatt tcgcccagca acaatttcag cccctcttgt 4440

gtattggaag gtctggaaga ttaccgttgc accgcctgcc caaggggcta tgaaggacag 4500

tactgtgaaa ggtgtgcccc aggctatact ggcagcccaa gcagccccgg aggctcctgc 4560

caagaatgtg agtgtgaccc ttatggctcc ctaccggttc cctgtgaccg ggtcacggga 4620

ctctgcacgt gccgccctgg agccacagga aggaagtgtg atggctgcga gcactggcat 4680

gcacgcgagg gtgcagagtg tgtcttttgt ggagacgagt gtacaggcct tcttcttggt 4740

gacctggctc gtctagagca gatgaccatg aacatcaacc tcacgggccc actgcctgct 4800

ccatataaaa ttctgtatgg tcttgaaaat acaactcagg aactcaagca cctgctatca 4860

ccgcaacggg caccagagag gctcattcag ttggcagagg gcaacgtgaa cacacttgtg 4920

atggaaacaa atgagctgct aaccagagca accaaagtga cagcagatgg tgagcaaaca 4980

ggacaagatg ctgagaggac caactccaga gcagaatcct tggaagaatt cattaaaggg 5040

cttgtccagg atgctgaagc cataaatgaa aaagctgtac aactaaatga aaccttagga 5100

aatcaagata agacagcaga gagaaacttg gaggagcttc aaaaggaaat cgaccggatg 5160

ctgaaggaac tgagaagtaa agatcttcaa acacagaagg aagttgctga ggatgagctc 5220

gtggcagcag aaggccttct gaagagagta aacaagctgt ttggagagcc cagagcccag 5280

aatgaagata tggaaaagga tctccagcag aaactggcag agtacaagaa caaacttgat 5340

gatgcttggg atctattgag agaagccact gataaaactc gagatgctaa tcgtttgtct 5400

gctgccaatc aaaaaaacat gaccatactg gagacaaaga aggaggctat tgaaggtagc 5460

aaacgacaaa tagagaacac tttaaaggaa ggcaatgaca tccttgatga agccaatcga 5520

ctcttaggtg aaatcaactc agtcatagat tatgtcgacg acattaaaac taagttgcca 5580

ccaatgtccg aggagctgag tgacaaaata gatgacctcg cccaggaaat aaaggacaga 5640

aggcttgctg agaaggtgtt ccaggctgag agccatgctg ctcagctgaa cgactcgtct 5700

gctgtacttg atggaatcct ggatgaggct aagaacatct ctttcaatgc cacggcagcc 5760

ttcagagctt acagtaatat taaagactac attgatgaag ctgagaaagt ggccagagaa 5820

gccaaagagc ttgcccaagg ggctacaaaa ctggcaacaa gtcctcaggg cttattaaaa 5880

gaagatgcca aaggctccct tcagaaaagc ttcaggatcc tcaatgaagc caagaagcta 5940

gcaaacgatg tgaaaggaaa tcacaatgat ctaaatgacc tgaaaaccag gttagaaact 6000

gctgacctta gaaacagtgg acttctagga gctctaaatg acaccatgga caagttatca 6060

gccattacaa atgacacggc tgctaaactg caggctgtca aagagaaagc cagagaagcc 6120

aatgacacag caaaagctgt cctggcccag gttaaggacc tgcatcagaa cctagatggc 6180

ctgaagcaaa actacaataa actggcagac agcgtggcca aaacgaacgc tgtggtgaaa 6240

gatccttcca aaaacaaaat cattgcagat gcaggcactt ccgtgagaaa tctagaacag 6300

gaagctgacc ggctaatcga caaactcaag cccatcaagg agcttgagga caacctaaag 6360

aaaaacattt ctgaaataaa ggaactgatc aaccaagctc ggaaacaagc taactctatc 6420

aaagtatctg tttcttcggg aggtgactgt gttcgaacat acaggccaga aatcaagaaa 6480

ggaagctaca ataacatcgt tgtccatgtc aagaccgctg ttgccgacaa cctccttttt 6540

tatcttggaa gtgccaaatt tattgacttt cttgctatag aaatgcgcaa aggcaaagtc 6600

agcttcctct gggatgttgg ctctggagtt ggccgagtag agtatccaga cttgaccatc 6660

gacgactcct attggtaccg tattgaagca tcaagaacgg gaagaaatgg atctatttct 6720

gtgagagctt tagatggacc caaagccagt atggtaccca gcacctacca ttcagtgtct 6780

cctcccgggt acactatcct agatgtggat gcaaatgcaa tgctgtttgt tggtggcctg 6840

accggaaaaa taaagaaggc cgatgctgta cgtgtgatca ccttcaccgg ctgtatggga 6900

gaaacatact ttgacaacaa acctataggt ttatggaact tccgggagaa agaaggcgac 6960

tgtaagggat gtactgtcag cccacaagtg gaagatagtg aggggactat tcagtttgat 7020

ggtgaaggct atgcattagt gagccgccct atccgctggt accccaacat ctccacagtc 7080

atgttcaagt tccggacatt ttcatcaagt gctctcctga tgtatcttgc cacacgagac 7140

ctgaaagatt tcatgagtgt agagctcagt gatggacatg tgaaagtcag ctatgacctg 7200

ggctcaggaa tgacttccgt tgtcagcaat caaaaccata atgatgggaa atggaaagca 7260

ttcacgctgt cgcggattca gaaacaagcc aacatatcga ttgtcgacat cgattctaac 7320

caggaggaga atgtagctac ttcatcttct ggaaacaact ttggtcttga cttgaaagca 7380

gatgacaaaa tatattttgg tggcctgcca actctgagaa acttgagtat gaaagcaagg 7440

ccagaagtca atgtgaagaa atactccggc tgcctcaaag atattgaaat ttcaagaaca 7500

ccttacaata tactcagcag ccctgattat gttggtgtga ccaaaggctg ttcactggag 7560

aatgtttata cagttagttt ccccaagcct ggttttgtgg agcttgccgc tgtgtctatt 7620

gatgttggaa cagaaatcaa tctgtccttt agtaccagga acgagtctgg gatcattctc 7680

ttgggaagtg gagggacact cacaccaccc aggagaaaac ggagacaaac cacacaggct 7740

tattatgcca tattcctcaa caagggccgc ttggaagtgc atctctcctc ggggacacgg 7800

acaatgagga aaattgtcat caaaccggag ccaaatttgt ttcatgatgg gagagaacat 7860

tctgtccacg tagaaagaac cagaggcatc ttcactgttc aaattgatga agacagaaga 7920

catatgcaaa acctgacaga ggaacagccc atcgaagtga aaaagctctt tgtcgggggt 7980

gctcctcctg aatttcagcc ctccccactc aggaatattc cggcctttca aggctgtgtg 8040

tggaaccttg ttattaactc catccccatg gactttgcgc agcctatagc cttcaaaaat 8100

gccgacattg gccgctgtac ctatcaaaag ccccgggaag atgagagtga agcagttcca 8160

gctgaagtta ttgtccagcc tcagccggtg cccacccctg ccttcccttt cccggccccc 8220

accatggtgc atggcccttg tgttgcagaa tcagaaccag ctcttctgac agggagcaag 8280

cagtttgggc tttccagaaa cagccacatt gcaattgctt ttgatgacac caaagttaaa 8340

aatcgcctca ccattgagct ggaggtacga actgaagctg aatcaggctt gctcttctac 8400

atggctcgga tcaatcatgc tgattttgct actgttcagc tgaggaatgg cttcccgtac 8460

ttcagttatg atttggggag tggggacaca agcaccatga tccccacaaa aatcaacgat 8520

ggccagtggc acaagattaa gattgtgaga gtgaagcagg agggaattct ttatgtggat 8580

gatgcctcca gccaaaccat cagtcccaag aaagctgaca tcctggatgt cgtggggatt 8640

ctgtatgtcg gtggattgcc gatcaactat accacacgca gaattggtcc agtgacttac 8700

agcctggatg gctgtgttag gaatcttcac atggaacaag cccctgttga tctggaccag 8760

cctacctcca gctttcacgt tgggacatgc tttgcgaatg cagagagtgg gacttacttt 8820

gatggaaccg gttttgctaa agcagttggt gggttcaaag ttggattgga ccttcttgtg 8880

gaatttgaat tccgtaccac aagacccact ggggtcctcc tgggagtcag cagtcagaag 8940

atggatggaa tgggtattga aatgatcgac gagaagctta tgttccacgt ggataatggc 9000

gctggccgat tcactgcaat ctatgatgct gggatcccag gccacatgtg caatggacag 9060

tggcataaag tcactgccaa gaagatcaaa aaccgtcttg agctggtggt agatgggaac 9120

caggtggatg cccagagccc aaactcagca tcgacatcag ctgatacaaa cgaccctgtt 9180

tttgttggcg gtttcccagg tggcctcaat cagtttggcc tgaccaccaa cattaggttc 9240

cgaggctgca tccgatctct gaagctcacc aaaggcactg gcaaaccgct ggaggttaat 9300

tttgccaagg ccctggaact gaggggtgtt caacctgtat catgcccgac tac 9353

<210> 100

<211> 21

<212> DNA

<213> 人工序列

<220>

<223> SYBR-Mm_Tfrc-F qPCR

<400> 100

tgggcactag attggatacc t 21

<210> 101

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> SYBR-Mm_Tfrc-R qPCR

<400> 101

atgagctgac cagccacttc 20

<210> 102

<211> 19

<212> DNA

<213> 人工序列

<220>

<223> SYBR-RFP-F qPCR

<400> 102

atggccagct ccgaggatg 19

<210> 103

<211> 22

<212> DNA

<213> 人工序列

<220>

<223> SYBR-RFP-R qPCR

<400> 103

gaactgaggg ctcagaatat cc 22

<210> 104

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> SYBR-hLama F qPCR

<400> 104

tagaacatgt ccctgggcag 20

<210> 105

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> SYBR-hLama r qPCR

<400> 105

atacgcgatc tggaacacct 20

<210> 106

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> SYBR-Mm-GAPDH-F qPCR

<400> 106

cgtcccgtag acaaaatggt 20

<210> 107

<211> 20

<212> DNA

<213> 人工序列

<220>

<223> SYBR-Mm-GAPDH-R qPCR

<400> 107

tggaagatgg tgatgggctt 20

<210> 108

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac R275A/N347S/K375A/D450N/S592G

<400> 108

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly

580 585 590

Cys Phe

<210> 109

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac N347S/D450N/T560A/S573A/F594L

<400> 109

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Leu

<210> 110

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac N347A/D450N

<400> 110

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 111

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac R275A/N347S/R372A/D450N/T560A/F594L

<400> 111

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Leu

<210> 112

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

R202K/R275A/N347S/R372A/D450N/T560A/F594L

<400> 112

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Lys Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Leu

<210> 113

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L

<400> 113

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Leu

<210> 114

<211> 5

<212> PRT

<213> 人工序列

<220>

<221> SITE

<222> 1..5

<223> 其中序列可以重复1至50次

<220>

<223>接头

<400> 114

Gly Gly Gly Gly Ser

1 5

<210> 115

<211> 5

<212> PRT

<213> 人工序列

<220>

<221> SITE

<222> 1..5

<223> 其中序列可以重复1至50次

<220>

<223>接头

<400> 115

Glu Ala Ala Ala Lys

1 5

<210> 116

<211> 3210

<212> PRT

<213> 人工序列

<220>

<223> 层粘连蛋白亚基α2同种型X1

<400> 116

Met Pro Gly Ala Ala Gly Val Leu Leu Leu Leu Leu Leu Ser Gly Gly

1 5 10 15

Leu Gly Gly Val Gln Ala Gln Arg Pro Gln Gln Gln Arg Gln Ser Gln

20 25 30

Ala His Gln Gln Arg Gly Leu Phe Pro Ala Val Leu Asn Leu Ala Ser

35 40 45

Asn Ala Leu Ile Thr Thr Asn Ala Thr Cys Gly Glu Lys Gly Pro Glu

50 55 60

Met Tyr Cys Lys Leu Val Glu His Val Pro Gly Gln Pro Val Arg Asn

65 70 75 80

Pro Gln Cys Arg Ile Cys Asn Gln Asn Ser Ser Asn Pro Asn Gln Arg

85 90 95

His Pro Ile Thr Asn Ala Ile Asp Gly Lys Asn Thr Trp Trp Gln Ser

100 105 110

Pro Ser Ile Lys Asn Gly Ile Glu Tyr His Tyr Val Thr Ile Thr Leu

115 120 125

Asp Leu Gln Gln Val Phe Gln Ile Ala Tyr Val Ile Val Lys Ala Ala

130 135 140

Asn Ser Pro Arg Pro Gly Asn Trp Ile Leu Glu Arg Ser Leu Asp Asp

145 150 155 160

Val Glu Tyr Lys Pro Trp Gln Tyr His Ala Val Thr Asp Thr Glu Cys

165 170 175

Leu Thr Leu Tyr Asn Ile Tyr Pro Arg Thr Gly Pro Pro Ser Tyr Ala

180 185 190

Lys Asp Asp Glu Val Ile Cys Thr Ser Phe Tyr Ser Lys Ile His Pro

195 200 205

Leu Glu Asn Gly Glu Ile His Ile Ser Leu Ile Asn Gly Arg Pro Ser

210 215 220

Ala Asp Asp Pro Ser Pro Glu Leu Leu Glu Phe Thr Ser Ala Arg Tyr

225 230 235 240

Ile Arg Leu Arg Phe Gln Arg Ile Arg Thr Leu Asn Ala Asp Leu Met

245 250 255

Met Phe Ala His Lys Asp Pro Arg Glu Ile Asp Pro Ile Val Thr Arg

260 265 270

Arg Tyr Tyr Tyr Ser Val Lys Asp Ile Ser Val Gly Gly Met Cys Ile

275 280 285

Cys Tyr Gly His Ala Arg Ala Cys Pro Leu Asp Pro Ala Thr Asn Lys

290 295 300

Ser Arg Cys Glu Cys Glu His Asn Thr Cys Gly Asp Ser Cys Asp Gln

305 310 315 320

Cys Cys Pro Gly Phe His Gln Lys Pro Trp Arg Ala Gly Thr Phe Leu

325 330 335

Thr Lys Thr Glu Cys Glu Ala Cys Asn Cys His Gly Lys Ala Glu Glu

340 345 350

Cys Tyr Tyr Asp Glu Asn Val Ala Arg Arg Asn Leu Ser Leu Asn Ile

355 360 365

Arg Gly Lys Tyr Ile Gly Gly Gly Val Cys Ile Asn Cys Thr Gln Asn

370 375 380

Thr Ala Gly Ile Asn Cys Glu Thr Cys Thr Asp Gly Phe Phe Arg Pro

385 390 395 400

Lys Gly Val Ser Pro Asn Tyr Pro Arg Pro Cys Gln Pro Cys His Cys

405 410 415

Asp Pro Ile Gly Ser Leu Asn Glu Val Cys Val Lys Asp Glu Lys His

420 425 430

Ala Arg Arg Gly Leu Ala Pro Gly Ser Cys His Cys Lys Thr Gly Phe

435 440 445

Gly Gly Val Ser Cys Asp Arg Cys Ala Arg Gly Tyr Thr Gly Tyr Pro

450 455 460

Asp Cys Lys Ala Cys Asn Cys Ser Gly Leu Gly Ser Lys Asn Glu Asp

465 470 475 480

Pro Cys Phe Gly Pro Cys Ile Cys Lys Glu Asn Val Glu Gly Gly Asp

485 490 495

Cys Ser Arg Cys Lys Ser Gly Phe Phe Asn Leu Gln Glu Asp Asn Trp

500 505 510

Lys Gly Cys Asp Glu Cys Phe Cys Ser Gly Val Ser Asn Arg Cys Gln

515 520 525

Ser Ser Tyr Trp Thr Tyr Gly Lys Ile Gln Asp Met Ser Gly Trp Tyr

530 535 540

Leu Thr Asp Leu Pro Gly Arg Ile Arg Val Ala Pro Gln Gln Asp Asp

545 550 555 560

Leu Asp Ser Pro Gln Gln Ile Ser Ile Ser Asn Ala Glu Ala Arg Gln

565 570 575

Ala Leu Pro His Ser Tyr Tyr Trp Ser Ala Pro Ala Pro Tyr Leu Gly

580 585 590

Asn Lys Leu Pro Ala Val Gly Gly Gln Leu Thr Phe Thr Ile Ser Tyr

595 600 605

Asp Leu Glu Glu Glu Glu Glu Asp Thr Glu Arg Val Leu Gln Leu Met

610 615 620

Ile Ile Leu Glu Gly Asn Asp Leu Ser Ile Ser Thr Ala Gln Asp Glu

625 630 635 640

Val Tyr Leu His Pro Ser Glu Glu His Thr Asn Val Leu Leu Leu Lys

645 650 655

Glu Glu Ser Phe Thr Ile His Gly Thr His Phe Pro Val Arg Arg Lys

660 665 670

Glu Phe Met Thr Val Leu Ala Asn Leu Lys Arg Val Leu Leu Gln Ile

675 680 685

Thr Tyr Ser Phe Gly Met Asp Ala Ile Phe Arg Leu Ser Ser Val Asn

690 695 700

Leu Glu Ser Ala Val Ser Tyr Pro Thr Asp Gly Ser Ile Ala Ala Ala

705 710 715 720

Val Glu Val Cys Gln Cys Pro Pro Gly Tyr Thr Gly Ser Ser Cys Glu

725 730 735

Ser Cys Trp Pro Arg His Arg Arg Val Asn Gly Thr Ile Phe Gly Gly

740 745 750

Ile Cys Glu Pro Cys Gln Cys Phe Gly His Ala Glu Ser Cys Asp Asp

755 760 765

Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro Tyr

770 775 780

Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys Gly Thr

785 790 795 800

Ser Glu Asp Cys Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser Asn

805 810 815

Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile Cys

820 825 830

Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys Ala

835 840 845

Glu Gly Tyr Phe Gly Gln Pro Ser Val Pro Gly Gly Ser Cys Gln Pro

850 855 860

Cys Gln Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser Cys Asp

865 870 875 880

Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly Arg

885 890 895

Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asp Ala

900 905 910

Lys Asn Cys Gln Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe Ser Glu

915 920 925

Val Cys His Ser Gln Thr Gly Gln Cys Glu Cys Arg Ala Asn Val Gln

930 935 940

Gly Gln Arg Cys Asp Lys Cys Lys Pro Asn Met Trp Arg Asp Pro Glu

945 950 955 960

Lys Arg Phe Cys Val Leu Cys Asp Cys Asp Pro Val Gly Ser Val Ser

965 970 975

Pro Gln Cys Asp Ile Thr Gly Arg Cys Val Cys Lys Ser Gly Phe Val

980 985 990

Gly Lys Gln Cys Asn Leu Gly Arg Gln Val His Gln Gln Glu Glu Gln

995 1000 1005

Pro Arg Arg Ala Gln Arg Val Leu Gly Ser Pro Gln Arg Trp Ala Ile

1010 1015 1020

Gly Ser Ser Ser Gly Cys Pro Arg Gly Ala Tyr Arg Ala Pro Ala Pro

1025 1030 1035 1040

Ala Gly Thr Phe Gly Leu Gln Ser Ala Arg Gly Cys Val Pro Cys Asn

1045 1050 1055

Cys Asn Ser Phe Gly Ser Lys Ser Phe Asp Cys Glu Glu Ser Gly Gln

1060 1065 1070

Cys Trp Cys Gln Pro Gly Val Thr Gly Lys Lys Cys Asp Arg Cys Ala

1075 1080 1085

His Gly Tyr Phe Asn Phe Gln Glu Gly Gly Cys Thr Ala Cys Glu Cys

1090 1095 1100

Ser His Leu Gly Asn Asn Cys Asp Pro Lys Thr Gly Arg Cys Ile Cys

1105 1110 1115 1120

Pro Pro Asn Thr Ile Gly Glu Lys Cys Ser Lys Cys Ala Pro Asn Thr

1125 1130 1135

Trp Gly His Ser Ile Thr Thr Gly Cys Lys Ala Cys Asn Cys Ser Thr

1140 1145 1150

Val Gly Ser Leu Asp Phe Gln Cys Asn Val Asn Thr Gly Gln Cys Asn

1155 1160 1165

Cys His Pro Lys Phe Ser Gly Ala Lys Cys Thr Glu Cys Ser Arg Gly

1170 1175 1180

His Trp Asn Tyr Pro Arg Cys Asn Leu Cys Asp Cys Phe Leu Pro Gly

1185 1190 1195 1200

Thr Asp Ala Thr Thr Cys Asp Ser Glu Thr Lys Lys Cys Ser Cys Ser

1205 1210 1215

Asp Gln Thr Gly Gln Cys Thr Cys Lys Val Asn Val Glu Gly Ile His

1220 1225 1230

Cys Asp Arg Cys Arg Pro Gly Lys Phe Gly Leu Asp Ala Lys Asn Pro

1235 1240 1245

Leu Gly Cys Ser Ser Cys Tyr Cys Phe Gly Thr Thr Thr Gln Cys Ser

1250 1255 1260

Glu Ala Lys Gly Leu Ile Arg Thr Trp Val Thr Leu Lys Ala Glu Gln

1265 1270 1275 1280

Thr Ile Leu Pro Leu Val Asp Glu Ala Leu Gln His Thr Thr Thr Lys

1285 1290 1295

Gly Ile Val Phe Gln His Pro Glu Ile Val Ala His Met Asp Leu Met

1300 1305 1310

Arg Glu Asp Leu His Leu Glu Pro Phe Tyr Trp Lys Leu Pro Glu Gln

1315 1320 1325

Phe Glu Gly Lys Lys Leu Met Ala Tyr Gly Gly Lys Leu Lys Tyr Ala

1330 1335 1340

Ile Tyr Phe Glu Ala Arg Glu Glu Thr Gly Phe Ser Thr Tyr Asn Pro

1345 1350 1355 1360

Gln Val Ile Ile Arg Gly Gly Thr Pro Thr His Ala Arg Ile Ile Val

1365 1370 1375

Arg His Met Ala Ala Pro Leu Ile Gly Gln Leu Thr Arg His Glu Ile

1380 1385 1390

Glu Met Thr Glu Lys Glu Trp Lys Tyr Tyr Gly Asp Asp Pro Arg Val

1395 1400 1405

His Arg Thr Val Thr Arg Glu Asp Phe Leu Asp Ile Leu Tyr Asp Ile

1410 1415 1420

His Tyr Ile Leu Ile Lys Ala Thr Tyr Gly Asn Phe Met Arg Gln Ser

1425 1430 1435 1440

Arg Ile Ser Glu Ile Ser Met Glu Val Ala Glu Gln Gly Arg Gly Thr

1445 1450 1455

Thr Met Thr Pro Pro Ala Asp Leu Ile Glu Lys Cys Asp Cys Pro Leu

1460 1465 1470

Gly Tyr Ser Gly Leu Ser Cys Glu Ala Cys Leu Pro Gly Phe Tyr Arg

1475 1480 1485

Leu Arg Ser Gln Pro Gly Gly Arg Thr Pro Gly Pro Thr Leu Gly Thr

1490 1495 1500

Cys Val Pro Cys Gln Cys Asn Gly His Ser Ser Leu Cys Asp Pro Glu

1505 1510 1515 1520

Thr Ser Ile Cys Gln Asn Cys Gln His His Thr Ala Gly Asp Phe Cys

1525 1530 1535

Glu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val Lys Gly Leu Pro Asn

1540 1545 1550

Asp Cys Gln Gln Cys Ala Cys Pro Leu Ile Ser Ser Ser Asn Asn Phe

1555 1560 1565

Ser Pro Ser Cys Val Ala Glu Gly Leu Asp Asp Tyr Arg Cys Thr Ala

1570 1575 1580

Cys Pro Arg Gly Tyr Glu Gly Gln Tyr Cys Glu Arg Cys Ala Pro Gly

1585 1590 1595 1600

Tyr Thr Gly Ser Pro Gly Asn Pro Gly Gly Ser Cys Gln Glu Cys Glu

1605 1610 1615

Cys Asp Pro Tyr Gly Ser Leu Pro Val Pro Cys Asp Pro Val Thr Gly

1620 1625 1630

Phe Cys Thr Cys Arg Pro Gly Ala Thr Gly Arg Lys Cys Asp Gly Cys

1635 1640 1645

Lys His Trp His Ala Arg Glu Gly Trp Glu Cys Val Phe Cys Gly Asp

1650 1655 1660

Glu Cys Thr Gly Leu Leu Leu Gly Asp Leu Ala Arg Leu Glu Gln Met

1665 1670 1675 1680

Val Met Ser Ile Asn Leu Thr Gly Pro Leu Pro Ala Pro Tyr Lys Met

1685 1690 1695

Leu Tyr Gly Leu Glu Asn Met Thr Gln Glu Leu Lys His Leu Leu Ser

1700 1705 1710

Pro Gln Arg Ala Pro Glu Arg Leu Ile Gln Leu Ala Glu Gly Asn Leu

1715 1720 1725

Asn Thr Leu Val Thr Glu Met Asn Glu Leu Leu Thr Arg Ala Thr Lys

1730 1735 1740

Val Thr Ala Asp Gly Glu Gln Thr Gly Gln Asp Ala Glu Arg Thr Asn

1745 1750 1755 1760

Thr Arg Ala Lys Ser Leu Gly Glu Phe Ile Lys Glu Leu Ala Arg Asp

1765 1770 1775

Ala Glu Ala Val Asn Glu Lys Ala Ile Lys Leu Asn Glu Thr Leu Gly

1780 1785 1790

Thr Arg Asp Glu Ala Phe Glu Arg Asn Leu Glu Gly Leu Gln Lys Glu

1795 1800 1805

Ile Asp Gln Met Ile Lys Glu Leu Arg Arg Lys Asn Leu Glu Thr Gln

1810 1815 1820

Lys Glu Ile Ala Glu Asp Glu Leu Val Ala Ala Glu Ala Leu Leu Lys

1825 1830 1835 1840

Lys Val Lys Lys Leu Phe Gly Glu Ser Arg Gly Glu Asn Glu Glu Met

1845 1850 1855

Glu Lys Asp Leu Arg Glu Lys Leu Ala Asp Tyr Lys Asn Lys Val Asp

1860 1865 1870

Asp Ala Trp Asp Leu Leu Arg Glu Ala Thr Asp Lys Ile Arg Glu Ala

1875 1880 1885

Asn Arg Leu Phe Ala Val Asn Gln Lys Asn Met Thr Ala Leu Glu Lys

1890 1895 1900

Lys Lys Glu Ala Val Glu Ser Gly Lys Arg Gln Ile Glu Asn Thr Leu

1905 1910 1915 1920

Lys Glu Gly Asn Asp Ile Leu Asp Glu Ala Asn Arg Leu Ala Asp Glu

1925 1930 1935

Ile Asn Ser Ile Ile Asp Tyr Val Glu Asp Ile Gln Thr Lys Leu Pro

1940 1945 1950

Pro Met Ser Glu Glu Leu Asn Asp Lys Ile Asp Asp Leu Ser Gln Glu

1955 1960 1965

Ile Lys Asp Arg Lys Leu Ala Glu Lys Val Ser Gln Ala Glu Ser His

1970 1975 1980

Ala Ala Gln Leu Asn Asp Ser Ser Ala Val Leu Asp Gly Ile Leu Asp

1985 1990 1995 2000

Glu Ala Lys Asn Ile Ser Phe Asn Ala Thr Ala Ala Phe Lys Ala Tyr

2005 2010 2015

Ser Asn Ile Lys Asp Tyr Ile Asp Glu Ala Glu Lys Val Ala Lys Glu

2020 2025 2030

Ala Lys Asp Leu Ala His Glu Ala Thr Lys Leu Ala Thr Gly Pro Arg

2035 2040 2045

Gly Leu Leu Lys Glu Asp Ala Lys Gly Cys Leu Gln Lys Ser Phe Arg

2050 2055 2060

Ile Leu Asn Glu Ala Lys Lys Leu Ala Asn Asp Val Lys Glu Asn Glu

2065 2070 2075 2080

Asp His Leu Asn Gly Leu Lys Thr Arg Ile Glu Asn Ala Asp Ala Arg

2085 2090 2095

Asn Gly Asp Leu Leu Arg Thr Leu Asn Asp Thr Leu Gly Lys Leu Ser

2100 2105 2110

Ala Ile Pro Asn Asp Thr Ala Ala Lys Leu Gln Ala Val Lys Asp Lys

2115 2120 2125

Ala Arg Gln Ala Asn Asp Thr Ala Lys Asp Val Leu Ala Gln Ile Thr

2130 2135 2140

Glu Leu His Gln Asn Leu Asp Gly Leu Lys Lys Asn Tyr Asn Lys Leu

2145 2150 2155 2160

Ala Asp Ser Val Ala Lys Thr Asn Ala Val Val Lys Asp Pro Ser Lys

2165 2170 2175

Asn Lys Ile Ile Ala Asp Ala Asp Ala Thr Val Lys Asn Leu Glu Gln

2180 2185 2190

Glu Ala Asp Arg Leu Ile Asp Lys Leu Lys Pro Ile Lys Glu Leu Glu

2195 2200 2205

Asp Asn Leu Lys Lys Asn Ile Ser Glu Ile Lys Glu Leu Ile Asn Gln

2210 2215 2220

Ala Arg Lys Gln Ala Asn Ser Ile Lys Val Ser Val Ser Ser Gly Gly

2225 2230 2235 2240

Asp Cys Ile Arg Thr Tyr Lys Pro Glu Ile Lys Lys Gly Ser Tyr Asn

2245 2250 2255

Asn Ile Val Val Asn Val Lys Thr Ala Val Ala Asp Asn Leu Leu Phe

2260 2265 2270

Tyr Leu Gly Ser Ala Lys Phe Ile Asp Phe Leu Ala Ile Glu Met Arg

2275 2280 2285

Lys Gly Lys Val Ser Phe Leu Trp Asp Val Gly Ser Gly Val Gly Arg

2290 2295 2300

Val Glu Tyr Pro Asp Leu Thr Ile Asp Asp Ser Tyr Trp Tyr Arg Ile

2305 2310 2315 2320

Val Ala Ser Arg Thr Gly Arg Asn Gly Thr Ile Ser Val Arg Ala Leu

2325 2330 2335

Asp Gly Pro Lys Ala Ser Ile Val Pro Ser Thr His His Ser Thr Ser

2340 2345 2350

Pro Pro Gly Tyr Thr Ile Leu Asp Val Asp Ala Asn Ala Met Leu Phe

2355 2360 2365

Val Gly Gly Leu Thr Gly Lys Leu Lys Lys Ala Asp Ala Val Arg Val

2370 2375 2380

Ile Thr Phe Thr Gly Cys Met Gly Glu Thr Tyr Phe Asp Asn Lys Pro

2385 2390 2395 2400

Ile Gly Leu Trp Asn Phe Arg Glu Lys Glu Gly Asp Cys Lys Gly Cys

2405 2410 2415

Thr Val Ser Pro Gln Val Glu Asp Ser Glu Gly Thr Ile Gln Phe Asp

2420 2425 2430

Gly Glu Gly Tyr Ala Leu Val Ser Arg Pro Ile Arg Trp Tyr Pro Asn

2435 2440 2445

Ile Ser Thr Val Met Phe Lys Phe Arg Thr Phe Ser Ser Ser Ala Leu

2450 2455 2460

Leu Met Tyr Leu Ala Thr Arg Asp Leu Arg Asp Phe Met Ser Val Glu

2465 2470 2475 2480

Leu Thr Asp Gly His Ile Lys Val Ser Tyr Asp Leu Gly Ser Gly Met

2485 2490 2495

Ala Ser Val Val Ser Asn Gln Asn His Asn Asp Gly Lys Trp Lys Ser

2500 2505 2510

Phe Thr Leu Ser Arg Ile Gln Lys Gln Ala Asn Ile Ser Ile Val Asp

2515 2520 2525

Ile Asp Thr Asn Gln Glu Glu Asn Ile Ala Thr Ser Ser Ser Gly Asn

2530 2535 2540

Asn Phe Gly Leu Asp Leu Lys Ala Asp Asp Lys Ile Tyr Phe Gly Gly

2545 2550 2555 2560

Leu Pro Thr Leu Arg Asn Leu Ser Met Lys Ala Arg Pro Glu Val Asn

2565 2570 2575

Leu Lys Lys Tyr Ser Gly Cys Leu Lys Asp Ile Glu Ile Ser Arg Thr

2580 2585 2590

Pro Tyr Asn Ile Leu Ser Ser Pro Asp Tyr Val Gly Val Thr Lys Gly

2595 2600 2605

Cys Ser Leu Glu Asn Val Tyr Thr Val Ser Phe Pro Lys Pro Gly Phe

2610 2615 2620

Val Glu Leu Ser Pro Val Pro Ile Asp Val Gly Thr Glu Ile Asn Leu

2625 2630 2635 2640

Ser Phe Ser Thr Lys Asn Glu Ser Gly Ile Ile Leu Leu Gly Ser Gly

2645 2650 2655

Gly Thr Pro Ala Pro Pro Arg Arg Lys Arg Arg Gln Thr Gly Gln Ala

2660 2665 2670

Tyr Tyr Ala Ile Leu Leu Asn Arg Gly Arg Leu Glu Val His Leu Ser

2675 2680 2685

Thr Gly Ala Arg Thr Met Arg Lys Ile Val Ile Arg Pro Glu Pro Asn

2690 2695 2700

Leu Phe His Asp Gly Arg Glu His Ser Val His Val Glu Arg Thr Arg

2705 2710 2715 2720

Gly Ile Phe Thr Val Gln Val Asp Glu Asn Arg Arg Tyr Met Gln Asn

2725 2730 2735

Leu Thr Val Glu Gln Pro Ile Glu Val Lys Lys Leu Phe Val Gly Gly

2740 2745 2750

Ala Pro Pro Glu Phe Gln Pro Ser Pro Leu Arg Asn Ile Pro Pro Phe

2755 2760 2765

Glu Gly Cys Ile Trp Asn Leu Val Ile Asn Ser Val Pro Met Asp Phe

2770 2775 2780

Ala Arg Pro Val Ser Phe Lys Asn Ala Asp Ile Gly Arg Cys Ala His

2785 2790 2795 2800

Gln Lys Leu Arg Glu Asp Glu Asp Gly Ala Ala Pro Ala Glu Ile Val

2805 2810 2815

Ile Gln Pro Glu Pro Val Pro Thr Pro Ala Phe Pro Thr Pro Thr Pro

2820 2825 2830

Val Leu Thr His Gly Pro Cys Ala Ala Glu Ser Glu Pro Ala Leu Leu

2835 2840 2845

Ile Gly Ser Lys Gln Phe Gly Leu Ser Arg Asn Ser His Ile Ala Ile

2850 2855 2860

Ala Phe Asp Asp Thr Lys Val Lys Asn Arg Leu Thr Ile Glu Leu Glu

2865 2870 2875 2880

Val Arg Thr Glu Ala Glu Ser Gly Leu Leu Phe Tyr Met Ala Arg Ile

2885 2890 2895

Asn His Ala Asp Phe Ala Thr Val Gln Leu Arg Asn Gly Leu Pro Tyr

2900 2905 2910

Phe Ser Tyr Asp Leu Gly Ser Gly Asp Thr His Thr Met Ile Pro Thr

2915 2920 2925

Lys Ile Asn Asp Gly Gln Trp His Lys Ile Lys Ile Met Arg Ser Lys

2930 2935 2940

Gln Glu Gly Ile Leu Tyr Val Asp Gly Ala Ser Asn Arg Thr Ile Ser

2945 2950 2955 2960

Pro Lys Lys Ala Asp Ile Leu Asp Val Val Gly Met Leu Tyr Val Gly

2965 2970 2975

Gly Leu Pro Ile Asn Tyr Thr Thr Arg Arg Ile Gly Pro Val Thr Tyr

2980 2985 2990

Ser Ile Asp Gly Cys Val Arg Asn Leu His Met Ala Glu Ala Pro Ala

2995 3000 3005

Asp Leu Glu Gln Pro Thr Ser Ser Phe His Val Gly Thr Cys Phe Ala

3010 3015 3020

Asn Ala Gln Arg Gly Thr Tyr Phe Asp Gly Thr Gly Phe Ala Lys Ala

3025 3030 3035 3040

Val Gly Gly Phe Lys Val Gly Leu Asp Leu Leu Val Glu Phe Glu Phe

3045 3050 3055

Arg Thr Thr Thr Thr Thr Gly Val Leu Leu Gly Ile Ser Ser Gln Lys

3060 3065 3070

Met Asp Gly Met Gly Ile Glu Met Ile Asp Glu Lys Leu Met Phe His

3075 3080 3085

Val Asp Asn Gly Ala Gly Arg Phe Thr Ala Val Tyr Asp Ala Gly Val

3090 3095 3100

Pro Gly His Leu Cys Asp Gly Gln Trp His Lys Val Thr Ala Asn Lys

3105 3110 3115 3120

Ile Lys His Arg Ile Glu Leu Thr Val Asp Gly Asn Gln Val Glu Ala

3125 3130 3135

Gln Ser Pro Asn Pro Ala Ser Thr Ser Ala Asp Thr Asn Asp Pro Val

3140 3145 3150

Phe Val Gly Gly Phe Pro Asp Asp Leu Lys Gln Phe Gly Leu Thr Thr

3155 3160 3165

Ser Ile Pro Phe Arg Gly Cys Ile Arg Ser Leu Lys Leu Thr Lys Gly

3170 3175 3180

Thr Gly Lys Pro Leu Glu Val Asn Phe Ala Lys Ala Leu Glu Leu Arg

3185 3190 3195 3200

Gly Val Gln Pro Val Ser Cys Pro Ala Asn

3205 3210

<210> 117

<211> 3207

<212> PRT

<213> 人工序列

<220>

<223> 层粘连蛋白亚基α2同种型X2

<400> 117

Met Pro Gly Ala Ala Gly Val Leu Leu Leu Leu Leu Leu Ser Gly Gly

1 5 10 15

Leu Gly Gly Val Gln Ala Gln Arg Pro Gln Gln Gln Arg Gln Ser Gln

20 25 30

Ala His Gln Gln Arg Gly Leu Phe Pro Ala Val Leu Asn Leu Ala Ser

35 40 45

Asn Ala Leu Ile Thr Thr Asn Ala Thr Cys Gly Glu Lys Gly Pro Glu

50 55 60

Met Tyr Cys Lys Leu Val Glu His Val Pro Gly Gln Pro Val Arg Asn

65 70 75 80

Pro Gln Cys Arg Ile Cys Asn Gln Asn Ser Ser Asn Pro Asn Gln Arg

85 90 95

His Pro Ile Thr Asn Ala Ile Asp Gly Lys Asn Thr Trp Trp Gln Ser

100 105 110

Pro Ser Ile Lys Asn Gly Ile Glu Tyr His Tyr Val Thr Ile Thr Leu

115 120 125

Asp Leu Gln Gln Val Phe Gln Ile Ala Tyr Val Ile Val Lys Ala Ala

130 135 140

Asn Ser Pro Arg Pro Gly Asn Trp Ile Leu Glu Arg Ser Leu Asp Asp

145 150 155 160

Val Glu Tyr Lys Pro Trp Gln Tyr His Ala Val Thr Asp Thr Glu Cys

165 170 175

Leu Thr Leu Tyr Asn Ile Tyr Pro Arg Thr Gly Pro Pro Ser Tyr Ala

180 185 190

Lys Asp Asp Glu Val Ile Cys Thr Ser Phe Tyr Ser Lys Ile His Pro

195 200 205

Leu Glu Asn Gly Glu Ile His Ile Ser Leu Ile Asn Gly Arg Pro Ser

210 215 220

Ala Asp Asp Pro Ser Pro Glu Leu Leu Glu Phe Thr Ser Ala Arg Tyr

225 230 235 240

Ile Arg Leu Arg Phe Gln Arg Ile Arg Thr Leu Asn Ala Asp Leu Met

245 250 255

Met Phe Ala His Lys Asp Pro Arg Glu Ile Asp Pro Ile Val Thr Arg

260 265 270

Arg Tyr Tyr Tyr Ser Val Lys Asp Ile Ser Val Gly Gly Met Cys Ile

275 280 285

Cys Tyr Gly His Ala Arg Ala Cys Pro Leu Asp Pro Ala Thr Asn Lys

290 295 300

Ser Arg Cys Glu Cys Glu His Asn Thr Cys Gly Asp Ser Cys Asp Gln

305 310 315 320

Cys Cys Pro Gly Phe His Gln Lys Pro Trp Arg Ala Gly Thr Phe Leu

325 330 335

Thr Lys Thr Glu Cys Glu Ala Cys Asn Cys His Gly Lys Ala Glu Glu

340 345 350

Cys Tyr Tyr Asp Glu Asn Val Ala Arg Arg Asn Leu Ser Leu Asn Ile

355 360 365

Arg Gly Lys Tyr Ile Gly Gly Gly Val Cys Ile Asn Cys Thr Gln Asn

370 375 380

Thr Ala Gly Ile Asn Cys Glu Thr Cys Thr Asp Gly Phe Phe Arg Pro

385 390 395 400

Lys Gly Val Ser Pro Asn Tyr Pro Arg Pro Cys Gln Pro Cys His Cys

405 410 415

Asp Pro Ile Gly Ser Leu Asn Glu Val Cys Val Lys Asp Glu Lys His

420 425 430

Ala Arg Arg Gly Leu Ala Pro Gly Ser Cys His Cys Lys Thr Gly Phe

435 440 445

Gly Gly Val Ser Cys Asp Arg Cys Ala Arg Gly Tyr Thr Gly Tyr Pro

450 455 460

Asp Cys Lys Ala Cys Asn Cys Ser Gly Leu Gly Ser Lys Asn Glu Asp

465 470 475 480

Pro Cys Phe Gly Pro Cys Ile Cys Lys Glu Asn Val Glu Gly Gly Asp

485 490 495

Cys Ser Arg Cys Lys Ser Gly Phe Phe Asn Leu Gln Glu Asp Asn Trp

500 505 510

Lys Gly Cys Asp Glu Cys Phe Cys Ser Gly Val Ser Asn Arg Cys Gln

515 520 525

Ser Ser Tyr Trp Thr Tyr Gly Lys Ile Gln Asp Met Ser Gly Trp Tyr

530 535 540

Leu Thr Asp Leu Pro Gly Arg Ile Arg Val Ala Pro Gln Gln Asp Asp

545 550 555 560

Leu Asp Ser Pro Gln Gln Ile Ser Ile Ser Asn Ala Glu Ala Arg Gln

565 570 575

Ala Leu Pro His Ser Tyr Tyr Trp Ser Ala Pro Ala Pro Tyr Leu Gly

580 585 590

Asn Lys Leu Pro Ala Val Gly Gly Gln Leu Thr Phe Thr Ile Ser Tyr

595 600 605

Asp Leu Glu Glu Glu Glu Glu Asp Thr Glu Arg Val Leu Gln Leu Met

610 615 620

Ile Ile Leu Glu Gly Asn Asp Leu Ser Ile Ser Thr Ala Gln Asp Glu

625 630 635 640

Val Tyr Leu His Pro Ser Glu Glu His Thr Asn Val Leu Leu Leu Lys

645 650 655

Glu Glu Ser Phe Thr Ile His Gly Thr His Phe Pro Val Arg Arg Lys

660 665 670

Glu Phe Met Thr Val Leu Ala Asn Leu Lys Arg Val Leu Leu Gln Ile

675 680 685

Thr Tyr Ser Phe Gly Met Asp Ala Ile Phe Arg Leu Ser Ser Val Asn

690 695 700

Leu Glu Ser Ala Val Ser Tyr Pro Thr Asp Gly Ser Ile Ala Ala Ala

705 710 715 720

Val Glu Val Cys Gln Cys Pro Pro Gly Tyr Thr Gly Ser Ser Cys Glu

725 730 735

Ser Cys Trp Pro Arg His Arg Arg Val Asn Gly Thr Ile Phe Gly Gly

740 745 750

Ile Cys Glu Pro Cys Gln Cys Phe Gly His Ala Glu Ser Cys Asp Asp

755 760 765

Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro Tyr

770 775 780

Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys Gly Thr

785 790 795 800

Ser Glu Asp Cys Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser Asn

805 810 815

Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile Cys

820 825 830

Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys Ala

835 840 845

Glu Gly Tyr Phe Gly Gln Pro Ser Val Pro Gly Gly Ser Cys Gln Pro

850 855 860

Cys Gln Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser Cys Asp

865 870 875 880

Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly Arg

885 890 895

Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asp Ala

900 905 910

Lys Asn Cys Gln Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe Ser Glu

915 920 925

Val Cys His Ser Gln Thr Gly Gln Cys Glu Cys Arg Ala Asn Val Gln

930 935 940

Gly Gln Arg Cys Asp Lys Cys Lys Pro Asn Met Trp Arg Asp Pro Glu

945 950 955 960

Lys Arg Phe Cys Val Leu Cys Asp Cys Asp Pro Val Gly Ser Val Ser

965 970 975

Pro Gln Cys Asp Ile Thr Gly Arg Cys Val Cys Lys Ser Gly Phe Val

980 985 990

Gly Lys Gln Cys Asn Leu Gly Arg Gln Val His Gln Gln Glu Glu Gln

995 1000 1005

Pro Arg Arg Ala Gln Arg Val Leu Gly Ser Pro Gln Arg Trp Ala Ile

1010 1015 1020

Gly Ser Ser Ser Gly Cys Pro Arg Gly Ala Tyr Arg Ala Pro Ala Pro

1025 1030 1035 1040

Ala Gly Thr Phe Gly Leu Gln Ser Ala Arg Gly Cys Val Pro Cys Asn

1045 1050 1055

Cys Asn Ser Phe Gly Ser Lys Ser Phe Asp Cys Glu Glu Ser Gly Gln

1060 1065 1070

Cys Trp Cys Gln Pro Gly Val Thr Gly Lys Lys Cys Asp Arg Cys Ala

1075 1080 1085

His Gly Tyr Phe Asn Phe Gln Glu Gly Gly Cys Thr Ala Cys Glu Cys

1090 1095 1100

Ser His Leu Gly Asn Asn Cys Asp Pro Lys Thr Gly Arg Cys Ile Cys

1105 1110 1115 1120

Pro Pro Asn Thr Ile Gly Glu Lys Cys Ser Lys Cys Ala Pro Asn Thr

1125 1130 1135

Trp Gly His Ser Ile Thr Thr Gly Cys Lys Ala Cys Asn Cys Ser Thr

1140 1145 1150

Val Gly Ser Leu Asp Phe Gln Cys Asn Val Asn Thr Gly Gln Cys Asn

1155 1160 1165

Cys His Pro Lys Phe Ser Gly Ala Lys Cys Thr Glu Cys Ser Arg Gly

1170 1175 1180

His Trp Asn Tyr Pro Arg Cys Asn Leu Cys Asp Cys Phe Leu Pro Gly

1185 1190 1195 1200

Thr Asp Ala Thr Thr Cys Asp Ser Glu Thr Lys Lys Cys Ser Cys Ser

1205 1210 1215

Asp Gln Thr Gly Gln Cys Thr Cys Lys Val Asn Val Glu Gly Ile His

1220 1225 1230

Cys Asp Arg Cys Arg Pro Gly Lys Phe Gly Leu Asp Ala Lys Asn Pro

1235 1240 1245

Leu Gly Cys Ser Ser Cys Tyr Cys Phe Gly Thr Thr Thr Gln Cys Ser

1250 1255 1260

Glu Ala Lys Gly Leu Ile Arg Thr Trp Val Thr Leu Lys Ala Glu Gln

1265 1270 1275 1280

Thr Ile Leu Pro Leu Val Asp Glu Ala Leu Gln His Thr Thr Thr Lys

1285 1290 1295

Gly Ile Val Phe Gln His Pro Glu Ile Val Ala His Met Asp Leu Met

1300 1305 1310

Arg Glu Asp Leu His Leu Glu Pro Phe Tyr Trp Lys Leu Pro Glu Gln

1315 1320 1325

Phe Glu Gly Lys Lys Leu Met Ala Tyr Gly Gly Lys Leu Lys Tyr Ala

1330 1335 1340

Ile Tyr Phe Glu Ala Arg Glu Glu Thr Gly Phe Ser Thr Tyr Asn Pro

1345 1350 1355 1360

Gln Val Ile Ile Arg Gly Gly Thr Pro Thr His Ala Arg Ile Ile Val

1365 1370 1375

Arg His Met Ala Ala Pro Leu Ile Gly Gln Leu Thr Arg His Glu Ile

1380 1385 1390

Glu Met Thr Glu Lys Glu Trp Lys Tyr Tyr Gly Asp Asp Pro Arg Val

1395 1400 1405

His Arg Thr Val Thr Arg Glu Asp Phe Leu Asp Ile Leu Tyr Asp Ile

1410 1415 1420

His Tyr Ile Leu Ile Lys Ala Thr Tyr Gly Asn Phe Met Arg Gln Ser

1425 1430 1435 1440

Arg Ile Ser Glu Ile Ser Met Glu Val Ala Glu Gln Gly Arg Gly Thr

1445 1450 1455

Thr Met Thr Pro Pro Ala Asp Leu Ile Glu Lys Cys Asp Cys Pro Leu

1460 1465 1470

Gly Tyr Ser Gly Leu Ser Cys Glu Ala Cys Leu Pro Gly Phe Tyr Arg

1475 1480 1485

Leu Arg Ser Gln Pro Gly Gly Arg Thr Pro Gly Pro Thr Leu Gly Thr

1490 1495 1500

Cys Val Pro Cys Gln Cys Asn Gly His Ser Ser Leu Cys Asp Pro Glu

1505 1510 1515 1520

Thr Ser Ile Cys Gln Asn Cys Gln His His Thr Ala Gly Asp Phe Cys

1525 1530 1535

Glu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val Lys Gly Leu Pro Asn

1540 1545 1550

Asp Cys Gln Gln Cys Ala Cys Pro Leu Ile Ser Ser Ser Asn Asn Phe

1555 1560 1565

Ser Pro Ser Cys Val Ala Glu Gly Leu Asp Asp Tyr Arg Cys Thr Ala

1570 1575 1580

Cys Pro Arg Gly Tyr Glu Gly Gln Tyr Cys Glu Arg Cys Ala Pro Gly

1585 1590 1595 1600

Tyr Thr Gly Ser Pro Gly Asn Pro Gly Ser Cys Gln Glu Cys Glu Cys

1605 1610 1615

Asp Pro Tyr Gly Ser Leu Pro Val Pro Cys Asp Pro Val Thr Gly Phe

1620 1625 1630

Cys Thr Cys Arg Pro Gly Ala Thr Gly Arg Lys Cys Asp Gly Cys Lys

1635 1640 1645

His Trp His Ala Arg Glu Gly Trp Glu Cys Val Phe Cys Gly Asp Glu

1650 1655 1660

Cys Thr Gly Leu Leu Leu Gly Asp Leu Ala Arg Leu Glu Gln Met Val

1665 1670 1675 1680

Met Ser Ile Asn Leu Thr Gly Pro Leu Pro Ala Pro Tyr Lys Met Leu

1685 1690 1695

Tyr Gly Leu Glu Asn Met Thr Gln Glu Leu Lys His Leu Leu Ser Pro

1700 1705 1710

Gln Arg Ala Pro Glu Arg Leu Ile Gln Leu Ala Glu Gly Asn Leu Asn

1715 1720 1725

Thr Leu Val Thr Glu Met Asn Glu Leu Leu Thr Arg Ala Thr Lys Val

1730 1735 1740

Thr Ala Asp Gly Glu Gln Thr Gly Gln Asp Ala Glu Arg Thr Asn Thr

1745 1750 1755 1760

Arg Ala Lys Ser Leu Gly Glu Phe Ile Lys Glu Leu Ala Arg Asp Ala

1765 1770 1775

Glu Ala Val Asn Glu Lys Ala Ile Lys Leu Asn Glu Thr Leu Gly Thr

1780 1785 1790

Arg Asp Glu Ala Phe Glu Arg Asn Leu Glu Gly Leu Gln Lys Glu Ile

1795 1800 1805

Asp Gln Met Ile Lys Glu Leu Arg Arg Lys Asn Leu Glu Thr Gln Lys

1810 1815 1820

Glu Ile Ala Glu Asp Glu Leu Val Ala Ala Glu Ala Leu Leu Lys Lys

1825 1830 1835 1840

Val Lys Lys Leu Phe Gly Glu Ser Arg Gly Glu Asn Glu Glu Met Glu

1845 1850 1855

Lys Asp Leu Arg Glu Lys Leu Ala Asp Tyr Lys Asn Lys Val Asp Asp

1860 1865 1870

Ala Trp Asp Leu Leu Arg Glu Ala Thr Asp Lys Ile Arg Glu Ala Asn

1875 1880 1885

Arg Leu Phe Ala Val Asn Gln Lys Asn Met Thr Ala Leu Glu Lys Lys

1890 1895 1900

Lys Glu Ala Val Glu Ser Gly Lys Arg Gln Ile Glu Asn Thr Leu Lys

1905 1910 1915 1920

Glu Gly Asn Asp Ile Leu Asp Glu Ala Asn Arg Leu Ala Asp Glu Ile

1925 1930 1935

Asn Ser Ile Ile Asp Tyr Val Glu Asp Ile Gln Thr Lys Leu Pro Pro

1940 1945 1950

Met Ser Glu Glu Leu Asn Asp Lys Ile Asp Asp Leu Ser Gln Glu Ile

1955 1960 1965

Lys Asp Arg Lys Leu Ala Glu Lys Val Ser Gln Ala Glu Ser His Ala

1970 1975 1980

Ala Gln Leu Asn Asp Ser Ser Ala Val Leu Asp Gly Ile Leu Asp Glu

1985 1990 1995 2000

Ala Lys Asn Ile Ser Phe Asn Ala Thr Ala Ala Phe Lys Ala Tyr Ser

2005 2010 2015

Asn Ile Lys Asp Tyr Ile Asp Glu Ala Glu Lys Val Ala Lys Glu Ala

2020 2025 2030

Lys Asp Leu Ala His Glu Ala Thr Lys Leu Ala Thr Gly Pro Arg Gly

2035 2040 2045

Leu Leu Lys Glu Asp Ala Lys Gly Cys Leu Gln Lys Ser Phe Arg Ile

2050 2055 2060

Leu Asn Glu Ala Lys Lys Leu Ala Asn Asp Val Lys Glu Asn Glu Asp

2065 2070 2075 2080

His Leu Asn Gly Leu Lys Thr Arg Ile Glu Asn Ala Asp Ala Arg Asn

2085 2090 2095

Gly Asp Leu Leu Arg Thr Leu Asn Asp Thr Leu Gly Lys Leu Ser Ala

2100 2105 2110

Ile Pro Asn Asp Thr Ala Ala Lys Leu Gln Ala Val Lys Asp Lys Ala

2115 2120 2125

Arg Gln Ala Asn Asp Thr Ala Lys Asp Val Leu Ala Gln Ile Thr Glu

2130 2135 2140

Leu His Gln Asn Leu Asp Gly Leu Lys Lys Asn Tyr Asn Lys Leu Ala

2145 2150 2155 2160

Asp Ser Val Ala Lys Thr Asn Ala Val Val Lys Asp Pro Ser Lys Asn

2165 2170 2175

Ile Ala Asp Ala Asp Ala Thr Val Lys Asn Leu Glu Gln Glu Ala Asp

2180 2185 2190

Arg Leu Ile Asp Lys Leu Lys Pro Ile Lys Glu Leu Glu Asp Asn Leu

2195 2200 2205

Lys Lys Asn Ile Ser Glu Ile Lys Glu Leu Ile Asn Gln Ala Arg Lys

2210 2215 2220

Gln Ala Asn Ser Ile Lys Val Ser Val Ser Ser Gly Gly Asp Cys Ile

2225 2230 2235 2240

Arg Thr Tyr Lys Pro Glu Ile Lys Lys Gly Ser Tyr Asn Asn Ile Val

2245 2250 2255

Val Asn Val Lys Thr Ala Val Ala Asp Asn Leu Leu Phe Tyr Leu Gly

2260 2265 2270

Ser Ala Lys Phe Ile Asp Phe Leu Ala Ile Glu Met Arg Lys Gly Lys

2275 2280 2285

Val Ser Phe Leu Trp Asp Val Gly Ser Gly Val Gly Arg Val Glu Tyr

2290 2295 2300

Pro Asp Leu Thr Ile Asp Asp Ser Tyr Trp Tyr Arg Ile Val Ala Ser

2305 2310 2315 2320

Arg Thr Gly Arg Asn Gly Thr Ile Ser Val Arg Ala Leu Asp Gly Pro

2325 2330 2335

Lys Ala Ser Ile Val Pro Ser Thr His His Ser Thr Ser Pro Pro Gly

2340 2345 2350

Tyr Thr Ile Leu Asp Val Asp Ala Asn Ala Met Leu Phe Val Gly Gly

2355 2360 2365

Leu Thr Gly Lys Leu Lys Lys Ala Asp Ala Val Arg Val Ile Thr Phe

2370 2375 2380

Thr Gly Cys Met Gly Glu Thr Tyr Phe Asp Asn Lys Pro Ile Gly Leu

2385 2390 2395 2400

Trp Asn Phe Arg Glu Lys Glu Gly Asp Cys Lys Gly Cys Thr Val Ser

2405 2410 2415

Pro Gln Val Glu Asp Ser Glu Gly Thr Ile Gln Phe Asp Gly Glu Gly

2420 2425 2430

Tyr Ala Leu Val Ser Arg Pro Ile Arg Trp Tyr Pro Asn Ile Ser Thr

2435 2440 2445

Val Met Phe Lys Phe Arg Thr Phe Ser Ser Ser Ala Leu Leu Met Tyr

2450 2455 2460

Leu Ala Thr Arg Asp Leu Arg Asp Phe Met Ser Val Glu Leu Thr Asp

2465 2470 2475 2480

Gly His Ile Lys Val Ser Tyr Asp Leu Gly Ser Gly Met Ala Ser Val

2485 2490 2495

Val Ser Asn Gln Asn His Asn Asp Gly Lys Trp Lys Ser Phe Thr Leu

2500 2505 2510

Ser Arg Ile Gln Lys Gln Ala Asn Ile Ser Ile Val Asp Ile Asp Thr

2515 2520 2525

Asn Gln Glu Glu Asn Ile Ala Thr Ser Ser Ser Gly Asn Asn Phe Gly

2530 2535 2540

Leu Asp Leu Lys Ala Asp Asp Lys Ile Tyr Phe Gly Gly Leu Pro Thr

2545 2550 2555 2560

Leu Arg Asn Leu Ser Met Lys Ala Arg Pro Glu Val Asn Leu Lys Lys

2565 2570 2575

Tyr Ser Gly Cys Leu Lys Asp Ile Glu Ile Ser Arg Thr Pro Tyr Asn

2580 2585 2590

Ile Leu Ser Ser Pro Asp Tyr Val Gly Val Thr Lys Gly Cys Ser Leu

2595 2600 2605

Glu Asn Val Tyr Thr Val Ser Phe Pro Lys Pro Gly Phe Val Glu Leu

2610 2615 2620

Ser Pro Val Pro Ile Asp Val Gly Thr Glu Ile Asn Leu Ser Phe Ser

2625 2630 2635 2640

Thr Lys Asn Glu Ser Gly Ile Ile Leu Leu Gly Ser Gly Gly Thr Pro

2645 2650 2655

Ala Pro Pro Arg Arg Lys Arg Arg Gln Thr Gly Gln Ala Tyr Tyr Ala

2660 2665 2670

Ile Leu Leu Asn Arg Gly Arg Leu Glu Val His Leu Ser Thr Gly Ala

2675 2680 2685

Arg Thr Met Arg Lys Ile Val Ile Arg Pro Glu Pro Asn Leu Phe His

2690 2695 2700

Asp Gly Arg Glu His Ser Val His Val Glu Arg Thr Arg Gly Ile Phe

2705 2710 2715 2720

Thr Val Gln Val Asp Glu Asn Arg Arg Tyr Met Gln Asn Leu Thr Val

2725 2730 2735

Glu Gln Pro Ile Glu Val Lys Lys Leu Phe Val Gly Gly Ala Pro Pro

2740 2745 2750

Glu Phe Gln Pro Ser Pro Leu Arg Asn Ile Pro Pro Phe Glu Gly Cys

2755 2760 2765

Ile Trp Asn Leu Val Ile Asn Ser Val Pro Met Asp Phe Ala Arg Pro

2770 2775 2780

Val Ser Phe Lys Asn Ala Asp Ile Gly Arg Cys Ala His Gln Lys Leu

2785 2790 2795 2800

Arg Glu Asp Glu Asp Gly Ala Ala Pro Ala Glu Ile Val Ile Gln Pro

2805 2810 2815

Glu Pro Val Pro Thr Pro Ala Phe Pro Thr Pro Thr Pro Val Leu Thr

2820 2825 2830

His Gly Pro Cys Ala Ala Glu Ser Glu Pro Ala Leu Leu Ile Gly Ser

2835 2840 2845

Lys Gln Phe Gly Leu Ser Arg Asn Ser His Ile Ala Ile Ala Phe Asp

2850 2855 2860

Asp Thr Lys Val Lys Asn Arg Leu Thr Ile Glu Leu Glu Val Arg Thr

2865 2870 2875 2880

Glu Ala Glu Ser Gly Leu Leu Phe Tyr Met Ala Arg Ile Asn His Ala

2885 2890 2895

Asp Phe Ala Thr Val Gln Leu Arg Asn Gly Leu Pro Tyr Phe Ser Tyr

2900 2905 2910

Asp Leu Gly Ser Gly Asp Thr His Thr Met Ile Pro Thr Lys Ile Asn

2915 2920 2925

Asp Gly Gln Trp His Lys Ile Lys Ile Met Arg Ser Lys Gln Glu Gly

2930 2935 2940

Ile Leu Tyr Val Asp Gly Ala Ser Asn Arg Thr Ile Ser Pro Lys Lys

2945 2950 2955 2960

Ala Asp Ile Leu Asp Val Val Gly Met Leu Tyr Val Gly Gly Leu Pro

2965 2970 2975

Ile Asn Tyr Thr Thr Arg Arg Ile Gly Pro Val Thr Tyr Ser Ile Asp

2980 2985 2990

Gly Cys Val Arg Asn Leu His Met Ala Glu Ala Pro Ala Asp Leu Glu

2995 3000 3005

Gln Pro Thr Ser Ser Phe His Val Gly Thr Cys Phe Ala Asn Ala Gln

3010 3015 3020

Arg Gly Thr Tyr Phe Asp Gly Thr Gly Phe Ala Lys Ala Val Gly Gly

3025 3030 3035 3040

Phe Lys Val Gly Leu Asp Leu Leu Val Glu Phe Glu Phe Arg Thr Thr

3045 3050 3055

Thr Thr Thr Gly Val Leu Leu Gly Ile Ser Ser Gln Lys Met Asp Gly

3060 3065 3070

Met Gly Ile Glu Met Ile Asp Glu Lys Leu Met Phe His Val Asp Asn

3075 3080 3085

Gly Ala Gly Arg Phe Thr Ala Val Tyr Asp Ala Gly Val Pro Gly His

3090 3095 3100

Leu Cys Asp Gly Gln Trp His Lys Val Thr Ala Asn Lys Ile Lys His

3105 3110 3115 3120

Arg Ile Glu Leu Thr Val Asp Gly Asn Gln Val Glu Ala Gln Ser Pro

3125 3130 3135

Asn Pro Ala Ser Thr Ser Ala Asp Thr Asn Asp Pro Val Phe Val Gly

3140 3145 3150

Gly Phe Pro Asp Asp Leu Lys Gln Phe Gly Leu Thr Thr Ser Ile Pro

3155 3160 3165

Phe Arg Gly Cys Ile Arg Ser Leu Lys Leu Thr Lys Gly Thr Gly Lys

3170 3175 3180

Pro Leu Glu Val Asn Phe Ala Lys Ala Leu Glu Leu Arg Gly Val Gln

3185 3190 3195 3200

Pro Val Ser Cys Pro Ala Asn

3205

<210> 118

<211> 3206

<212> PRT

<213> 人工序列

<220>

<223> 层粘连蛋白亚基α2同种型X3

<400> 118

Met Pro Gly Ala Ala Gly Val Leu Leu Leu Leu Leu Leu Ser Gly Gly

1 5 10 15

Leu Gly Gly Val Gln Ala Gln Arg Pro Gln Gln Gln Arg Gln Ser Gln

20 25 30

Ala His Gln Gln Arg Gly Leu Phe Pro Ala Val Leu Asn Leu Ala Ser

35 40 45

Asn Ala Leu Ile Thr Thr Asn Ala Thr Cys Gly Glu Lys Gly Pro Glu

50 55 60

Met Tyr Cys Lys Leu Val Glu His Val Pro Gly Gln Pro Val Arg Asn

65 70 75 80

Pro Gln Cys Arg Ile Cys Asn Gln Asn Ser Ser Asn Pro Asn Gln Arg

85 90 95

His Pro Ile Thr Asn Ala Ile Asp Gly Lys Asn Thr Trp Trp Gln Ser

100 105 110

Pro Ser Ile Lys Asn Gly Ile Glu Tyr His Tyr Val Thr Ile Thr Leu

115 120 125

Asp Leu Gln Gln Val Phe Gln Ile Ala Tyr Val Ile Val Lys Ala Ala

130 135 140

Asn Ser Pro Arg Pro Gly Asn Trp Ile Leu Glu Arg Ser Leu Asp Asp

145 150 155 160

Val Glu Tyr Lys Pro Trp Gln Tyr His Ala Val Thr Asp Thr Glu Cys

165 170 175

Leu Thr Leu Tyr Asn Ile Tyr Pro Arg Thr Gly Pro Pro Ser Tyr Ala

180 185 190

Lys Asp Asp Glu Val Ile Cys Thr Ser Phe Tyr Ser Lys Ile His Pro

195 200 205

Leu Glu Asn Gly Glu Ile His Ile Ser Leu Ile Asn Gly Arg Pro Ser

210 215 220

Ala Asp Asp Pro Ser Pro Glu Leu Leu Glu Phe Thr Ser Ala Arg Tyr

225 230 235 240

Ile Arg Leu Arg Phe Gln Arg Ile Arg Thr Leu Asn Ala Asp Leu Met

245 250 255

Met Phe Ala His Lys Asp Pro Arg Glu Ile Asp Pro Ile Val Thr Arg

260 265 270

Arg Tyr Tyr Tyr Ser Val Lys Asp Ile Ser Val Gly Gly Met Cys Ile

275 280 285

Cys Tyr Gly His Ala Arg Ala Cys Pro Leu Asp Pro Ala Thr Asn Lys

290 295 300

Ser Arg Cys Glu Cys Glu His Asn Thr Cys Gly Asp Ser Cys Asp Gln

305 310 315 320

Cys Cys Pro Gly Phe His Gln Lys Pro Trp Arg Ala Gly Thr Phe Leu

325 330 335

Thr Lys Thr Glu Cys Glu Ala Cys Asn Cys His Gly Lys Ala Glu Glu

340 345 350

Cys Tyr Tyr Asp Glu Asn Val Ala Arg Arg Asn Leu Ser Leu Asn Ile

355 360 365

Arg Gly Lys Tyr Ile Gly Gly Gly Val Cys Ile Asn Cys Thr Gln Asn

370 375 380

Thr Ala Gly Ile Asn Cys Glu Thr Cys Thr Asp Gly Phe Phe Arg Pro

385 390 395 400

Lys Gly Val Ser Pro Asn Tyr Pro Arg Pro Cys Gln Pro Cys His Cys

405 410 415

Asp Pro Ile Gly Ser Leu Asn Glu Val Cys Val Lys Asp Glu Lys His

420 425 430

Ala Arg Arg Gly Leu Ala Pro Gly Ser Cys His Cys Lys Thr Gly Phe

435 440 445

Gly Gly Val Ser Cys Asp Arg Cys Ala Arg Gly Tyr Thr Gly Tyr Pro

450 455 460

Asp Cys Lys Ala Cys Asn Cys Ser Gly Leu Gly Ser Lys Asn Glu Asp

465 470 475 480

Pro Cys Phe Gly Pro Cys Ile Cys Lys Glu Asn Val Glu Gly Gly Asp

485 490 495

Cys Ser Arg Cys Lys Ser Gly Phe Phe Asn Leu Gln Glu Asp Asn Trp

500 505 510

Lys Gly Cys Asp Glu Cys Phe Cys Ser Gly Val Ser Asn Arg Cys Gln

515 520 525

Ser Ser Tyr Trp Thr Tyr Gly Lys Ile Gln Asp Met Ser Gly Trp Tyr

530 535 540

Leu Thr Asp Leu Pro Gly Arg Ile Arg Val Ala Pro Gln Gln Asp Asp

545 550 555 560

Leu Asp Ser Pro Gln Gln Ile Ser Ile Ser Asn Ala Glu Ala Arg Gln

565 570 575

Ala Leu Pro His Ser Tyr Tyr Trp Ser Ala Pro Ala Pro Tyr Leu Gly

580 585 590

Asn Lys Leu Pro Ala Val Gly Gly Gln Leu Thr Phe Thr Ile Ser Tyr

595 600 605

Asp Leu Glu Glu Glu Glu Glu Asp Thr Glu Arg Val Leu Gln Leu Met

610 615 620

Ile Ile Leu Glu Gly Asn Asp Leu Ser Ile Ser Thr Ala Gln Asp Glu

625 630 635 640

Val Tyr Leu His Pro Ser Glu Glu His Thr Asn Val Leu Leu Leu Lys

645 650 655

Glu Glu Ser Phe Thr Ile His Gly Thr His Phe Pro Val Arg Arg Lys

660 665 670

Glu Phe Met Thr Val Leu Ala Asn Leu Lys Arg Val Leu Leu Gln Ile

675 680 685

Thr Tyr Ser Phe Gly Met Asp Ala Ile Phe Arg Leu Ser Ser Val Asn

690 695 700

Leu Glu Ser Ala Val Ser Tyr Pro Thr Asp Gly Ser Ile Ala Ala Ala

705 710 715 720

Val Glu Val Cys Gln Cys Pro Pro Gly Tyr Thr Gly Ser Ser Cys Glu

725 730 735

Ser Cys Trp Pro Arg His Arg Arg Val Asn Gly Thr Ile Phe Gly Gly

740 745 750

Ile Cys Glu Pro Cys Gln Cys Phe Gly His Ala Glu Ser Cys Asp Asp

755 760 765

Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro Tyr

770 775 780

Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys Gly Thr

785 790 795 800

Ser Glu Asp Cys Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser Asn

805 810 815

Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile Cys

820 825 830

Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys Ala

835 840 845

Glu Gly Tyr Phe Gly Gln Pro Ser Val Pro Gly Gly Ser Cys Gln Pro

850 855 860

Cys Gln Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser Cys Asp

865 870 875 880

Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly Arg

885 890 895

Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asp Ala

900 905 910

Lys Asn Cys Gln Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe Ser Glu

915 920 925

Val Cys His Ser Gln Thr Gly Gln Cys Glu Cys Arg Ala Asn Val Gln

930 935 940

Gly Gln Arg Cys Asp Lys Cys Lys Pro Asn Met Trp Arg Asp Pro Glu

945 950 955 960

Lys Arg Phe Cys Val Leu Cys Asp Cys Asp Pro Val Gly Ser Val Ser

965 970 975

Pro Gln Cys Asp Ile Thr Gly Arg Cys Val Cys Lys Ser Gly Phe Val

980 985 990

Gly Lys Gln Cys Asn Leu Gly Arg Gln Val His Gln Gln Glu Glu Gln

995 1000 1005

Pro Arg Arg Ala Gln Arg Val Leu Gly Ser Pro Gln Arg Trp Ala Ile

1010 1015 1020

Gly Ser Ser Ser Gly Cys Pro Arg Gly Ala Tyr Arg Ala Pro Ala Pro

1025 1030 1035 1040

Ala Gly Thr Phe Gly Leu Gln Ser Ala Arg Gly Cys Val Pro Cys Asn

1045 1050 1055

Cys Asn Ser Phe Gly Ser Lys Ser Phe Asp Cys Glu Glu Ser Gly Gln

1060 1065 1070

Cys Trp Cys Gln Pro Gly Val Thr Gly Lys Lys Cys Asp Arg Cys Ala

1075 1080 1085

His Gly Tyr Phe Asn Phe Gln Glu Gly Gly Cys Thr Ala Cys Glu Cys

1090 1095 1100

Ser His Leu Gly Asn Asn Cys Asp Pro Lys Thr Gly Arg Cys Ile Cys

1105 1110 1115 1120

Pro Pro Asn Thr Ile Gly Glu Lys Cys Ser Lys Cys Ala Pro Asn Thr

1125 1130 1135

Trp Gly His Ser Ile Thr Thr Gly Cys Lys Ala Cys Asn Cys Ser Thr

1140 1145 1150

Val Gly Ser Leu Asp Phe Gln Cys Asn Val Asn Thr Gly Gln Cys Asn

1155 1160 1165

Cys His Pro Lys Phe Ser Gly Ala Lys Cys Thr Glu Cys Ser Arg Gly

1170 1175 1180

His Trp Asn Tyr Pro Arg Cys Asn Leu Cys Asp Cys Phe Leu Pro Gly

1185 1190 1195 1200

Thr Asp Ala Thr Thr Cys Asp Ser Glu Thr Lys Lys Cys Ser Cys Ser

1205 1210 1215

Asp Gln Thr Gly Gln Cys Thr Cys Lys Val Asn Val Glu Gly Ile His

1220 1225 1230

Cys Asp Arg Cys Arg Pro Gly Lys Phe Gly Leu Asp Ala Lys Asn Pro

1235 1240 1245

Leu Gly Cys Ser Ser Cys Tyr Cys Phe Gly Thr Thr Thr Gln Cys Ser

1250 1255 1260

Glu Ala Lys Gly Leu Ile Arg Thr Trp Val Thr Leu Lys Ala Glu Gln

1265 1270 1275 1280

Thr Ile Leu Pro Leu Val Asp Glu Ala Leu Gln His Thr Thr Thr Lys

1285 1290 1295

Gly Ile Val Phe Gln His Pro Glu Ile Val Ala His Met Asp Leu Met

1300 1305 1310

Arg Glu Asp Leu His Leu Glu Pro Phe Tyr Trp Lys Leu Pro Glu Gln

1315 1320 1325

Phe Glu Gly Lys Lys Leu Met Ala Tyr Gly Gly Lys Leu Lys Tyr Ala

1330 1335 1340

Ile Tyr Phe Glu Ala Arg Glu Glu Thr Gly Phe Ser Thr Tyr Asn Pro

1345 1350 1355 1360

Gln Val Ile Ile Arg Gly Gly Thr Pro Thr His Ala Arg Ile Ile Val

1365 1370 1375

Arg His Met Ala Ala Pro Leu Ile Gly Gln Leu Thr Arg His Glu Ile

1380 1385 1390

Glu Met Thr Glu Lys Glu Trp Lys Tyr Tyr Gly Asp Asp Pro Arg Val

1395 1400 1405

His Arg Thr Val Thr Arg Glu Asp Phe Leu Asp Ile Leu Tyr Asp Ile

1410 1415 1420

His Tyr Ile Leu Ile Lys Ala Thr Tyr Gly Asn Phe Met Arg Gln Ser

1425 1430 1435 1440

Arg Ile Ser Glu Ile Ser Met Glu Val Ala Glu Gln Gly Arg Gly Thr

1445 1450 1455

Thr Met Thr Pro Pro Ala Asp Leu Ile Glu Lys Cys Asp Cys Pro Leu

1460 1465 1470

Gly Tyr Ser Gly Leu Ser Cys Glu Ala Cys Leu Pro Gly Phe Tyr Arg

1475 1480 1485

Leu Arg Ser Gln Pro Gly Gly Arg Thr Pro Gly Pro Thr Leu Gly Thr

1490 1495 1500

Cys Val Pro Cys Gln Cys Asn Gly His Ser Ser Leu Cys Asp Pro Glu

1505 1510 1515 1520

Thr Ser Ile Cys Gln Asn Cys Gln His His Thr Ala Gly Asp Phe Cys

1525 1530 1535

Glu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val Lys Gly Leu Pro Asn

1540 1545 1550

Asp Cys Gln Gln Cys Ala Cys Pro Leu Ile Ser Ser Ser Asn Asn Phe

1555 1560 1565

Ser Pro Ser Cys Val Ala Glu Gly Leu Asp Asp Tyr Arg Cys Thr Ala

1570 1575 1580

Cys Pro Arg Gly Tyr Glu Gly Gln Tyr Cys Glu Arg Cys Ala Pro Gly

1585 1590 1595 1600

Tyr Thr Gly Ser Pro Gly Asn Pro Gly Gly Ser Cys Gln Glu Cys Glu

1605 1610 1615

Cys Asp Pro Tyr Gly Ser Leu Pro Val Pro Cys Asp Pro Val Thr Gly

1620 1625 1630

Phe Cys Thr Cys Arg Pro Gly Ala Thr Gly Arg Lys Cys Asp Gly Cys

1635 1640 1645

Lys His Trp His Ala Arg Glu Gly Trp Glu Cys Val Phe Cys Gly Asp

1650 1655 1660

Glu Cys Thr Gly Leu Leu Leu Gly Asp Leu Ala Arg Leu Glu Gln Met

1665 1670 1675 1680

Val Met Ser Ile Asn Leu Thr Gly Pro Leu Pro Ala Pro Tyr Lys Met

1685 1690 1695

Leu Tyr Gly Leu Glu Asn Met Thr Gln Glu Leu Lys His Leu Leu Ser

1700 1705 1710

Pro Gln Arg Ala Pro Glu Arg Leu Ile Gln Leu Ala Glu Gly Asn Leu

1715 1720 1725

Asn Thr Leu Val Thr Glu Met Asn Glu Leu Leu Thr Arg Ala Thr Lys

1730 1735 1740

Val Thr Ala Asp Gly Glu Gln Thr Gly Gln Asp Ala Glu Arg Thr Asn

1745 1750 1755 1760

Thr Arg Ala Lys Ser Leu Gly Glu Phe Ile Lys Glu Leu Ala Arg Asp

1765 1770 1775

Ala Glu Ala Val Asn Glu Lys Ala Ile Lys Leu Asn Glu Thr Leu Gly

1780 1785 1790

Thr Arg Asp Glu Ala Phe Glu Arg Asn Leu Glu Gly Leu Gln Lys Glu

1795 1800 1805

Ile Asp Gln Met Ile Lys Glu Leu Arg Arg Lys Asn Leu Glu Thr Gln

1810 1815 1820

Lys Glu Ile Ala Glu Asp Glu Leu Val Ala Ala Glu Ala Leu Leu Lys

1825 1830 1835 1840

Lys Val Lys Lys Leu Phe Gly Glu Ser Arg Gly Glu Asn Glu Glu Met

1845 1850 1855

Glu Lys Asp Leu Arg Glu Lys Leu Ala Asp Tyr Lys Asn Lys Val Asp

1860 1865 1870

Asp Ala Trp Asp Leu Leu Arg Glu Ala Thr Asp Lys Ile Arg Glu Ala

1875 1880 1885

Asn Arg Leu Phe Ala Val Asn Gln Lys Asn Met Thr Ala Leu Glu Lys

1890 1895 1900

Lys Lys Glu Ala Val Glu Ser Gly Lys Arg Gln Ile Glu Asn Thr Leu

1905 1910 1915 1920

Lys Glu Gly Asn Asp Ile Leu Asp Glu Ala Asn Arg Leu Ala Asp Glu

1925 1930 1935

Ile Asn Ser Ile Ile Asp Tyr Val Glu Asp Ile Gln Thr Lys Leu Pro

1940 1945 1950

Pro Met Ser Glu Glu Leu Asn Asp Lys Ile Asp Asp Leu Ser Gln Glu

1955 1960 1965

Ile Lys Asp Arg Lys Leu Ala Glu Lys Val Ser Gln Ala Glu Ser His

1970 1975 1980

Ala Ala Gln Leu Asn Asp Ser Ser Ala Val Leu Asp Gly Ile Leu Asp

1985 1990 1995 2000

Glu Ala Lys Asn Ile Ser Phe Asn Ala Thr Ala Ala Phe Lys Ala Tyr

2005 2010 2015

Ser Asn Ile Lys Asp Tyr Ile Asp Glu Ala Glu Lys Val Ala Lys Glu

2020 2025 2030

Ala Lys Asp Leu Ala His Glu Ala Thr Lys Leu Ala Thr Gly Pro Arg

2035 2040 2045

Gly Leu Leu Lys Glu Asp Ala Lys Gly Cys Leu Gln Lys Ser Phe Arg

2050 2055 2060

Ile Leu Asn Glu Ala Lys Lys Leu Ala Asn Asp Val Lys Glu Asn Glu

2065 2070 2075 2080

Asp His Leu Asn Gly Leu Lys Thr Arg Ile Glu Asn Ala Asp Ala Arg

2085 2090 2095

Asn Gly Asp Leu Leu Arg Thr Leu Asn Asp Thr Leu Gly Lys Leu Ser

2100 2105 2110

Ala Ile Pro Asn Asp Thr Ala Ala Lys Leu Gln Ala Val Lys Asp Lys

2115 2120 2125

Ala Arg Gln Ala Asn Asp Thr Ala Lys Asp Val Leu Ala Gln Ile Thr

2130 2135 2140

Glu Leu His Gln Asn Leu Asp Gly Leu Lys Lys Asn Tyr Asn Lys Leu

2145 2150 2155 2160

Ala Asp Ser Val Ala Lys Thr Asn Ala Val Val Lys Asp Pro Ser Lys

2165 2170 2175

Asn Lys Ile Ile Ala Asp Ala Asp Ala Thr Val Lys Asn Leu Glu Gln

2180 2185 2190

Glu Ala Asp Arg Leu Ile Asp Lys Leu Lys Pro Ile Lys Glu Leu Glu

2195 2200 2205

Asp Asn Leu Lys Lys Asn Ile Ser Glu Ile Lys Glu Leu Ile Asn Gln

2210 2215 2220

Ala Arg Lys Gln Ala Asn Ser Ile Lys Val Ser Val Ser Ser Gly Gly

2225 2230 2235 2240

Asp Cys Ile Arg Thr Tyr Lys Pro Glu Ile Lys Lys Gly Ser Tyr Asn

2245 2250 2255

Asn Ile Val Val Asn Val Lys Thr Ala Val Ala Asp Asn Leu Leu Phe

2260 2265 2270

Tyr Leu Gly Ser Ala Lys Phe Ile Asp Phe Leu Ala Ile Glu Met Arg

2275 2280 2285

Lys Gly Lys Val Ser Phe Leu Trp Asp Val Gly Ser Gly Val Gly Arg

2290 2295 2300

Val Glu Tyr Pro Asp Leu Thr Ile Asp Asp Ser Tyr Trp Tyr Arg Ile

2305 2310 2315 2320

Val Ala Ser Arg Thr Gly Arg Asn Gly Thr Ile Ser Val Arg Ala Leu

2325 2330 2335

Asp Gly Pro Lys Ala Ser Ile Val Pro Ser Thr His His Ser Thr Ser

2340 2345 2350

Pro Pro Gly Tyr Thr Ile Leu Asp Val Asp Ala Asn Ala Met Leu Phe

2355 2360 2365

Val Gly Gly Leu Thr Gly Lys Leu Lys Lys Ala Asp Ala Val Arg Val

2370 2375 2380

Ile Thr Phe Thr Gly Cys Met Gly Glu Thr Tyr Phe Asp Asn Lys Pro

2385 2390 2395 2400

Ile Gly Leu Trp Asn Phe Arg Glu Lys Glu Gly Asp Cys Lys Gly Cys

2405 2410 2415

Thr Val Ser Pro Gln Val Glu Asp Ser Glu Gly Thr Ile Gln Phe Asp

2420 2425 2430

Gly Glu Gly Tyr Ala Leu Val Ser Arg Pro Ile Arg Trp Tyr Pro Asn

2435 2440 2445

Ile Ser Thr Val Met Phe Lys Phe Arg Thr Phe Ser Ser Ser Ala Leu

2450 2455 2460

Leu Met Tyr Leu Ala Thr Arg Asp Leu Arg Asp Phe Met Ser Val Glu

2465 2470 2475 2480

Leu Thr Asp Gly His Ile Lys Val Ser Tyr Asp Leu Gly Ser Gly Met

2485 2490 2495

Ala Ser Val Val Ser Asn Gln Asn His Asn Asp Gly Lys Trp Lys Ser

2500 2505 2510

Phe Thr Leu Ser Arg Ile Gln Lys Gln Ala Asn Ile Ser Ile Val Asp

2515 2520 2525

Ile Asp Thr Asn Gln Glu Glu Asn Ile Ala Thr Ser Ser Ser Gly Asn

2530 2535 2540

Asn Phe Gly Leu Asp Leu Lys Ala Asp Asp Lys Ile Tyr Phe Gly Gly

2545 2550 2555 2560

Leu Pro Thr Leu Arg Asn Leu Arg Pro Glu Val Asn Leu Lys Lys Tyr

2565 2570 2575

Ser Gly Cys Leu Lys Asp Ile Glu Ile Ser Arg Thr Pro Tyr Asn Ile

2580 2585 2590

Leu Ser Ser Pro Asp Tyr Val Gly Val Thr Lys Gly Cys Ser Leu Glu

2595 2600 2605

Asn Val Tyr Thr Val Ser Phe Pro Lys Pro Gly Phe Val Glu Leu Ser

2610 2615 2620

Pro Val Pro Ile Asp Val Gly Thr Glu Ile Asn Leu Ser Phe Ser Thr

2625 2630 2635 2640

Lys Asn Glu Ser Gly Ile Ile Leu Leu Gly Ser Gly Gly Thr Pro Ala

2645 2650 2655

Pro Pro Arg Arg Lys Arg Arg Gln Thr Gly Gln Ala Tyr Tyr Ala Ile

2660 2665 2670

Leu Leu Asn Arg Gly Arg Leu Glu Val His Leu Ser Thr Gly Ala Arg

2675 2680 2685

Thr Met Arg Lys Ile Val Ile Arg Pro Glu Pro Asn Leu Phe His Asp

2690 2695 2700

Gly Arg Glu His Ser Val His Val Glu Arg Thr Arg Gly Ile Phe Thr

2705 2710 2715 2720

Val Gln Val Asp Glu Asn Arg Arg Tyr Met Gln Asn Leu Thr Val Glu

2725 2730 2735

Gln Pro Ile Glu Val Lys Lys Leu Phe Val Gly Gly Ala Pro Pro Glu

2740 2745 2750

Phe Gln Pro Ser Pro Leu Arg Asn Ile Pro Pro Phe Glu Gly Cys Ile

2755 2760 2765

Trp Asn Leu Val Ile Asn Ser Val Pro Met Asp Phe Ala Arg Pro Val

2770 2775 2780

Ser Phe Lys Asn Ala Asp Ile Gly Arg Cys Ala His Gln Lys Leu Arg

2785 2790 2795 2800

Glu Asp Glu Asp Gly Ala Ala Pro Ala Glu Ile Val Ile Gln Pro Glu

2805 2810 2815

Pro Val Pro Thr Pro Ala Phe Pro Thr Pro Thr Pro Val Leu Thr His

2820 2825 2830

Gly Pro Cys Ala Ala Glu Ser Glu Pro Ala Leu Leu Ile Gly Ser Lys

2835 2840 2845

Gln Phe Gly Leu Ser Arg Asn Ser His Ile Ala Ile Ala Phe Asp Asp

2850 2855 2860

Thr Lys Val Lys Asn Arg Leu Thr Ile Glu Leu Glu Val Arg Thr Glu

2865 2870 2875 2880

Ala Glu Ser Gly Leu Leu Phe Tyr Met Ala Arg Ile Asn His Ala Asp

2885 2890 2895

Phe Ala Thr Val Gln Leu Arg Asn Gly Leu Pro Tyr Phe Ser Tyr Asp

2900 2905 2910

Leu Gly Ser Gly Asp Thr His Thr Met Ile Pro Thr Lys Ile Asn Asp

2915 2920 2925

Gly Gln Trp His Lys Ile Lys Ile Met Arg Ser Lys Gln Glu Gly Ile

2930 2935 2940

Leu Tyr Val Asp Gly Ala Ser Asn Arg Thr Ile Ser Pro Lys Lys Ala

2945 2950 2955 2960

Asp Ile Leu Asp Val Val Gly Met Leu Tyr Val Gly Gly Leu Pro Ile

2965 2970 2975

Asn Tyr Thr Thr Arg Arg Ile Gly Pro Val Thr Tyr Ser Ile Asp Gly

2980 2985 2990

Cys Val Arg Asn Leu His Met Ala Glu Ala Pro Ala Asp Leu Glu Gln

2995 3000 3005

Pro Thr Ser Ser Phe His Val Gly Thr Cys Phe Ala Asn Ala Gln Arg

3010 3015 3020

Gly Thr Tyr Phe Asp Gly Thr Gly Phe Ala Lys Ala Val Gly Gly Phe

3025 3030 3035 3040

Lys Val Gly Leu Asp Leu Leu Val Glu Phe Glu Phe Arg Thr Thr Thr

3045 3050 3055

Thr Thr Gly Val Leu Leu Gly Ile Ser Ser Gln Lys Met Asp Gly Met

3060 3065 3070

Gly Ile Glu Met Ile Asp Glu Lys Leu Met Phe His Val Asp Asn Gly

3075 3080 3085

Ala Gly Arg Phe Thr Ala Val Tyr Asp Ala Gly Val Pro Gly His Leu

3090 3095 3100

Cys Asp Gly Gln Trp His Lys Val Thr Ala Asn Lys Ile Lys His Arg

3105 3110 3115 3120

Ile Glu Leu Thr Val Asp Gly Asn Gln Val Glu Ala Gln Ser Pro Asn

3125 3130 3135

Pro Ala Ser Thr Ser Ala Asp Thr Asn Asp Pro Val Phe Val Gly Gly

3140 3145 3150

Phe Pro Asp Asp Leu Lys Gln Phe Gly Leu Thr Thr Ser Ile Pro Phe

3155 3160 3165

Arg Gly Cys Ile Arg Ser Leu Lys Leu Thr Lys Gly Thr Gly Lys Pro

3170 3175 3180

Leu Glu Val Asn Phe Ala Lys Ala Leu Glu Leu Arg Gly Val Gln Pro

3185 3190 3195 3200

Val Ser Cys Pro Ala Asn

3205

<210> 119

<211> 3212

<212> PRT

<213> 人工序列

<220>

<223> 层粘连蛋白亚基α2同种型X4

<400> 119

Met Gln Trp Cys Phe Ser Val Leu Met Trp Arg Ser Gln Glu Thr Ser

1 5 10 15

Tyr Asn Ser Glu Lys His Leu Met Leu Ala Ala Val Ala Val Arg Leu

20 25 30

Ala His Glu Leu Arg Ala Arg Gly Leu Phe Pro Ala Val Leu Asn Leu

35 40 45

Ala Ser Asn Ala Leu Ile Thr Thr Asn Ala Thr Cys Gly Glu Lys Gly

50 55 60

Pro Glu Met Tyr Cys Lys Leu Val Glu His Val Pro Gly Gln Pro Val

65 70 75 80

Arg Asn Pro Gln Cys Arg Ile Cys Asn Gln Asn Ser Ser Asn Pro Asn

85 90 95

Gln Arg His Pro Ile Thr Asn Ala Ile Asp Gly Lys Asn Thr Trp Trp

100 105 110

Gln Ser Pro Ser Ile Lys Asn Gly Ile Glu Tyr His Tyr Val Thr Ile

115 120 125

Thr Leu Asp Leu Gln Gln Val Phe Gln Ile Ala Tyr Val Ile Val Lys

130 135 140

Ala Ala Asn Ser Pro Arg Pro Gly Asn Trp Ile Leu Glu Arg Ser Leu

145 150 155 160

Asp Asp Val Glu Tyr Lys Pro Trp Gln Tyr His Ala Val Thr Asp Thr

165 170 175

Glu Cys Leu Thr Leu Tyr Asn Ile Tyr Pro Arg Thr Gly Pro Pro Ser

180 185 190

Tyr Ala Lys Asp Asp Glu Val Ile Cys Thr Ser Phe Tyr Ser Lys Ile

195 200 205

His Pro Leu Glu Asn Gly Glu Ile His Ile Ser Leu Ile Asn Gly Arg

210 215 220

Pro Ser Ala Asp Asp Pro Ser Pro Glu Leu Leu Glu Phe Thr Ser Ala

225 230 235 240

Arg Tyr Ile Arg Leu Arg Phe Gln Arg Ile Arg Thr Leu Asn Ala Asp

245 250 255

Leu Met Met Phe Ala His Lys Asp Pro Arg Glu Ile Asp Pro Ile Val

260 265 270

Thr Arg Arg Tyr Tyr Tyr Ser Val Lys Asp Ile Ser Val Gly Gly Met

275 280 285

Cys Ile Cys Tyr Gly His Ala Arg Ala Cys Pro Leu Asp Pro Ala Thr

290 295 300

Asn Lys Ser Arg Cys Glu Cys Glu His Asn Thr Cys Gly Asp Ser Cys

305 310 315 320

Asp Gln Cys Cys Pro Gly Phe His Gln Lys Pro Trp Arg Ala Gly Thr

325 330 335

Phe Leu Thr Lys Thr Glu Cys Glu Ala Cys Asn Cys His Gly Lys Ala

340 345 350

Glu Glu Cys Tyr Tyr Asp Glu Asn Val Ala Arg Arg Asn Leu Ser Leu

355 360 365

Asn Ile Arg Gly Lys Tyr Ile Gly Gly Gly Val Cys Ile Asn Cys Thr

370 375 380

Gln Asn Thr Ala Gly Ile Asn Cys Glu Thr Cys Thr Asp Gly Phe Phe

385 390 395 400

Arg Pro Lys Gly Val Ser Pro Asn Tyr Pro Arg Pro Cys Gln Pro Cys

405 410 415

His Cys Asp Pro Ile Gly Ser Leu Asn Glu Val Cys Val Lys Asp Glu

420 425 430

Lys His Ala Arg Arg Gly Leu Ala Pro Gly Ser Cys His Cys Lys Thr

435 440 445

Gly Phe Gly Gly Val Ser Cys Asp Arg Cys Ala Arg Gly Tyr Thr Gly

450 455 460

Tyr Pro Asp Cys Lys Ala Cys Asn Cys Ser Gly Leu Gly Ser Lys Asn

465 470 475 480

Glu Asp Pro Cys Phe Gly Pro Cys Ile Cys Lys Glu Asn Val Glu Gly

485 490 495

Gly Asp Cys Ser Arg Cys Lys Ser Gly Phe Phe Asn Leu Gln Glu Asp

500 505 510

Asn Trp Lys Gly Cys Asp Glu Cys Phe Cys Ser Gly Val Ser Asn Arg

515 520 525

Cys Gln Ser Ser Tyr Trp Thr Tyr Gly Lys Ile Gln Asp Met Ser Gly

530 535 540

Trp Tyr Leu Thr Asp Leu Pro Gly Arg Ile Arg Val Ala Pro Gln Gln

545 550 555 560

Asp Asp Leu Asp Ser Pro Gln Gln Ile Ser Ile Ser Asn Ala Glu Ala

565 570 575

Arg Gln Ala Leu Pro His Ser Tyr Tyr Trp Ser Ala Pro Ala Pro Tyr

580 585 590

Leu Gly Asn Lys Leu Pro Ala Val Gly Gly Gln Leu Thr Phe Thr Ile

595 600 605

Ser Tyr Asp Leu Glu Glu Glu Glu Glu Asp Thr Glu Arg Val Leu Gln

610 615 620

Leu Met Ile Ile Leu Glu Gly Asn Asp Leu Ser Ile Ser Thr Ala Gln

625 630 635 640

Asp Glu Val Tyr Leu His Pro Ser Glu Glu His Thr Asn Val Leu Leu

645 650 655

Leu Lys Glu Glu Ser Phe Thr Ile His Gly Thr His Phe Pro Val Arg

660 665 670

Arg Lys Glu Phe Met Thr Val Leu Ala Asn Leu Lys Arg Val Leu Leu

675 680 685

Gln Ile Thr Tyr Ser Phe Gly Met Asp Ala Ile Phe Arg Leu Ser Ser

690 695 700

Val Asn Leu Glu Ser Ala Val Ser Tyr Pro Thr Asp Gly Ser Ile Ala

705 710 715 720

Ala Ala Val Glu Val Cys Gln Cys Pro Pro Gly Tyr Thr Gly Ser Ser

725 730 735

Cys Glu Ser Cys Trp Pro Arg His Arg Arg Val Asn Gly Thr Ile Phe

740 745 750

Gly Gly Ile Cys Glu Pro Cys Gln Cys Phe Gly His Ala Glu Ser Cys

755 760 765

Asp Asp Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly

770 775 780

Pro Tyr Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys

785 790 795 800

Gly Thr Ser Glu Asp Cys Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro

805 810 815

Ser Asn Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu

820 825 830

Ile Cys Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg

835 840 845

Cys Ala Glu Gly Tyr Phe Gly Gln Pro Ser Val Pro Gly Gly Ser Cys

850 855 860

Gln Pro Cys Gln Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser

865 870 875 880

Cys Asp Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr

885 890 895

Gly Arg Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val

900 905 910

Asp Ala Lys Asn Cys Gln Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe

915 920 925

Ser Glu Val Cys His Ser Gln Thr Gly Gln Cys Glu Cys Arg Ala Asn

930 935 940

Val Gln Gly Gln Arg Cys Asp Lys Cys Lys Pro Asn Met Trp Arg Asp

945 950 955 960

Pro Glu Lys Arg Phe Cys Val Leu Cys Asp Cys Asp Pro Val Gly Ser

965 970 975

Val Ser Pro Gln Cys Asp Ile Thr Gly Arg Cys Val Cys Lys Ser Gly

980 985 990

Phe Val Gly Lys Gln Cys Asn Leu Gly Arg Gln Val His Gln Gln Glu

995 1000 1005

Glu Gln Pro Arg Arg Ala Gln Arg Val Leu Gly Ser Pro Gln Arg Trp

1010 1015 1020

Ala Ile Gly Ser Ser Ser Gly Cys Pro Arg Gly Ala Tyr Arg Ala Pro

1025 1030 1035 1040

Ala Pro Ala Gly Thr Phe Gly Leu Gln Ser Ala Arg Gly Cys Val Pro

1045 1050 1055

Cys Asn Cys Asn Ser Phe Gly Ser Lys Ser Phe Asp Cys Glu Glu Ser

1060 1065 1070

Gly Gln Cys Trp Cys Gln Pro Gly Val Thr Gly Lys Lys Cys Asp Arg

1075 1080 1085

Cys Ala His Gly Tyr Phe Asn Phe Gln Glu Gly Gly Cys Thr Ala Cys

1090 1095 1100

Glu Cys Ser His Leu Gly Asn Asn Cys Asp Pro Lys Thr Gly Arg Cys

1105 1110 1115 1120

Ile Cys Pro Pro Asn Thr Ile Gly Glu Lys Cys Ser Lys Cys Ala Pro

1125 1130 1135

Asn Thr Trp Gly His Ser Ile Thr Thr Gly Cys Lys Ala Cys Asn Cys

1140 1145 1150

Ser Thr Val Gly Ser Leu Asp Phe Gln Cys Asn Val Asn Thr Gly Gln

1155 1160 1165

Cys Asn Cys His Pro Lys Phe Ser Gly Ala Lys Cys Thr Glu Cys Ser

1170 1175 1180

Arg Gly His Trp Asn Tyr Pro Arg Cys Asn Leu Cys Asp Cys Phe Leu

1185 1190 1195 1200

Pro Gly Thr Asp Ala Thr Thr Cys Asp Ser Glu Thr Lys Lys Cys Ser

1205 1210 1215

Cys Ser Asp Gln Thr Gly Gln Cys Thr Cys Lys Val Asn Val Glu Gly

1220 1225 1230

Ile His Cys Asp Arg Cys Arg Pro Gly Lys Phe Gly Leu Asp Ala Lys

1235 1240 1245

Asn Pro Leu Gly Cys Ser Ser Cys Tyr Cys Phe Gly Thr Thr Thr Gln

1250 1255 1260

Cys Ser Glu Ala Lys Gly Leu Ile Arg Thr Trp Val Thr Leu Lys Ala

1265 1270 1275 1280

Glu Gln Thr Ile Leu Pro Leu Val Asp Glu Ala Leu Gln His Thr Thr

1285 1290 1295

Thr Lys Gly Ile Val Phe Gln His Pro Glu Ile Val Ala His Met Asp

1300 1305 1310

Leu Met Arg Glu Asp Leu His Leu Glu Pro Phe Tyr Trp Lys Leu Pro

1315 1320 1325

Glu Gln Phe Glu Gly Lys Lys Leu Met Ala Tyr Gly Gly Lys Leu Lys

1330 1335 1340

Tyr Ala Ile Tyr Phe Glu Ala Arg Glu Glu Thr Gly Phe Ser Thr Tyr

1345 1350 1355 1360

Asn Pro Gln Val Ile Ile Arg Gly Gly Thr Pro Thr His Ala Arg Ile

1365 1370 1375

Ile Val Arg His Met Ala Ala Pro Leu Ile Gly Gln Leu Thr Arg His

1380 1385 1390

Glu Ile Glu Met Thr Glu Lys Glu Trp Lys Tyr Tyr Gly Asp Asp Pro

1395 1400 1405

Arg Val His Arg Thr Val Thr Arg Glu Asp Phe Leu Asp Ile Leu Tyr

1410 1415 1420

Asp Ile His Tyr Ile Leu Ile Lys Ala Thr Tyr Gly Asn Phe Met Arg

1425 1430 1435 1440

Gln Ser Arg Ile Ser Glu Ile Ser Met Glu Val Ala Glu Gln Gly Arg

1445 1450 1455

Gly Thr Thr Met Thr Pro Pro Ala Asp Leu Ile Glu Lys Cys Asp Cys

1460 1465 1470

Pro Leu Gly Tyr Ser Gly Leu Ser Cys Glu Ala Cys Leu Pro Gly Phe

1475 1480 1485

Tyr Arg Leu Arg Ser Gln Pro Gly Gly Arg Thr Pro Gly Pro Thr Leu

1490 1495 1500

Gly Thr Cys Val Pro Cys Gln Cys Asn Gly His Ser Ser Leu Cys Asp

1505 1510 1515 1520

Pro Glu Thr Ser Ile Cys Gln Asn Cys Gln His His Thr Ala Gly Asp

1525 1530 1535

Phe Cys Glu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val Lys Gly Leu

1540 1545 1550

Pro Asn Asp Cys Gln Gln Cys Ala Cys Pro Leu Ile Ser Ser Ser Asn

1555 1560 1565

Asn Phe Ser Pro Ser Cys Val Ala Glu Gly Leu Asp Asp Tyr Arg Cys

1570 1575 1580

Thr Ala Cys Pro Arg Gly Tyr Glu Gly Gln Tyr Cys Glu Arg Cys Ala

1585 1590 1595 1600

Pro Gly Tyr Thr Gly Ser Pro Gly Asn Pro Gly Gly Ser Cys Gln Glu

1605 1610 1615

Cys Glu Cys Asp Pro Tyr Gly Ser Leu Pro Val Pro Cys Asp Pro Val

1620 1625 1630

Thr Gly Phe Cys Thr Cys Arg Pro Gly Ala Thr Gly Arg Lys Cys Asp

1635 1640 1645

Gly Cys Lys His Trp His Ala Arg Glu Gly Trp Glu Cys Val Phe Cys

1650 1655 1660

Gly Asp Glu Cys Thr Gly Leu Leu Leu Gly Asp Leu Ala Arg Leu Glu

1665 1670 1675 1680

Gln Met Val Met Ser Ile Asn Leu Thr Gly Pro Leu Pro Ala Pro Tyr

1685 1690 1695

Lys Met Leu Tyr Gly Leu Glu Asn Met Thr Gln Glu Leu Lys His Leu

1700 1705 1710

Leu Ser Pro Gln Arg Ala Pro Glu Arg Leu Ile Gln Leu Ala Glu Gly

1715 1720 1725

Asn Leu Asn Thr Leu Val Thr Glu Met Asn Glu Leu Leu Thr Arg Ala

1730 1735 1740

Thr Lys Val Thr Ala Asp Gly Glu Gln Thr Gly Gln Asp Ala Glu Arg

1745 1750 1755 1760

Thr Asn Thr Arg Ala Lys Ser Leu Gly Glu Phe Ile Lys Glu Leu Ala

1765 1770 1775

Arg Asp Ala Glu Ala Val Asn Glu Lys Ala Ile Lys Leu Asn Glu Thr

1780 1785 1790

Leu Gly Thr Arg Asp Glu Ala Phe Glu Arg Asn Leu Glu Gly Leu Gln

1795 1800 1805

Lys Glu Ile Asp Gln Met Ile Lys Glu Leu Arg Arg Lys Asn Leu Glu

1810 1815 1820

Thr Gln Lys Glu Ile Ala Glu Asp Glu Leu Val Ala Ala Glu Ala Leu

1825 1830 1835 1840

Leu Lys Lys Val Lys Lys Leu Phe Gly Glu Ser Arg Gly Glu Asn Glu

1845 1850 1855

Glu Met Glu Lys Asp Leu Arg Glu Lys Leu Ala Asp Tyr Lys Asn Lys

1860 1865 1870

Val Asp Asp Ala Trp Asp Leu Leu Arg Glu Ala Thr Asp Lys Ile Arg

1875 1880 1885

Glu Ala Asn Arg Leu Phe Ala Val Asn Gln Lys Asn Met Thr Ala Leu

1890 1895 1900

Glu Lys Lys Lys Glu Ala Val Glu Ser Gly Lys Arg Gln Ile Glu Asn

1905 1910 1915 1920

Thr Leu Lys Glu Gly Asn Asp Ile Leu Asp Glu Ala Asn Arg Leu Ala

1925 1930 1935

Asp Glu Ile Asn Ser Ile Ile Asp Tyr Val Glu Asp Ile Gln Thr Lys

1940 1945 1950

Leu Pro Pro Met Ser Glu Glu Leu Asn Asp Lys Ile Asp Asp Leu Ser

1955 1960 1965

Gln Glu Ile Lys Asp Arg Lys Leu Ala Glu Lys Val Ser Gln Ala Glu

1970 1975 1980

Ser His Ala Ala Gln Leu Asn Asp Ser Ser Ala Val Leu Asp Gly Ile

1985 1990 1995 2000

Leu Asp Glu Ala Lys Asn Ile Ser Phe Asn Ala Thr Ala Ala Phe Lys

2005 2010 2015

Ala Tyr Ser Asn Ile Lys Asp Tyr Ile Asp Glu Ala Glu Lys Val Ala

2020 2025 2030

Lys Glu Ala Lys Asp Leu Ala His Glu Ala Thr Lys Leu Ala Thr Gly

2035 2040 2045

Pro Arg Gly Leu Leu Lys Glu Asp Ala Lys Gly Cys Leu Gln Lys Ser

2050 2055 2060

Phe Arg Ile Leu Asn Glu Ala Lys Lys Leu Ala Asn Asp Val Lys Glu

2065 2070 2075 2080

Asn Glu Asp His Leu Asn Gly Leu Lys Thr Arg Ile Glu Asn Ala Asp

2085 2090 2095

Ala Arg Asn Gly Asp Leu Leu Arg Thr Leu Asn Asp Thr Leu Gly Lys

2100 2105 2110

Leu Ser Ala Ile Pro Asn Asp Thr Ala Ala Lys Leu Gln Ala Val Lys

2115 2120 2125

Asp Lys Ala Arg Gln Ala Asn Asp Thr Ala Lys Asp Val Leu Ala Gln

2130 2135 2140

Ile Thr Glu Leu His Gln Asn Leu Asp Gly Leu Lys Lys Asn Tyr Asn

2145 2150 2155 2160

Lys Leu Ala Asp Ser Val Ala Lys Thr Asn Ala Val Val Lys Asp Pro

2165 2170 2175

Ser Lys Asn Lys Ile Ile Ala Asp Ala Asp Ala Thr Val Lys Asn Leu

2180 2185 2190

Glu Gln Glu Ala Asp Arg Leu Ile Asp Lys Leu Lys Pro Ile Lys Glu

2195 2200 2205

Leu Glu Asp Asn Leu Lys Lys Asn Ile Ser Glu Ile Lys Glu Leu Ile

2210 2215 2220

Asn Gln Ala Arg Lys Gln Ala Asn Ser Ile Lys Val Ser Val Ser Ser

2225 2230 2235 2240

Gly Gly Asp Cys Ile Arg Thr Tyr Lys Pro Glu Ile Lys Lys Gly Ser

2245 2250 2255

Tyr Asn Asn Ile Val Val Asn Val Lys Thr Ala Val Ala Asp Asn Leu

2260 2265 2270

Leu Phe Tyr Leu Gly Ser Ala Lys Phe Ile Asp Phe Leu Ala Ile Glu

2275 2280 2285

Met Arg Lys Gly Lys Val Ser Phe Leu Trp Asp Val Gly Ser Gly Val

2290 2295 2300

Gly Arg Val Glu Tyr Pro Asp Leu Thr Ile Asp Asp Ser Tyr Trp Tyr

2305 2310 2315 2320

Arg Ile Val Ala Ser Arg Thr Gly Arg Asn Gly Thr Ile Ser Val Arg

2325 2330 2335

Ala Leu Asp Gly Pro Lys Ala Ser Ile Val Pro Ser Thr His His Ser

2340 2345 2350

Thr Ser Pro Pro Gly Tyr Thr Ile Leu Asp Val Asp Ala Asn Ala Met

2355 2360 2365

Leu Phe Val Gly Gly Leu Thr Gly Lys Leu Lys Lys Ala Asp Ala Val

2370 2375 2380

Arg Val Ile Thr Phe Thr Gly Cys Met Gly Glu Thr Tyr Phe Asp Asn

2385 2390 2395 2400

Lys Pro Ile Gly Leu Trp Asn Phe Arg Glu Lys Glu Gly Asp Cys Lys

2405 2410 2415

Gly Cys Thr Val Ser Pro Gln Val Glu Asp Ser Glu Gly Thr Ile Gln

2420 2425 2430

Phe Asp Gly Glu Gly Tyr Ala Leu Val Ser Arg Pro Ile Arg Trp Tyr

2435 2440 2445

Pro Asn Ile Ser Thr Val Met Phe Lys Phe Arg Thr Phe Ser Ser Ser

2450 2455 2460

Ala Leu Leu Met Tyr Leu Ala Thr Arg Asp Leu Arg Asp Phe Met Ser

2465 2470 2475 2480

Val Glu Leu Thr Asp Gly His Ile Lys Val Ser Tyr Asp Leu Gly Ser

2485 2490 2495

Gly Met Ala Ser Val Val Ser Asn Gln Asn His Asn Asp Gly Lys Trp

2500 2505 2510

Lys Ser Phe Thr Leu Ser Arg Ile Gln Lys Gln Ala Asn Ile Ser Ile

2515 2520 2525

Val Asp Ile Asp Thr Asn Gln Glu Glu Asn Ile Ala Thr Ser Ser Ser

2530 2535 2540

Gly Asn Asn Phe Gly Leu Asp Leu Lys Ala Asp Asp Lys Ile Tyr Phe

2545 2550 2555 2560

Gly Gly Leu Pro Thr Leu Arg Asn Leu Ser Met Lys Ala Arg Pro Glu

2565 2570 2575

Val Asn Leu Lys Lys Tyr Ser Gly Cys Leu Lys Asp Ile Glu Ile Ser

2580 2585 2590

Arg Thr Pro Tyr Asn Ile Leu Ser Ser Pro Asp Tyr Val Gly Val Thr

2595 2600 2605

Lys Gly Cys Ser Leu Glu Asn Val Tyr Thr Val Ser Phe Pro Lys Pro

2610 2615 2620

Gly Phe Val Glu Leu Ser Pro Val Pro Ile Asp Val Gly Thr Glu Ile

2625 2630 2635 2640

Asn Leu Ser Phe Ser Thr Lys Asn Glu Ser Gly Ile Ile Leu Leu Gly

2645 2650 2655

Ser Gly Gly Thr Pro Ala Pro Pro Arg Arg Lys Arg Arg Gln Thr Gly

2660 2665 2670

Gln Ala Tyr Tyr Ala Ile Leu Leu Asn Arg Gly Arg Leu Glu Val His

2675 2680 2685

Leu Ser Thr Gly Ala Arg Thr Met Arg Lys Ile Val Ile Arg Pro Glu

2690 2695 2700

Pro Asn Leu Phe His Asp Gly Arg Glu His Ser Val His Val Glu Arg

2705 2710 2715 2720

Thr Arg Gly Ile Phe Thr Val Gln Val Asp Glu Asn Arg Arg Tyr Met

2725 2730 2735

Gln Asn Leu Thr Val Glu Gln Pro Ile Glu Val Lys Lys Leu Phe Val

2740 2745 2750

Gly Gly Ala Pro Pro Glu Phe Gln Pro Ser Pro Leu Arg Asn Ile Pro

2755 2760 2765

Pro Phe Glu Gly Cys Ile Trp Asn Leu Val Ile Asn Ser Val Pro Met

2770 2775 2780

Asp Phe Ala Arg Pro Val Ser Phe Lys Asn Ala Asp Ile Gly Arg Cys

2785 2790 2795 2800

Ala His Gln Lys Leu Arg Glu Asp Glu Asp Gly Ala Ala Pro Ala Glu

2805 2810 2815

Ile Val Ile Gln Pro Glu Pro Val Pro Thr Pro Ala Phe Pro Thr Pro

2820 2825 2830

Thr Pro Val Leu Thr His Gly Pro Cys Ala Ala Glu Ser Glu Pro Ala

2835 2840 2845

Leu Leu Ile Gly Ser Lys Gln Phe Gly Leu Ser Arg Asn Ser His Ile

2850 2855 2860

Ala Ile Ala Phe Asp Asp Thr Lys Val Lys Asn Arg Leu Thr Ile Glu

2865 2870 2875 2880

Leu Glu Val Arg Thr Glu Ala Glu Ser Gly Leu Leu Phe Tyr Met Ala

2885 2890 2895

Arg Ile Asn His Ala Asp Phe Ala Thr Val Gln Leu Arg Asn Gly Leu

2900 2905 2910

Pro Tyr Phe Ser Tyr Asp Leu Gly Ser Gly Asp Thr His Thr Met Ile

2915 2920 2925

Pro Thr Lys Ile Asn Asp Gly Gln Trp His Lys Ile Lys Ile Met Arg

2930 2935 2940

Ser Lys Gln Glu Gly Ile Leu Tyr Val Asp Gly Ala Ser Asn Arg Thr

2945 2950 2955 2960

Ile Ser Pro Lys Lys Ala Asp Ile Leu Asp Val Val Gly Met Leu Tyr

2965 2970 2975

Val Gly Gly Leu Pro Ile Asn Tyr Thr Thr Arg Arg Ile Gly Pro Val

2980 2985 2990

Thr Tyr Ser Ile Asp Gly Cys Val Arg Asn Leu His Met Ala Glu Ala

2995 3000 3005

Pro Ala Asp Leu Glu Gln Pro Thr Ser Ser Phe His Val Gly Thr Cys

3010 3015 3020

Phe Ala Asn Ala Gln Arg Gly Thr Tyr Phe Asp Gly Thr Gly Phe Ala

3025 3030 3035 3040

Lys Ala Val Gly Gly Phe Lys Val Gly Leu Asp Leu Leu Val Glu Phe

3045 3050 3055

Glu Phe Arg Thr Thr Thr Thr Thr Gly Val Leu Leu Gly Ile Ser Ser

3060 3065 3070

Gln Lys Met Asp Gly Met Gly Ile Glu Met Ile Asp Glu Lys Leu Met

3075 3080 3085

Phe His Val Asp Asn Gly Ala Gly Arg Phe Thr Ala Val Tyr Asp Ala

3090 3095 3100

Gly Val Pro Gly His Leu Cys Asp Gly Gln Trp His Lys Val Thr Ala

3105 3110 3115 3120

Asn Lys Ile Lys His Arg Ile Glu Leu Thr Val Asp Gly Asn Gln Val

3125 3130 3135

Glu Ala Gln Ser Pro Asn Pro Ala Ser Thr Ser Ala Asp Thr Asn Asp

3140 3145 3150

Pro Val Phe Val Gly Gly Phe Pro Asp Asp Leu Lys Gln Phe Gly Leu

3155 3160 3165

Thr Thr Ser Ile Pro Phe Arg Gly Cys Ile Arg Ser Leu Lys Leu Thr

3170 3175 3180

Lys Gly Thr Gly Lys Pro Leu Glu Val Asn Phe Ala Lys Ala Leu Glu

3185 3190 3195 3200

Leu Arg Gly Val Gln Pro Val Ser Cys Pro Ala Asn

3205 3210

<210> 120

<211> 2587

<212> PRT

<213> 人工序列

<220>

<223> 层粘连蛋白亚基α2同种型X5

<400> 120

Met Ile Ile Leu Glu Gly Asn Asp Leu Ser Ile Ser Thr Ala Gln Asp

1 5 10 15

Glu Val Tyr Leu His Pro Ser Glu Glu His Thr Asn Val Leu Leu Leu

20 25 30

Lys Glu Glu Ser Phe Thr Ile His Gly Thr His Phe Pro Val Arg Arg

35 40 45

Lys Glu Phe Met Thr Val Leu Ala Asn Leu Lys Arg Val Leu Leu Gln

50 55 60

Ile Thr Tyr Ser Phe Gly Met Asp Ala Ile Phe Arg Leu Ser Ser Val

65 70 75 80

Asn Leu Glu Ser Ala Val Ser Tyr Pro Thr Asp Gly Ser Ile Ala Ala

85 90 95

Ala Val Glu Val Cys Gln Cys Pro Pro Gly Tyr Thr Gly Ser Ser Cys

100 105 110

Glu Ser Cys Trp Pro Arg His Arg Arg Val Asn Gly Thr Ile Phe Gly

115 120 125

Gly Ile Cys Glu Pro Cys Gln Cys Phe Gly His Ala Glu Ser Cys Asp

130 135 140

Asp Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro

145 150 155 160

Tyr Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys Gly

165 170 175

Thr Ser Glu Asp Cys Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser

180 185 190

Asn Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile

195 200 205

Cys Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys

210 215 220

Ala Glu Gly Tyr Phe Gly Gln Pro Ser Val Pro Gly Gly Ser Cys Gln

225 230 235 240

Pro Cys Gln Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser Cys

245 250 255

Asp Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly

260 265 270

Arg Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asp

275 280 285

Ala Lys Asn Cys Gln Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe Ser

290 295 300

Glu Val Cys His Ser Gln Thr Gly Gln Cys Glu Cys Arg Ala Asn Val

305 310 315 320

Gln Gly Gln Arg Cys Asp Lys Cys Lys Pro Asn Met Trp Arg Asp Pro

325 330 335

Glu Lys Arg Phe Cys Val Leu Cys Asp Cys Asp Pro Val Gly Ser Val

340 345 350

Ser Pro Gln Cys Asp Ile Thr Gly Arg Cys Val Cys Lys Ser Gly Phe

355 360 365

Val Gly Lys Gln Cys Asn Leu Gly Arg Gln Val His Gln Gln Glu Glu

370 375 380

Gln Pro Arg Arg Ala Gln Arg Val Leu Gly Ser Pro Gln Arg Trp Ala

385 390 395 400

Ile Gly Ser Ser Ser Gly Cys Pro Arg Gly Ala Tyr Arg Ala Pro Ala

405 410 415

Pro Ala Gly Thr Phe Gly Leu Gln Ser Ala Arg Gly Cys Val Pro Cys

420 425 430

Asn Cys Asn Ser Phe Gly Ser Lys Ser Phe Asp Cys Glu Glu Ser Gly

435 440 445

Gln Cys Trp Cys Gln Pro Gly Val Thr Gly Lys Lys Cys Asp Arg Cys

450 455 460

Ala His Gly Tyr Phe Asn Phe Gln Glu Gly Gly Cys Thr Ala Cys Glu

465 470 475 480

Cys Ser His Leu Gly Asn Asn Cys Asp Pro Lys Thr Gly Arg Cys Ile

485 490 495

Cys Pro Pro Asn Thr Ile Gly Glu Lys Cys Ser Lys Cys Ala Pro Asn

500 505 510

Thr Trp Gly His Ser Ile Thr Thr Gly Cys Lys Ala Cys Asn Cys Ser

515 520 525

Thr Val Gly Ser Leu Asp Phe Gln Cys Asn Val Asn Thr Gly Gln Cys

530 535 540

Asn Cys His Pro Lys Phe Ser Gly Ala Lys Cys Thr Glu Cys Ser Arg

545 550 555 560

Gly His Trp Asn Tyr Pro Arg Cys Asn Leu Cys Asp Cys Phe Leu Pro

565 570 575

Gly Thr Asp Ala Thr Thr Cys Asp Ser Glu Thr Lys Lys Cys Ser Cys

580 585 590

Ser Asp Gln Thr Gly Gln Cys Thr Cys Lys Val Asn Val Glu Gly Ile

595 600 605

His Cys Asp Arg Cys Arg Pro Gly Lys Phe Gly Leu Asp Ala Lys Asn

610 615 620

Pro Leu Gly Cys Ser Ser Cys Tyr Cys Phe Gly Thr Thr Thr Gln Cys

625 630 635 640

Ser Glu Ala Lys Gly Leu Ile Arg Thr Trp Val Thr Leu Lys Ala Glu

645 650 655

Gln Thr Ile Leu Pro Leu Val Asp Glu Ala Leu Gln His Thr Thr Thr

660 665 670

Lys Gly Ile Val Phe Gln His Pro Glu Ile Val Ala His Met Asp Leu

675 680 685

Met Arg Glu Asp Leu His Leu Glu Pro Phe Tyr Trp Lys Leu Pro Glu

690 695 700

Gln Phe Glu Gly Lys Lys Leu Met Ala Tyr Gly Gly Lys Leu Lys Tyr

705 710 715 720

Ala Ile Tyr Phe Glu Ala Arg Glu Glu Thr Gly Phe Ser Thr Tyr Asn

725 730 735

Pro Gln Val Ile Ile Arg Gly Gly Thr Pro Thr His Ala Arg Ile Ile

740 745 750

Val Arg His Met Ala Ala Pro Leu Ile Gly Gln Leu Thr Arg His Glu

755 760 765

Ile Glu Met Thr Glu Lys Glu Trp Lys Tyr Tyr Gly Asp Asp Pro Arg

770 775 780

Val His Arg Thr Val Thr Arg Glu Asp Phe Leu Asp Ile Leu Tyr Asp

785 790 795 800

Ile His Tyr Ile Leu Ile Lys Ala Thr Tyr Gly Asn Phe Met Arg Gln

805 810 815

Ser Arg Ile Ser Glu Ile Ser Met Glu Val Ala Glu Gln Gly Arg Gly

820 825 830

Thr Thr Met Thr Pro Pro Ala Asp Leu Ile Glu Lys Cys Asp Cys Pro

835 840 845

Leu Gly Tyr Ser Gly Leu Ser Cys Glu Ala Cys Leu Pro Gly Phe Tyr

850 855 860

Arg Leu Arg Ser Gln Pro Gly Gly Arg Thr Pro Gly Pro Thr Leu Gly

865 870 875 880

Thr Cys Val Pro Cys Gln Cys Asn Gly His Ser Ser Leu Cys Asp Pro

885 890 895

Glu Thr Ser Ile Cys Gln Asn Cys Gln His His Thr Ala Gly Asp Phe

900 905 910

Cys Glu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val Lys Gly Leu Pro

915 920 925

Asn Asp Cys Gln Gln Cys Ala Cys Pro Leu Ile Ser Ser Ser Asn Asn

930 935 940

Phe Ser Pro Ser Cys Val Ala Glu Gly Leu Asp Asp Tyr Arg Cys Thr

945 950 955 960

Ala Cys Pro Arg Gly Tyr Glu Gly Gln Tyr Cys Glu Arg Cys Ala Pro

965 970 975

Gly Tyr Thr Gly Ser Pro Gly Asn Pro Gly Gly Ser Cys Gln Glu Cys

980 985 990

Glu Cys Asp Pro Tyr Gly Ser Leu Pro Val Pro Cys Asp Pro Val Thr

995 1000 1005

Gly Phe Cys Thr Cys Arg Pro Gly Ala Thr Gly Arg Lys Cys Asp Gly

1010 1015 1020

Cys Lys His Trp His Ala Arg Glu Gly Trp Glu Cys Val Phe Cys Gly

1025 1030 1035 1040

Asp Glu Cys Thr Gly Leu Leu Leu Gly Asp Leu Ala Arg Leu Glu Gln

1045 1050 1055

Met Val Met Ser Ile Asn Leu Thr Gly Pro Leu Pro Ala Pro Tyr Lys

1060 1065 1070

Met Leu Tyr Gly Leu Glu Asn Met Thr Gln Glu Leu Lys His Leu Leu

1075 1080 1085

Ser Pro Gln Arg Ala Pro Glu Arg Leu Ile Gln Leu Ala Glu Gly Asn

1090 1095 1100

Leu Asn Thr Leu Val Thr Glu Met Asn Glu Leu Leu Thr Arg Ala Thr

1105 1110 1115 1120

Lys Val Thr Ala Asp Gly Glu Gln Thr Gly Gln Asp Ala Glu Arg Thr

1125 1130 1135

Asn Thr Arg Ala Lys Ser Leu Gly Glu Phe Ile Lys Glu Leu Ala Arg

1140 1145 1150

Asp Ala Glu Ala Val Asn Glu Lys Ala Ile Lys Leu Asn Glu Thr Leu

1155 1160 1165

Gly Thr Arg Asp Glu Ala Phe Glu Arg Asn Leu Glu Gly Leu Gln Lys

1170 1175 1180

Glu Ile Asp Gln Met Ile Lys Glu Leu Arg Arg Lys Asn Leu Glu Thr

1185 1190 1195 1200

Gln Lys Glu Ile Ala Glu Asp Glu Leu Val Ala Ala Glu Ala Leu Leu

1205 1210 1215

Lys Lys Val Lys Lys Leu Phe Gly Glu Ser Arg Gly Glu Asn Glu Glu

1220 1225 1230

Met Glu Lys Asp Leu Arg Glu Lys Leu Ala Asp Tyr Lys Asn Lys Val

1235 1240 1245

Asp Asp Ala Trp Asp Leu Leu Arg Glu Ala Thr Asp Lys Ile Arg Glu

1250 1255 1260

Ala Asn Arg Leu Phe Ala Val Asn Gln Lys Asn Met Thr Ala Leu Glu

1265 1270 1275 1280

Lys Lys Lys Glu Ala Val Glu Ser Gly Lys Arg Gln Ile Glu Asn Thr

1285 1290 1295

Leu Lys Glu Gly Asn Asp Ile Leu Asp Glu Ala Asn Arg Leu Ala Asp

1300 1305 1310

Glu Ile Asn Ser Ile Ile Asp Tyr Val Glu Asp Ile Gln Thr Lys Leu

1315 1320 1325

Pro Pro Met Ser Glu Glu Leu Asn Asp Lys Ile Asp Asp Leu Ser Gln

1330 1335 1340

Glu Ile Lys Asp Arg Lys Leu Ala Glu Lys Val Ser Gln Ala Glu Ser

1345 1350 1355 1360

His Ala Ala Gln Leu Asn Asp Ser Ser Ala Val Leu Asp Gly Ile Leu

1365 1370 1375

Asp Glu Ala Lys Asn Ile Ser Phe Asn Ala Thr Ala Ala Phe Lys Ala

1380 1385 1390

Tyr Ser Asn Ile Lys Asp Tyr Ile Asp Glu Ala Glu Lys Val Ala Lys

1395 1400 1405

Glu Ala Lys Asp Leu Ala His Glu Ala Thr Lys Leu Ala Thr Gly Pro

1410 1415 1420

Arg Gly Leu Leu Lys Glu Asp Ala Lys Gly Cys Leu Gln Lys Ser Phe

1425 1430 1435 1440

Arg Ile Leu Asn Glu Ala Lys Lys Leu Ala Asn Asp Val Lys Glu Asn

1445 1450 1455

Glu Asp His Leu Asn Gly Leu Lys Thr Arg Ile Glu Asn Ala Asp Ala

1460 1465 1470

Arg Asn Gly Asp Leu Leu Arg Thr Leu Asn Asp Thr Leu Gly Lys Leu

1475 1480 1485

Ser Ala Ile Pro Asn Asp Thr Ala Ala Lys Leu Gln Ala Val Lys Asp

1490 1495 1500

Lys Ala Arg Gln Ala Asn Asp Thr Ala Lys Asp Val Leu Ala Gln Ile

1505 1510 1515 1520

Thr Glu Leu His Gln Asn Leu Asp Gly Leu Lys Lys Asn Tyr Asn Lys

1525 1530 1535

Leu Ala Asp Ser Val Ala Lys Thr Asn Ala Val Val Lys Asp Pro Ser

1540 1545 1550

Lys Asn Lys Ile Ile Ala Asp Ala Asp Ala Thr Val Lys Asn Leu Glu

1555 1560 1565

Gln Glu Ala Asp Arg Leu Ile Asp Lys Leu Lys Pro Ile Lys Glu Leu

1570 1575 1580

Glu Asp Asn Leu Lys Lys Asn Ile Ser Glu Ile Lys Glu Leu Ile Asn

1585 1590 1595 1600

Gln Ala Arg Lys Gln Ala Asn Ser Ile Lys Val Ser Val Ser Ser Gly

1605 1610 1615

Gly Asp Cys Ile Arg Thr Tyr Lys Pro Glu Ile Lys Lys Gly Ser Tyr

1620 1625 1630

Asn Asn Ile Val Val Asn Val Lys Thr Ala Val Ala Asp Asn Leu Leu

1635 1640 1645

Phe Tyr Leu Gly Ser Ala Lys Phe Ile Asp Phe Leu Ala Ile Glu Met

1650 1655 1660

Arg Lys Gly Lys Val Ser Phe Leu Trp Asp Val Gly Ser Gly Val Gly

1665 1670 1675 1680

Arg Val Glu Tyr Pro Asp Leu Thr Ile Asp Asp Ser Tyr Trp Tyr Arg

1685 1690 1695

Ile Val Ala Ser Arg Thr Gly Arg Asn Gly Thr Ile Ser Val Arg Ala

1700 1705 1710

Leu Asp Gly Pro Lys Ala Ser Ile Val Pro Ser Thr His His Ser Thr

1715 1720 1725

Ser Pro Pro Gly Tyr Thr Ile Leu Asp Val Asp Ala Asn Ala Met Leu

1730 1735 1740

Phe Val Gly Gly Leu Thr Gly Lys Leu Lys Lys Ala Asp Ala Val Arg

1745 1750 1755 1760

Val Ile Thr Phe Thr Gly Cys Met Gly Glu Thr Tyr Phe Asp Asn Lys

1765 1770 1775

Pro Ile Gly Leu Trp Asn Phe Arg Glu Lys Glu Gly Asp Cys Lys Gly

1780 1785 1790

Cys Thr Val Ser Pro Gln Val Glu Asp Ser Glu Gly Thr Ile Gln Phe

1795 1800 1805

Asp Gly Glu Gly Tyr Ala Leu Val Ser Arg Pro Ile Arg Trp Tyr Pro

1810 1815 1820

Asn Ile Ser Thr Val Met Phe Lys Phe Arg Thr Phe Ser Ser Ser Ala

1825 1830 1835 1840

Leu Leu Met Tyr Leu Ala Thr Arg Asp Leu Arg Asp Phe Met Ser Val

1845 1850 1855

Glu Leu Thr Asp Gly His Ile Lys Val Ser Tyr Asp Leu Gly Ser Gly

1860 1865 1870

Met Ala Ser Val Val Ser Asn Gln Asn His Asn Asp Gly Lys Trp Lys

1875 1880 1885

Ser Phe Thr Leu Ser Arg Ile Gln Lys Gln Ala Asn Ile Ser Ile Val

1890 1895 1900

Asp Ile Asp Thr Asn Gln Glu Glu Asn Ile Ala Thr Ser Ser Ser Gly

1905 1910 1915 1920

Asn Asn Phe Gly Leu Asp Leu Lys Ala Asp Asp Lys Ile Tyr Phe Gly

1925 1930 1935

Gly Leu Pro Thr Leu Arg Asn Leu Ser Met Lys Ala Arg Pro Glu Val

1940 1945 1950

Asn Leu Lys Lys Tyr Ser Gly Cys Leu Lys Asp Ile Glu Ile Ser Arg

1955 1960 1965

Thr Pro Tyr Asn Ile Leu Ser Ser Pro Asp Tyr Val Gly Val Thr Lys

1970 1975 1980

Gly Cys Ser Leu Glu Asn Val Tyr Thr Val Ser Phe Pro Lys Pro Gly

1985 1990 1995 2000

Phe Val Glu Leu Ser Pro Val Pro Ile Asp Val Gly Thr Glu Ile Asn

2005 2010 2015

Leu Ser Phe Ser Thr Lys Asn Glu Ser Gly Ile Ile Leu Leu Gly Ser

2020 2025 2030

Gly Gly Thr Pro Ala Pro Pro Arg Arg Lys Arg Arg Gln Thr Gly Gln

2035 2040 2045

Ala Tyr Tyr Ala Ile Leu Leu Asn Arg Gly Arg Leu Glu Val His Leu

2050 2055 2060

Ser Thr Gly Ala Arg Thr Met Arg Lys Ile Val Ile Arg Pro Glu Pro

2065 2070 2075 2080

Asn Leu Phe His Asp Gly Arg Glu His Ser Val His Val Glu Arg Thr

2085 2090 2095

Arg Gly Ile Phe Thr Val Gln Val Asp Glu Asn Arg Arg Tyr Met Gln

2100 2105 2110

Asn Leu Thr Val Glu Gln Pro Ile Glu Val Lys Lys Leu Phe Val Gly

2115 2120 2125

Gly Ala Pro Pro Glu Phe Gln Pro Ser Pro Leu Arg Asn Ile Pro Pro

2130 2135 2140

Phe Glu Gly Cys Ile Trp Asn Leu Val Ile Asn Ser Val Pro Met Asp

2145 2150 2155 2160

Phe Ala Arg Pro Val Ser Phe Lys Asn Ala Asp Ile Gly Arg Cys Ala

2165 2170 2175

His Gln Lys Leu Arg Glu Asp Glu Asp Gly Ala Ala Pro Ala Glu Ile

2180 2185 2190

Val Ile Gln Pro Glu Pro Val Pro Thr Pro Ala Phe Pro Thr Pro Thr

2195 2200 2205

Pro Val Leu Thr His Gly Pro Cys Ala Ala Glu Ser Glu Pro Ala Leu

2210 2215 2220

Leu Ile Gly Ser Lys Gln Phe Gly Leu Ser Arg Asn Ser His Ile Ala

2225 2230 2235 2240

Ile Ala Phe Asp Asp Thr Lys Val Lys Asn Arg Leu Thr Ile Glu Leu

2245 2250 2255

Glu Val Arg Thr Glu Ala Glu Ser Gly Leu Leu Phe Tyr Met Ala Arg

2260 2265 2270

Ile Asn His Ala Asp Phe Ala Thr Val Gln Leu Arg Asn Gly Leu Pro

2275 2280 2285

Tyr Phe Ser Tyr Asp Leu Gly Ser Gly Asp Thr His Thr Met Ile Pro

2290 2295 2300

Thr Lys Ile Asn Asp Gly Gln Trp His Lys Ile Lys Ile Met Arg Ser

2305 2310 2315 2320

Lys Gln Glu Gly Ile Leu Tyr Val Asp Gly Ala Ser Asn Arg Thr Ile

2325 2330 2335

Ser Pro Lys Lys Ala Asp Ile Leu Asp Val Val Gly Met Leu Tyr Val

2340 2345 2350

Gly Gly Leu Pro Ile Asn Tyr Thr Thr Arg Arg Ile Gly Pro Val Thr

2355 2360 2365

Tyr Ser Ile Asp Gly Cys Val Arg Asn Leu His Met Ala Glu Ala Pro

2370 2375 2380

Ala Asp Leu Glu Gln Pro Thr Ser Ser Phe His Val Gly Thr Cys Phe

2385 2390 2395 2400

Ala Asn Ala Gln Arg Gly Thr Tyr Phe Asp Gly Thr Gly Phe Ala Lys

2405 2410 2415

Ala Val Gly Gly Phe Lys Val Gly Leu Asp Leu Leu Val Glu Phe Glu

2420 2425 2430

Phe Arg Thr Thr Thr Thr Thr Gly Val Leu Leu Gly Ile Ser Ser Gln

2435 2440 2445

Lys Met Asp Gly Met Gly Ile Glu Met Ile Asp Glu Lys Leu Met Phe

2450 2455 2460

His Val Asp Asn Gly Ala Gly Arg Phe Thr Ala Val Tyr Asp Ala Gly

2465 2470 2475 2480

Val Pro Gly His Leu Cys Asp Gly Gln Trp His Lys Val Thr Ala Asn

2485 2490 2495

Lys Ile Lys His Arg Ile Glu Leu Thr Val Asp Gly Asn Gln Val Glu

2500 2505 2510

Ala Gln Ser Pro Asn Pro Ala Ser Thr Ser Ala Asp Thr Asn Asp Pro

2515 2520 2525

Val Phe Val Gly Gly Phe Pro Asp Asp Leu Lys Gln Phe Gly Leu Thr

2530 2535 2540

Thr Ser Ile Pro Phe Arg Gly Cys Ile Arg Ser Leu Lys Leu Thr Lys

2545 2550 2555 2560

Gly Thr Gly Lys Pro Leu Glu Val Asn Phe Ala Lys Ala Leu Glu Leu

2565 2570 2575

Arg Gly Val Gln Pro Val Ser Cys Pro Ala Asn

2580 2585

<210> 121

<211> 1916

<212> PRT

<213> 人工序列

<220>

<223> 层粘连蛋白亚基α2同种型X6

<400> 121

Met Pro Gly Ala Ala Gly Val Leu Leu Leu Leu Leu Leu Ser Gly Gly

1 5 10 15

Leu Gly Gly Val Gln Ala Gln Arg Pro Gln Gln Gln Arg Gln Ser Gln

20 25 30

Ala His Gln Gln Arg Gly Leu Phe Pro Ala Val Leu Asn Leu Ala Ser

35 40 45

Asn Ala Leu Ile Thr Thr Asn Ala Thr Cys Gly Glu Lys Gly Pro Glu

50 55 60

Met Tyr Cys Lys Leu Val Glu His Val Pro Gly Gln Pro Val Arg Asn

65 70 75 80

Pro Gln Cys Arg Ile Cys Asn Gln Asn Ser Ser Asn Pro Asn Gln Arg

85 90 95

His Pro Ile Thr Asn Ala Ile Asp Gly Lys Asn Thr Trp Trp Gln Ser

100 105 110

Pro Ser Ile Lys Asn Gly Ile Glu Tyr His Tyr Val Thr Ile Thr Leu

115 120 125

Asp Leu Gln Gln Val Phe Gln Ile Ala Tyr Val Ile Val Lys Ala Ala

130 135 140

Asn Ser Pro Arg Pro Gly Asn Trp Ile Leu Glu Arg Ser Leu Asp Asp

145 150 155 160

Val Glu Tyr Lys Pro Trp Gln Tyr His Ala Val Thr Asp Thr Glu Cys

165 170 175

Leu Thr Leu Tyr Asn Ile Tyr Pro Arg Thr Gly Pro Pro Ser Tyr Ala

180 185 190

Lys Asp Asp Glu Val Ile Cys Thr Ser Phe Tyr Ser Lys Ile His Pro

195 200 205

Leu Glu Asn Gly Glu Ile His Ile Ser Leu Ile Asn Gly Arg Pro Ser

210 215 220

Ala Asp Asp Pro Ser Pro Glu Leu Leu Glu Phe Thr Ser Ala Arg Tyr

225 230 235 240

Ile Arg Leu Arg Phe Gln Arg Ile Arg Thr Leu Asn Ala Asp Leu Met

245 250 255

Met Phe Ala His Lys Asp Pro Arg Glu Ile Asp Pro Ile Val Thr Arg

260 265 270

Arg Tyr Tyr Tyr Ser Val Lys Asp Ile Ser Val Gly Gly Met Cys Ile

275 280 285

Cys Tyr Gly His Ala Arg Ala Cys Pro Leu Asp Pro Ala Thr Asn Lys

290 295 300

Ser Arg Cys Glu Cys Glu His Asn Thr Cys Gly Asp Ser Cys Asp Gln

305 310 315 320

Cys Cys Pro Gly Phe His Gln Lys Pro Trp Arg Ala Gly Thr Phe Leu

325 330 335

Thr Lys Thr Glu Cys Glu Ala Cys Asn Cys His Gly Lys Ala Glu Glu

340 345 350

Cys Tyr Tyr Asp Glu Asn Val Ala Arg Arg Asn Leu Ser Leu Asn Ile

355 360 365

Arg Gly Lys Tyr Ile Gly Gly Gly Val Cys Ile Asn Cys Thr Gln Asn

370 375 380

Thr Ala Gly Ile Asn Cys Glu Thr Cys Thr Asp Gly Phe Phe Arg Pro

385 390 395 400

Lys Gly Val Ser Pro Asn Tyr Pro Arg Pro Cys Gln Pro Cys His Cys

405 410 415

Asp Pro Ile Gly Ser Leu Asn Glu Val Cys Val Lys Asp Glu Lys His

420 425 430

Ala Arg Arg Gly Leu Ala Pro Gly Ser Cys His Cys Lys Thr Gly Phe

435 440 445

Gly Gly Val Ser Cys Asp Arg Cys Ala Arg Gly Tyr Thr Gly Tyr Pro

450 455 460

Asp Cys Lys Ala Cys Asn Cys Ser Gly Leu Gly Ser Lys Asn Glu Asp

465 470 475 480

Pro Cys Phe Gly Pro Cys Ile Cys Lys Glu Asn Val Glu Gly Gly Asp

485 490 495

Cys Ser Arg Cys Lys Ser Gly Phe Phe Asn Leu Gln Glu Asp Asn Trp

500 505 510

Lys Gly Cys Asp Glu Cys Phe Cys Ser Gly Val Ser Asn Arg Cys Gln

515 520 525

Ser Ser Tyr Trp Thr Tyr Gly Lys Ile Gln Asp Met Ser Gly Trp Tyr

530 535 540

Leu Thr Asp Leu Pro Gly Arg Ile Arg Val Ala Pro Gln Gln Asp Asp

545 550 555 560

Leu Asp Ser Pro Gln Gln Ile Ser Ile Ser Asn Ala Glu Ala Arg Gln

565 570 575

Ala Leu Pro His Ser Tyr Tyr Trp Ser Ala Pro Ala Pro Tyr Leu Gly

580 585 590

Asn Lys Leu Pro Ala Val Gly Gly Gln Leu Thr Phe Thr Ile Ser Tyr

595 600 605

Asp Leu Glu Glu Glu Glu Glu Asp Thr Glu Arg Val Leu Gln Leu Met

610 615 620

Ile Ile Leu Glu Gly Asn Asp Leu Ser Ile Ser Thr Ala Gln Asp Glu

625 630 635 640

Val Tyr Leu His Pro Ser Glu Glu His Thr Asn Val Leu Leu Leu Lys

645 650 655

Glu Glu Ser Phe Thr Ile His Gly Thr His Phe Pro Val Arg Arg Lys

660 665 670

Glu Phe Met Thr Val Leu Ala Asn Leu Lys Arg Val Leu Leu Gln Ile

675 680 685

Thr Tyr Ser Phe Gly Met Asp Ala Ile Phe Arg Leu Ser Ser Val Asn

690 695 700

Leu Glu Ser Ala Val Ser Tyr Pro Thr Asp Gly Ser Ile Ala Ala Ala

705 710 715 720

Val Glu Val Cys Gln Cys Pro Pro Gly Tyr Thr Gly Ser Ser Cys Glu

725 730 735

Ser Cys Trp Pro Arg His Arg Arg Val Asn Gly Thr Ile Phe Gly Gly

740 745 750

Ile Cys Glu Pro Cys Gln Cys Phe Gly His Ala Glu Ser Cys Asp Asp

755 760 765

Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro Tyr

770 775 780

Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys Gly Thr

785 790 795 800

Ser Glu Asp Cys Gln Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser Asn

805 810 815

Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile Cys

820 825 830

Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys Ala

835 840 845

Glu Gly Tyr Phe Gly Gln Pro Ser Val Pro Gly Gly Ser Cys Gln Pro

850 855 860

Cys Gln Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser Cys Asp

865 870 875 880

Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly Arg

885 890 895

Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asp Ala

900 905 910

Lys Asn Cys Gln Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe Ser Glu

915 920 925

Val Cys His Ser Gln Thr Gly Gln Cys Glu Cys Arg Ala Asn Val Gln

930 935 940

Gly Gln Arg Cys Asp Lys Cys Lys Pro Asn Met Trp Arg Asp Pro Glu

945 950 955 960

Lys Arg Phe Cys Val Leu Cys Asp Cys Asp Pro Val Gly Ser Val Ser

965 970 975

Pro Gln Cys Asp Ile Thr Gly Arg Cys Val Cys Lys Ser Gly Phe Val

980 985 990

Gly Lys Gln Cys Asn Leu Gly Arg Gln Val His Gln Gln Glu Glu Gln

995 1000 1005

Pro Arg Arg Ala Gln Arg Val Leu Gly Ser Pro Gln Arg Trp Ala Ile

1010 1015 1020

Gly Ser Ser Ser Gly Cys Pro Arg Gly Ala Tyr Arg Ala Pro Ala Pro

1025 1030 1035 1040

Ala Gly Thr Phe Gly Leu Gln Ser Ala Arg Gly Cys Val Pro Cys Asn

1045 1050 1055

Cys Asn Ser Phe Gly Ser Lys Ser Phe Asp Cys Glu Glu Ser Gly Gln

1060 1065 1070

Cys Trp Cys Gln Pro Gly Val Thr Gly Lys Lys Cys Asp Arg Cys Ala

1075 1080 1085

His Gly Tyr Phe Asn Phe Gln Glu Gly Gly Cys Thr Ala Cys Glu Cys

1090 1095 1100

Ser His Leu Gly Asn Asn Cys Asp Pro Lys Thr Gly Arg Cys Ile Cys

1105 1110 1115 1120

Pro Pro Asn Thr Ile Gly Glu Lys Cys Ser Lys Cys Ala Pro Asn Thr

1125 1130 1135

Trp Gly His Ser Ile Thr Thr Gly Cys Lys Ala Cys Asn Cys Ser Thr

1140 1145 1150

Val Gly Ser Leu Asp Phe Gln Cys Asn Val Asn Thr Gly Gln Cys Asn

1155 1160 1165

Cys His Pro Lys Phe Ser Gly Ala Lys Cys Thr Glu Cys Ser Arg Gly

1170 1175 1180

His Trp Asn Tyr Pro Arg Cys Asn Leu Cys Asp Cys Phe Leu Pro Gly

1185 1190 1195 1200

Thr Asp Ala Thr Thr Cys Asp Ser Glu Thr Lys Lys Cys Ser Cys Ser

1205 1210 1215

Asp Gln Thr Gly Gln Cys Thr Cys Lys Val Asn Val Glu Gly Ile His

1220 1225 1230

Cys Asp Arg Cys Arg Pro Gly Lys Phe Gly Leu Asp Ala Lys Asn Pro

1235 1240 1245

Leu Gly Cys Ser Ser Cys Tyr Cys Phe Gly Thr Thr Thr Gln Cys Ser

1250 1255 1260

Glu Ala Lys Gly Leu Ile Arg Thr Trp Val Thr Leu Lys Ala Glu Gln

1265 1270 1275 1280

Thr Ile Leu Pro Leu Val Asp Glu Ala Leu Gln His Thr Thr Thr Lys

1285 1290 1295

Gly Ile Val Phe Gln His Pro Glu Ile Val Ala His Met Asp Leu Met

1300 1305 1310

Arg Glu Asp Leu His Leu Glu Pro Phe Tyr Trp Lys Leu Pro Glu Gln

1315 1320 1325

Phe Glu Gly Lys Lys Leu Met Ala Tyr Gly Gly Lys Leu Lys Tyr Ala

1330 1335 1340

Ile Tyr Phe Glu Ala Arg Glu Glu Thr Gly Phe Ser Thr Tyr Asn Pro

1345 1350 1355 1360

Gln Val Ile Ile Arg Gly Gly Thr Pro Thr His Ala Arg Ile Ile Val

1365 1370 1375

Arg His Met Ala Ala Pro Leu Ile Gly Gln Leu Thr Arg His Glu Ile

1380 1385 1390

Glu Met Thr Glu Lys Glu Trp Lys Tyr Tyr Gly Asp Asp Pro Arg Val

1395 1400 1405

His Arg Thr Val Thr Arg Glu Asp Phe Leu Asp Ile Leu Tyr Asp Ile

1410 1415 1420

His Tyr Ile Leu Ile Lys Ala Thr Tyr Gly Asn Phe Met Arg Gln Ser

1425 1430 1435 1440

Arg Ile Ser Glu Ile Ser Met Glu Val Ala Glu Gln Gly Arg Gly Thr

1445 1450 1455

Thr Met Thr Pro Pro Ala Asp Leu Ile Glu Lys Cys Asp Cys Pro Leu

1460 1465 1470

Gly Tyr Ser Gly Leu Ser Cys Glu Ala Cys Leu Pro Gly Phe Tyr Arg

1475 1480 1485

Leu Arg Ser Gln Pro Gly Gly Arg Thr Pro Gly Pro Thr Leu Gly Thr

1490 1495 1500

Cys Val Pro Cys Gln Cys Asn Gly His Ser Ser Leu Cys Asp Pro Glu

1505 1510 1515 1520

Thr Ser Ile Cys Gln Asn Cys Gln His His Thr Ala Gly Asp Phe Cys

1525 1530 1535

Glu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val Lys Gly Leu Pro Asn

1540 1545 1550

Asp Cys Gln Gln Cys Ala Cys Pro Leu Ile Ser Ser Ser Asn Asn Phe

1555 1560 1565

Ser Pro Ser Cys Val Ala Glu Gly Leu Asp Asp Tyr Arg Cys Thr Ala

1570 1575 1580

Cys Pro Arg Gly Tyr Glu Gly Gln Tyr Cys Glu Arg Cys Ala Pro Gly

1585 1590 1595 1600

Tyr Thr Gly Ser Pro Gly Asn Pro Gly Gly Ser Cys Gln Glu Cys Glu

1605 1610 1615

Cys Asp Pro Tyr Gly Ser Leu Pro Val Pro Cys Asp Pro Val Thr Gly

1620 1625 1630

Phe Cys Thr Cys Arg Pro Gly Ala Thr Gly Arg Lys Cys Asp Gly Cys

1635 1640 1645

Lys His Trp His Ala Arg Glu Gly Trp Glu Cys Val Phe Cys Gly Asp

1650 1655 1660

Glu Cys Thr Gly Leu Leu Leu Gly Asp Leu Ala Arg Leu Glu Gln Met

1665 1670 1675 1680

Val Met Ser Ile Asn Leu Thr Gly Pro Leu Pro Ala Pro Tyr Lys Met

1685 1690 1695

Leu Tyr Gly Leu Glu Asn Met Thr Gln Glu Leu Lys His Leu Leu Ser

1700 1705 1710

Pro Gln Arg Ala Pro Glu Arg Leu Ile Gln Leu Ala Glu Gly Asn Leu

1715 1720 1725

Asn Thr Leu Val Thr Glu Met Asn Glu Leu Leu Thr Arg Ala Thr Lys

1730 1735 1740

Val Thr Ala Asp Gly Glu Gln Thr Gly Gln Asp Ala Glu Arg Thr Asn

1745 1750 1755 1760

Thr Arg Ala Lys Ser Leu Gly Glu Phe Ile Lys Glu Leu Ala Arg Asp

1765 1770 1775

Ala Glu Ala Val Asn Glu Lys Ala Ile Lys Leu Asn Glu Thr Leu Gly

1780 1785 1790

Thr Arg Asp Glu Ala Phe Glu Arg Asn Leu Glu Gly Leu Gln Lys Glu

1795 1800 1805

Ile Asp Gln Met Ile Lys Glu Leu Arg Arg Lys Asn Leu Glu Thr Gln

1810 1815 1820

Lys Glu Ile Ala Glu Asp Glu Leu Val Ala Ala Glu Ala Leu Leu Lys

1825 1830 1835 1840

Lys Val Lys Lys Leu Phe Gly Glu Ser Arg Gly Glu Asn Glu Glu Met

1845 1850 1855

Glu Lys Asp Leu Arg Glu Lys Leu Ala Asp Tyr Lys Asn Lys Val Asp

1860 1865 1870

Asp Ala Trp Asp Leu Leu Arg Glu Ala Thr Asp Lys Ile Arg Glu Ala

1875 1880 1885

Asn Arg Leu Phe Ala Val Asn Gln Lys Asn Met Thr Ala Leu Glu Gln

1890 1895 1900

Leu Pro Ala Lys Val Ile Lys Thr Asn Gln Ser Ile

1905 1910 1915

<210> 122

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性Piggybac三重突变体

D450N/R372A/K375A/R376A

<400> 122

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Ala Ser Asn Ala Ala Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 123

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G

<400> 123

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Ala Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly

580 585 590

Cys Phe

<210> 124

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L

<400> 124

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ala Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly

580 585 590

Cys Leu

<210> 125

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P

<400> 125

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Met Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ala Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Ala Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 126

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

G325A/N347S/K375A/D450N/S573A/M589V/S592G

<400> 126

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Gly

580 585 590

Cys Phe

<210> 127

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<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac S230N/R277A/N347S/K375A/D450N

<400> 127

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Asn Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Ala Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 128

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac T43I/R372A/K375A/A411T/D450N

<400> 128

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Ile Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Ala Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Thr Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser

580 585 590

Cys Phe

<210> 129

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G

<400> 129

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ala Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr

545 550 555 560

Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Gly

580 585 590

Cys Phe

<210> 130

<211> 594

<212> PRT

<213> 人工序列

<220>

<223> 修饰的高活性PiggyBac

Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G

<400> 130

Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln

1 5 10 15

Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp

20 25 30

His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile

35 40 45

Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu

50 55 60

Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn

65 70 75 80

Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His

85 90 95

Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu

100 105 110

Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile

115 120 125

Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile

130 135 140

Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg

145 150 155 160

Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile

165 170 175

His Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn

180 185 190

His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr

195 200 205

Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu

210 215 220

Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val

225 230 235 240

Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile

245 250 255

Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu

260 265 270

Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro

275 280 285

Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys

290 295 300

Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn

305 310 315 320

Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val

325 330 335

His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile

340 345 350

Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val

355 360 365

Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn

370 375 380

Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro

385 390 395 400

Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu

405 410 415

Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys

420 425 430

Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr

435 440 445

Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg

450 455 460

Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn

465 470 475 480

Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val

485 490 495

Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser

500 505 510

Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu

515 520 525

Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser

530 535 540

Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala

545 550 555 560

Tyr Cys Pro Gly Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys

565 570 575

Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly

580 585 590

Cys Phe

<210> 131

<211> 390

<212> DNA

<213> 人工序列

<220>

<223> MCP N55K

<400> 131

atggcttcaa actttactca gttcgtgctc gtggacaatg gtgggacagg ggatgtgaca 60

gtggctcctt ctaatttcgc taatggggtg gcagagtgga tcagctccaa ctcacggagc 120

caggcctaca aggtgacatg cagcgtcagg cagtctagtg cccagaagag aaagtatacc 180

atcaaggtgg aggtccccaa agtggctacc cagacagtgg gcggagtcga actgcctgtc 240

gccgcttgga ggtcctacct gaacatggag ctcactatcc caattttcgc taccaattct 300

gactgtgaac tcatcgtgaa ggcaatgcag gggctcctca aagacggtaa tcctatccct 360

tccgccatcg ccgctaactc aggtatctac 390

<210> 132

<211> 130

<212> PRT

<213> 人工序列

<220>

<223> MCP N55K

<400> 132

Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr

1 5 10 15

Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu

20 25 30

Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser

35 40 45

Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu

50 55 60

Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val

65 70 75 80

Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe

85 90 95

Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu

100 105 110

Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly

115 120 125

Ile Tyr

130

<210> 133

<211> 162

<212> DNA

<213> 人工序列

<220>

<223> gRNA-MS2(四环)-AAVS1-3间隔区

<400> 133

ggggccacta gggacaggat gttttagagc taggccaaca tgaggatcac ccatgtctgc 60

agggcctagc aagttaaaat aaggctagtc cgttatcaac ttggccaaca tgaggatcac 120

ccatgtctgc agggccaagt ggcaccgagt cggtgctttt tt 162

<210> 134

<211> 162

<212> RNA

<213> 人工序列

<220>

<223> gRNA-MS2(四环)-AAVS1-3间隔区

<400> 134

ggggccacua gggacaggau guuuuagagc uaggccaaca ugaggaucac ccaugucugc 60

agggccuagc aaguuaaaau aaggcuaguc cguuaucaac uuggccaaca ugaggaucac 120

ccaugucugc agggccaagu ggcaccgagu cggugcuuuu uu 162

Claims (21)

1.组合物，所述组合物包含：

a)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；

b)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由转座酶组成；以及

c)包含编码层粘连蛋白-α2蛋白或其功能变体或片段的转基因的核酸构建体，所述层粘连蛋白-α2蛋白优选是SEQ ID NO：74的层粘连蛋白-α2蛋白。

2.根据权利要求1所述的组合物，其中a)和b)的所述第一蛋白和所述第二蛋白融合在一起，任选地通过接头融合在一起。

3.根据权利要求1或2所述的组合物，其中c)的所述核酸构建体包含选自以下的启动子：SEQ ID NO：76的CMV启动子、SEQ ID NO：77的CAG启动子、SEQ ID NO：78的EF1-α启动子、SEQ ID NO：79的SV-40启动子和SEQ ID NO：80的EalbAAT启动子。

4.根据权利要求1至3中任一项所述的组合物，其中c)的所述核酸构建体包含SEQ IDNO：81的剪接受体。

5.根据权利要求1至4中任一项所述的组合物，其中c)的所述核酸构建体包含ploy(A)信号序列和/或隔绝元件，所述ploy(A)信号序列优选地选自SEQ ID NO：83-85，所述隔绝元件优选地选自SEQ ID NO：86-87。

6.根据权利要求1至5中任一项所述的组合物，其中c)的所述核酸构建体的侧翼是反向末端重复序列(ITR)，优选是SEQ ID NO：88和89的5'-ITR和3'-ITR。

7.根据权利要求1至6中任一项所述的组合物，其中c)的所述核酸构建体包含在选自以下的载体中：质粒载体、微环载体、犬骨DNA供体载体、慢病毒载体和逆转录病毒载体。

8.根据权利要求1至7中任一项所述的组合物，其中所述位点特异性DNA结合蛋白是包含Cas蛋白的RNA引导的核酸酶，并且其中所述组合物还包含引导RNA，所述引导RNA包含用于将所述LAMA2转基因整合在细胞基因组的特定位点中的靶核酸序列的互补序列，优选地，所述Cas蛋白是酿脓链球菌(S.pyogenes)Cas 9蛋白。

9.根据权利要求8所述的组合物，其中所述引导RNA包含SEQ ID NO：90至97的任一者。

10.根据权利要求1至9中任一项所述的组合物，其中所述转座酶是修饰的高活性PiggyBac转座酶或睡美人转座酶，优选地是修饰的高活性PiggyBac转座酶，其与未修饰的高活性PiggyBac相比包含一个或多个增加切除活性的氨基酸突变，和与未修饰的高活性PiggyBac相比包含一个或多个降低DNA结合活性的氨基酸突变。

11.根据权利要求10所述的组合物，其中所述高活性PiggyBac转座酶是修饰的高活性PiggyBac转座酶，其包含至少一个选自V34、T43、Y177、M194、R202、S230、R245、R275、R277、G325、S351、N347、R372、K375、R376、E377、E380、A411、D450、T560、S564、S573、M589、S592和F594的氨基酸的突变，优选包含氨基酸Y177、R202、S230、R245、R275、R277、G325、N347、S351、E377、D450、R372和K375、E377、T560、S564、S573、M589、S592、F594的突变，所述位置编号对应于SEQ ID NO：9的未修饰的高活性PiggyBac的氨基酸编号。

12.根据权利要求10或11所述的组合物，其中所述高活性PiggyBac转座酶是修饰的高活性PiggyBac转座酶，其包含至少一个选自M194、R245、R275、R277、G325、R372、K375、R376、E377、E380、D450和S573的氨基酸的突变，优选包含氨基酸D450、R372和R375的突变，所述位置编号对应于SEQ ID NO：9的未修饰的高活性PiggyBac的氨基酸编号。

13.根据权利要求1至12中任一项所述的组合物，其中所述转座酶通过接头在N-末端与所述位点特异性DNA结合蛋白融合，所述接头优选是包括GGS、XTEN或FOKI的肽接头，更优选是SEQ ID NO：53的XTEN。

14.根据权利要求1至13中任一项所述的组合物，其中所述组合物被包装在纳米颗粒内。

15.一种用于将LAMA2转基因整合至细胞基因组内的靶核酸序列中的体外方法，其包括将权利要求1至14中任一项所述的组合物引入细胞中。

16.通过权利要求15所述的方法可获得的工程化细胞，其中所述工程化细胞包含整合在其基因组内的核酸，所述核酸包含编码层粘连蛋白-α2蛋白的转基因，所述转基因的侧翼是用于整合酶和/或转座酶介导的基因插入的操作序列。

17.根据权利要求16所述的工程化细胞，其中所述转基因侧翼的所述操作序列包含SEQID NO：88和89或由SEQ ID NO：88和89组成。

18.根据权利要求16或17所述的工程化细胞，其中inter-ITR的大小为至少300bp。

19.根据权利要求16至18中任一项所述的工程化细胞，其中所述转基因编码全长层粘连蛋白-α2蛋白。

20.药物组合物，其包含权利要求1至14中任一项所定义的组合物或权利要求16至19中任一项所述的工程化细胞，任选地与一种或多种药学上可接受的赋形剂组合。

21.根据权利要求1至14中任一项所述的组合物、根据权利要求14至19中任一项所述的工程化细胞或根据权利要求20所述的药物组合物，用于治疗，特别是用于治疗有此需要的受试者中的分区蛋白缺陷型先天性肌营养不良1A型(MDC1A)。