本发明涉及切割来自HPRT基因的DNA靶序列的大范围核酸酶(meganuclease)变体、编码所述变体的载体、经所述载体修饰的细胞、动物或植物以及所述大范围核酸酶变体及衍生产物用于基因组工程(genome engineering)和基因组疗法的用途。
大范围核酸酶定义为具有大(>14bp)切割位点的序列特异性内切核酸酶,其可向活细胞的特定基因座中递送DNA双链断裂(double-strand break,DSB)(Thierry和Dujon,Nucleic Acids Res.,1992,20,5625-5631)。大范围核酸酶已经用于在培养细胞和植物中刺激其靶序列附近的同源重组(Rouet等,Mol.Cell.Biol.,1994,14,8096-106;Choulika等,Mol.Cell.Biol.,1995,15,1968-73;Donoho等,Mol.Cell.Biol,1998,18,4070-8;Elliott等,Mol.Cell.Biol.,1998,18,93-101;Sargent等,Mol.Cell.Biol.,1997,17,267-77;Puchta等,Proc.Natl.Acad.Sci.USA,1996,93,5055-60;Chiurazzi等,Plant Cell,1996,8,2057-2066),使得大范围核酸酶诱导的重组成为用于基因组工程有效并有力的方法。
大范围核酸酶诱导重组的使用长期受到天然大范围核酸酶种类的限制,当前技术的主要限制是需要事先在目的基因座中引入大范围核酸酶切割位点。因此,进行了大量研究来产生具有定制的特异性的大范围核酸酶。这些蛋白质可以用于切割天然的染色体序列,并展现了将基因组工程用于更广泛应用的新前景。例如,大范围核酸酶可用于在染色体中敲除内源基因或敲入外源基因。其同样也可以用于基因矫正,并原则上用于矫正与单基因疾病相关的突变。
在自然界中,大范围核酸酶基本上由归巢内切核酸酶(HomingEndonuclease,HE)代表,其为可动遗传因子编码的内切核酸酶家族,其功能是在称作归巢的过程中启动DNA双链断裂(DSB)诱导的重组事件(Chevalier和Stoddard,Nucleic Acids Res.,2001,29,3757-74;Kostriken等,Cell;1983,35,167-74;Jacquier和Dujon,Cell,1985,41,383-94)。在细菌、真核生物和古细菌(archea)中已经鉴定了数百种HE(上文引用的Chevalier和Stoddard);然而在选定的基因中发现HE切割位点的可能性非常低。
HE属于四个主要的家族。以涉及催化中心的保守肽基序命名的LAGLIDADG家族是分布最广和最充分表征的一组(上文引用的Chevalier和Stoddard)。目前能够获得七种结构。尽管来自该家族的大部分蛋白质是单体的并展示两种LAGLIDADG基序,但是少数仅具有一个基序,并发生二聚化以切割回文或假回文的靶序列。
尽管LAGLIDADG肽是该家族成员中唯一的保守区,但是这些蛋白质共有非常类似的结构(图1A)。催化核心的侧翼是两个DNA结合结构域,对同二聚体(如I-CreI(Chevalier等,Nat.Struct.Biol.,2001,8,312-6)和I-MsoI(Chevalier等,J.Mol.Biol.,2003,329,253-69))而言具有完全的二重对称性,对单体(如I-SceI(Moure等,J.Mol.Biol.,2003,334,685-95)、I-DmoI(Silva等,J.Mol.Biol.,1999,286,1123-36)或I-AniI(Bolduc等,Genes Dev.,2003,17,2875-88))而言具有假对称性。这两个单体或两个结构域(对于单体蛋白质而言)组成组织在二价阳离子周围的催化核心。就在催化核心上方的两个LAGLIDADG肽也在二聚化界面中起关键作用。DNA结合依赖于位于DNA大沟上的两个鞍形ββαββ折叠。对与其天然靶标结合的I-CreI结构进行的分析显示,在每个单体中,八个残基(Y33、Q38、N30、K28、Q26、Q44、R68和R70)与±3、4、5、6、7、9和10位的七个碱基建立直接相互作用(Jurica等,Mol.Cell.,1998,2,469-76)。另外,一些残基与若干碱基建立由水介导的接触;例如S40、K28和N30与+8和-8位的碱基对建立接触(上文引用的Chevalier等,2003)。其他结构域例如可见于内蛋白(如PI-PfuI(Ichiyanagi等,J.Mol.Biol.,2000,300,889-901)和PI-SceI(Moure等,Nat.Struct.Biol.,2002,9,764-70))中,其蛋白质剪接结构域也参与DNA结合。
通过将N端I-DmoI结构域与I-CreI单体融合产生功能性嵌合的单链人工HE(Epinat等,Nucleic Acids Res.,2003,31,2952-62;Chevalier等,Mol.Cell.,2002,10,895-905;Steuer等,Chembiochem.,2004,5,206-13;国际PCT申请WO 03/078619和WO 2004/031346)已证明了LAGLIDADG蛋白的可塑性:不同的单体或核心结构域可融合到单个蛋白质中,以获得切割新的(非回文)靶序列的新大范围核酸酶。
此外,不同研究组还使用推理性方法来局部改变I-CreI(Seligman等,Genetics,1997,147,1653-64;Sussman等,J.Mol.Biol.,2004,342,31-41;Seligman等,Nucleic Acids Res.,2006年9月13日发表;Arnould等,J.Mol.Biol.,2006,355,443-458及国际PCT申请WO 2006/097853和WO 2006/097784)、I-SceI(Doyon等,J.Am.Chem.Soc.,2006,128,2477-2484)、PI-SceI(Gimble等,J.Mol.Biol.,2003,334,993-1008)和I-MsoI(Ashworth等,Nature,2006,441,656-659)的特异性。
通过将半推理性方法与高通量筛选(High Throughput Screening,HTS;Arnould等(上文引用);国际PCT申请WO 2006/097853和WO 2006/097784)组合而改造出了具有改变的特异性的数百种I-CreI衍生物;诱变了I-CreI的残基Q44、R68和R70或Q44、R68、D75和I77并通过筛选鉴定了在±3到5位(5NNN DNA靶标)中具有改变的特异性的变体集合。
然后,将两种不同的变体(图1B;右上和左下)组合并装配在能切割嵌合靶标的功能性异二聚体内切核酸酶(图1B;右下)中,所述嵌合靶标得自每个变体DNA靶序列的不同一半的融合物(图1B;上文引用的Arnould等;国际PCT申请WO 2006/097854)。有趣的是,新蛋白保持了正确的折叠和稳定性、高活性和窄特异性。
因此,可使用两步策略来定制天然LAGLIDADG大范围核酸酶的特异性。第一步是局部诱变天然LAGLIDADG大范围核酸酶如I-CreI,并通过筛选来鉴定具有改变的特异性的变体集合。第二步是依赖于这些蛋白质的模块性,使用组合方法产生切割选定位点的新的大范围核酸酶(图1B)。
然而,在单个单体或结构域内应用组合方法比在单体之间难得多,因为结合界面的结构非常紧密,并且负责基本上所有碱基特异性相互作用的两个不同ββ发夹不构成独立的亚结构域,而是单个折叠的一部分。例如,在I-CreI DNA结合区的内部部分中,gtc三联体与来自第一发夹的一个残基(Q44)和来自第二发夹的两个残基(R68和R70;见前文引用的Chevalier等,2003的图1B)结合。此外,一系列突变的累积影响最终会破坏正确的折叠。
尽管在结构水平上缺乏明显的模块性,但是本发明人已证实,I-CreI的残基28到40和44到77形成两个可分离的功能性亚结构域,其能够结合归巢内切核酸酶半位点的不同部分。
通过将来自不同单体或核心结构域的两个亚结构域装配在相同的单体内,本发明人已改造出能够切割回文嵌合靶标的功能性归巢内切核酸酶(同二聚体)变体(图1C)。另外,通过装配四个不同的亚结构域形成能够切割非回文嵌合靶标的新异二聚体分子,可以进行更大的组合方法(图1D)。不同的亚结构域可被独立修饰以改造出新的切割特异性,并组合在能够切割来自目的基因的靶标的一个大范围核酸酶变体(同二聚体、异二聚体、单链分子)中。
次黄嘌呤磷酸核糖转移酶(HPRT)基因是位于X染色体上的单拷贝基因,并因此以单拷贝存在(XY细胞)或仅从一个等位基因表达(XX细胞)。例如,可分别在NCBI数据库登录号NC_000086和NC_000023下获得小鼠和人的HPRT基因。两个基因均具有9个外显子(图2),其转录成1289个碱基的mRNA(小鼠;登录号NM_013556)或1331个碱基的mRNA(人;登录号NM_000194),含有来自88到744位(小鼠)或86到742位(人)的HPRT ORF。中国仓鼠(Criteculussp.)mRNA是1301个碱基的序列(登录号J00060.1),其含有来自91到747位的HPRT ORF。
次黄嘌呤磷酸核糖转移酶是催化5-磷酸核糖-1-焦磷酸酯和次黄嘌呤、鸟嘌呤或6-巯基嘌呤转化成相应的5’-单核苷酸和焦磷酸酯的酶。该酶在嘌呤生物合成及中枢神经系统功能中是很重要的。考虑到其生物学功能,HPRT基因可用作基因靶向实验的选择标记。与其他选择标记相比,HPRT具有同时作为阳性及阴性选择标记的优势。此外,HPRT基因中的突变与自毁容貌综合征相关。
小鼠HPRT的基因靶向首先由Thomas,K.R.和M.R.Cappechi(Cell,1987,51,503-512)在来自胚胎的干细胞(ES细胞)中进行。然而,效应非常低(约10-7的转染细胞)。在HPRT基因座处产生双链断裂的能力提供了在该基因座处显著提高同源重组的手段。使用经典的基因靶向,Donoho等(Mol.Cell.Biol.,1998,18,4070-4078)向小鼠HPRT基因中引入了I-SceI大范围核酸酶的切割位点。在第二步中,他们通过用修复基质和I-SceI表达载体进行共转化而可在1%的细胞中诱导基因靶向。
此外,如已对小鼠描述的(van der Lugt等Gene,1991,105,263-267;Selfridge等,Somat.Cell.Mol.Genet.,1992,18,325-336),HPRT可用作基因靶向实验的选择标记。
通过Reid和同事首先提出的双置换基因靶向操作(Proc.Natl.Acad.Sci.USA,1990,87,4299-4303)是基于HPRT选择标记的(Magin等,Gene,1992,122,289-296),以产生具有微小的基因改变的小鼠。该操作基于次黄嘌呤磷酸核糖转移酶(HPRT)小基因在HPRT缺陷型胚胎干细胞中的使用及对HPRT表达进行正选择和负选择的能力。
在第一步中,为使靶标失活,用HPRT小基因代替靶基因座的区域,以HAT(次黄嘌呤/氨基喋呤/胸苷;Littlefield J.W.,Science,1964,145,709-)对HPRT标记的表达进行选择。HAT是次黄嘌呤钠、氨基喋呤和胸苷的混合物。氨基喋呤是有效的叶酸拮抗剂,其抑制二氢叶酸还原酶,阻断核苷从头合成。细胞仅在嘌呤和嘧啶补救途径有活性时才能在HAT中存活。次黄嘌呤是嘌呤补救途径的底物。因此,HPRT突变体不能利用嘌呤补救途径,并对HAT选择敏感。
自毁容貌综合征是作为伴性性状传递的遗传病,其由HPRT的缺少引起,特征是高尿酸血、严重的运动性残疾和自我损害行为。已经鉴定了与自毁容貌疾病或者仅具有完整综合征中一部分的较轻临床表型相关的高度异质的突变集合。目前的基因治疗策略基于回补(complementation)方法,其中随机插入但是有功能的额外基因拷贝提供了突变的内源拷贝的功能。相反,大范围核酸酶诱导的重组将允许在原位精确地矫正突变(图3C),并因此避免目前基因治疗方法遇到的因随机插入转基因而引起的风险(Hacein-Bey-Abina等Science,2003,302,415-419)。
矫正遗传缺陷的最准确方法是使用带有未突变基因拷贝的修复基质(repair matrix),得到突变的回复(图3C)。然而,基因矫正的效率随着突变与DSB之间距离的延长而降低,200bp的距离降低五倍。因此,仅可使用给定的大范围核酸酶矫正其DNA靶标附近的突变。图3D中展示了称为“外显子敲入”的替代方案。在该情况下,可以使用在基因5′部分进行切割的大范围核酸酶将功能性外显子序列敲入有害突变的上游。尽管该方法将转基因置于其常规位置,但是它也引起外显子重复(exons duplication),其长期影响尚待评价。另外,如将天然顺式作用元件置于切割下游的内含子中,则其直接环境将被改变,其正确的功能也有待于探索。然而,该方法具有一个巨大的优点:单个大范围核酸酶可用于许多不同的患者。
本发明人已鉴定了HPRT基因中可被I-CreI变体切割的一系列DNA靶标(图2和19)。图1D中描述的组合方法用于将四组突变装配到具有完全改造特异性的异二聚体归巢内切核酸酶中,以切割来自HPRT基因的DNA靶标。能够切割来自HPRT基因的基因组DNA靶标的这些I-CreI变体可用于在HPRT基因座处进行基因组改造(敲除和敲入),并用于使用HPRT作为选择标记来进行任何基因座处的基因组改造(图3A和3B)。
本发明涉及I-CreI变体或单链衍生物用于在HPRT基因中诱导位点特异性修饰的非治疗目的用途,其中所述I-CreI变体或单链衍生物在位于I-CreI 26到40位和44到77位的LAGLIDADG核心结构域的两个功能性亚结构域之一中含有至少一个替换,并能够切割选自序列SEQ ID NO:1到14的DNA靶序列。
可通过任何公知的体外或体内切割测定法来测量本发明所定义变体的切割活性,例如在国际PCT申请WO 2004/067736、Arnould等(J.Mol.Biol.,2006,355,443-458)、Epinat等(Nucleic Acids Res.,2003,31,2952-2962)和Chames等(Nucleic Acids Res.,2005,33,e178)中所述的那些。例如,通过直接重复重组测定法,使用报告载体在酵母或哺乳动物细胞中测量本发明变体的切割活性。所述报告载体在间插序列内包含报告基因的两个截短的非功能性拷贝(直接重复)和基因组DNA靶序列,所述间插序列克隆到酵母或哺乳动物表达载体中。变体的表达产生能切割基因组DNA靶序列的功能性内切核酸酶。该切割诱导直接重复之间的同源重组,产生功能性报告基因,其表达可通过合适的测定法监测。
-核苷酸如下命名:使用单字母代码命名核苷的碱基:a为腺嘌呤,t为胸腺嘧啶,c为胞嘧啶,g为鸟嘌呤。对于简并核苷酸而言,r表示g或a(嘌呤核苷酸),k表示g或t,s表示g或c,w表示a或t,m表示a或c,y表示t或c(嘧啶核苷酸),d表示g、a或t,v表示g、a或c,b表示g、t或c,h表示a、t或c,n表示g、a、t或c。
-“大范围核酸酶”是指具有14到40pb的双链DNA靶序列的内切核酸酶。所述大范围核酸酶是其中各结构域位于单体上的二聚体酶,或是在单个多肽上包含两个结构域的单体酶。
-“大范围核酸酶结构域”是指这样的区域,其与大范围核酸酶DNA靶标的一半相互作用,并能够与相同大范围核酸酶中与所述DNA靶标的另一半相互作用的另一结构域结合,形成能够切割DNA靶标的功能性大范围核酸酶。
-“功能性变体”是指能够切割DNA靶序列的变体,优选地所述靶标是不被母体大范围核酸酶切割的新靶标。例如,这些变体在接触DNA靶序列的位置或与所述DNA靶标直接或间接相互作用的位置上具有氨基酸改变。
-“具有新特异性的大范围核酸酶变体”是指具有与母体归巢内切核酸酶不同的切割靶标模式的变体。等价且无差异地使用的术语“新特异性”、“经修饰的特异性”、“新切割特异性”、“新底物特异性”是指变体针对DNA靶序列中核苷酸的特异性。
-“I-CreI”是指具有序列SWISSPROT P05725(SEQ ID NO:143)或pdb登录号1g9y(SEQ ID NO:144)的野生型I-CreI。
-“单链大范围核酸酶”、“单链嵌合大范围核酸酶”、“单链内切核酸酶”、“单链大范围核酸酶衍生物”、“单链嵌合大范围核酸酶衍生物”或“单链衍生物”是指包含通过肽间隔区连接的两个LAGLIDADG归巢内切核酸酶结构域或核心结构域的大范围核酸酶。单链大范围核酸酶能够切割嵌合DNA靶序列,所述靶序列包含各母体大范围核酸酶靶序列中不同的一半。
-“亚结构域”是指LAGLIDADG归巢内切核酸酶核心结构域中的区域,其与归巢内切核酸酶DNA靶标半位点的不同部分相互作用。两个不同的亚结构域独立作用,并且一个亚结构域中的突变不改变另一亚结构域的结合和切割特性。因此,两个亚结构域结合归巢内切核酸酶DNA靶标半位点的不同部分。
-“I-CreI位点”是指被I-CreI切割的22到24bp双链DNA序列。I-CreI位点包括野生型(天然)非回文I-CreI归巢位点和衍生的回文序列,如序列5’-t-12c-11a-10a-9a-8a-7c-6g-5t-4c-3g-2t-1a+1c+2g+3a+4c+5g+6t+7t+8t+9t+10g+11a+12,也称为C1221(SEQ ID NO:16;图10)。
-“DNA靶标”、“DNA靶序列”、“靶序列”、“靶位点”、“靶标”、“位点”;“目的位点”;“识别位点”、“识别序列”、“归巢识别位点”、“归巢位点”、“切割位点”是指被LAGLIDADG归巢内切核酸酶(如I-CreI)或者来自I-CreI的变体或单链嵌合大范围核酸酶识别并切割的20到24bp的双链回文、局部回文(假回文)或非回文的多核苷酸序列。这些术语是指要通过内切核酸酶在其中诱导双链断裂(切割)的独特DNA位置,优选地为基因组位置。DNA靶标通过双链多核苷酸中一条链的5′到3′序列来定义,如上文针对C1221所指出的。DNA靶标的切割发生在有义和反义链中分别为+2和-2位的核苷酸处。除非另有说明,否则I-Cre I大范围核酸酶变体对DNA靶标进行切割的位置对应于DNA靶标有义链上的切割位点。
-“嵌合DNA靶标”或“杂合DNA靶标”是指两条母体大范围核酸酶靶序列中不同的一半的融合物。另外,所述靶标的至少一半可包含与至少两个独立的亚结构域结合的核苷酸组合(组合DNA靶标)。
-“同一性”是指两条核酸分子或多肽之间的序列同一性。可通过比较各序列中可为比较目的而排列的位置来测定同一性。当所比较序列中的一个位置被相同的碱基占据时,则这些分子在该位置上是相同的。核酸或氨基酸序列之间的相似性或同一性程度是核酸序列间共有的位置上相同或匹配的核苷酸数量的函数。可使用多种比对算法和/或程序计算两条序列之间的同一性,包括FASTA或BLAST,它们可作为GCG序列分析包(University of Wisconsin,Madison,Wis.)的一部分获得,并可以例如默认设定使用。
-“个体”包括哺乳动物以及其他脊椎动物(例如鸟类、鱼和爬行动物)。本文使用的术语“哺乳动物”是指任何这样的脊椎动物,包括单孔类、有袋类和有胎盘的动物,它们对其幼仔哺乳,并且分娩活的幼仔(真哺乳亚纲(eutharian)或胎盘哺乳动物)或者产卵(后兽亚纲(metatharian)或无胎盘(nonplacental)哺乳动物)。哺乳动物物种的实例包括人和其他灵长类(例如猴、猩猩)、啮齿动物(例如大鼠、小鼠、豚鼠)和其他,例如:牛、猪和马。
-突变是指在多核苷酸(cDNA、基因)或多肽序列中替换、缺失、添加一个或多个核苷酸/氨基酸。所述突变可影响基因的编码序列或其调节序列。其还可影响基因组序列的结构或所编码mRNA的结构/稳定性。
在本发明用途的另一优选实施方案中,所述I-CreI变体或单链衍生物包含接触DNA靶序列或与DNA主链或核苷酸碱基直接或经水分子进行相互作用的其他氨基酸残基的替换;这些I-CreI相互作用残基为本领域所公知。
在根据本发明的用途的另一优选实施方案中,所述I-CreI变体或单链衍生物包含一个或更多额外的替换,所述替换改善所述变体对上文所定义HPRT基因的DNA靶标的结合和/或切割活性;这些替换位于整个I-CreI序列上或仅位于I-CreI的C端的一半中(80到163位)。
优选地,所述额外的替换位于选自以下位置的I-CreI的位置中:2、9、19、42、43、54、66、69、72、81、82、86、90、92、96、100、103、104、105、107、108、109、110、113、120、125、129、130、131、132、135、136、137、140、143、151、154、155、157、158、159、161和162位。
更优选地,所述替换是G19S或G19A突变,其提高I-CreI变体/单链衍生物的切割活性。更优选地,所述突变是G19S突变,其进一步破坏功能性同二聚体的形成。G19S突变被有利地引入异二聚体I-CreI变体的两个单体之一,以获得具有提高的切割活性和增强的切割特异性的大范围核酸酶。
如在本发明中定义的I-CreI变体可以是同二聚体或异二聚体,其得自在I-CreI 26到40位或44到77位中含有至少一个突变的第一单体与第二单体(I-CreI或I-CreI变体)的结合。
在根据本发明的用途的另一优选实施方案中,所述I-CreI变体是异二聚体,其得自在I-CreI中26到40位和/或44到77位含有不同突变的第一单体和第二单体的结合。
更优选地,所述异二聚体由选自以下序列对的第一和第二单体组成:SEQ ID NO:83与97,SEQ ID NO:84与98,SEQ ID NO:85与99,SEQ ID NO:32与52,SEQ ID NO:32和53,SEQ ID NO:32与54,SEQ ID NO:32与55,SEQ ID NO:32与56,SEQ ID NO:32与57,SEQ ID NO:32与58,SEQ ID NO:32与60,SEQ ID NO:32与65,SEQ ID NO:32与66,SEQ ID NO:32与67,SEQ ID NO:32与68,SEQ ID NO:32与69,SEQ ID NO:32与70,SEQ ID NO:32与71,SEQ ID NO:32与72,SEQ ID NO:32与73,SEQ ID NO:32与74,SEQ ID NO:75与56,SEQ ID NO:76与56,SEQ ID NO:77与56,SEQ ID NO:78与56,SEQ ID NO:79与56,SEQ ID NO:80与56,SEQ ID NO:81与56,SEQ ID NO:82与56,SEQ ID NO:86与96,SEQ ID NO:87与100,SEQ ID NO:88与101,SEQ ID NO:89与102,SEQ ID NO:90与103,SEQ ID NO:91与104,SEQ ID NO:92与105,SEQ ID NO:93与106,SEQ ID NO:94与107,SEQ ID NO:95与108,SEQ ID NO:147与148。
如在本发明中定义的I-CreI变体的单链衍生物是包含LAGLIDADG大范围核酸酶的两个单体或两个核心结构域或二者之组合的融合蛋白,其中至少一个单体或核心结构域具有I-CreI变体的序列,所述I-CreI变体在I-CreI的26到40位和/或44到77位中含有至少一个替换,如上文所述。
另外,与序列SEQ ID NO:8和14的±3到5位及±8到10位中的核苷酸具有序列同一性的靶序列至少在人和小鼠HPRT基因中存在。与序列SEQ ID NO:10和11中±3到5位及±8到10位的核苷酸具有序列同一性的靶序列至少在小鼠HPRT基因中存在(没有发现与人HPRT基因具有序列同一性)。与序列SEQ ID NO:9中±3到5位及±8到10位的核苷酸具有序列同一性的靶序列至少在人HPRT基因中存在。
因此,切割SEQ ID NO:6和12中一个DNA靶序列的I-CreI变体能够至少在人、小鼠和中国仓鼠HPRT基因中诱导位点特异性的修饰。另外,切割DNA靶序列SEQ ID NO:9的I-CreI变体能够在中国仓鼠和小鼠HPRT基因中诱导位点特异性的修饰,并且对它们中的一些而言,也能够在人HPRT基因中进行诱导。切割DNA靶序列SEQ IDNO:8的I-CreI变体能够在中国仓鼠HPRT基因中诱导位点特异性修饰,并且对它们中的一些而言,也能够在人和/或小鼠HPRT基因中进行诱导;HPRT基因中修饰的位置对应于基因组DNA切割位点的位置(基因组DNA靶标有义链上的+2位(即,对序列SEQ ID NO:6、12、9和8而言分别为以下位置:101(外显子3)、16(外显子8)、21(外显子6)、150(外显子3))。
切割DNA靶序列SEQ ID NO:7的I-CreI变体能够至少在小鼠和中国仓鼠HPRT基因中(但不在人HPRT基因中的相应位置)诱导位点特异性的修饰。另外,切割DNA靶序列SEQ ID NO:10和11的I-CreI变体能够在中国仓鼠HPRT基因中诱导位点特异性修饰,并且对它们中的一些而言,也能够在小鼠HPRT基因中(但不是在人HPRT基因中的相应位置)进行诱导;HPRT基因中的修饰位置分别对应于106(外显子3)、51(外显子6)和52(外显子6)位。
切割DNA靶序列SEQ ID NO:14的I-CreI变体能够在中国仓鼠HPRT基因中诱导位点特异性修饰,并且对它们中的一些而言,也能够在人HPRT基因中(但不是在小鼠HPRT基因中的相应位置)进行诱导;HPRT基因中的修饰位置对应于68位(外显子9)。
切割DNA靶序列SEQ ID NO:1到5和13的I-CreI变体能够至少在中国仓鼠HPRT基因(但不是在人或小鼠HPRT基因中的相应位置)中诱导位点特异性修饰;HPRT基因中的修饰位置分别对应于从ATG(外显子1)开始的-7、54(外显子2)、93(外显子2)、29(外显子3)、69(外显子3)、93(外显子9)和21位(外显子9)。
各变体的序列通过指定位置处的氨基酸残基来定义。例如,表II的第一个异二聚体变体由第一单体和第二单体组成,所述第一单体在28、30、32、33、38、40、44、68、70、75和77位分别具有K、Q、D、Y、Q、S、N、K、S、R和T,所述第二单体在28、30、32、33、38、40、44、68、70、75和77位分别具有K、N、S、G、C、S、Q、R、R、N和I。所述位置通过参考I-CreI序列SWISSPROT P05725或pdb登录号1g9y来指定;I-CreI在28、30、32、33、38、40、44、68、70、75和77位分别具有K、N、S、Y、Q、S、Q、R、R、D和I。未指出的位置未发生突变,并因此与野生型I-CreI序列一致。
在本发明用途的另一优选实施方案中,所述I-CreI变体或单链衍生物与包含待引入序列的靶向DNA构建体组合,所述待引入序列的侧翼是与所述I-CreI变体或单链衍生物基因组DNA切割位点周围的HPRT基因区域共有同源性的序列,如上所述。
优选使用至少50bp,优选多于100bp,并最优选多于200bp的同源序列。事实上,共有的DNA同源性位于断裂位点上游和下游的侧翼区域中,且待引入的DNA序列应位于两臂之间。待引入的序列包含外源目的基因或序列或者用于失活或缺失HPRT基因或其部分的序列。
另外,内源HPRT基因失活之后,HPRT在对HPRT缺陷型细胞/动物染色体中任何基因座进行的进一步基因靶向操作中可用作阳性选择标记(用HAT选择HPRT标记表达)。
(a)如上述,将I-CreI变体或单链衍生物引入动物的多能前体细胞或胚胎中,以在包含所述I-CreI变体或单链衍生物的DNA识别和切割位点的HPRT基因目的位点处同时或连续诱导双链断裂,
(b)将靶向DNA引入步骤(a)的动物前体细胞或胚胎中,以产生基因组被修饰的动物前体细胞或胚胎(其通过同源重组而修复了目的位点),其中所述靶向DNA包含(1)与切割位点周围区域共有同源性的DNA和(2)在该靶向DNA与染色体DNA之间发生重组后修复该目的位点的DNA,
(a)如上述,将I-CreI变体或单链衍生物引入细胞中,以在包含所述I-CreI变体或单链衍生物的DNA识别和切割位点的HPRT基因目的位点处同时或连续诱导双链断裂,
(b)将靶向DNA引入步骤(a)的细胞中,以产生基因组被修饰的细胞(其通过同源重组而修复了目的位点),其中所述靶向DNA包含(1)与切割位点周围的区域共有同源性的DNA和(2)在该靶向DNA与染色体DNA之间发生重组后修复该目的位点的DNA,
(a)如上述,将I-CreI变体或单链衍生物引入动物的多能前体细胞或胚胎中,以在包含所述I-CreI变体或单链衍生物的DNA识别和切割位点的HPRT基因目的位点处诱导双链断裂,从而产生基因组被修饰的前体细胞或胚胎,所述前体细胞或胚胎具有通过非同源末端接合修复的双链断裂,
(a)如上述,将I-CreI变体或单链衍生物引入细胞中,以在包含所述I-CreI变体或单链衍生物的DNA识别和切割位点的HPRT基因目的位点处诱导双链断裂,从而产生基因组被修饰的HPRT缺陷型细胞,所述细胞具有通过非同源末端接合被修复的双链断裂,和
被修饰的细胞可以是任何目的细胞。对产生转基因/敲除动物而言,所述细胞是多能前体细胞,如本领域所公知的胚胎衍生干细胞(ES细胞)。所述I-CreI变体/单链衍生物可直接提供给细胞,或通过包含编码所述大范围核酸酶的多核苷酸序列并适于在所用细胞中表达的表达载体提供给细胞。
在本发明用途的另一优选实施方案中,所述I-CreI变体或单链衍生物由多核苷酸片段编码。所述多核苷酸可编码同二聚体或异二聚体变体中的一个单体,或编码单链嵌合内切核酸酶的两个结构域/单体。
在一个更优选的实施方案中,将所述多核苷酸片段插入适于在所用细胞中表达的载体中。所述载体有利地包含如上述的靶向DNA构建体。优选地,所述载体包含两个不同的多核苷酸片段,每个片段各编码如上述的异二聚体I-Cre I变体的一个单体。
可在本发明中使用的载体包括但不限于病毒载体、质粒、RNA载体,或者可由染色体、非染色体、半合成或合成的核酸所组成的线性或环形DNA或RNA分子。优选的载体是能够自主复制与之连接的核酸的载体(附加型载体)和/或表达与之连接的核酸的载体(表达载体)。大量的合适载体是本领域技术人员已知的,并可以商品获得。
病毒载体包括逆转录病毒、腺病毒、细小病毒(parvovirus)(例如腺相关病毒)、冠形病毒、负链RNA病毒如正粘病毒(orthomyxovirus)(例如流感病毒)、杆状病毒(例如狂犬病毒和疱疹性口腔炎病毒(vesicular stomatitis virus))、副粘病毒(paramyxovirus)(例如麻疹病毒和仙台病毒(Sendai))、正链RNA病毒如小RNA病毒(picornavirus)和α病毒,以及双链DNA病毒,其包括腺病毒、疱疹病毒(例如1和2型单纯疱疹病毒、EB病毒、巨细胞病毒)和痘病毒(例如牛痘病毒、禽痘病毒和金丝雀痘病毒)。其他病毒包括例如诺瓦克病毒、披膜病毒(togavirus)、黄病毒(flavivirus)、呼肠病毒(reoviruses)、乳多空病毒(papovavirus)、肝DNA病毒(hepadnavirus)和肝炎病毒。逆转录病毒的实例包括:禽白血病-肉瘤、哺乳动物C型、B型病毒、D型病毒、HTLV-BLV组、慢病毒、泡沫病毒(spumavirus)(Coffin,J.M.,Retroviridae:The viruses and their replication,In FundamentalVirology,第三版,B.N.Fields,等编辑,Lippincott-Raven Publishers,Philadelphia,1996)。
载体可包含选择标记,例如:用于真核细胞培养的新霉素磷酸转移酶、组氨醇脱氢酶、二氢叶酸还原酶、潮霉素磷酸转移酶、单纯疱疹病毒胸苷激酶、腺苷脱氨酶、谷氨酰胺合成酶和次黄嘌呤-鸟嘌呤磷酸核糖基转移酶;用于酿酒酵母(S.cerevisiae)的TRP1;大肠杆菌(E.coli)中的四环素、利福平或氨苄青霉素抗性。
优选地,所述载体是表达载体,其中编码本发明变体/单链衍生物的序列置于适当的转录和翻译控制元件的控制下,以允许产生或合成所述变体。因此,所述多核苷酸包含在表达盒中。更具体地,载体包含复制起点、与所述编码多核苷酸有效连接的启动子、核糖体结合位点、RNA剪接位点(使用基因组DNA时)、多腺苷酸化位点和转录终止位点。其还可包含增强子。启动子的选择将取决于多肽在其中表达的细胞。优选地,所述变体是异二聚体,编码各个单体的两多核苷酸包含在一个载体中,所述载体能够驱动两多核苷酸同时表达。合适的启动子包括组织特异性和/或诱导型启动子。诱导型启动子的例子是:由提高的重金属水平诱导的真核金属硫堇(metallothionine)启动子,应答于异丙基-β-D-硫代半乳吡喃糖苷(IPTG)诱导的原核lacZ启动子,和由提高的温度诱导的真核热休克启动子。组织特异性启动子的实例是骨骼肌肌酸激酶、前列腺特异性抗原(PSA)、α-抗胰蛋白酶、人表面活性剂(SP)A和B蛋白、β-酪蛋白和酸性乳清蛋白基因。
本发明的主题还包括如上述的I-CreI变体或单链衍生物用于制备药物中的用途,所述药物用于在有此需要的个体中预防、改善或治疗与HPRT基因中突变相关的遗传性疾病,所述药物通过任何手段施用给所述个体。
在该情况下,如上述I-CreI变体或单链衍生物的使用至少包括以下步骤:(a)在个体的躯体组织中,在包含所述变体的至少一个识别和切割位点的HPRT基因的目的位点处诱导双链切割,和(b)向该个体中引入靶向DNA,其中所述靶向DNA包含(1)与切割位点周围的区域共有同源性的DNA,和(2)在该靶向DNA与染色体DNA之间发生重组后修复目的位点的DNA。在适于将靶向DNA引入目的位点的条件下将靶向DNA引入所述个体。
根据本发明,所述双链切割通过对个体施用所述大范围核酸酶而整体(in toto)诱导,或者通过将所述大范围核酸酶引入从个体中取出的体细胞内并在修饰后放回个体内而进行离体诱导。
在所述用途的一个优选实施方案中,I-CreI变体或单链衍生物与靶向DNA构建体组合,所述靶向DNA构建体包含修复HPRT基因中突变的序列,其侧翼是与所述I-CreI变体或单链衍生物的基因组DNA切割位点周围的HPRT基因区域共有同源性的序列,如上述。
就矫正HPRT基因而言,基因的切割发生在突变附近,优选在突变的500bp范围内(图3C)。靶向构建体包含HPRT基因片段,其具有基因组DNA切割位点侧翼至少200bp的同源序列(最小修复基质)用于修复切割,并含有HPRT基因的正确序列用于修复突变(图3C)。因此,用于基因矫正的靶向构建体包含最小修复基质或由其组成;其优选地为200pb到6000pb,更优选地为1000pb到2000pb。
为了恢复功能基因(图3D),基因的切割发生在突变的上游。优选所述突变是基因序列中第一个已知的突变,从而该基因中所有下游突变可同时被矫正。靶向构建体包含基因组DNA切割位点下游的外显子,它们框内融合(像在cDNA中一样),并在3′具有多聚腺苷酸化位点以终止转录。待引入的序列(外显子敲入构建体)的侧翼是切割位点周围的内含子或外显子序列,从而允许改造基因(外显子敲入基因)转录成为能编码功能性蛋白质的mRNA(图3D)。例如,外显子敲入构建体的侧翼是上游和下游的序列。
在所述组合物的一个优选的实施方案中,其包含靶向DNA构建体,所述构建体包含修复HPRT基因中突变的序列,其侧翼是与所述变体的基因组DNA切割位点共有同源性的序列,如上所述。所述修复突变的序列是具有正确序列的基因片段或者是外显子敲入构建体,如上所述。
本发明的主题还包括产品,其含有上述至少一种I-CreI变体/单链衍生物或编码所述大范围核酸酶的一种表达载体,及上述包含靶向构建体的载体,所述产品作为组合制剂用于在与HPRT基因中突变相关的遗传性疾病的预防或治疗中同时、分别或依次使用。
本发明的主题还包括用于在有此需要的个体中预防、改善或治疗与HPRT基因中突变相关的遗传性疾病的方法,所述方法至少包括通过任何手段对所述个体施用上文所述组合物的步骤。
对治疗的目的而言,以治疗有效量施用I-CreI变体/单链衍生物和可药用赋形剂。如果施用量是有生理意义的,则这样的组合被称作以“治疗有效量”施用。如果一种药剂的存在导致接受者生理状况可检测的改变,则它是有生理意义的。在本文中,如果药剂的存在导致目标疾病的一个或多个症状的严重程度减轻以及对损伤或异常的基因组矫正,则它是有生理意义的。
在本发明用途的一个实施方案中,I-CreI变体/单链衍生物基本上是非免疫原性的,即不引发或几乎不引发不利的免疫应答。根据本发明可使用大量改善或消除这类有害免疫反应的方法。在一个优选的实施方案中,I-CreI变体/单链衍生物基本不含N-甲酰基甲硫氨酸。避免不想要的免疫反应的另一种方式是将大范围核酸酶与聚乙二醇(″PEG″)或聚丙二醇(″PPG″)(优选500到20,000道尔顿的平均分子量(MW))缀合。与PEG或PPG缀合(例如Davis等(US 4,179,337)所述)例如能够提供具有抗病毒活性的非免疫原性的生理活性水溶性内切核酸酶缀合物。Saifer等(US 5,006,333)中还描述了使用聚乙-聚丙二醇共聚物的类似方法。
I-CreI变体或单链衍生物可作为多肽使用或作为编码所述多肽的多核苷酸构建体/载体使用。其通过本领域技术人员公知的任何适用于特定细胞种类的便利手段而(单独地或与至少一种合适的运载体或运载体和/或与靶向DNA组合)在体外、离体或在体内将其引入细胞中。一旦进入细胞内,大范围核酸酶和包含靶向DNA和/或编码大范围核酸酶的核酸的载体(如果存在的话)被细胞从胞质输入或易位至核内的作用位点。
I-CreI变体或单链衍生物(多肽)可有利地与以下结合:脂质体、聚乙烯亚胺(PEI)、和/或膜易位肽(Bonetta,The Scientist,2002,16,38;Ford等,Gene Ther.,2001,8,1-4;Wadia和Dowdy,Curr.Opin.Biotechnol.,2002,13,52-56);在后一种情况下,I-CreI变体/单链衍生物的序列与膜易位肽的序列融合(融合蛋白)。
可通过大量方法(例如注射、直接摄入、射弹轰击、脂质体、电穿孔)将包含靶向DNA和/或编码大范围核酸酶的核酸的载体引入细胞中。可使用表达载体在细胞中稳定或瞬时地表达大范围核酸酶。在真核细胞中进行表达的技术是本领域技术人员公知的(见CurrentProtocols in Human Genetics:第12章“Vectors For Gene Therapy”&第13章“Delivery Systems for Gene Therapy”)。任选地,可优选在重组蛋白中掺入核定位信号以确保其在核内表达。
本发明的主题还包括I-CreI变体或单链衍生物、编码所述变体或单链衍生物的多核苷酸片段、包含所述多核苷酸片段和/或DNA靶向构建体的载体、经上述多核苷酸或载体(优选表达载体)修饰的原核或真核宿主细胞。
(a)构建第一系列I-CreI变体,其在LAGLIDADG核心结构域的第一功能性亚结构域中含有至少一个替换,所述第一功能性亚结构域位于I-CreI的26到40位,
(b)构建第二系列I-CreI变体,其在LAGLIDADG核心结构域的第二功能性亚结构域中具有至少一个替换,所述第二功能性亚结构域位于I-CreI的44到77位,
(c)从步骤(a)的第一系列中选择和/或筛选能够切割突变体I-CreI位点的变体,所述突变体位点中(i)I-CreI位点-10到-8位的核苷酸三联体已被替换为选自cag、att、cct、ttg、gac、atg、ttt、ttc、tgg、gtc、aag、gag的核苷酸三联体,且(ii)+8到+10位的核苷酸三联体已被替换为所述I-CreI位点-10到-8位替换的所述核苷酸三联体的反向互补序列(即:分别为ctg、aat、agg、caa、gtc、cat、aaa、gaa、cca、gac、ctt和ctc),
(d)从来自步骤(b)的第二系列中选择和/或筛选能够切割突变体I-CreI位点的变体,所述突变体位点中(i)I-CreI位点-5到-3位的核苷酸三联体已被替换为选自gac、taa、tca、gtg、gct、tgt、tgg、ctg、ttg、tag和gag的核苷酸三联体,且(ii)+3到+5位的核苷酸三联体已被替换为所述I-CreI位点-5到-3位替换的所述核苷酸三联体的反向互补序列(即:分别为gtc、tta、tga、cac、agc、aca、cca、cag、caa、cta和ctc),
(e)从来自步骤(a)的第一系列中选择和/或筛选能够切割突变体I-CreI位点的变体,所述突变体位点中(i)I-CreI位点+8到+10位的核苷酸三联体已被替换为选自cat、cga、tat、ggg、tac、taa、cag、gca、aca、gaa、tga、atg的核苷酸三联体,且(ii)-10到-8位的核苷酸三联体已被替换为所述I-CreI位点+8到+10位替换的所述核苷酸三联体的反向互补序列(即:分别为atg、tcg、ata、ccc、gta、tta、ctg、tgc、tgt、ttc、tca和cat),
(f)从来自步骤(b)的第二系列中选择和/或筛选能够切割突变体I-CreI位点的变体,所述突变体位点中(i)I-CreI位点+3到+5位的核苷酸三联体已被替换为选自tcc、tat、gtg、gaa、tgg、tac、ttt、aca、agc、gcg、tcc、act、caa和aag的核苷酸三联体,且(ii)-5到-3位的核苷酸三联体已被替换为所述I-CreI位点+3到+5位替换的反向互补序列(即:分别是gga、ata、cac、ttc、cca、gta、aaa、tgt、gct、cgc、gga、agt、ttg和ctt),
可如国际PCT申请WO 2004/067736、Epinat等(Nucleic AcidsRes.,2003,31,2952-2962)、Chames等(Nucleic Acids Res.,2005,33,e178)和Arnould等(J.Mol.Biol.,2006,355,443-458)中所述,通过使用体外或体内切割测定来进行步骤(c)、(d)、(e)、(f)、(g1)、(h2)、(i3)和(j4)中的选择和/或筛选。优选地,如在国际PCT申请WO 2004/067736、Epinat等(Nucleic Acids Res.,2003,31,2952-2962)、Chames等(NucleicAcids Res.,2005,33,e178)和Arnould等(J.Mol.Biol.,2006,355,443-458)中所述,通过重组介导的所述DNA双链断裂的修复在下述条件下在体内进行步骤(c)、(d)、(e)、(f)、(g1)、(h2)、(i3)和/或(j4),在所述条件下,由所述变体产生的突变DNA靶序列中的双链断裂导致阳性选择标记或报告基因的激活或者阴性选择标记或报告基因的失活。
步骤(a)和(b)可包括引入额外的突变,以改善突变体的结合和/或切割特性,尤其是在其他位置接触DNA靶序列或者直接或间接与所述DNA靶标相互作用。可如国际PCT申请WO 2004/067736和Arnould等(J.Mol.Biol.,2006,355,443-458)中所述,通过产生组合文库来进行这些步骤。
本发明的主题还包括在I-CreI的26到40位和/或44到77位含有突变的I-CreI变体,所述变体可用于根据本发明改造出能够切割来自HPRT基因的DNA靶标的变体。具体地,本发明包括如上述改造I-CreI变体的方法的步骤(c)到(f)中定义的I-CreI变体,包括序列SEQID NO:24到47和129到142的变体。本发明还包括如上述改造I-CreI变体的方法的步骤(g3)和(h3)中定义的I-CreI变体,包括序列SEQ IDNO:52到60的变体。
能够切割来自目的基因的DNA靶标的单链嵌合内切核酸酶根据本领域公知的方法衍生自本发明的变体(Epinat等,Nucleic Acids Res.,2003,31,2952-62;Chevalier等,Mol.Cell.,2002,10,895-905;Steuer等,Chembiochem.,2004,5,206-13;国际PCT申请WO 03/078619和WO 2004/031346)。任何这些方法都可用于构建衍生自本发明所述变体的单链嵌合内切核酸酶。
可通过本领域技术人员已知任何方法制备多核苷酸片段,其含有靶向DNA构建体的序列或编码本发明所述I-CreI变体或单链衍生物的序列。例如,通过使用特定引物的聚合酶链式反应从DNA模板扩增它们。优选将编码I-CreI变体或单链衍生物的cDNA的密码子选择成有助于所述蛋白质在期望的表达系统中表达。
通过表达如上所述的多肽来产生本发明所述的I-CreI变体或单链衍生物;优选在适合多肽表达或共表达的条件下在经一种或两种表达载体(仅在变体的情况下)修饰的宿主细胞或转基因动物/植物中表达或共表达(仅在变体的情况下)所述多肽,并从宿主细胞培养物或从转基因动物/植物中回收变体或单链衍生物。
除非另有说明,否则本发明的实施将使用本领域内的细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的常规技术。这些技术在文献中有详尽解释。参阅,例如CurrentProtocols in Molecular Biology(Frederick M.AUSUBEL,2000,Wiley和son Inc,Library of Congress,USA);Molecular Cloning:实验室操作手册,第三版,(Sambrook等,2001,Cold Spring Harbor,New York:冷泉港实验室出版);Oligonucleotide Synthesis(M.J.Gait编辑,1984);Mullis等美国专利号4,683,195;Nucleic Acid Hybridization(B.D.Harries&S.J.Higgins编辑1984);Transcription And Translation(B.D.Hames&S.J.Higgins编辑1984);Culture Of Animal Cells(R.I.Freshney,Alan R.Liss,Inc.,1987);Immobilized Cells And Enzymes(IRL Press,1986);B.Perbal,A Practical Guide To Molecular Cloning(1984);the series,Methods In ENZYMOLOGY(J.Abelson和M.Simon,编辑-in-chief,Academic Press,Inc.,New York),尤其是,第154和155卷(Wu等编辑)和第185卷,″Gene Expression Technology″(D.Goeddel,编辑);Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Calos编辑,1987,Cold Spring Harbor Laboratory);Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker,编辑,Academic Press,London,1987);Handbook OfExperimental Immunology,第I-IV卷(D.M.Weir和C.C.Blackwell,编辑,1986);和Manipulating the Mouse Embryo,(Cold Spring HarborLaboratory Press,Cold Spring Harbor,N.Y.,1986)。
-图1显示了归巢内切核酸酶的模块结构和用于设计定制大范围核酸酶的组合方法。A.与其DNA靶标结合的I-CreI归巢内切核酸酶的三维结构。催化核心被两个αββαββα折叠包围,所述折叠在DNA大沟上形成鞍形相互作用界面。B.可以将与衍生自I-CreI靶序列的不同结合序列(右上和左下)相组合,以获得切割非回文嵌合靶标的异二聚体或单链融合分子(右下)。C.鉴定更小的独立亚基(即在单个单体或αββαββα折叠内的亚基(右上和左下))会允许通过将突变在同一单体内组合来设计新的嵌合分子(右下)。这样的分子会切割回文嵌合靶标(右下)。D.之前两个步骤的组合会允许进行涉及四个不同亚结构域的更大的组合方法。在第一步骤中,可将几个新的大范围核酸酶组合成新的分子(“半-大范围核酸酶”),该分子切割来自想要切割之靶标的回文靶标。然后,这样的“半-大范围核酸酶”的组合可产生切割目的靶标的异二聚体种类。因此,针对各亚结构域鉴定少量新的切割酶(cleaver)会允许设计非常大量的具有定制特异性的新内切核酸酶。
-图2显示了次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因及相应的mRNA。外显子以框显示,并标出了小鼠基因(登录号NC 000086)中各外显子的大小;也标出了与人基因(NC 000023)的大小差异。在外显子上标出了I-CreI变体的切割位点(SEQ ID NO:1到14)。基因下方显示中国仓鼠HPRT mRNA(登录号J00060.1;SEQ ID NO:15)。ORF用灰色框表示。标出了HprCH3靶位点及其序列(SEQ IDNO:4)和位置。
-图3显示了使用切割次黄嘌呤-鸟嘌呤磷酸核糖转移酶(HPRT)基因的大范围核酸酶的四种不同策略。A.基因插入和/或基因失活。用大范围核酸酶切割并与含有目的基因(基因插入)或失活盒(基因失活)的修复基质重组后,会发生基因插入或基因失活,所述目的基因或失活表达盒侧翼是与切割位点周围的序列共有同源性的序列。B.通过非同源末端接合产生的基因失活。用大范围核酸酶切割后,DNA末端发生降解并通过非同源末端接合(Non-Homologous-End-Joining,NHEJ)重新连接,然后发生基因失活。C.基因矫正。突变发生在HPRT基因内。用大范围核酸酶切割并与修复基质重组后,有害突变被矫正。D.外显子序列敲入。突变发生在HPRT基因内。突变的mRNA转录本示于基因下方。在修复基质中,位于切割位点下游的外显子与多聚腺苷酸化位点框内融合(像cDNA中那样),以在3′终止转录。可使用内含子和外显子序列作为同源区。外显子序列敲入产生被改造的基因,其转录为能够编码功能性蛋白质的mRNA。
-图4显示了编码I-CreI N75骨架蛋白的核苷酸序列和用于Ulib4和Ulib5文库构建的简并引物。A.骨架(SEQ ID NO:111)是I-CreIORF,其包含D75N密码子替换、在ATG起始密码子后插入丙氨酸(A)密码子和3′端三个额外的密码子(AAD)。B.引物(SEQ ID NO:112、113、114)。
-图5显示了模式的实例和切割各靶标的突变体数。A.模式分析(profiling)的实例。在酵母中以一系列64种回文靶标对每个新内切核酸酶进行模式分析,所述靶标如图5B排列中,并在±8、±9和±10位与序列C1221(SEQ ID NO:16;图8B)不同。各靶序列以-10、-9、-8三联体(10NNN)命名。例如,GGG对应于tcgggacgtcgtacgacgtcccga靶标(SEQ ID NO:122;图8B)。针对64种靶标对大范围核酸酶测试4次。被I-CreI(D75)、I-CreI N75或10种衍生变体切割的靶标以黑色或灰色点显示。B.切割各靶标的突变体数以及切割的平均强度。各序列以-10、-9、-8三联体(10NNN)命名。切割各靶标的蛋白质数在下方显示,并且灰色程度与在酵母中用这些切割酶获得的平均信号强度成正比。
-图6显示了I-CreI变体在28、30、33、38和/或40位的切割模式。对于在筛选后获得并由28、30、33、38、40、70和75位残基定义的141种I-CreI变体中的每一种而言,用64种靶标在酵母中监测切割,所述靶标从被I-CreI切割的C1221回文靶标通过替换±8到10位的核苷酸衍生而来。靶标用对应于-10、-9和-8位核苷酸的三个字母来命名。例如,GGG对应于tcgggacgtcgtacgacgtcccga靶标(SEQ ID NO:122)。数值(框内的)对应于切割强度,所述强度在扫描滤纸后通过适当的软件评价,而(0)表示没有切割。
-图7显示了与其靶标结合的I-CreI同二聚体上蛋白质和DNA靶标中突变的定位。两组突变(残基44、68和70;残基30、33和38)在左侧的单体上用黑色显示。这两组突变明显在空间上是分离的。然而,并没有不同亚结构域的结构证据。DNA靶位点中的对应区(-5到-3区;-10到-8区)在一个半位点上以灰色显示。
-图8:I-CreI衍生靶标的定义(A和B)及模式分析(C和D)。所有靶标均衍生自被I-CreI野生型切割的回文靶标C1221,并示于A和B的上部。A.第一系列的64种靶标衍生自±5到±3位(灰色框中)的诱变。少数实例示于下文中。显示了与I-CreI残基44、68和70之间的相互作用。B.第二系列的64种靶标衍生自±10到±8位(灰色框中)的诱变。少数实例示于下文中。±8、±9和±10位不与44、68和70位残基接触。C.如图8D中组织靶标。对左图而言,表中的三字母表示-5、-4、-3位的碱基(例如,GGG指tcaaaacggggtaccccgttttga(SEQID NO:115))。对右图而言,三字母表示-10、-9、-8位的碱基(例如,GGG指tcgggacgtcgtacgacgtcccga(SEQ ID NO:122))。D.模式分析。将切割C1221靶标的10种I-CreI变体(包括I-CreI N75(QRR))针对两组64种靶标(左侧的±5到±3,和右侧的±10到±8)进行模式分析。靶标如图8C排列。在两组中均存在C1221靶标(正方形内)。通过对应于在44、68和70位所存在残基的三字母(例如:QRR是Q44、R68、R70)来标识突变体,并且其全部具有额外的D75N突变。
-图9显示了与其靶标结合的I-CreI同二聚体上蛋白质和DNA靶标中突变的定位。两组突变(残基44、68和70;残基28、30、33、38和40)在左侧的单体上用黑色显示。这两组突变明显在空间上是分离的。然而,并没有不同亚结构域的结构证据。DNA靶位点中的对应区(-5到-3区;-10到-8区)在一个半位点上以灰色显示。
-图10显示了HprCH3系列靶标和近缘衍生物。10GAG_P、10CAT_P和5CTT_P(SEQ ID NO:17到19)是发现被I-CreI突变体切割的近缘衍生物。它们与C1221(SEQ ID NO:16)的区别在于加框的基序。C1221、10GAG_P、10CAT_P和5CTT_P是最早被描述为24bp的序列,但是结构数据提示,只有22bp与蛋白质/DNA相互作用相关。然而,在括号中标出了±12位。在HprCH3.2靶标(SEQ ID NO:20)中,靶标中间的atga序列被替换为可见于C1221中的碱基gtac。HprCH3.3(SEQ ID NO:21)是衍生自HprCH3.2左侧部分的回文序列,HprCH3.4(SEQ ID NO:22)是衍生自HprCH3.2右侧部分的回文序列。如图中所示,来自10GAG_P、10CAT_P和5CTT_P的加框的基序可见于HprCH3系列靶标中。
-图11显示了10NNN_P突变体对HprCH3.3的切割。该图展示了用HprCH3.3靶标初步筛选I_CreI的实例。加框的是阳性克隆。G1、H6和H7位置处的阳性突变体的序列分别是KNDTQS/QRRDI(SEQ IDNO:24)、KNTPQS/QRRDI(SEQ ID NO:44)和KNTTQS/QRRDI(SEQ IDNO:45)(与表III的命名法相同)。
-图12显示了组合突变体对HprCH3.4的切割。该图展示了用HprCH3.4靶标初步筛选I-CreI组合突变体的实例。A9和B1位置处的阳性突变体的序列分别是KNTHQS/RYSDN(SEQ ID NO:54)和KNSYQS/RYSNI(SEQ ID NO:60)(与表IV的命名法相同)。
-图13显示了异二聚体组合突变体对HprCH3.2和HprCH3的切割。A.用HprCH3.2靶标再次筛选I-CreI突变体的组合。B.用HprCH3靶标再次筛选I-CreI突变体的相同组合。
-图14展示了HprCH3靶标的切割。将切割HprCH3.4的一系列I-CreI突变体优化并与切割HprCH3.3的突变体共表达。用HprCH3靶标测试切割。圆圈中是展示HprCH3切割提高的突变体。在所示滤纸中,C9对应于异二聚体28R,32S,33S,38Y,40Q,44R,68,70S,75N,77N(SEQ ID NO:65)+33H(SEQ ID NO:32),E6对应于28R,32S33S,38Y,40Q,44R,68A,70S,75H,77Y(SEQ ID NO:66)+33H(SEQ ID NO:32),F3对应于28K,32T,33H,38Q,40S,44K,68Y,70S,75D,77R,92R,96R,107R,132V,140A,143A(SEQ IDNO:74)+33H(SEQ ID NO:32)。H11是原始异二聚体(与切割HprCH3.3的突变体KNSHQS/QRRDI(SEQ ID NO:32)共表达的切割HprCH3.4的突变体KSSQQS/RYSDN(SEQ ID NO:53))。H12是阳性对照。
-图15展示了HprCH3靶标的切割。将切割HprCH3.3的一系列I-CreI突变体优化并与切割HprCH3.4的突变体共表达。用HprCH3靶标测试切割。圆圈中是显示有效切割HprCH3的突变体。在第一个滤纸上,B10对应于异二聚体33H、71R、103I、129A和130G(SEQ IDNO:80)+33T、38Y、44K、68Y、70S、75E和77V(SEQ ID NO:56)。在第二个滤纸上,H3对应于异二聚体2I、33H、81V、86I、110G、131R、135Q、151A和157V(SEQ ID NO:79)+33T、38Y、44K、68Y、70S、75E和77V(SEQ ID NO:56)。H12是阳性对照。
-图19显示了在中国仓鼠(Criteculus griseus)HPRT基因中存在的DNA靶序列及能够切割所述DNA靶标的相应I-CreI变体。DNA靶标(第3列)通过其第一个核苷酸(起点,第1列)和最后一个核苷酸(终点,第2列)显示;位置相对于HPRT mRNA序列(登录号J00060.1)而标出。各异二聚体变体的序列通过第一单体(第4列)和第二单体(第5列)中指定位置处的氨基酸残基来定义。例如,图19的第一个异二聚体变体由在28、30、32、33、38、40、44、68、70、75和77位分别具有K、Q、D、Y、Q、S、N、K、S、R和T的第一单体和在28、30、32、38、40、44、68、70、75和77位分别具有K、N、S、G、C、S、Q、R、R、N和I的第二单体组成。上述位置参考I-CreI序列SWISSPROT P05725或pdb登录号1g9y标出;I-CreI在28、30、32、33、38、40、44、68、70、75和77位分别具有K、N、S、Y、Q、S、Q、R、R、D、I。未标出的位置未突变,并因此对应于野生型I-CreI序列。
-图20展示了哺乳动物细胞中报告系统的设计。在起始密码子下游132bp处被I-SceI切割位点中断的嘌呤霉素抗性基因处于EFIα启动子的控制下(1)。该转基因已在CHO-K1细胞中以单拷贝稳定表达。为了在同一染色体环境中引入大范围核酸酶靶位点,修复基质包含i)无启动子的潮霉素抗性基因、(ii)完整的lacZ表达盒和iii)两条同源序列臂(1.1kb和2.3kb)。已构建了若干修复基质,其区别仅在于中断lacZ基因的识别位点(2)。因此,已生产了非常类似的细胞系:A1细胞系、I-SceI细胞系和I-CreI细胞系。当lacZ修复基质(长度2kb)与表达切割该识别位点的大范围核酸酶的载体一起共转染时,恢复了功能性lacZ基因(3)。大范围核酸酶诱导的重组水平可根据转染后蓝色集落或灶点(foci)的数量来推断。
-图22显示了切割HprCH3DNA靶序列的大范围核酸酶的切割效率。在转染CHO细胞后检测LacZ基因的修复频率,所述细胞含有HprCH3染色体报告系统、修复基质和不同量的大范围核酸酶表达载体(其编码原始经改造的异二聚体(HprCH3.3/HprCH3.4)或其G19S衍生物(HprCH3.3/HprCh3.4G19S或HprCH3.3G19S/HprCh3.4))。
实施例9:通过导致甘氨酸-19替换丝氨酸(G19S)的定点诱变对切割HprCH3靶标的大范围核酸酶的精制
为了验证G19S替换提高I-CreI衍生大范围核酸酶切割活性的能力,将该突变整合进异二聚体HprCH3.3(KNSHQS/QRRDI/42A43L,SEQ ID NO:147)/HprCH3.4(KNTHQS/RYSNN/72T,SEQ ID NO:148)的两种蛋白质的每一种中。如实施例7和8中所述,切割HprCH3靶标的该异二聚体通过随机诱变获得。
为了评价原始及G19S衍生突变体的切割活性,使用CHO细胞中的染色体报告系统(图20)。在该系统中,由CMV启动子驱动的单拷贝LacZ基因被HprCH3位点中断,因此是非功能性的。用HprCH3.3/HprCH3.4的CHO表达质粒和LacZ修复质粒转染细胞系允许通过同源重组恢复功能性LacZ基因。先前已经显示双链断裂可诱导同源重组;因此修复LacZ基因的频率指示了基因组HprCH3靶位点的切割效率。
1)材料和方法
a)定点诱变
为了向HprCH3.3和HprCH3.4编码序列中引入G19S替换,进行扩增I-CreI编码序列5’末端(残基1-24)或3’末端(残基14-167)的两次独立重叠PCR反应。对5’末端和3’末端而言,PCR扩增都使用与载体具有同源性的引物(CCM2For
5’-aagcagagctctctggctaactagagaacccactgcttactggcttatcgaccatggccaataccaaatataacaaagagttcc-3’(SEQ ID NO:149)或CCMRev
5’-tctgatcgattcaagtcagtgtctctctagatagcgagtcggccgccggggaggatttcttcttctcgc-3’:SEQ ID NO:150))和对含有替换突变G19S的14-24位氨基酸的I-CreI编码序列具有特异性的引物(G19SF 5’-gccggctttgtggactctgacggtagcatcatc-3’(SEQ IDNO:151)或G19SR 5’-gatgatgctaccgtcagagtccacaaagccggc-3’(SEQ ID NO:152))进行。
所得PCR产物彼此间具有33bp的同源性。接着使用两个片段中每一个的等分试样和CCM2For或CCMRev引物通过PCR来装配片段。
b)在CHO表达载体中克隆突变体
将经SacI-XbaI消化的PCR产物克隆进质粒pCLS1088中相应的SacI-XbaI位点中(图21),所述质粒是含有I-CreI N75编码序列的CHO gateway表达载体pCDNA6.2(INVITROGEN)。通过测序(MILLEGEN)来验证HprCH3.3-G19S或HprCH3.4-G19S编码序列对I-CreI N75编码序列的替换。
c)哺乳动物细胞中的染色体测定
将带有报告系统的CHO细胞系以每个10cm平皿2×105个细胞的密度接种在完全培养基(Kaighn’s改良F-12培养基(F12-K),补充有2mM L-谷氨酰胺、青霉素(100UI/ml)、链霉素(100μg/ml)、两性霉素B(Fongizone)(0.25μg/ml)(INVITROGEN-LIFE SCIENCE)和10%FBS(SIGMA-ALDRICH CHIMIE))中。第二天,用Polyfect转染试剂(QIAGEN)转染细胞。简言之,将2μglacz修复基质载体与多种量的大范围核酸酶表达载体共转染。在37℃孵育72小时后,在0.5%戊二醛中于4℃下将细胞固定10分钟,在含0.02%NP40的100mM磷酸盐缓冲液中洗涤两次,并用以下染色缓冲液染色(10mM磷酸盐缓冲液、1mM MgCl2、33mM铁氰化钾(K hexacyanoferrate(III))、33mM亚铁氰化钾(K hexacyanoferrate(II))、0.1%(v/v)X-Gal)。在37℃孵育过夜后,在光学显微镜下检查平板,并计数LacZ阳性细胞克隆的数目。LacZ修复的频率表述为LacZ+灶点数除以转染细胞数(5×105),并通过转染效率进行修正。
2)结果
在含有HprCH3靶标的CHO细胞系中使用染色体测定来测试异二聚体的活性,所述异二聚体含有两种原始突变体(HprCH3.3/HprCH3.4)或者与相应原始突变体组合的两种G19S衍生突变体之一(HprCH3.3/HprCh3.4G19S 或HprCH3.3G19S/HprCh3.4)。该染色体测定已经广泛描述于最近的出版物中(Arnould等,J.Mol.Biol.Epub May 10,2007)。简言之,首先建立携带单拷贝转基因的CHO细胞系。所述转基因在I-SceI切割位点上游含有人EF1α启动子(图20,步骤1)。接着,使用I-SceI大范围核酸酶在该基因座处引发DSB诱导的同源重组,并插入带有新大范围核酸酶切割位点的5.5kb盒(图20,步骤2)。该盒含有由CMV启动子驱动的非功能性LacZ开放读码框,以及无启动子的潮霉素标记基因。LacZ基因本身被含有待测试大范围核酸酶切割位点(此处为HprCH3切割位点)的50bp插入所失活。该细胞系继而可用于使用切割HprCH3靶标的改造I-CreI衍生物来评价DSB诱导的基因靶向效率(LacZ修复)(图20,步骤3)。
用修复基质和多种量的表达大范围核酸酶的载体共转染该细胞系。LacZ基因修复的频率从使用最初改造的异二聚体(HprCH3.3/HprCH3.4)时2.0×10-3的最大值提高至使用HprCH3.3-G19S衍生突变体时1.15×10-2的最大值和和使用HprCH3.4-G19S衍生突变体时1.2×10-2的最大值(图22)。
这些发现证实,当仅在其单体之一中存在时,G19S突变本身就能够提高异二聚体的活性。单个G19S替换显示可以提高完全不同的异二聚体切割其他靶标的活性。因此,所述G19S突变的作用类似于“便携式”基序,其自身或与其他突变组合能够提高不同I-CreI衍生物的活性。
然而,当HprCH3.3G19S/HprCH3.4G19S异二聚体与修复基质一起转化时,没有检测到LacZ+灶点,显示了低于6.0×10-6的重组频率。这些发现表明单个G19S替换提高了活性,而异二聚体的每个单体中均有G19S替换则导致活性非常强的降低。
序列表
<110>CELLECTIS
SMITH,Julianne
GRIZOT,Sylvestre
GOUBLE,Agnès
<120>切割来自HPRT基因的DNA靶序列的大范围核酸酶变体及其用途
<130>BLOcpHLP1546-13PCT
<160>152
<170>PatentIn version 3.3
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<212>DNA
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ttacctcacc gctttctcgt gcctcggcgc ctcctctgcg ggcttcctcc tcacaccgct 60
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agtgatgatg aaccaggcta tgacctagat ttattttgta ttcctaatca ctatgtcgag 180
gatttggaaa aggtgtttat tcctcatgga gtgattatgg acaggactga aagacttgcc 240
cgagatgtca tgaaagagat gggaggccat cacattgtgg ccctctgtgt gctgaagggg 300
ggctataaat tctttgctga cctgctggat tacattaaag cactgaatag aaatagtgat 360
agatccattc ccatgactgt agattttatc agactgaaga gctactgtaa tgatcagtca 420
acaggggaca taaaagttat tggtggggat gatctctcaa ctttaactgg aaagaatgtc 480
ttgattgttg aggacataat tgacactggt aaaacaatgc aaactctgct ttccctggtc 540
aagcggtaca acctcaaaat ggttaaggtt gcaagcttgc tggtgaaaag gacctctcga 600
agtgttggat ataggccaga ctttgttgga tttgaaattc cagacaagtt tgttgttgga 660
tatgcccttg actataatga gtacttcagg gatttgaatc atatttgtgt cattagtgaa 720
actgggaaag ccaaatacaa agcctaagat gagagttcaa gttgaatctg caaacacgag 780
gagtcccatt catgttccca gtaaaattac caagcattct agttctgcag ccatctgctt 840
agtacagctt tttgcatgaa ccttctaaga attttatggt tttttatttt tagaaatgtc 900
agttgctgca ttcttaaact ttttatttgc actatgagcc ttcgatagat tgtcgcttac 960
cttgtgagca aagtaaatct cttaaattac cactattaaa aatgctgaga ttgtatctgt 1020
aagaaacatt taaagaggag atatattagt tttttaattg gtattttaat ttttatatat 1080
tcgggaaaga aagctgtgat tgaatattgt taattatacc accatgtgtt tagaacagtg 1140
tgaagcactc agtttcatgt cagtaaccat ctaagaagtt ttgctcagtg gaataaacat 1200
atgttttatc agtgttgcct atattttccc acttggattc tttcagtgag tcattgtcaa 1260
cagttccttt aaatgcaaat aaattctaaa aatttaccac t 1301
<210>16
<211>24
<212>DNA
<213>人工序列
<220>
<223>C1221I-CreI靶标
<400>16
tcaaaacgtc gtacgacgtt ttga 24
<210>17
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GAG_P靶标
<400>17
tcgagacgtc gtacgacgtc tcga 24
<210>18
<211>24
<212>DNA
<213>人工序列
<220>
<223>10CAT_P靶标
<400>18
tccatacgtc gtacgacgtatgga 24
<210>19
<211>24
<212>DNA
<213>人工序列
<220>
<223>5CTT_P靶标
<400>19
tcaaaacctt gtacaaggtt ttga 24
<210>20
<211>22
<212>DNA
<213>人工序列
<220>
<223>HprCH3.2靶标
<400>20
cgagatgtcg tacaagagat gg 22
<210>21
<211>22
<212>DNA
<213>人工序列
<220>
<223>HprCH3.3靶标
<400>21
cgagatgtcg tacgacatct cg 22
<210>22
<211>22
<212>DNA
<213>人工序列
<220>
<223>HprCH3.4靶标
<400>22
ccatctcttg tacaagagat gg 22
<210>23
<211>50
<212>DNA
<213>人工序列
<220>
<223>HprCH3.3靶标寡核苷酸
<400>23
tggcatacaa gtttcgagat gtcgtacgac atctcgacaa tcgtctgtca 50
<210>24
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>24
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Asp
20 25 30
Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>25
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>25
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Glu
20 25 30
Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>26
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>26
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Pro
20 25 30
Ala Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>27
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>27
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Arg
20 25 30
Asp Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>28
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>28
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Cys Lys Phe Lys His Lys Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>29
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>29
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Cys Lys Phe Lys His Ser Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>30
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>30
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Cys Lys Phe Lys His Thr Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>31
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>31
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Gly Lys Phe Lys His Ala Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Ihr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>32
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>32
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>33
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>33
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Pro Lys Phe Lys His His Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>34
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>34
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Pro Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>35
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>35
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Gln Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>36
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>36
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Gln Lys Phe Lys His Tyr Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
IleLys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>37
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>37
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Arg Lys Phe Lys His Gln Leu Tyr Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>38
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>38
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Ser Lys Phe Lys His Asp Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>39
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>39
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Gly Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>40
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>40
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Asn Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>41
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>41
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Ser Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>42
<211>163
<212>PRT
<213>人工的
<220>
<223>切割HprCH3.3的I-CreI变体
<400>42
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Thr Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>43
<211>163
<212>PRT
<213>人工的
<220>
<223>切割HprCH3.3的I-CreI变体
<400>43
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Val Lys Phe Lys His His Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>44
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>44
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
Pro Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>45
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>45
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>46
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>46
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln Ser
20 25 30
Thr Lys Phe Lys His Asn Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>47
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.3的I-CreI变体
<400>47
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Thr Pro Asn Gln Ser
20 25 30
Arg Lys Phe Lys His Gln Leu Arg Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr ValArg Asp Ser Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>48
<211>26
<212>DNA
<213>人工序列
<220>
<223>Gal10F引物
<400>48
gcaactttag tgctgacaca tacagg 26
<210>49
<211>24
<212>DNA
<213>人工序列
<220>
<223>Gal10R引物
<400>49
acaaccttga ttggagactt gacc 24
<210>50
<211>15
<212>DNA
<213>人工序列
<220>
<223>assF引物
<220>
<221>misc_feature
<222>(4)..(6)
<223>n为a、c、g或t
<400>50
ctannnttga ccttt 15
<210>51
<211>15
<212>DNA
<213>人工序列
<220>
<223>assR引物
<220>
<221>misc_feature
<222>(10)..(12)
<223>n为a、c、g或t
<400>51
aaaggtcaan nntag 15
<210>52
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>52
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Ala Asp Ser Gly Ser Val Ser Asn Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>53
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>53
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ser Gln Ser
20 25 30
Gln Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>54
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>54
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>55
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>55
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>56
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>56
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Tyr Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Glu Tyr Val Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>57
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>57
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Arg
20 25 30
Asp Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>58
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>58
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Ser Lys Phe Lys His Asp Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>59
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.4的I-CreI变体
<400>59
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asn Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>60
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>60
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>61
<211>59
<212>DNA
<213>人工序列
<220>
<223>preATGCreFor引物
<400>61
gcataaatta ctatacttct atagacacgc aaacacaaat acacagcggc cttgccacc
<210>62
<211>48
<212>DNA
<213>人工序列
<220>
<223>ICreIpostRev 引物
<400>62
ggctcgagga gctcgtctag aggatcgctc gagttatcag tcggccgc
<210>63
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.4的I-CreI变体
<400>63
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser His Tyr Tyr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>64
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3.4的I-CreI变体
<400>64
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Asn Asp Ser Gly Ser Val Ser Gln Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>65
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>65
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Arg Pro Asn Gln Ser
20 25 30
Ser Lys Phe Lys His Tyr Leu Gln Leu Thr Phe Arg ValThr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Ala Asp Ser Gly Ser Val Ser Asn Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>66
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>66
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Arg Pro Asn Gln Ser
20 25 30
Ser Lys Phe Lys His Tyr Leu Gln Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Ala Asp Ser Gly Ser Val Ser His Tyr Tyr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>67
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>67
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Arg Pro Asn Gln Thr
20 25 30
Ser Lys Phe Lys His Tyr Leu Gln Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asn Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Met Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>68
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>68
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His His Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>69
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>69
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>70
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>70
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Asn Asp Ser Gly Ser Val Ser His Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>71
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>71
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr ValArg Asp Ser Gly Ser Val Ser His Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>72
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>72
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val His Asp Ser Gly Ser Val Ser His Tyr His Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>73
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>73
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val His Asp Ser Gly Ser Val Ser His Tyr His Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Arg Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>74
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>74
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Arg Pro Phe Leu Arg
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Arg Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Val Ala Ala Leu Asn Asp Ser Lys Ala Arg Lys Ala Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>75
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>75
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Cys Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Val Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Arg Ser Glu Lys Lys Lys
145 150 155 160
Ser Pro Pro
<210>76
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>76
Met Asn Thr Lys Tyr Asn Lys Glu Leu Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Ile Gln Ala Asn Leu Val Leu Lys Val Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Gly Pro Ser Glu Lys Lys Lys
145 150 155 160
Pro Ser Pro
<210>77
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>77
Met Tyr Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Val Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Ala Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>78
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>78
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Ser Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Ser Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>79
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>79
Met Ile Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Val Lys Pro Leu His Ile Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Gly Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Arg Ile Ala Ala Gln Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Ala Leu Asp Ser Leu Ser Val Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>80
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>80
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Arg Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Ile Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Ala Gly Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>81
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>81
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Val Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Arg Pro Leu His Asn Phe Leu Thr Arg Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Val Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Arg Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Met Lys Lys
145 150 155 160
Ser Ser Pro
<210>82
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>82
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Leu Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asp Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Arg Gln Ala Asn Met Ala Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Ser Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Arg Lys
145 150 155 160
Ser Ser Pro
<210>83
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>83
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Gln Gln Asp
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Lys Asp Ser Gly Ser Val Ser Arg Tyr Thr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>84
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>84
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asp Gln Ser
20 25 30
Arg Lys Phe Lys His Gln Leu Ser Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Gln Asp Ser Gly Ser Val Ser Ser Tyr Val Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>85
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>85
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Thr Gln Ser
20 25 30
Gly Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Arg Tyr Tyr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>86
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>86
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
Tyr Lys Phe Lys His Trp Leu Ser Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Tyr Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>87
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>87
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Gln Leu Ala Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Lys Asp Ser Gly Ser Val Ser Arg Tyr Thr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>88
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>88
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Ser Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Asn Tyr Asp Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glo Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>89
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>89
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Gly Lys Phe Lys His Thr Leu Ser Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Lys Asp Ser Gly Ser Val Ser His Tyr Phe Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>90
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>90
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Lys Gln Ser
20 25 30
Arg Lys Phe Lys His Gln Leu Ser Leu Thr Phe Asp Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Ser Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>91
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>91
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Gln Leu Thr Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Glu Tyr Tyr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>92
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>92
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Tyr Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Tyr Tyr Val Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>93
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>93
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Tyr Gln Thr
20 25 30
Cys Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Asp Tyr Lys Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>94
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>94
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gly Leu Ser Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Tyr Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>95
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>95
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Ala Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Gln Tyr Glu Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>96
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>96
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Arg Gln Ser
20 25 30
Tyr Lys Phe Lys His Glu Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val His Asp His Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>97
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>97
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Gly Lys Phe Lys His Cys Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>98
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>98
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Gly Leu Ser Leu Thr Phe Thr Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Tyr Tyr Thr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>99
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>99
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Gln Leu Arg Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Glu Asp Ser Gly Ser Val Ser Arg Tyr Lys Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>100
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>100
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Thr Leu Ser Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Asn Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>101
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>101
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Gly Lys Phe Lys His Gln Leu Gln Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Ala Asp Lys Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>102
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>102
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Cys Gln Ser
20 25 30
Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Arg Tyr Gln Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>103
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>103
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Gln Leu Gln Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Lys Asp Ser Gly Ser Val Ser His Tyr Phe Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>104
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>104
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Arg Lys Phe Lys His Thr Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Thr Asp Ser Gly Ser Val Ser Asn Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>105
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>105
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Gly
20 25 30
Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Asn Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr ValTyr Asp Ser Gly Ser Val Ser Arg Tyr Tyr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>106
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>106
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Trp
20 25 30
Thr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>107
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>107
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Ala Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser Asn Tyr Leu Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>108
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割Hpr靶标的I-CreI变体
<400>108
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Cys Lys Phe Lys His Tyr Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>109
<211>49
<212>DNA
<213>人工序列
<220>
<223>10NNN靶标寡核苷酸
<220>
<221>misc_feature
<222>(15)..(17)
<223>n为a、c、g或t
<220>
<221>misc_feature
<222>(32)..(34)
<223>n为a、c、g或t
<400>109
ggcatacaag tttcnnnacg tcgtacgacg tnnngacaat cgtctgtca 49
<210>110
<211>49
<212>DNA
<213>人工序列
<220>
<223>5NNN靶标寡核苷酸
<220>
<221>misc_feature
<222>(20)..(22)
<223>n为a、c、g或t
<220>
<221>misc_feature
<222>(27)..(29)
<223>n为a、c、g或t
<400>110
ggcatacaag tttcaaaacn nngtacnnng ttttgacaat cgtctgtca 49
<210>111
<211>501
<212>DNA
<213>人工序列
<220>
<223>I-CreIN75cDNA
<400>111
atggccaata ccaaatataa caaagagttc ctgctgtacc tggccggctt tgtggacggt 60
gacggtagca tcatcgctca gattaaacca aaccagtctt ataagtttaa acatcagcta 120
agcttgacct ttcaggtgac tcaaaagacc cagcgccgtt ggtttctgga caaactagtg 180
gatgaaattg gcgttggtta cgtacgtgat cgcggatccg tttccaacta catcttaagc 240
gaaatcaagc cgctgcacaa cttcctgact caactgcagc cgtttctgaa actgaaacag 300
aaacaggcaa acctggttct gaaaattatc gaacagctgc cgtctgcaaa agaatccccg 360
gacaaattcc tggaagtttg tacctgggtg gatcagattg cagctctgaa cgattctaag 420
acgcgtaaaa ccacttctga aaccgttcgt gctgtgctgg acagcctgag cgagaagaag 480
aaatcctccc cggcggccga c 501
<210>112
<211>68
<212>DNA
<213>人工序列
<220>
<223>Ulib4rev引物
<400>112
ctgaaaggtc aagcttagmb batgtttaaa cttmbbagac tgmbbtggtt taatctgagc 60
gatgatgc 68
<210>113
<211>68
<212>DNA
<213>人工序列
<220>
<223>Ulib5rev引物
<400>113
ctgaaaggtc aagcttagmb batgtttaaa cttataagac tgmbbtggmb baatctgagc 60
gatgatgc 68
<210>114
<211>18
<212>DNA
<213>人工序列
<220>
<223>Ulib456for引物
<400>114
ctaagcttga cctttcag 18
<210>115
<211>24
<212>DNA
<213>人工序列
<220>
<223>5GGG_P靶标
<400>115
tcaaaacggg gtaccccgtt ttga 24
<210>116
<211>24
<212>DNA
<213>人工序列
<220>
<223>5GGA_P靶标
<400>116
tcaaaacgga gtactccgtt ttga 24
<210>117
<211>24
<212>DNA
<213>人工序列
<220>
<223>5GGT_P靶标
<400>117
tcaaaacggt gtacaccgtt ttga 24
<210>118
<211>24
<212>DNA
<213>人工序列
<220>
<223>5GGC_P DNA靶标
<400>118
tcaaaacggc gtacgccgtt ttga 24
<210>119
<211>24
<212>DNA
<213>人工序列
<220>
<223>5GAG_P DNA靶标
<400>119
tcaaaacgag gtacctcgtt ttga 24
<210>120
<211>24
<212>DNA
<213>人工序列
<220>
<223>5GAA_P DNA靶标
<400>120
tcaaaacgaa gtacttcgtt ttga 24
<210>121
<211>24
<212>DNA
<213>人工序列
<220>
<223>5GAT_P DNA靶标
<400>121
tcaaaacgat gtacatcgtt ttga 24
<210>122
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GGG_P靶标
<400>122
tcgggacgtc gtacgacgtc ccga 24
<210>123
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GGA_PDNA靶标
<400>123
tcggaacgtc gtacgacgtt ccga 24
<210>124
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GGT_P DNA靶标
<400>124
tcggtacgtc gtacgacgta ccga 24
<210>125
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GGC_P DNA靶标
<400>125
tcggcacgtc gtacgacgtg ccga 24
<210>126
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GGA_P DNA靶标
<400>126
tcgagacgtc gtacgacgtc tcga 24
<210>127
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GAA_P DNA靶标
<400>127
tcgaaacgtc gtacgacgtt tcga 24
<210>128
<211>24
<212>DNA
<213>人工序列
<220>
<223>10GAT_P DNA靶标
<400>128
tcgatacgtc gtacgacgta tcga 24
<210>129
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>129
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Ser Gln Ser
20 25 30
Gln Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>130
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>130
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>131
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>131
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Thr Lys Phe Lys His Tyr Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>132
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>132
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Arg
20 25 30
Asp Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>133
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>133
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Ser Lys Phe Lys His Asp Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>134
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>134
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Ala Asp Ser Gly Ser Val Ser Ser Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>135
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>135
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>136
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>136
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>137
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>137
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Asn Asp Ser Gly Ser Val Ser Gln Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>138
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>138
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Ser Gly Ser Val Ser His Tyr Tyr Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>139
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>139
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Glu Tyr Val Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>140
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>140
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asp Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>141
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>141
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Lys Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asn Tyr Arg Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>142
<211>163
<212>PRT
<213>人工序列
<220>
<223>I-CreI变体
<400>142
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Ser Val Ser Asn Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>143
<211>163
<212>PRT
<213>莱茵衣藻
<400>143
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Ala Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Trp Arg Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>144
<211>163
<212>PRT
<213>莱茵衣藻
<400>144
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
Tyr Lys Phe Lys His Gln Leu Ser Leu Thr Phe Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>145
<211>1289
<212>DNA
<213>小鼠
<400>145
ttacctcact gctttccgga gcggtagcac ctcctccgcc ggcttcctcc tcagaccgct 60
ttttgccgcg agccgaccgg tcccgtcatg ccgacccgca gtcccagcgt cgtgattagc 120
gatgatgaac caggttatga cctagatttg ttttgtatac ctaatcatta tgccgaggat 180
ttggaaaaag tgtttattcc tcatggactg attatggaca ggactgaaag acttgctcga 240
gatgtcatga aggagatggg aggccatcac attgtggccc tctgtgtgct caaggggggc 300
tataagttct ttgctgacct gctggattac attaaagcac tgaatagaaa tagtgataga 360
tccattccta tgactgtaga ttttatcaga ctgaagagct actgtaatga tcagtcaacg 420
ggggacataa aagttattgg tggagatgat ctctcaactt taactggaaa gaatgtcttg 480
attgttgaag atataattga cactggtaaa acaatgcaaa ctttgctttc cctggttaag 540
cagtacagcc ccaaaatggt taaggttgca agcttgctgg tgaaaaggac ctctcgaagt 600
gttggataca ggccagactt tgttggattt gaaattccag acaagtttgt tgttggatat 660
gcccttgact ataatgagta cttcaggaat ttgaatcacg tttgtgtcat tagtgaaact 720
ggaaaagcca aatacaaagc ctaagatgag cgcaagttga atctgcaaat acgaggagtc 780
ctgttgatgt tgccagtaaa attagcaggt gttctagtcc tgtggccatc tgcctagtaa 840
agctttttgc atgaaccttc tatgaatgtt actgttttat ttttagaaat gtcagttgct 900
gcgtccccag acttttgatt tgcactatga gcctataggc cagcctaccc tctggtagat 960
tgtcgcttat cttgtaagaa aaacaaatct cttaaattac cacttttaaa taataatact 1020
gagattgtat ctgtaagaag gatttaaaga gaagctatat tagtttttta attggtattt 1080
taatttttat atattcagga gagaaagatg tgattgatat tgttaattta gacgagtctg 1140
aagctctcga tttcctatca gtaacagcat ctaagaggtt ttgctcagtg gaataaacat 1200
gtttcagcag tgttggctgt attttcccac tttcagtaaa tcgttgtcaa cagttccttt 1260
taaatgcaaa taaataaatt ctaaaaatt 1289
<210>146
<211>1331
<212>DNA
<213>人
<400>146
tcttgctgcg cctccgcctc ctcctctgct ccgccaccgg cttcctcctc ctgagcagtc 60
agcccgcgcg ccggccggct ccgttatggc gacccgcagc cctggcgtcg tgattagtga 120
tgatgaacca ggttatgacc ttgatttatt ttgcatacct aatcattatg ctgaggattt 180
ggaaagggtg tttattcctc atggactaat tatggacagg actgaacgtc ttgctcgaga 240
tgtgatgaag gagatgggag gccatcacat tgtagccctc tgtgtgctca aggggggcta 300
taaattcttt gctgacctgc tggattacat caaagcactg aatagaaata gtgatagatc 360
cattcctatg actgtagatt ttatcagact gaagagctat tgtaatgacc agtcaacagg 420
ggacataaaa gtaattggtg gagatgatct ctcaacttta actggaaaga atgtcttgat 480
tgtggaagat ataattgaca ctggcaaaac aatgcagact ttgctttcct tggtcaggca 540
gtataatcca aagatggtca aggtcgcaag cttgctggtg aaaaggaccc cacgaagtgt 600
tggatataag ccagactttg ttggatttga aattccagac aagtttgttg taggatatgc 660
ccttgactat aatgaatact tcagggattt gaatcatgtt tgtgtcatta gtgaaactgg 720
aaaagcaaaa tacaaagcct aagatgagag ttcaagttga gtttggaaac atctggagtc 780
ctattgacat cgccagtaaa attatcaatg ttctagttct gtggccatct gcttagtaga 840
gctttttgca tgtatcttct aagaatttta tctgttttgt actttagaaa tgtcagttgc 900
tgcattccta aactgtttat ttgcactatg agcctataga ctatcagttc cctttgggcg 960
gattgttgtt taacttgtaa atgaaaaaat tctcttaaac cacagcacta ttgagtgaaa 1020
cattgaactc atatctgtaa gaaataaaga gaagatatat tagtttttta attggtattt 1080
taatttttat atatgcagga aagaatagaa gtgattgaat attgttaatt ataccaccgt 1140
gtgttagaaa agtaagaagc agtcaatttt cacatcaaag acagcatcta agaagttttg 1200
ttctgtcctg gaattatttt agtagtgttt cagtaatgtt gactgtattt tccaacttgt 1260
tcaaattatt accagtgaat ctttgtcagc agttcccttt taaatgcaaa tcaataaatt 1320
cccaaaaatt t 1331
<210>147
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>147
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Ser
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Ala Leu Gln Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Arg Asp Arg Gly Ser Val Ser Asp Tyr Ile Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>148
<211>163
<212>PRT
<213>人工序列
<220>
<223>切割HprCH3的I-CreI变体
<400>148
Met Asn Thr Lys Tyr Asn Lys Glu Phe Leu Leu Tyr Leu Ala Gly Phe
1 5 10 15
Val Asp Gly Asp Gly Ser Ile Ile Ala Gln Ile Lys Pro Asn Gln Thr
20 25 30
His Lys Phe Lys His Gln Leu Ser Leu Thr Phe Arg Val Thr Gln Lys
35 40 45
Thr Gln Arg Arg Trp Phe Leu Asp Lys Leu Val Asp Glu Ile Gly Val
50 55 60
Gly Tyr Val Tyr Asp Ser Gly Thr Val Ser Asn Tyr Asn Leu Ser Glu
65 70 75 80
Ile Lys Pro Leu His Asn Phe Leu Thr Gln Leu Gln Pro Phe Leu Lys
85 90 95
Leu Lys Gln Lys Gln Ala Asn Leu Val Leu Lys Ile Ile Glu Gln Leu
100 105 110
Pro Ser Ala Lys Glu Ser Pro Asp Lys Phe Leu Glu Val Cys Thr Trp
115 120 125
Val Asp Gln Ile Ala Ala Leu Asn Asp Ser Lys Thr Arg Lys Thr Thr
130 135 140
Ser Glu Thr Val Arg Ala Val Leu Asp Ser Leu Ser Glu Lys Lys Lys
145 150 155 160
Ser Ser Pro
<210>149
<211>84
<212>DNA
<213>人工序列
<220>
<223>CCM2For引物
<400>149
aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg accatggcca 60
ataccaaata taacaaagag ttcc 84
<210>150
<211>69
<212>DNA
<213>人工序列
<220>
<223>CCMRev引物
<400>150
tctgatcgat tcaagtcagt gtctctctag atagcgagtc ggccgccggg gaggatttct 60
tcttctcgc 69
<210>151
<211>33
<212>DNA
<213>人工序列
<220>
<223>G19SF引物
<400>151
gccggctttg tggactctga cggtagcatc atc 33
<210>152
<211>33
<212>DNA
<213>人工序列
<220>
<223>G19SR引物
<400>152
gatgatgcta ccgtcagagt ccacaaagcc ggc 33