CN107384920A - 一套基于化脓性链球菌的碱基编辑系统及其在基因编辑中的应用 - Google Patents

一套基于化脓性链球菌的碱基编辑系统及其在基因编辑中的应用 Download PDF

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CN107384920A
CN107384920A CN201710326650.9A CN201710326650A CN107384920A CN 107384920 A CN107384920 A CN 107384920A CN 201710326650 A CN201710326650 A CN 201710326650A CN 107384920 A CN107384920 A CN 107384920A
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黄军就
松阳洲
梁普平
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Microlight Gene Suzhou Co ltd
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Abstract

本发明公开了一套基于化脓性链球菌Cas9的精确的碱基编辑系统及其在哺乳动物细胞和/或胚胎基因编辑中的应用。所述碱基编辑系统由rAPOBEC1:Cas9:UGI表达载体和gRNA表达载体两部分组分组成；所述rAPOBEC1:Cas9:UGI表达载体是通过基因合成和分子克隆的方法将rAPOBEC1:Cas9:UGI的编码基因克隆到pcDNA3.1（‑）载体中得到；所述gRNA表达载体是通过酶切连接的方式将gRNA序列克隆到包含T7启动子的pDR274载体中得到。该系统能够应用于基因修饰、哺乳动物细胞模型、动物模型的构建，以及用于哺乳动物细胞和胚胎的基因治疗，具有很好的应用前景。

Description

一套基于化脓性链球菌的碱基编辑系统及其在基因编辑中的 应用
技术领域
本发明属于分子生物学技术领域。更具体地，涉及一套基于化脓性链球菌(Streptococcus pyogenes SF370)Cas9的精确的碱基编辑系统(Base editing,BE) 及其在哺乳动物细胞和胚胎基因编辑中的应用。
背景技术
2003年完成的人类基因组计划掀起了基因功能研究的热潮，使得功能基因 组学成为生命科学研究的焦点。大量的测序结果显示了人群中基因的多样性，尤 其是单核苷酸多态性(single nucleotide variations,SNVs)，研究这些SNV的生理 功能将有助于阐释人与人在生理甚至心理上的差异的遗传学基础。在此之前，构 建SNV的动物或细胞模型都依赖于基因打靶技术，基因打靶技术由于细胞利用 细胞自身的自发同源重组，导致其效率非常低(<10-5)，需要耗费大量的人力和 时间，并且其应用范围优先，只能用于与少部分细胞(如HCT116，胚胎干细胞 等)和少部分物种(小鼠、大鼠等)。
虽然，基因编辑技术，如锌指核酸酶、TALEN核酸酶和CRISPR/Cas9的出 现，极大促进了同源重组的发生。但是，要构建单个位点的突变仍然效率不高。 而新一代的碱基编辑技术(base editing,BE)则改变了这一情况。与CRISPR/Cas9 技术类似，碱基编辑技术利用了CRISPR/Cas9的平台，也是通过gRNA的碱基 互补配对找到特定的DNA靶标。但又与CRISPR/Cas9技术不同，碱基编辑技术 主要是利用rAPOBEC1:Cas9:UGI构成的融合蛋白来将靶位点的胞嘧啶 (Cystidine,C)脱氨基，从而形成尿嘧啶(Uridine,U)。rAPOBEC1:Cas9:UGI 的融合蛋白中的Cas9通过与gRNA结合，并利用gRNA的序列，通过碱基互补 配对，将融合蛋白靶向到靶DNA上。然后，利用rAPOBEC1的胞嘧啶(Cystidine, C)脱氨酶活性将靶位点区的C转变成尿嘧啶(Uridine,U)，UGI是尿嘧啶DNA 糖基化酶抑制剂(Uracil DNAglycosylase inhibitor,UGI)，其通过抑制U的切除， 导致DNA在复制时U与A(腺嘌呤,Adenine)配对，然后再通过DNA复制， 使得U变成T(Thymine,胸腺嘧啶)，从而最终将C转变成T。而只有靶位点特 定区域内的C才能转变成T，这一区域称为脱氨基的窗口，通常这个区域在gRNA 靶向区域远离PAM(Protospacer adjacent motif)的这一端的2号至8号位的C。因此，碱基编辑技术能够高效地对特定位点进行单个碱基的突变，在基因治疗等 领域有广泛的应用前景。
然而，科学家发现，普通的Cas9蛋白在基因组上会结合到很多与靶DNA 序列类似的位点上，这就使得基于普通Cas9蛋白的碱基编辑系统有明显的脱靶 效应，阻碍了碱基编辑技术在疾病模型构建及基因治疗领域的应用。
发明内容
本发明要解决的技术问题是克服上述现有技术中碱基编辑技术的缺陷和不 足，提供一套更精确的碱基编辑系统。具体涉及通过基因合成、分子克隆等技术 构建一套基于化脓性链球菌(Streptococcus pyogenes SF370)Cas9的精确的碱基 编辑系统(Baseediting,BE)的载体序列信息，并将该碱基编辑系统应用到小鼠 细胞和胚胎基因编辑中。
本发明的目的是提供一套基于化脓性链球菌(Streptococcus pyogenes SF370)Cas9的精确的碱基编辑系统。
本发明另一目的是提供上述碱基编辑系统在哺乳动物细胞和胚胎基因编辑 中的应用。
本发明上述目的通过以下技术方案实现：
一套基于化脓性链球菌的碱基编辑系统，所述碱基编辑系统由 rAPOBEC1:Cas9:UGI和gRNA表达载体两部分组分组成。
所述rAPOBEC1:Cas9:UGI为rAPOBEC1:Cas9:UGI表达载体、 rAPOBEC1:Cas9:UGImRNA或rAPOBEC1:Cas9:UGI蛋白。所述 rAPOBEC1:Cas9:UGI表达载体是通过基因合成和分子克隆的方法将 rAPOBEC1:Cas9:UGI的编码基因克隆到pcDNA3.1(-)载体(购自Invitrogen) 中得到。
所述gRNA表达载体是通过酶切连接的方式将SEQ ID NO.15所示RNA序 列(gRNA)克隆到包含T7启动子的pDR274载体(购自Addgene)中，并将其 线性化，然后再以该线性化载体为模板制备得到。其中，克隆gRNA所用的引物 分别为：gRNA上游引物(SEQ ID NO.1)和gRNA下游引物(SEQ ID NO.2)。
另外，优选地，所述rAPOBEC1:Cas9:UGI表达载体为HF1-BE2表达载体、 HF1-BE3表达载体、HF2-BE2表达载体或HF2-BE3表达载体，序列分别如SEQ ID NO.3～6所示。
本发明合成的SpCas9基因是特异性更高的SpCas9-HF1和SpCas9-HF2，制 备了具有更高特异性的rAPOBEC1:Cas9:UGI融合基因的表达载体，分别命名为 HF1-BE2，HF1-BE3,HF2-BE2,HF2-BE3(HF1-BE2,BF1-BE3,HF2-BE2和 HF2-BE3碱基编辑蛋白结构示意图如图5所示)。该表达载体包含了可以用于 真核细胞表达的CMV启动子和体外转录的T7启动子，为了在真核细胞中表达， 只需要将该载体导入到真核细胞中，而做体外转录时，只需要将该载体用KpnI 酶切，将该载体线性化，然后进行体外转录；为了表达和纯化这些蛋白，只需要将rAPOBEC1:Cas9:UGI融合基因从以上载体克隆出来，然后连入到蛋白表达载 体中，利用原核或者真核表达系统表达rAPOBEC1:Cas9:UGI融合蛋白，并纯化。
优选地，所述rAPOBEC1:Cas9:UGI mRNA是用限制性内切酶切割所述 rAPOBEC1:Cas9:UGI表达载体，酶切产物纯化获得转录模板DNA，然后转录生 产mRNA。
更优选地，所述rAPOBEC1:Cas9:UGI mRNA是用KpnI切割 rAPOBEC1:Cas9:UGI表达载体，酶切产物纯化后用不含核酸酶的水洗脱，得转 录模板DNA；然后转录生产mRNA，纯化并用不含核酸酶的水洗脱mRNA得到。
本发明所制备的所述rAPOBEC1:Cas9:UGI mRNA包括HF1-BE2mRNA、 HF1-BE3mRNA、HF2-BE2mRNA或HF2-BE3mRNA，序列分别如SEQ ID NO.7～10所示。
优选地，所述rAPOBEC1:Cas9:UGI蛋白是先通过PCR的方式将 rAPOBEC1:Cas9:UGI融合基因克隆到pET28a载体中，然后将其转化到大肠杆菌 中表达并纯化获得。
更优选地，所述rAPOBEC1:Cas9:UGI蛋白是以所述rAPOBEC1:Cas9:UGI 表达载体为模板，利用SEQ ID NO.16～17所示mRNA上下游引物进行扩增，然 后克隆到用NotI和AscI酶切过的pET28a(购自Novagen)载体，获得 APOBEC1:Cas9:UGI蛋白表达载体，表达纯化获得APOBEC1:Cas9:UGI蛋白。
本发明所获得的所述APOBEC1:Cas9:UGI蛋白包括HF1-BE2蛋白、 HF1-BE3蛋白、HF2-BE2蛋白或HF2-BE3蛋白，序列分别如SEQ ID NO.11～14。
本发明制备rAPOBEC1:Cas9:UGI融合基因的mRNA的方法为将载体线性化 后进行体外转录。制备rAPOBEC1:Cas9:UGI融合蛋白的方法是通过将融合基因 克隆到蛋白表达载体中，利用原核或者真核表达系统表达rAPOBEC1:Cas9:UGI 融合蛋白。所得rAPOBEC1:Cas9:UGI mRNA的长度为5133nt，所得 rAPOBEC1:Cas9:UGI蛋白的大小约为197kDa：所得gRNA长度为104nt。
作为一种优选的具体实施方案，本发明的基于化脓性链球菌的碱基编辑系统 的构建方法如下：
S1.构建rAPOBEC1:Cas9:UGI
S11.制备rAPOBEC1:Cas9:UGI表达载体，为HF1-BE2表达载体、HF1-BE3 表达载体、HF2-BE2表达载体或HF2-BE3表达载体，序列分别如SEQ ID NO.3～ 6所示；
S12.制备化脓性链球菌rAPOBEC1:Cas9:UGI mRNA：以线性化的HF1-BE2 表达载体、HF1-BE3表达载体、HF2-BE2表达载体或HF2-BE3的表达载体为模 板，转录生产rAPOBEC1:Cas9:UGI mRNA，然后纯化并用不含核酸酶的水洗脱；
S13.制备APOBEC1:Cas9:UGI蛋白：以HF1-BE2表达载体、HF1-BE3表达 载体、HF2-BE2表达载体或HF2-BE3表达载体为模板，利用SEQ ID NO.16～17 所示mRNA上下游引物进行扩增，然后克隆到用NotI和AscI酶切过的pET28a (购自Novagen)载体，然后转化到大肠杆菌中，通过诱导表达，层析柱纯化获得 rAPOBEC1:Cas9:UGI蛋白；
S2.构建gRNA表达载体
S21.制备化脓性链球菌gRNA的转录载体：将SEQ ID NO.1和SEQ ID NO.2 所示的gRNA上游引物和gRNA下游引物退火成双链DNA，同时用BasI酶切 pDR274载体，然后将退火产物克隆到该载体中，获得gRNA的转录载体；然后 将转录载体用DraI酶切，纯化后用不含核酸酶的水洗脱，获得包含T7启动子的 化脓性链球菌gRNA转录模板DNA；
S22.制备化脓性链球菌gRNA：以包含T7启动子的化脓性链球菌gRNA转 录模板DNA为模板，转录生产化脓性链球菌gRNA；纯化并用不含核酸酶的水 洗脱gRNA，获得化脓性链球菌gRNA。
本发明构建了基于化脓性链球菌的更精确的碱基编辑系统，HF1-BE2， HF1-BE3,HF2-BE2,HF2-BE3，制备了碱基编辑系统的表达载体，通过体外转录 的方式制备了剪辑编辑系统rAPOBEC1:Cas9:UGI的mRNA和蛋白，以及化脓性 链球菌的gRNA；所述碱基编辑系统可应用于小鼠、人等哺乳动物的基因修饰， 比如如下的应用：1)在哺乳动物细胞和胚胎中进行单基因的定点突变；2)在哺 乳动物细胞和胚胎中进行多基因的定点突变；3)在哺乳动物细胞和胚胎中进行 基因突变的矫正。
因此，本发明所构建的基于化脓性链球菌的碱基编辑系统在哺乳动物细胞和 /或胚胎的基因编辑中的应用，也在本发明的保护范围之内。
具体地，所述基因编辑是指在哺乳动物细胞和/或胚胎中进行精确的单基因 敲除、多基因敲除和/或基因突变。所述基因突变包括单基因突变和多基因突变。
作为一种具体的可实施方案，所述应用的方法是将rAPOBEC1:Cas9:UGI表 达载体、rAPOBEC1:Cas9:UGI mRNA或rAPOBEC1:Cas9:UGI蛋白，以及gRNA 导入(包括但不限定于通过脂质体转染、电转、病毒感染、显微注射、电转等方 式)到哺乳动物细胞或胚胎中。将碱基编辑系统导入到哺乳动物细胞和胚胎中， 可以实现单基因、多基因的定点突变或敲除。
因此，本发明将促进以基于化脓性链球菌的更精确的碱基编辑系统为工具， 进行哺乳动物细胞和胚胎基因编辑的工作，促进疾病细胞模型、疾病动物模型的 构建以及基因治疗的应用。本发明制备的基于化脓性链球菌的更精确的碱基编辑 系统在哺乳动物细胞和胚胎基因编辑中的工作、动物模型的构建以及基因治疗方 面的应用，都应在本发明的保护范围之内。
本发明公开了一套基于化脓性链球菌(Streptococcus pyogenes SF370)Cas9 的精确的碱基编辑系统(Base editing,BE)及其在哺乳动物细胞和胚胎基因编辑 中的应用。所述化脓性链球菌碱基编辑系统是由rAPOBEC1:Cas9:UGI融合蛋白 和gRNA两部分组分组成，其中Cas9是没有核酸酶活性的失活的Cas9(dead Cas9, dCas9)或者只能切割DNA双链中一条链的Cas9缺刻酶(Cas9nickase,Cas9n)， 而rAPOBEC1:dCas9:UGI名为BE2，rAPOBEC1:Cas9n:UGI名为BE3； APOBEC1:Cas9:UGI的融合蛋白中的Cas9通过与gRNA结合，并利用gRNA的 序列，通过碱基互补配对，将融合蛋白靶向到靶DNA上，然后，利用rAPOBEC1的胞嘧啶(Cystidine,C)脱氨酶活性将靶位点区的C转变成尿嘧啶(Uridine,U)， UGI是尿嘧啶DNA糖基化酶抑制剂(Uracil DNA glycosylase inhibitor,UGI),其 通过抑制U的切除，导致DNA在复制时U与A(腺嘌呤,Adenine)配对，然后 再通过DNA复制，使得U变成T(Thymine,胸腺嘧啶)，从而最终将C转变成 T。由于野生型的Cas9蛋白的特异性不高，其与gRNA结合后，容易将rAPOBEC1:Cas9:UGI融合蛋白靶向到与gRNA不完全匹配的脱靶位点上，导致 脱靶，从而严重制约碱基编辑在疾病细胞模型，疾病动物模型及基因治疗领域的 应用,为了提高碱基编辑的特异性，结合已经报道的具有更高特异性的高保真 (High-fidelity,HF)Cas9蛋白HF1和HF2，我们构建了更精确的碱基编辑系统， HF1-BE2,HF-BE3,HF2-BE2和HF2-BE3。将该基因编辑系统导入到胚胎中，可 以对胚胎的基因组进行精确的单碱基水平的基因编辑，在疾病细胞模型构建和基 因治疗领域，具有广泛的应用前景。
本发明具有以下有益效果：
本发明提供了一套基于化脓性链球菌的更精确的碱基编辑系统，并提供了一 种制备基于化脓性链球菌的更精确的碱基编辑系统组分的方法，该系统能够应用 于基因修饰、哺乳动物细胞模型、动物模型的构建，以及用于哺乳动物细胞和胚 胎的基因治疗。
本发明研究结果显示，将该剪辑编辑系统导入到哺乳动物细胞和胚胎中，可 以对哺乳动物细胞和胚胎进行精确的定点突变，该突变可以是造成氨基酸的突 变，也可以形成一个终止密码子，从而破坏靶基因的表达，在哺乳动物模型以及 哺乳动物受精卵基因治疗方面，具有很好的应用前景。
附图说明
图1为化脓性链球菌的更精确的碱基编辑系统的表达载体图谱(pcDNA3.1 (-)-HF1-BE2)。
图2为化脓性链球菌的更精确的碱基编辑系统的表达载体图谱(pcDNA3.1 (-)-HF1-BE3)
图3为化脓性链球菌的更精确的碱基编辑系统的表达载体图谱(pcDNA3.1 (-)-HF2-BE2)
图4为化脓性链球菌的更精确的碱基编辑系统的表达载体图谱(pcDNA3.1 (-)-HF2-BE3)
图5为HF1-BE2,BF1-BE3,HF2-BE2和HF2-BE3碱基编辑蛋白结构示意图。
图6为化脓性链球菌的更精确的碱基编辑系统rAPOBEC1:Cas9:UGI mRNA 和gRNA的制备结果(琼脂糖凝胶电泳结果)；图6中A图为HF1-BE2,HF1-BE3, HF2-BE2,HF2-BE3mRNA电泳图，左侧的泳道为DNA分子Marker；图6中B 图为gRNA电泳图，左侧的泳道为DNA分子Marker，右侧2条泳道为gRNA。
图7为化脓性链球菌的更精确的碱基编辑系统介导的Tyr基因定点突变；图 7中A图为通过Sanger测序鉴定Tyr突变的胚胎，WT是野生型胚胎对照，Edited 是被编辑的胚胎，红色三角标注的为被编辑的碱基；图7中B图为Tyr碱基编辑 系统在小鼠胚胎基因编辑中的统计结果。图7中C图为通过Sanger测序鉴定Tyr 突变的首建鼠，WT是野生型小鼠对照,Edited是被编辑的首建鼠，红色三角标 注的为被编辑的碱基；图7中D图为Tyr碱基编辑系统在小鼠胚胎基因编辑中的 统计结果。
图8为通过碱基编辑系统构建的嵌合体和完全白化的首建鼠。野生型小鼠为 黑色，部分突变的为黑白相间，完全突变为白化小鼠。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本 发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技 术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1基于化脓性链球菌的碱基编辑系统组分的制备方法
图1～4为四种化脓性链球菌的更精确的碱基编辑系统的表达载体图谱。
本实施例提供基于化脓性链球菌的碱基编辑系统组分rAPOBEC1:Cas9:UGI mRNA和gRNA的制备方法。
1、化脓性链球菌rAPOBEC1:Cas9:UGI mRNA的制备，方法如下：
(1)制备HF1-BE2、HF1-BE3、HF2-BE2或HF2-BE3的转录载体，序列见 SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6所示。
(2)制备包含T7启动子的化脓性链球菌APOBEC1:Cas9:UGI的转录模板：
用KpnI切割HF1-BE2、HF1-BE3、HF2-BE2或HF2-BE3的转录载体，然后 再将酶切产物用PCR产物纯化试剂盒(Axygen)进行过柱纯化，然后用不含核 酸酶的水洗脱，即可获得包含T7启动子的化脓性链球菌APOBEC1:Cas9:UGI的 转录模板DNA；
(3)制备rAPOBEC1:Cas9:UGI mRNA：
以步骤(1)制备的包含T7启动子的化脓性链球菌APOBEC1:Cas9:UGI的 转录模板DNA为模板，用mMESSAGEmMACHINE T7ULTRA kit(Life Technologies)转录生产mRNA；然后，再用RNA纯化试剂盒纯化mRNA(Qiagen)， 并用不含核酸酶的水洗脱mRNA，即可获得化脓性链球菌APOBEC1:Cas9:UGI 的mRNA。
2、化脓性链球菌APOBEC1:Cas9:UGI蛋白的制备：
(1)利用合成的mRNA上游引物(SEQ ID NO.16)和mRNA下游引物(SEQ ID NO.17)，以HF1-BE2(SEQ ID NO.3)、HF1-BE3(SEQ ID NO.4)、HF2-BE2(SEQ ID NO.5)、HF2-BE3(SEQID NO.6)为模板，然后克隆到用NotI和AscI酶切过的 pET28a(购自Novagen)载体，从而获得APOBEC1:Cas9:UGI蛋白的表达载体；
(2)表达纯化APOBEC1:Cas9:UGI蛋白，包括HF1-BE2,HF1-BE3, HF2-BE2和HF2-BE3等；
具体方法是：将表达载体转化到大肠杆菌BL21中，然后，用isopropyl- β-d-1-thiogalactopyranoside(IPTG)诱导表达，然后将菌液裂解，并过镍柱纯化。
3、gRNA的制备
(1)制备化脓性链球菌gRNA的转录载体：
利用合成的gRNA上游引物和gRNA下游引物(序列分别如SEQ ID NO.1 和SEQ IDNO.2所示)，首先用不含核酸酶的水溶解成100μM的母液，然后将 两条引物退火成双链DNA。同时用BasI酶切pDR274载体，然后将退火产物克 隆到该载体中，从而获得gRNA的转录载体。然后将转录载体用DraI酶切，然 后用PCR产物纯化试剂盒(Axygen)进行过柱纯化，然后用不含核酸酶的水洗 脱，即可获得包含T7启动子的化脓性链球菌gRNA转录模板DNA；
(2)制备化脓性链球菌gRNA：
以包含T7启动子的化脓性链球菌gRNA转录模板DNA为模板，用 MEGAshortscriptT7kit(Life Technologies)转录生产化脓性链球菌gRNA。再用 RNA纯化试剂盒纯化gRNA(Qiagen)，并用不含核酸酶的水洗脱gRNA，即可获 得化脓性链球菌gRNA(RNA序列如SEQ IDNO.15所示)。
实施例2基于化脓性链球菌的碱基编辑系统组分rAPOBEC1:Cas9:UGI mRNA和gRNA的制备案例
具体地，上述实施例1所述化脓性链球菌APOBEC1:Cas9:UGI mRNA、 APOBEC1:Cas9:UGI蛋白和gRNA的制备方法的操作程序如下：
1、APOBEC1:Cas9:UGI mRNA和gRNA转录模板DNA的制备：
(1)化脓性链球菌APOBEC1:Cas9:UGI mRNA转录模板DNA的制备
通过质粒提取制备HF1-BE2、HF1-BE3、HF2-BE2或HF2-BE3的转录载体(自主研发)，然后用KpnI酶切该转录载体，按照如下表1所示反应体系进行：
表1反应体系
成分 用量
mRNA转录载体 2000ng
10X NEBuffer 1.1 5μl
Kpn I 5μl
ddH2O 补足至50μl
37℃酶切过夜。
(2)化脓性链球菌gRNA转录模板DNA的制备。
将gRNA上游引物和gRNA下游引物作用不含核酸酶的水稀释至100μM，
然后将5μl gRNA上游引物与5μl gRNA下游引物混匀在一起，95℃变性5 min，然后室温复性3h。同时，用BasI酶切pDR274载体(购自Addgene)，酶 切体系如下：
表2反应体系
成分 用量
pDR274 2000ng
10X Cutsmart buffer 5μl
BsaI 5μl
ddH2O 补足至50μl
再将gRNA上游引物与下游引物的退火产物连入BsaI酶切过的载体中，连 接体系如下：
表3反应体系
成分 用量
pDR274(BsaI) 25ng
退火产物 1μl
10X T4DNA ligase buffer 0.5μl
T4DNA ligase 0.25μl
ddH2O 补足至5μl
22℃连接3h，然后转化大肠杆菌，并测序验证后，提取质粒，并用DraI酶 切质粒，按照表4所示酶切体系，37℃酶切过夜。
表4反应体系
成分 用量
gRNA转录载体 2000ng
10X Cutsmart buffer 5μl
DraI 5μl
ddH2O 补足至50μl
2、APOBEC1:Cas9:UGI mRNA和gRNA转录模板DNA的纯化
按AxyPrep PCR cleanup kit的操作手册进行实验。
(1)在PCR反应液中，加3个体积的Buffer PCR-A并混匀，然后转移到 DNA制备管，将DNA制备管置于2ml离心管中，12,000g离心1min，将滤液 弃掉。
(2)将制备管放回2ml离心管中，加700μl Buffer W2，12000g离心1min， 将滤液弃掉。
(3)将制备管放回2ml离心管中，加400μl Buffer W2，12000g离心1min， 弃滤液。
(4)12,000g离心3min，使Buffer W2中的乙醇充分弃掉。
(5)将制备管置于新的1.5ml离心管中，在制备管中央加25-30μl的无核 酸酶的水，静置1min。
(6)12000g离心1min(无核酸酶的水用前先65℃预热)。
3、APOBEC1:Cas9:UGI mRNA和gRNA的制备和纯化。
(1)APOBEC1:Cas9:UGI mRNA的转录
以APOBEC1:Cas9:UGI mRNA转录模板DNA为模板，利用 mMESSAGEmMACHINET7ULTRA kit(Life Technologies)进行转录。
按照如下表5所示体系配制反应体系。
表5反应体系
37℃反应2h，然后往反应体系中加1μl TURBO DNase,37℃反应15min。终 止反应后，再往表6所示反应体系中加入如下成分加poly A尾。
表6反应体系
成分 用量
5×E-PAP Buffer 20μl
25mM MnCl2 10μl
ATP溶液 10μl
ddH2O 35μl
E-PAP 4μl
37℃反应45min，然后置于冰上。
(2)化脓性链球菌gRNA的转录
以化脓性链球菌gRNA转录模板DNA为模板，利用MEGAshortscript T7kit (LifeTechnologies)，按照如下表7所示体系配制反应体系。
表7反应体系
成分 用量
T7 10×反应液 2μl
T7ATP溶液 2μl
T7CTP溶液 2μl
T7GTP溶液 2μl
T7UTP溶液 2μl
模板DNA 1μg
T7RNA转录酶 2μl
ddH2O 加水至20μl
37℃反应2h，案后往反应体系中加1μl TURBO DNase,37℃反应15min。
(3)APOBEC1:Cas9:UGI mRNA和gRNA的纯化，用Qiagen的RNaeasy Kit 纯化，按照如下步骤进行：
a.加ddH2O使得起始RNA的体积为100μl，混匀。
b.加350μl Binding Solution Concentrate到RNA样品中，并混匀。
c.加250μl 100％乙醇，并混匀。
d.将样品转移到柱子中，12000g离心15s。
e.用500μl Wash Solution洗两次，12000g离心15s。
f.加50μlddH2O将RNA从柱子上洗脱下来。
(4)结果如图6所示，图6显示了化脓性链球菌rAPOBEC1:Cas9:UGI (HF1-BE2,HF1-BE3,HF2-BE2,HF2-BE3)mRNA和Tyr gRNA的琼脂糖凝胶电泳 结果。
4、rAPOBEC1:Cas9:UGI蛋白的表达和纯化
(1)利用合成的mRNA上游引物(SEQ ID NO.16)和mRNA下游引物(SEQ ID NO.17)，以HF1-BE2(SEQ ID NO.3),HF1-BE3(SEQ ID NO.4),HF2-BE2(SEQ ID NO.5),HF2-BE3(SEQID NO.6)为模板，然后克隆到用NotI和AscI酶切过的 pET28a(购自Novagen)载体，从而获得APOBEC1:Cas9:UGI蛋白的表达载体； PCR体系和程序如下：
表8反应体系
成分 用量
质粒PX601 50ng
5×HF缓冲液 10μl
gRNA上游引物(10μM) 1μl
gRNA下游引物(10μM) 1μl
10mM dNTP 1μl
Phusion DNA聚合酶 0.5μl
ddH2O 补足至50μl
表9反应程序
(2)表达纯化APOBEC1:Cas9:UGI蛋白：
a.将表达载体通过热激法转化到大肠杆菌BL21中，于含有100ug/ml的 Luria-Bertani(LB)培养基中37℃过夜。
b.第二天1:100加到同样的培养基中37℃摇至OD600＝～0.6。
c.加入isopropyl-β-d-1-thiogalactopyranoside(IPTG)至0.5mM，16℃诱导过夜。
d.第二天收菌，4000rpm 10min离心，再在Buffer I(50mM tris(hydroxymethyl)-aminomethane(Tris)HCl(pH 7.5),1M NaCl,20％ glycerol,20mM Imidazole)中重悬超声破碎(2s pulse-on,5s pulse-off for 5min total pulse-on)。
e.14000rpm，4℃离心15min，取上清0.45um过滤。
f.Ni柱先以超纯水洗柱，后以Buffer II(50mM tris(hydroxymethyl)-aminomethane(Tris)HCl(pH 7.5),1M NaCl,20％glycerol)平衡，滤后上清上柱流过Ni柱，Buffer II洗柱至无蛋白流出。
g.用Buffer III(50mM tris(hydroxymethyl)-aminomethane(Tris)HCl(pH7.5), 1M NaCl,20％glycerol,300mM Imidazole)将His标签的融合蛋白洗脱下柱子。
h.后用浓缩管(30-kDa molecular weight cut-off)将蛋白浓缩至300ul上分子筛，以BufferⅣ(50mM tris(hydroxymethyl)-aminomethane(Tris)-HCl(pH 7.0),0.5 MNaCl,5％glycerol)洗脱，SDS-PAGE蛋白胶检测。
实施例3基于化脓性链球菌的更精确的碱基编辑系统介导的Tyr基因定点突变
1、为了利用基于化脓性链球菌的更精确的碱基编辑系统在小鼠受精卵中实 现单基因定点突变，我们针对Tyr基因设计了2条gRNA(gRNA-1和gRNA-2)。 Tyr基因的定点突变会形成终止密码子，导致Tyr基因翻译提前终止，从而使得 崽鼠的毛色由黑色变成白色。
首先，我们转录了Tyr的gRNA，然后将Tyr gRNA(50ng/μl)与 rAPOBEC1:Cas9:UGI(HF2-BE2)mRNA(100ng/μl)混匀后一起注射到0.5天的 小鼠受精卵中。注射后48h检测定点突变效率，通过结合PCR及Sanger测序检 测，我们发现：对于gRNA-1，有11.6％的小鼠胚胎发生了突变，而对于gRNA-2， 则有50％的胚胎发生了突变。这远远高于通过同源重组引入定点突变的效率。
同时，我们还将另外一部分注射了的受精卵移植到0.5天假孕鼠的输卵管中， 待20天之后，假孕鼠便会产下崽鼠。通过将靶位点用PCR的方式扩增出来。然 后，利用Sanger测序技术检测PCR产物，我们发现：对于gRNA-1，有18.2％ 的首建鼠发生了突变，而对于gRNA-2，则有63.6％的首建鼠发生了突变。同时， 我们还观察了崽鼠的毛色，结果显示基于化脓性链球菌的更精确的碱基编辑系统 能够高效介导Tyr基因的定点突变，从毛色上我们发现黑白相间的首建鼠以及完 全白化的首建鼠。
2、结果如附图7和图8所示。
图7A通过Sanger测序检测定点突变的小鼠胚胎，WT是野生型小鼠对照， Edited是突变了的胚胎，红色三角标注的是突变了的碱基；图7B是小鼠胚胎碱 基编辑的统计结果；图7C通过Sanger测序检测定点突变的首建鼠，WT是野生 型小鼠对照，Edited是突变了的首建鼠；图7D是首建鼠碱基编辑的统计结果。
图8是Tyr碱基编辑小鼠的照片，Tyr基因敲除小鼠毛色为白色，野生型为 黑色，部分突变的为黑白相间；
图7和图8的结果显示了，本发明制备的基于化脓性链球菌的更精确的碱基 编辑系统能在小鼠受精卵中高效进行基因的定点突变。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施 例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替 代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 中山大学
<120> 一套基于化脓性链球菌的碱基编辑系统及其在基因编辑中的应用
<130>
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213> gRNA上游引物
<220>
<221> misc_feature
<222> (5)..(24)
<223> n is a, c, g, t or u
<400> 1
taggnnnnnn nnnnnnnnnn nnnn 24
<210> 2
<211> 24
<212> DNA
<213> gRNA下游引物
<220>
<221> misc_feature
<222> (5)..(24)
<223> n is a, c, g, t or u
<400> 2
aaacnnnnnn nnnnnnnnnn nnnn 24
<210> 3
<211> 10530
<212> DNA
<213> HF1-BE2表达载体
<400> 3
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaacgg gccctctaga gccaccatga gctcagagac tggcccagtg gctgtggacc 960
ccacattgag acggcggatc gagccccatg agtttgaggt attcttcgat ccgagagagc 1020
tccgcaagga gacctgcctg ctttacgaaa ttaattgggg gggccggcac tccatttggc 1080
gacatacatc acagaacact aacaagcacg tcgaagtcaa cttcatcgag aagttcacga 1140
cagaaagata tttctgtccg aacacaaggt gcagcattac ctggtttctc agctggagcc 1200
catgcggcga atgtagtagg gccatcactg aattcctgtc aaggtatccc cacgtcactc 1260
tgtttattta catcgcaagg ctgtaccacc acgctgaccc ccgcaatcga caaggcctgc 1320
gggatttgat ctcttcaggt gtgactatcc aaattatgac tgagcaggag tcaggatact 1380
gctggagaaa ctttgtgaat tatagcccga gtaatgaagc ccactggcct aggtatcccc 1440
atctgtgggt acgactgtac gttcttgaac tgtactgcat catactgggc ctgcctcctt 1500
gtctcaacat tctgagaagg aagcagccac agctgacatt ctttaccatc gctcttcagt 1560
cttgtcatta ccagcgactg cccccacaca ttctctgggc caccgggttg aaaagcggca 1620
gcgagactcc cgggacctca gagtccgcca cacccgaaag tgataaaaag tattctattg 1680
gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa tacaaagtac 1740
cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa aagaatctta 1800
tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg aaacgaaccg 1860
ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa atttttagca 1920
atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc ttccttgtcg 1980
aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat gaggtggcat 2040
atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac tcaactgata 2100
aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc cgtgggcact 2160
ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg ttcatccagt 2220
tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt ggcgtggatg 2280
cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac ctgatcgcac 2340
aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc tcactaggcc 2400
tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg cagcttagta 2460
aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat cagtatgcgg 2520
acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac atactgagag 2580
ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg tacgatgaac 2640
atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct gagaaatata 2700
aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac ggcggagcga 2760
gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat gggacggaag 2820
agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact ttcgacaacg 2880
gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga aggcaggagg 2940
atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta acctttcgca 3000
taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg atgacaagaa 3060
agtccgaaga aacgattact ccctggaatt ttgaggaagt tgtcgataaa ggtgcgtcag 3120
ctcaatcgtt catcgagagg atgaccgcct ttgacaagaa tttaccgaac gaaaaagtat 3180
tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc acgaaagtta 3240
agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag aagaaagcaa 3300
tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg aaagaggact 3360
actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa gatcgattta 3420
atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag gacttcctgg 3480
ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc ctctttgaag 3540
atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac gataaggtta 3600
tgaaacagtt aaagaggcgt cgctatacgg gctggggagc cttgtcgcgg aaacttatca 3660
acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag agcgacggct 3720
tcgccaatag gaactttatg gccctgatcc atgatgactc tttaaccttc aaagaggata 3780
tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt gcgaatcttg 3840
ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg gatgagctag 3900
ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca cgcgaaaatc 3960
aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata gaagagggta 4020
ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc caattgcaga 4080
acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt gatcaggaac 4140
tggacataaa ccgtttatct gattacgacg tcgatgccat tgtaccccaa tcctttttga 4200
aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga gggaaaagtg 4260
acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg cagctcctaa 4320
atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag aggggtggct 4380
tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc cgcgccatca 4440
caaagcatgt tgcccagata ctagattccc gaatgaatac gaaatacgac gagaacgata 4500
agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg gacttcagaa 4560
aggattttca attctataaa gttagggaga taaataacta ccaccatgcg cacgacgctt 4620
atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta gaaagtgagt 4680
ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa agcgaacagg 4740
agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat ttctttaaga 4800
cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa accaatgggg 4860
agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga aaagttttgt 4920
ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg ttttcaaagg 4980
aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag gactgggacc 5040
cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta gtagtggcaa 5100
aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg gggataacga 5160
ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg aaaggttaca 5220
aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt gagttagaaa 5280
atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac gaactcgcac 5340
taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag ttgaaaggtt 5400
cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat tatctcgacg 5460
aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat gccaatctgg 5520
acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag caggcggaaa 5580
atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc aagtattttg 5640
acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac gcgacactga 5700
ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag cttgggggtg 5760
actctggtgg ttctactaat ctgtcagata ttattgaaaa ggagaccggt aagcaactgg 5820
ttatccagga atccatcctc atgctcccag aggaggtgga agaagtcatt gggaacaagc 5880
cggaaagcga tatactcgtg cacaccgcct acgacgagag caccgacgag aatgtcatgc 5940
ttctgactag cgacgcccct gaatacaagc cttgggctct ggtcatacag gatagcaacg 6000
gtgagaacaa gattaagatg ctctctggtg gttctcccaa gaagaagagg aaagtctaat 6060
tccaccacac tggactagtg gatccgagct cggtaccaag cttaagttta aaccgctgat 6120
cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 6180
ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 6240
cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 6300
gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 6360
aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 6420
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6480
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6540
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6600
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6660
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6720
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6780
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 6840
tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 6900
catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 6960
aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 7020
catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 7080
ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 7140
aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 7200
cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 7260
cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 7320
aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 7380
tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 7440
gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 7500
aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 7560
tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 7620
ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 7680
ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 7740
cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 7800
tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 7860
ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 7920
tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 7980
tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 8040
ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 8100
accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 8160
atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 8220
gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 8280
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 8340
ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 8400
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 8460
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 8520
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 8580
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 8640
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 8700
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 8760
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 8820
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 8880
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 8940
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 9000
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 9060
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 9120
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 9180
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 9240
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 9300
accgctggta gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 9360
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 9420
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 9480
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 9540
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 9600
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 9660
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 9720
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 9780
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 9840
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 9900
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 9960
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 10020
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 10080
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 10140
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 10200
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 10260
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 10320
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 10380
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 10440
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 10500
acatttcccc gaaaagtgcc acctgacgtc 10530
<210> 4
<211> 10530
<212> DNA
<213> HF1-BE3表达载体
<400> 4
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaacgg gccctctaga gccaccatga gctcagagac tggcccagtg gctgtggacc 960
ccacattgag acggcggatc gagccccatg agtttgaggt attcttcgat ccgagagagc 1020
tccgcaagga gacctgcctg ctttacgaaa ttaattgggg gggccggcac tccatttggc 1080
gacatacatc acagaacact aacaagcacg tcgaagtcaa cttcatcgag aagttcacga 1140
cagaaagata tttctgtccg aacacaaggt gcagcattac ctggtttctc agctggagcc 1200
catgcggcga atgtagtagg gccatcactg aattcctgtc aaggtatccc cacgtcactc 1260
tgtttattta catcgcaagg ctgtaccacc acgctgaccc ccgcaatcga caaggcctgc 1320
gggatttgat ctcttcaggt gtgactatcc aaattatgac tgagcaggag tcaggatact 1380
gctggagaaa ctttgtgaat tatagcccga gtaatgaagc ccactggcct aggtatcccc 1440
atctgtgggt acgactgtac gttcttgaac tgtactgcat catactgggc ctgcctcctt 1500
gtctcaacat tctgagaagg aagcagccac agctgacatt ctttaccatc gctcttcagt 1560
cttgtcatta ccagcgactg cccccacaca ttctctgggc caccgggttg aaaagcggca 1620
gcgagactcc cgggacctca gagtccgcca cacccgaaag tgataaaaag tattctattg 1680
gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa tacaaagtac 1740
cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa aagaatctta 1800
tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg aaacgaaccg 1860
ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa atttttagca 1920
atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc ttccttgtcg 1980
aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat gaggtggcat 2040
atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac tcaactgata 2100
aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc cgtgggcact 2160
ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg ttcatccagt 2220
tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt ggcgtggatg 2280
cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac ctgatcgcac 2340
aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc tcactaggcc 2400
tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg cagcttagta 2460
aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat cagtatgcgg 2520
acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac atactgagag 2580
ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg tacgatgaac 2640
atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct gagaaatata 2700
aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac ggcggagcga 2760
gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat gggacggaag 2820
agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact ttcgacaacg 2880
gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga aggcaggagg 2940
atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta acctttcgca 3000
taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg atgacaagaa 3060
agtccgaaga aacgattact ccctggaatt ttgaggaagt tgtcgataaa ggtgcgtcag 3120
ctcaatcgtt catcgagagg atgaccgcct ttgacaagaa tttaccgaac gaaaaagtat 3180
tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc acgaaagtta 3240
agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag aagaaagcaa 3300
tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg aaagaggact 3360
actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa gatcgattta 3420
atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag gacttcctgg 3480
ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc ctctttgaag 3540
atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac gataaggtta 3600
tgaaacagtt aaagaggcgt cgctatacgg gctggggagc cttgtcgcgg aaacttatca 3660
acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag agcgacggct 3720
tcgccaatag gaactttatg gccctgatcc atgatgactc tttaaccttc aaagaggata 3780
tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt gcgaatcttg 3840
ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg gatgagctag 3900
ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca cgcgaaaatc 3960
aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata gaagagggta 4020
ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc caattgcaga 4080
acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt gatcaggaac 4140
tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa tcctttttga 4200
aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga gggaaaagtg 4260
acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg cagctcctaa 4320
atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag aggggtggct 4380
tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc cgcgccatca 4440
caaagcatgt tgcccagata ctagattccc gaatgaatac gaaatacgac gagaacgata 4500
agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg gacttcagaa 4560
aggattttca attctataaa gttagggaga taaataacta ccaccatgcg cacgacgctt 4620
atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta gaaagtgagt 4680
ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa agcgaacagg 4740
agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat ttctttaaga 4800
cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa accaatgggg 4860
agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga aaagttttgt 4920
ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg ttttcaaagg 4980
aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag gactgggacc 5040
cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta gtagtggcaa 5100
aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg gggataacga 5160
ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg aaaggttaca 5220
aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt gagttagaaa 5280
atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac gaactcgcac 5340
taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag ttgaaaggtt 5400
cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat tatctcgacg 5460
aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat gccaatctgg 5520
acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag caggcggaaa 5580
atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc aagtattttg 5640
acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac gcgacactga 5700
ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag cttgggggtg 5760
actctggtgg ttctactaat ctgtcagata ttattgaaaa ggagaccggt aagcaactgg 5820
ttatccagga atccatcctc atgctcccag aggaggtgga agaagtcatt gggaacaagc 5880
cggaaagcga tatactcgtg cacaccgcct acgacgagag caccgacgag aatgtcatgc 5940
ttctgactag cgacgcccct gaatacaagc cttgggctct ggtcatacag gatagcaacg 6000
gtgagaacaa gattaagatg ctctctggtg gttctcccaa gaagaagagg aaagtctaat 6060
tccaccacac tggactagtg gatccgagct cggtaccaag cttaagttta aaccgctgat 6120
cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 6180
ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 6240
cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 6300
gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 6360
aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 6420
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6480
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6540
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6600
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6660
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6720
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6780
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 6840
tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 6900
catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 6960
aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 7020
catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 7080
ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 7140
aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 7200
cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 7260
cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 7320
aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 7380
tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 7440
gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 7500
aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 7560
tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 7620
ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 7680
ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 7740
cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 7800
tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 7860
ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 7920
tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 7980
tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 8040
ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 8100
accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 8160
atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 8220
gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 8280
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 8340
ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 8400
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 8460
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 8520
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 8580
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 8640
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 8700
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 8760
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 8820
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 8880
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 8940
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 9000
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 9060
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 9120
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 9180
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 9240
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 9300
accgctggta gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 9360
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 9420
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 9480
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 9540
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 9600
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 9660
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 9720
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 9780
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 9840
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 9900
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 9960
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 10020
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 10080
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 10140
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 10200
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 10260
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 10320
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 10380
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 10440
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 10500
acatttcccc gaaaagtgcc acctgacgtc 10530
<210> 5
<211> 10530
<212> DNA
<213> HF2-BE2表达载体
<400> 5
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaacgg gccctctaga gccaccatga gctcagagac tggcccagtg gctgtggacc 960
ccacattgag acggcggatc gagccccatg agtttgaggt attcttcgat ccgagagagc 1020
tccgcaagga gacctgcctg ctttacgaaa ttaattgggg gggccggcac tccatttggc 1080
gacatacatc acagaacact aacaagcacg tcgaagtcaa cttcatcgag aagttcacga 1140
cagaaagata tttctgtccg aacacaaggt gcagcattac ctggtttctc agctggagcc 1200
catgcggcga atgtagtagg gccatcactg aattcctgtc aaggtatccc cacgtcactc 1260
tgtttattta catcgcaagg ctgtaccacc acgctgaccc ccgcaatcga caaggcctgc 1320
gggatttgat ctcttcaggt gtgactatcc aaattatgac tgagcaggag tcaggatact 1380
gctggagaaa ctttgtgaat tatagcccga gtaatgaagc ccactggcct aggtatcccc 1440
atctgtgggt acgactgtac gttcttgaac tgtactgcat catactgggc ctgcctcctt 1500
gtctcaacat tctgagaagg aagcagccac agctgacatt ctttaccatc gctcttcagt 1560
cttgtcatta ccagcgactg cccccacaca ttctctgggc caccgggttg aaaagcggca 1620
gcgagactcc cgggacctca gagtccgcca cacccgaaag tgataaaaag tattctattg 1680
gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa tacaaagtac 1740
cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa aagaatctta 1800
tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg aaacgaaccg 1860
ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa atttttagca 1920
atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc ttccttgtcg 1980
aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat gaggtggcat 2040
atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac tcaactgata 2100
aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc cgtgggcact 2160
ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg ttcatccagt 2220
tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt ggcgtggatg 2280
cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac ctgatcgcac 2340
aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc tcactaggcc 2400
tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg cagcttagta 2460
aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat cagtatgcgg 2520
acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac atactgagag 2580
ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg tacgatgaac 2640
atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct gagaaatata 2700
aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac ggcggagcga 2760
gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat gggacggaag 2820
agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact ttcgacaacg 2880
gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga aggcaggagg 2940
atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta acctttcgca 3000
taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg atgacaagaa 3060
agtccgaaga aacgattact ccctggaatt ttgaggaagt tgtcgataaa ggtgcgtcag 3120
ctcaatcgtt catcgagagg atgaccgcct ttgacaagaa tttaccgaac gaaaaagtat 3180
tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc acgaaagtta 3240
agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag aagaaagcaa 3300
tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg aaagaggact 3360
actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa gatcgattta 3420
atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag gacttcctgg 3480
ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc ctctttgaag 3540
atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac gataaggtta 3600
tgaaacagtt aaagaggcgt cgctatacgg gctggggagc cttgtcgcgg aaacttatca 3660
acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag agcgacggct 3720
tcgccaatag gaactttatg gccctgatcc atgatgactc tttaaccttc aaagaggata 3780
tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt gcgaatcttg 3840
ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg gatgagctag 3900
ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca cgcgaaaatc 3960
aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata gaagagggta 4020
ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc caattgcaga 4080
acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt gatcaggaac 4140
tggacataaa ccgtttatct gattacgacg tcgatgccat tgtaccccaa tcctttttga 4200
aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga gggaaaagtg 4260
acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg cagctcctaa 4320
atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag aggggtggct 4380
tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc cgcgccatca 4440
caaagcatgt tgcccagata ctagattccc gaatgaatac gaaatacgac gagaacgata 4500
agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg gacttcagaa 4560
aggattttca attctataaa gttagggaga taaataacta ccaccatgcg cacgacgctt 4620
atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta gaaagtgagt 4680
ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa agcgaacagg 4740
agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat ttctttaaga 4800
cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa accaatgggg 4860
agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga aaagttttgt 4920
ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg ttttcaaagg 4980
aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag gactgggacc 5040
cgaaaaagta cggtggcttc gagagcccta cagttgccta ttctgtccta gtagtggcaa 5100
aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg gggataacga 5160
ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg aaaggttaca 5220
aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt gagttagaaa 5280
atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac gaactcgcac 5340
taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag ttgaaaggtt 5400
cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat tatctcgacg 5460
aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat gccaatctgg 5520
acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag caggcggaaa 5580
atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc aagtattttg 5640
acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac gcgacactga 5700
ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag cttgggggtg 5760
actctggtgg ttctactaat ctgtcagata ttattgaaaa ggagaccggt aagcaactgg 5820
ttatccagga atccatcctc atgctcccag aggaggtgga agaagtcatt gggaacaagc 5880
cggaaagcga tatactcgtg cacaccgcct acgacgagag caccgacgag aatgtcatgc 5940
ttctgactag cgacgcccct gaatacaagc cttgggctct ggtcatacag gatagcaacg 6000
gtgagaacaa gattaagatg ctctctggtg gttctcccaa gaagaagagg aaagtctaat 6060
tccaccacac tggactagtg gatccgagct cggtaccaag cttaagttta aaccgctgat 6120
cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 6180
ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 6240
cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 6300
gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 6360
aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 6420
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6480
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6540
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6600
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6660
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6720
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6780
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 6840
tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 6900
catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 6960
aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 7020
catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 7080
ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 7140
aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 7200
cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 7260
cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 7320
aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 7380
tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 7440
gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 7500
aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 7560
tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 7620
ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 7680
ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 7740
cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 7800
tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 7860
ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 7920
tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 7980
tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 8040
ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 8100
accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 8160
atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 8220
gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 8280
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 8340
ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 8400
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 8460
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 8520
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 8580
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 8640
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 8700
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 8760
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 8820
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 8880
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 8940
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 9000
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 9060
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 9120
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 9180
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 9240
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 9300
accgctggta gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 9360
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 9420
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 9480
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 9540
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 9600
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 9660
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 9720
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 9780
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 9840
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 9900
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 9960
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 10020
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 10080
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 10140
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 10200
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 10260
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 10320
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 10380
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 10440
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 10500
acatttcccc gaaaagtgcc acctgacgtc 10530
<210> 6
<211> 10530
<212> DNA
<213> HF2-BE3表达载体
<400> 6
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaacgg gccctctaga gccaccatga gctcagagac tggcccagtg gctgtggacc 960
ccacattgag acggcggatc gagccccatg agtttgaggt attcttcgat ccgagagagc 1020
tccgcaagga gacctgcctg ctttacgaaa ttaattgggg gggccggcac tccatttggc 1080
gacatacatc acagaacact aacaagcacg tcgaagtcaa cttcatcgag aagttcacga 1140
cagaaagata tttctgtccg aacacaaggt gcagcattac ctggtttctc agctggagcc 1200
catgcggcga atgtagtagg gccatcactg aattcctgtc aaggtatccc cacgtcactc 1260
tgtttattta catcgcaagg ctgtaccacc acgctgaccc ccgcaatcga caaggcctgc 1320
gggatttgat ctcttcaggt gtgactatcc aaattatgac tgagcaggag tcaggatact 1380
gctggagaaa ctttgtgaat tatagcccga gtaatgaagc ccactggcct aggtatcccc 1440
atctgtgggt acgactgtac gttcttgaac tgtactgcat catactgggc ctgcctcctt 1500
gtctcaacat tctgagaagg aagcagccac agctgacatt ctttaccatc gctcttcagt 1560
cttgtcatta ccagcgactg cccccacaca ttctctgggc caccgggttg aaaagcggca 1620
gcgagactcc cgggacctca gagtccgcca cacccgaaag tgataaaaag tattctattg 1680
gtttagccat cggcactaat tccgttggat gggctgtcat aaccgatgaa tacaaagtac 1740
cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa aagaatctta 1800
tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg aaacgaaccg 1860
ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa atttttagca 1920
atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc ttccttgtcg 1980
aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat gaggtggcat 2040
atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac tcaactgata 2100
aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc cgtgggcact 2160
ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg ttcatccagt 2220
tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt ggcgtggatg 2280
cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac ctgatcgcac 2340
aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc tcactaggcc 2400
tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg cagcttagta 2460
aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat cagtatgcgg 2520
acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac atactgagag 2580
ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg tacgatgaac 2640
atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct gagaaatata 2700
aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac ggcggagcga 2760
gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat gggacggaag 2820
agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact ttcgacaacg 2880
gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga aggcaggagg 2940
atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta acctttcgca 3000
taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg atgacaagaa 3060
agtccgaaga aacgattact ccctggaatt ttgaggaagt tgtcgataaa ggtgcgtcag 3120
ctcaatcgtt catcgagagg atgaccgcct ttgacaagaa tttaccgaac gaaaaagtat 3180
tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc acgaaagtta 3240
agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag aagaaagcaa 3300
tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg aaagaggact 3360
actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa gatcgattta 3420
atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag gacttcctgg 3480
ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc ctctttgaag 3540
atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac gataaggtta 3600
tgaaacagtt aaagaggcgt cgctatacgg gctggggagc cttgtcgcgg aaacttatca 3660
acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag agcgacggct 3720
tcgccaatag gaactttatg gccctgatcc atgatgactc tttaaccttc aaagaggata 3780
tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt gcgaatcttg 3840
ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg gatgagctag 3900
ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca cgcgaaaatc 3960
aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata gaagagggta 4020
ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc caattgcaga 4080
acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt gatcaggaac 4140
tggacataaa ccgtttatct gattacgacg tcgatcacat tgtaccccaa tcctttttga 4200
aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga gggaaaagtg 4260
acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg cagctcctaa 4320
atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag aggggtggct 4380
tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc cgcgccatca 4440
caaagcatgt tgcccagata ctagattccc gaatgaatac gaaatacgac gagaacgata 4500
agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg gacttcagaa 4560
aggattttca attctataaa gttagggaga taaataacta ccaccatgcg cacgacgctt 4620
atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta gaaagtgagt 4680
ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa agcgaacagg 4740
agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat ttctttaaga 4800
cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa accaatgggg 4860
agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga aaagttttgt 4920
ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg ttttcaaagg 4980
aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag gactgggacc 5040
cgaaaaagta cggtggcttc gagagcccta cagttgccta ttctgtccta gtagtggcaa 5100
aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg gggataacga 5160
ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg aaaggttaca 5220
aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt gagttagaaa 5280
atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac gaactcgcac 5340
taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag ttgaaaggtt 5400
cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat tatctcgacg 5460
aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat gccaatctgg 5520
acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag caggcggaaa 5580
atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc aagtattttg 5640
acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac gcgacactga 5700
ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag cttgggggtg 5760
actctggtgg ttctactaat ctgtcagata ttattgaaaa ggagaccggt aagcaactgg 5820
ttatccagga atccatcctc atgctcccag aggaggtgga agaagtcatt gggaacaagc 5880
cggaaagcga tatactcgtg cacaccgcct acgacgagag caccgacgag aatgtcatgc 5940
ttctgactag cgacgcccct gaatacaagc cttgggctct ggtcatacag gatagcaacg 6000
gtgagaacaa gattaagatg ctctctggtg gttctcccaa gaagaagagg aaagtctaat 6060
tccaccacac tggactagtg gatccgagct cggtaccaag cttaagttta aaccgctgat 6120
cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 6180
ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 6240
cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 6300
gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg 6360
aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat 6420
taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6480
cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6540
aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6600
ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6660
ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6720
caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6780
cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa 6840
tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 6900
catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag 6960
aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc 7020
catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt 7080
ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg 7140
aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt 7200
cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 7260
cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 7320
aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 7380
tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc 7440
gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 7500
aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 7560
tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 7620
ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 7680
ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 7740
cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca 7800
tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 7860
ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 7920
tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 7980
tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact 8040
ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 8100
accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 8160
atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 8220
gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 8280
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 8340
ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 8400
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 8460
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 8520
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 8580
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 8640
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 8700
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 8760
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 8820
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 8880
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 8940
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 9000
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 9060
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 9120
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 9180
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 9240
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 9300
accgctggta gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 9360
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 9420
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 9480
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 9540
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 9600
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 9660
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 9720
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 9780
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 9840
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 9900
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 9960
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 10020
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 10080
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 10140
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 10200
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 10260
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 10320
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 10380
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 10440
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 10500
acatttcccc gaaaagtgcc acctgacgtc 10530
<210> 7
<211> 5133
<212> DNA
<213> rAPOBEC1:Cas9:UGI mRNA中HF1-BE2的序列
<400> 7
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720
gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780
ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840
aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900
gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960
aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020
ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080
cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140
cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200
gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260
gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320
gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380
tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440
ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500
gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560
aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620
agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680
ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740
gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800
aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860
aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920
gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980
ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040
cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100
cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccctgg 2160
aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220
gcctttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280
tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340
cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400
cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460
tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520
ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580
gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640
aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700
acgggctggg gagccttgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760
aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatggccctg 2820
atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880
ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940
atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000
gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060
agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120
aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180
caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240
gacgtcgatg ccattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300
cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360
aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420
ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480
attaaacgtc agctcgtgga aacccgcgcc atcacaaagc atgttgccca gatactagat 3540
tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600
actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660
gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 3720
ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780
gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840
ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900
atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960
ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020
aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080
gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc 4140
cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200
ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260
aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320
aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380
gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440
tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500
ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560
agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620
cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680
aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740
acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800
gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860
gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920
ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980
gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040
aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100
ggtggttctc ccaagaagaa gaggaaagtc taa 5133
<210> 8
<211> 5133
<212> DNA
<213> rAPOBEC1:Cas9:UGI mRNA中HF1-BE3的序列
<400> 8
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720
gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780
ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840
aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900
gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960
aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020
ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080
cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140
cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200
gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260
gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320
gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380
tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440
ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500
gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560
aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620
agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680
ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740
gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800
aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860
aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920
gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980
ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040
cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100
cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccctgg 2160
aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220
gcctttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280
tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340
cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400
cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460
tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520
ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580
gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640
aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700
acgggctggg gagccttgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760
aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatggccctg 2820
atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880
ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940
atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000
gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060
agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120
aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180
caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240
gacgtcgatc acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300
cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360
aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420
ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480
attaaacgtc agctcgtgga aacccgcgcc atcacaaagc atgttgccca gatactagat 3540
tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600
actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660
gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 3720
ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780
gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840
ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900
atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960
ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020
aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080
gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgatagc 4140
cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200
ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260
aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320
aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380
gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440
tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500
ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560
agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620
cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680
aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740
acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800
gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860
gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920
ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980
gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040
aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100
ggtggttctc ccaagaagaa gaggaaagtc taa 5133
<210> 9
<211> 5133
<212> DNA
<213> rAPOBEC1:Cas9:UGI mRNA中HF2-BE2的序列
<400> 9
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720
gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780
ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840
aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900
gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960
aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020
ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080
cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140
cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200
gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260
gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320
gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380
tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440
ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500
gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560
aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620
agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680
ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740
gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800
aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860
aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920
gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980
ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040
cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100
cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccctgg 2160
aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220
gcctttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280
tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340
cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400
cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460
tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520
ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580
gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640
aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700
acgggctggg gagccttgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760
aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatggccctg 2820
atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880
ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940
atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000
gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060
agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120
aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180
caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240
gacgtcgatg ccattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300
cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360
aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420
ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480
attaaacgtc agctcgtgga aacccgcgcc atcacaaagc atgttgccca gatactagat 3540
tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600
actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660
gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 3720
ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780
gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840
ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900
atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960
ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020
aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080
gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgagagc 4140
cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200
ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260
aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320
aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380
gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440
tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500
ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560
agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620
cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680
aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740
acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800
gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860
gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920
ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980
gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040
aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100
ggtggttctc ccaagaagaa gaggaaagtc taa 5133
<210> 10
<211> 5133
<212> DNA
<213> rAPOBEC1:Cas9:UGI mRNA中HF2-BE3的序列
<400> 10
atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60
catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120
gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180
cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240
aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300
actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360
caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420
atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480
ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540
gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600
ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660
cacattctct gggccaccgg gttgaaaagc ggcagcgaga ctcccgggac ctcagagtcc 720
gccacacccg aaagtgataa aaagtattct attggtttag ccatcggcac taattccgtt 780
ggatgggctg tcataaccga tgaatacaaa gtaccttcaa agaaatttaa ggtgttgggg 840
aacacagacc gtcattcgat taaaaagaat cttatcggtg ccctcctatt cgatagtggc 900
gaaacggcag aggcgactcg cctgaaacga accgctcgga gaaggtatac acgtcgcaag 960
aaccgaatat gttacttaca agaaattttt agcaatgaga tggccaaagt tgacgattct 1020
ttctttcacc gtttggaaga gtccttcctt gtcgaagagg acaagaaaca tgaacggcac 1080
cccatctttg gaaacatagt agatgaggtg gcatatcatg aaaagtaccc aacgatttat 1140
cacctcagaa aaaagctagt tgactcaact gataaagcgg acctgaggtt aatctacttg 1200
gctcttgccc atatgataaa gttccgtggg cactttctca ttgagggtga tctaaatccg 1260
gacaactcgg atgtcgacaa actgttcatc cagttagtac aaacctataa tcagttgttt 1320
gaagagaacc ctataaatgc aagtggcgtg gatgcgaagg ctattcttag cgcccgcctc 1380
tctaaatccc gacggctaga aaacctgatc gcacaattac ccggagagaa gaaaaatggg 1440
ttgttcggta accttatagc gctctcacta ggcctgacac caaattttaa gtcgaacttc 1500
gacttagctg aagatgccaa attgcagctt agtaaggaca cgtacgatga cgatctcgac 1560
aatctactgg cacaaattgg agatcagtat gcggacttat ttttggctgc caaaaacctt 1620
agcgatgcaa tcctcctatc tgacatactg agagttaata ctgagattac caaggcgccg 1680
ttatccgctt caatgatcaa aaggtacgat gaacatcacc aagacttgac acttctcaag 1740
gccctagtcc gtcagcaact gcctgagaaa tataaggaaa tattctttga tcagtcgaaa 1800
aacgggtacg caggttatat tgacggcgga gcgagtcaag aggaattcta caagtttatc 1860
aaacccatat tagagaagat ggatgggacg gaagagttgc ttgtaaaact caatcgcgaa 1920
gatctactgc gaaagcagcg gactttcgac aacggtagca ttccacatca aatccactta 1980
ggcgaattgc atgctatact tagaaggcag gaggattttt atccgttcct caaagacaat 2040
cgtgaaaaga ttgagaaaat cctaaccttt cgcatacctt actatgtggg acccctggcc 2100
cgagggaact ctcggttcgc atggatgaca agaaagtccg aagaaacgat tactccctgg 2160
aattttgagg aagttgtcga taaaggtgcg tcagctcaat cgttcatcga gaggatgacc 2220
gcctttgaca agaatttacc gaacgaaaaa gtattgccta agcacagttt actttacgag 2280
tatttcacag tgtacaatga actcacgaaa gttaagtatg tcactgaggg catgcgtaaa 2340
cccgcctttc taagcggaga acagaagaaa gcaatagtag atctgttatt caagaccaac 2400
cgcaaagtga cagttaagca attgaaagag gactacttta agaaaattga atgcttcgat 2460
tctgtcgaga tctccggggt agaagatcga tttaatgcgt cacttggtac gtatcatgac 2520
ctcctaaaga taattaaaga taaggacttc ctggataacg aagagaatga agatatctta 2580
gaagatatag tgttgactct taccctcttt gaagatcggg aaatgattga ggaaagacta 2640
aaaacatacg ctcacctgtt cgacgataag gttatgaaac agttaaagag gcgtcgctat 2700
acgggctggg gagccttgtc gcggaaactt atcaacggga taagagacaa gcaaagtggt 2760
aaaactattc tcgattttct aaagagcgac ggcttcgcca ataggaactt tatggccctg 2820
atccatgatg actctttaac cttcaaagag gatatacaaa aggcacaggt ttccggacaa 2880
ggggactcat tgcacgaaca tattgcgaat cttgctggtt cgccagccat caaaaagggc 2940
atactccaga cagtcaaagt agtggatgag ctagttaagg tcatgggacg tcacaaaccg 3000
gaaaacattg taatcgagat ggcacgcgaa aatcaaacga ctcagaaggg gcaaaaaaac 3060
agtcgagagc ggatgaagag aatagaagag ggtattaaag aactgggcag ccagatctta 3120
aaggagcatc ctgtggaaaa tacccaattg cagaacgaga aactttacct ctattaccta 3180
caaaatggaa gggacatgta tgttgatcag gaactggaca taaaccgttt atctgattac 3240
gacgtcgatc acattgtacc ccaatccttt ttgaaggacg attcaatcga caataaagtg 3300
cttacacgct cggataagaa ccgagggaaa agtgacaatg ttccaagcga ggaagtcgta 3360
aagaaaatga agaactattg gcggcagctc ctaaatgcga aactgataac gcaaagaaag 3420
ttcgataact taactaaagc tgagaggggt ggcttgtctg aacttgacaa ggccggattt 3480
attaaacgtc agctcgtgga aacccgcgcc atcacaaagc atgttgccca gatactagat 3540
tcccgaatga atacgaaata cgacgagaac gataagctga ttcgggaagt caaagtaatc 3600
actttaaagt caaaattggt gtcggacttc agaaaggatt ttcaattcta taaagttagg 3660
gagataaata actaccacca tgcgcacgac gcttatctta atgccgtcgt agggaccgca 3720
ctcattaaga aatacccgaa gctagaaagt gagtttgtgt atggtgatta caaagtttat 3780
gacgtccgta agatgatcgc gaaaagcgaa caggagatag gcaaggctac agccaaatac 3840
ttcttttatt ctaacattat gaatttcttt aagacggaaa tcactctggc aaacggagag 3900
atacgcaaac gacctttaat tgaaaccaat ggggagacag gtgaaatcgt atgggataag 3960
ggccgggact tcgcgacggt gagaaaagtt ttgtccatgc cccaagtcaa catagtaaag 4020
aaaactgagg tgcagaccgg agggttttca aaggaatcga ttcttccaaa aaggaatagt 4080
gataagctca tcgctcgtaa aaaggactgg gacccgaaaa agtacggtgg cttcgagagc 4140
cctacagttg cctattctgt cctagtagtg gcaaaagttg agaagggaaa atccaagaaa 4200
ctgaagtcag tcaaagaatt attggggata acgattatgg agcgctcgtc ttttgaaaag 4260
aaccccatcg acttccttga ggcgaaaggt tacaaggaag taaaaaagga tctcataatt 4320
aaactaccaa agtatagtct gtttgagtta gaaaatggcc gaaaacggat gttggctagc 4380
gccggagagc ttcaaaaggg gaacgaactc gcactaccgt ctaaatacgt gaatttcctg 4440
tatttagcgt cccattacga gaagttgaaa ggttcacctg aagataacga acagaagcaa 4500
ctttttgttg agcagcacaa acattatctc gacgaaatca tagagcaaat ttcggaattc 4560
agtaagagag tcatcctagc tgatgccaat ctggacaaag tattaagcgc atacaacaag 4620
cacagggata aacccatacg tgagcaggcg gaaaatatta tccatttgtt tactcttacc 4680
aacctcggcg ctccagccgc attcaagtat tttgacacaa cgatagatcg caaacgatac 4740
acttctacca aggaggtgct agacgcgaca ctgattcacc aatccatcac gggattatat 4800
gaaactcgga tagatttgtc acagcttggg ggtgactctg gtggttctac taatctgtca 4860
gatattattg aaaaggagac cggtaagcaa ctggttatcc aggaatccat cctcatgctc 4920
ccagaggagg tggaagaagt cattgggaac aagccggaaa gcgatatact cgtgcacacc 4980
gcctacgacg agagcaccga cgagaatgtc atgcttctga ctagcgacgc ccctgaatac 5040
aagccttggg ctctggtcat acaggatagc aacggtgaga acaagattaa gatgctctct 5100
ggtggttctc ccaagaagaa gaggaaagtc taa 5133
<210> 11
<211> 1710
<212> PRT
<213> rAPOBEC1:Cas9:UGI蛋白中HF1-BE2的序列
<400> 11
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
225 230 235 240
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
245 250 255
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
260 265 270
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
275 280 285
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
290 295 300
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
305 310 315 320
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
325 330 335
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
340 345 350
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
355 360 365
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
370 375 380
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
385 390 395 400
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
405 410 415
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
420 425 430
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
435 440 445
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
450 455 460
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
465 470 475 480
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
485 490 495
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
500 505 510
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
515 520 525
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
530 535 540
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
545 550 555 560
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
565 570 575
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
580 585 590
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
595 600 605
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
610 615 620
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
625 630 635 640
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
645 650 655
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
660 665 670
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
675 680 685
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
690 695 700
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
705 710 715 720
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
725 730 735
Glu Arg Met Thr Ala Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
740 745 750
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
755 760 765
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
770 775 780
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
785 790 795 800
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
805 810 815
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
820 825 830
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
835 840 845
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
850 855 860
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu
865 870 875 880
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
885 890 895
Arg Arg Arg Tyr Thr Gly Trp Gly Ala Leu Ser Arg Lys Leu Ile Asn
900 905 910
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys
915 920 925
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Ala Leu Ile His Asp Asp
930 935 940
Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln
945 950 955 960
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
965 970 975
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val
980 985 990
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala
995 1000 1005
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
1010 1015 1020
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
1025 1030 1035
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
1040 1045 1050
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1055 1060 1065
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
1070 1075 1080
Ala Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn
1085 1090 1095
Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
1100 1105 1110
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg
1115 1120 1125
Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
1130 1135 1140
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala
1145 1150 1155
Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Ala Ile Thr Lys
1160 1165 1170
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
1175 1180 1185
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
1190 1195 1200
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys
1205 1210 1215
Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1220 1225 1230
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu
1235 1240 1245
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg
1250 1255 1260
Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala
1265 1270 1275
Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1280 1285 1290
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1295 1300 1305
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp
1310 1315 1320
Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile
1325 1330 1335
Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser
1340 1345 1350
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys
1355 1360 1365
Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val
1370 1375 1380
Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
1385 1390 1395
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1400 1405 1410
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1415 1420 1425
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro
1430 1435 1440
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu
1445 1450 1455
Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1460 1465 1470
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys
1475 1480 1485
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
1490 1495 1500
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
1505 1510 1515
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1520 1525 1530
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1535 1540 1545
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly
1550 1555 1560
Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys
1565 1570 1575
Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
1580 1585 1590
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln
1595 1600 1605
Leu Gly Gly Asp Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile
1610 1615 1620
Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu
1625 1630 1635
Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu
1640 1645 1650
Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu
1655 1660 1665
Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp
1670 1675 1680
Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met
1685 1690 1695
Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1700 1705 1710
<210> 12
<211> 1710
<212> PRT
<213> rAPOBEC1:Cas9:UGI蛋白中HF1-BE3的序列
<400> 12
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
225 230 235 240
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
245 250 255
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
260 265 270
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
275 280 285
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
290 295 300
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
305 310 315 320
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
325 330 335
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
340 345 350
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
355 360 365
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
370 375 380
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
385 390 395 400
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
405 410 415
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
420 425 430
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
435 440 445
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
450 455 460
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
465 470 475 480
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
485 490 495
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
500 505 510
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
515 520 525
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
530 535 540
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
545 550 555 560
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
565 570 575
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
580 585 590
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
595 600 605
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
610 615 620
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
625 630 635 640
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
645 650 655
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
660 665 670
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
675 680 685
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
690 695 700
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
705 710 715 720
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
725 730 735
Glu Arg Met Thr Ala Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
740 745 750
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
755 760 765
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
770 775 780
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
785 790 795 800
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
805 810 815
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
820 825 830
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
835 840 845
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
850 855 860
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu
865 870 875 880
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
885 890 895
Arg Arg Arg Tyr Thr Gly Trp Gly Ala Leu Ser Arg Lys Leu Ile Asn
900 905 910
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys
915 920 925
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Ala Leu Ile His Asp Asp
930 935 940
Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln
945 950 955 960
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
965 970 975
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val
980 985 990
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala
995 1000 1005
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
1010 1015 1020
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
1025 1030 1035
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
1040 1045 1050
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1055 1060 1065
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
1070 1075 1080
His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn
1085 1090 1095
Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
1100 1105 1110
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg
1115 1120 1125
Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
1130 1135 1140
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala
1145 1150 1155
Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Ala Ile Thr Lys
1160 1165 1170
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
1175 1180 1185
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
1190 1195 1200
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys
1205 1210 1215
Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1220 1225 1230
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu
1235 1240 1245
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg
1250 1255 1260
Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala
1265 1270 1275
Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1280 1285 1290
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1295 1300 1305
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp
1310 1315 1320
Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile
1325 1330 1335
Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser
1340 1345 1350
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys
1355 1360 1365
Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val
1370 1375 1380
Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
1385 1390 1395
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1400 1405 1410
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1415 1420 1425
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro
1430 1435 1440
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu
1445 1450 1455
Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1460 1465 1470
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys
1475 1480 1485
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
1490 1495 1500
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
1505 1510 1515
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1520 1525 1530
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1535 1540 1545
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly
1550 1555 1560
Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys
1565 1570 1575
Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
1580 1585 1590
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln
1595 1600 1605
Leu Gly Gly Asp Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile
1610 1615 1620
Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu
1625 1630 1635
Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu
1640 1645 1650
Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu
1655 1660 1665
Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp
1670 1675 1680
Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met
1685 1690 1695
Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1700 1705 1710
<210> 13
<211> 1710
<212> PRT
<213> rAPOBEC1:Cas9:UGI蛋白中HF2-BE2的序列
<400> 13
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
225 230 235 240
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
245 250 255
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
260 265 270
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
275 280 285
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
290 295 300
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
305 310 315 320
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
325 330 335
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
340 345 350
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
355 360 365
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
370 375 380
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
385 390 395 400
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
405 410 415
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
420 425 430
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
435 440 445
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
450 455 460
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
465 470 475 480
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
485 490 495
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
500 505 510
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
515 520 525
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
530 535 540
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
545 550 555 560
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
565 570 575
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
580 585 590
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
595 600 605
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
610 615 620
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
625 630 635 640
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
645 650 655
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
660 665 670
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
675 680 685
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
690 695 700
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
705 710 715 720
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
725 730 735
Glu Arg Met Thr Ala Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
740 745 750
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
755 760 765
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
770 775 780
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
785 790 795 800
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
805 810 815
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
820 825 830
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
835 840 845
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
850 855 860
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu
865 870 875 880
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
885 890 895
Arg Arg Arg Tyr Thr Gly Trp Gly Ala Leu Ser Arg Lys Leu Ile Asn
900 905 910
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys
915 920 925
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Ala Leu Ile His Asp Asp
930 935 940
Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln
945 950 955 960
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
965 970 975
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val
980 985 990
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala
995 1000 1005
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
1010 1015 1020
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
1025 1030 1035
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
1040 1045 1050
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1055 1060 1065
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
1070 1075 1080
Ala Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn
1085 1090 1095
Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
1100 1105 1110
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg
1115 1120 1125
Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
1130 1135 1140
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala
1145 1150 1155
Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Ala Ile Thr Lys
1160 1165 1170
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
1175 1180 1185
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
1190 1195 1200
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys
1205 1210 1215
Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1220 1225 1230
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu
1235 1240 1245
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg
1250 1255 1260
Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala
1265 1270 1275
Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1280 1285 1290
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1295 1300 1305
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp
1310 1315 1320
Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile
1325 1330 1335
Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser
1340 1345 1350
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys
1355 1360 1365
Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Glu Ser Pro Thr Val
1370 1375 1380
Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
1385 1390 1395
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1400 1405 1410
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1415 1420 1425
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro
1430 1435 1440
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu
1445 1450 1455
Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1460 1465 1470
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys
1475 1480 1485
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
1490 1495 1500
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
1505 1510 1515
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1520 1525 1530
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1535 1540 1545
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly
1550 1555 1560
Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys
1565 1570 1575
Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
1580 1585 1590
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln
1595 1600 1605
Leu Gly Gly Asp Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile
1610 1615 1620
Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu
1625 1630 1635
Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu
1640 1645 1650
Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu
1655 1660 1665
Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp
1670 1675 1680
Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met
1685 1690 1695
Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1700 1705 1710
<210> 14
<211> 1710
<212> PRT
<213> rAPOBEC1:Cas9:UGI蛋白中HF2-BE3的序列
<400> 14
Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg
1 5 10 15
Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu
20 25 30
Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His
35 40 45
Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val
50 55 60
Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr
65 70 75 80
Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys
85 90 95
Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu
100 105 110
Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg
115 120 125
Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met
130 135 140
Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser
145 150 155 160
Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg
165 170 175
Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys
180 185 190
Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile
195 200 205
Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp
210 215 220
Ala Thr Gly Leu Lys Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser
225 230 235 240
Ala Thr Pro Glu Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
245 250 255
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
260 265 270
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
275 280 285
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
290 295 300
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
305 310 315 320
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
325 330 335
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
340 345 350
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
355 360 365
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
370 375 380
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
385 390 395 400
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
405 410 415
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
420 425 430
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
435 440 445
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
450 455 460
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
465 470 475 480
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
485 490 495
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
500 505 510
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
515 520 525
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
530 535 540
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
545 550 555 560
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
565 570 575
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
580 585 590
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
595 600 605
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
610 615 620
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
625 630 635 640
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
645 650 655
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
660 665 670
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
675 680 685
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
690 695 700
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
705 710 715 720
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
725 730 735
Glu Arg Met Thr Ala Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
740 745 750
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
755 760 765
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
770 775 780
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
785 790 795 800
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
805 810 815
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
820 825 830
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
835 840 845
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
850 855 860
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu
865 870 875 880
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
885 890 895
Arg Arg Arg Tyr Thr Gly Trp Gly Ala Leu Ser Arg Lys Leu Ile Asn
900 905 910
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys
915 920 925
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Ala Leu Ile His Asp Asp
930 935 940
Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln
945 950 955 960
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
965 970 975
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val
980 985 990
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala
995 1000 1005
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu
1010 1015 1020
Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln
1025 1030 1035
Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
1040 1045 1050
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val
1055 1060 1065
Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp
1070 1075 1080
His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn
1085 1090 1095
Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn
1100 1105 1110
Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg
1115 1120 1125
Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn
1130 1135 1140
Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala
1145 1150 1155
Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Ala Ile Thr Lys
1160 1165 1170
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
1175 1180 1185
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys
1190 1195 1200
Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys
1205 1210 1215
Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
1220 1225 1230
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu
1235 1240 1245
Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg
1250 1255 1260
Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala
1265 1270 1275
Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
1280 1285 1290
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu
1295 1300 1305
Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp
1310 1315 1320
Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile
1325 1330 1335
Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser
1340 1345 1350
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys
1355 1360 1365
Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Glu Ser Pro Thr Val
1370 1375 1380
Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser
1385 1390 1395
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1400 1405 1410
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1415 1420 1425
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro
1430 1435 1440
Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu
1445 1450 1455
Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
1460 1465 1470
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys
1475 1480 1485
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val
1490 1495 1500
Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser
1505 1510 1515
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
1520 1525 1530
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu
1535 1540 1545
Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly
1550 1555 1560
Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys
1565 1570 1575
Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His
1580 1585 1590
Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln
1595 1600 1605
Leu Gly Gly Asp Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile
1610 1615 1620
Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu
1625 1630 1635
Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu
1640 1645 1650
Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu
1655 1660 1665
Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp
1670 1675 1680
Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met
1685 1690 1695
Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val
1700 1705 1710
<210> 15
<211> 96
<212> DNA
<213> gRNA的RNA序列
<220>
<221> misc_feature
<222> (1)..(20)
<223> n is a, c, g, t or u
<400> 15
nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 96
<210> 16
<211> 34
<212> DNA
<213> mRNA上游引物
<400> 16
aaagcggccg caatgagctc agagactggc ccag 34
<210> 17
<211> 35
<212> DNA
<213> mRNA下游引物
<400> 17
aaaggcgcgc cagactttcc tcttcttctt gggag 35

Claims (10)

1.一套基于化脓性链球菌的碱基编辑系统，其特征在于，所述碱基编辑系统由rAPOBEC1:Cas9:UGI和gRNA表达载体两部分组分组成；所述rAPOBEC1:Cas9:UGI为rAPOBEC1:Cas9:UGI表达载体、rAPOBEC1:Cas9:UGI mRNA或rAPOBEC1:Cas9:UGI蛋白；所述rAPOBEC1:Cas9:UGI表达载体是通过基因合成和分子克隆的方法将rAPOBEC1:Cas9:UGI的编码基因克隆到pcDNA3.1（-）载体中得到；所述gRNA表达载体是通过酶切连接的方式将SEQID NO.15所示RNA序列克隆到包含T7启动子的pDR274载体中，并将其线性化，然后再以该线性化载体为模板制备得到。
2. 根据权利要求1所述基于化脓性链球菌的碱基编辑系统，其特征在于，所述rAPOBEC1:Cas9:UGI 表达载体为HF1-BE2表达载体、HF1-BE3表达载体、HF2-BE2表达载体或HF2-BE3表达载体，序列分别如SEQ ID NO.3～6所示。
3. 根据权利要求1所述基于化脓性链球菌的碱基编辑系统，其特征在于，所述rAPOBEC1:Cas9:UGI mRNA是用限制性内切酶切割所述rAPOBEC1:Cas9:UGI表达载体，酶切产物纯化获得转录模板DNA，然后转录生产mRNA。
4. 根据权利要求3所述基于化脓性链球菌的碱基编辑系统，其特征在于，所述rAPOBEC1:Cas9:UGI mRNA为HF1-BE2 mRNA、HF1-BE3 mRNA、HF2-BE2 mRNA或HF2-BE3 mRNA，序列分别如SEQ ID NO.7～10所示。
5.根据权利要求1所述基于化脓性链球菌的碱基编辑系统，其特征在于，所述rAPOBEC1:Cas9:UGI蛋白是先通过PCR的方式将rAPOBEC1:Cas9:UGI融合基因克隆到pET28a载体中，然后将其转化到大肠杆菌中表达并纯化获得。
6. 根据权利要求5所述基于化脓性链球菌的碱基编辑系统，其特征在于，所述APOBEC1:Cas9:UGI 蛋白为HF1-BE2蛋白、HF1-BE3蛋白、HF2-BE2蛋白或HF2-BE3蛋白，序列分别如SEQ ID NO.11～14所示。
7. 根据权利要求1所述基于化脓性链球菌的碱基编辑系统，其特征在于，构建方法如下：
S1.构建rAPOBEC1:Cas9:UGI
S11.制备rAPOBEC1:Cas9:UGI表达载体，为HF1-BE2表达载体、HF1-BE3表达载体、HF2-BE2表达载体或HF2-BE3表达载体，序列分别如SEQ ID NO.3～6所示；
S12.制备化脓性链球菌rAPOBEC1:Cas9:UGI mRNA：以线性化的HF1-BE2表达载体、HF1-BE3表达载体、HF2-BE2表达载体或HF2-BE3的表达载体为模板，转录生产rAPOBEC1:Cas9:UGI mRNA，然后纯化并用不含核酸酶的水洗脱；
S13.制备APOBEC1:Cas9:UGI 蛋白：以HF1-BE2表达载体、HF1-BE3表达载体、HF2-BE2表达载体或HF2-BE3表达载体为模板，利用SEQ ID NO.16～17所示mRNA上下游引物进行扩增，然后克隆到用NotI和AscI酶切过的pET28a载体，然后转化到大肠杆菌中，通过诱导表达，层析柱纯化获得rAPOBEC1:Cas9:UGI蛋白；
S2.构建gRNA表达载体
S21.制备化脓性链球菌gRNA的转录载体：将SEQ ID NO.1和SEQ ID NO.2所示的gRNA上游引物和gRNA下游引物退火成双链DNA，同时用BasI酶切pDR274载体，然后将退火产物克隆到该载体中，获得gRNA的转录载体；然后将转录载体用DraI酶切，纯化后用不含核酸酶的水洗脱，获得包含T7启动子的化脓性链球菌gRNA转录模板DNA；
S22.制备化脓性链球菌gRNA：以包含T7启动子的化脓性链球菌gRNA转录模板DNA为模板，转录生产化脓性链球菌gRNA。
8.权利要求1～7任一所述基于化脓性链球菌的碱基编辑系统在哺乳动物细胞和/或胚胎的基因编辑中的应用。
9.根据权利要求8所述的应用，其特征在于，所述基因编辑是指在哺乳动物细胞和/或胚胎中进行单基因敲除、多基因敲除和/或基因突变。
10. 根据权利要求8所述的应用，其特征在于，应用的方法是将rAPOBEC1:Cas9:UGI表达载体、rAPOBEC1:Cas9:UGI mRNA或rAPOBEC1:Cas9:UGI蛋白，以及gRNA导入到哺乳动物细胞或胚胎中。
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