CN110467679B - 一种融合蛋白、碱基编辑工具和方法及其应用 - Google Patents

一种融合蛋白、碱基编辑工具和方法及其应用 Download PDF

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CN110467679B
CN110467679B CN201910725037.3A CN201910725037A CN110467679B CN 110467679 B CN110467679 B CN 110467679B CN 201910725037 A CN201910725037 A CN 201910725037A CN 110467679 B CN110467679 B CN 110467679B
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乔云波
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Abstract

本发明公开了一种基因编辑工具，所述编辑工具为将碱基A转换为G的N‑ABEmax‑NG+C‑ABEmax‑NG编辑系统，所述编辑系统包括融合蛋白、sgRNA和sgRNA包装载体，以及腺病毒包装系统。本发明的基因编辑工具可以识别NG作为PAM，拓宽了碱基编辑的靶向范围，并且本发明的碱基编辑工具可适用于腺病毒的包装要求。

Description

一种融合蛋白、碱基编辑工具和方法及其应用
技术领域
本发明涉及基因编辑技术领域，尤其是一种基于腺病毒的碱基编辑工具和方法及其应用。
背景技术
基因编辑是通过在DNA上特定位点引入序列改变，达到基因序列改变或插入的技术手段。目前CRISPR/Cas9是应用最广的基因编辑技术1。该系统操作简单，仅需通过sgRNA的靶向序列就能在靶向位点上进行基因编辑，该技术被广泛应用于基因功能研究、疾病模拟以及基因治疗等。CRISPR/Cas9的原理是，在sgRNA的引导下Cas9到达指定DNA区域发挥酶切活性，CRISPR/Cas9系统的靶向识别需要靶位点旁边具有前间隔序列邻近基序(protospacer adjacent motif,PAM)，然后对PAM上游3bp和4bp间进行切割，切割后造成DNA的双链断裂(DSB)激发自身的DNA修复机制。基于CRISPR/Cas9的发现使得基因操作非常简易，但是对内源基因的精准编辑还是个巨大的问题，通过NHEJ(Non-Homologous EndJoin，非同源重组末端修复)随机引入插入或缺失只能引入随机突变，而切割后提供同源重组的载体或者单链DNA的方法，效率低且耗时。同时，Cas9切割造成的DSB可能引起基因组的大片段缺失和影响基因组稳定性。
鉴于上述问题，哈佛大学的David Liu等利用部分切割活性缺失的Cas9-D10Anickase(nCas9)融合脱氨酶的方法可以实现不造成DSB的情况下对基因组单碱基进行点突变(C-to-T或A-to-G)，目前开发的碱基编辑工具包括胞嘧啶碱基编辑工具(CytosineBase Editor，CBE)和腺嘌呤碱基编辑工具(Adenine Base Editor，ABE)两种2,3。其中胞嘧啶碱基编辑工具融合了nCas9和rat APOBEC1，腺嘌呤碱基编辑工具融合了nCas9和ecTad-ecTadA*二聚体片段。具体原理是，nCas9的融合蛋白在sgRNA的引导下到达靶向位点并与sgRNA互补的DNA链进行结合，胞嘧啶脱氨酶/腺嘌呤脱氨酶对sgRNA范围内的胞嘧啶/腺嘌呤进行脱氨，然后根据碱基互补配对原则，在DNA复制过程中最终达到C-to-T或A-to-G的目的。在经过核定位信号以及密码子的优化后，目前ancBE4max和ABEmax的效率最高，两者识别的PAM为NG，BE4max对应的编辑窗口是sgRNA 5’端的4-8位，ABEmax对应的编辑窗口是sgRNA 5’端的4-7位。然而，来自酿脓链球菌(Streptococcus pyogenes)的Cas9(SpCas9)仅识别NGG序列的PAM，大幅限制了基因组中能够被靶向的范围。来自日本东京大学的濡木教授等人构建出一种SpCas9变体(SpCas9-NG)，它能够识别NG序列作为PAM，我们基于此构建的ancBE4ma-NG和ABEmax-NG得到了能够识别NG PAM的碱基编辑器，可以大幅扩展碱基编辑的使用范围而不受PAM限制。
单碱基基因突变，可导致发育、癌症等多种疾病，因此利用碱基编辑工具可修饰疾病的点突变以达到治疗或者缓解疾病的目的。目前，最为接受的体内基因编辑工具为腺病毒，然而碱基编辑器的质粒大小远远超出腺病毒的包装范围(4.7Kb)。因此，如何利用腺病毒进行体内碱基编辑是目前的一项重大科学难题。
发明内容
基于上述问题，本发明的目的在于克服上述现有技术的不足之处而提供一种新的组合型胞嘧啶/腺嘌呤碱基编辑工具，可以识别NG作为PAM，拓宽了碱基编辑的靶向范围，并且本发明的碱基编辑工具可适用于腺病毒的包装要求，可获得高滴度的腺病毒。
为实现上述目的，本发明采取的技术方案包括如下几个方面：
在第一个方面，本发明提供了一种融合蛋白，包括蛋白质内含子intein N片段或C片段和碱基编辑器的N端或C端片段，所述碱基编辑器为多肽ancBE4max-NG或ABEmax-NG，所述多肽ancBE4max-NG包括APOBEC1多肽和SpCas9-NG D10A nickase多肽，所述多肽ABEmax-NG包括ecTad-ecTadA*二聚体多肽和SpCas9-NG D10A nickase多肽。需要说明的是，在下文的SEQ ID NO.17中分别示出了APOBEC1、SpCas9-NG D10A nickase、2*UGI相应的核苷酸序列，其中，APOBEC1碱基序列采用加粗和下划线显示，SpCas9-NG D10A nickase的碱基序列采用加粗和斜体示出，2*UGI相应的碱基序列采用下划线示出；在下文的SEQ ID NO.18中分别示出了ecTad-ecTadA*和SpCas9-NG相应的核苷酸序列，其中，ecTad-ecTadA*相应的碱基序列加粗和下划线示出，SpCas9-NG相应的碱基序列采用加粗和斜体示出。
在一些实施方案中，所述intein N片段的氨基酸序列为：
a)如SEQ ID NO.1所示的intein-N氨基酸序列；或
b)与SEQ ID NO.1具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上序列一致性的氨基酸序列、且具有a)所限定的氨基酸序列的功能，优选为能够作为内含子进行氨基酸序列剪切和拼接的功能；
所述intein C片段的氨基酸序列为：
c)如SEQ ID NO.2所示的intein-C氨基酸序列；或
d)与SEQ ID NO.2相比具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上序列一致性的氨基酸序列、且具有c)所限定的氨基酸序列的功能，优选为能够作为内含子进行氨基酸序列剪切和拼接的功能。
在一些实施方案中，所述碱基编辑器的N端片段为
e)由APOBEC1多肽和SpCas9-NG D10A nickase N端第2～573个氨基酸组成的多肽融合而成；
f)SEQ ID NO.3所示的氨基酸序列；或
g)与SEQ ID NO.3所示的氨基酸序列相比具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上序列一致性的氨基酸序列，且具有e)或f)所限定的氨基酸序列所具有的功能。
在一些实施方案中，所述碱基编辑器的C端片段为
h)由SpCas9-NG D10A nickase片段C端第574～1368个氨基酸组成的多肽与2*UGI、3*FLAG、BPNLS多肽序列依次融合而成；
i)SEQ ID NO.4所示的氨基酸序列；或
j)与SEQ ID NO.4所示的氨基酸序列相比具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上序列一致性的氨基酸序列，且具有h)或i)所限定的氨基酸序列所具有的功能，优选具有蛋白质水平剪切融合所得全长蛋白的胞嘧啶脱氨酶功能，更优选为能够识别NG作为PAM，N表示任意碱基。
在一些实施方案中，所述碱基编辑器的N端片段为
k)由ecTad-ecTadA*二聚体多肽片段和SpCas9-NG D10A nickase片段N端第2～573个氨基酸组成的多肽融合而成；
l)SEQ ID NO.5所示的氨基酸序列；或
m)与SEQ ID NO.5所示的氨基酸序列相比具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上序列一致性的氨基酸序列，且具有k)或l)所限定的功能。
在一些实施方案中，所述碱基编辑器的C端片段为
n)由SpCas9-NG D10A nickase多肽C端第574～1368个氨基酸组成的多肽与多肽3*FLAG、BPNLS依次融合而成；
o)SEQ ID NO.6所示的氨基酸序列；或
p)与SEQ ID NO.6所示的氨基酸序列相比具有90％、91％、92％、93％、94％、95％、96％、97％、98％或99％以上序列一致性的氨基酸序列，且具有n)或o)所限定的功能，优选具有蛋白质水平剪切融合所得全长蛋白的腺嘌呤脱氨酶功能，更优选为能够识别NG作为PAM。
在一些实施方案中，所述融合蛋白自N端至C端依次包括APOBEC1多肽片段、SpCas9-NG D10A nickase的N端第2～573个氨基酸组成的多肽片段和N-intein 多肽。
在一些实施方案中，所述融合蛋白自N端至C端依次包括C-intein多肽片段、SpCas9-NG D10A nickase的C端第574～1368个氨基酸组成的多肽片段、2*UGI多肽、3*FLAG多肽和BPNLS多肽。
在一些实施方案中，所述融合蛋白自N端至C端依次包括ecTadA-ecTadA*二聚体多肽片段、SpCas9-NG D10A nickase的N端第2～573个氨基酸组成的多肽片段和N-intein多肽。
在一些实施方案中，所述融合蛋白自N端至C端依次包括C-intein多肽、SpCas9-NGD10A nickase的C端第574～1368个氨基酸组成的多肽片段、3*FLAG多肽、BPNLS多肽。
在一些实施方案中，所述融合蛋白还包括核定位信号多肽片段，
优选的，所述核定位信号多肽片段位于上述融合蛋白的N端和/或C端，
更优选的，所述核定位信号多肽片段的氨基酸序列如SEQ ID NO.7所示。
在一些实施方案中，所述融合蛋白氨基酸序列如SEQ ID NO.8～11任一项所示。
在第二个方面，本发明提供了一种腺病毒包装系统，包括上述的融合蛋白相应的氨基酸序列或/和所述融合蛋白对应的如SEQ ID NO.12～15任一项所示的核苷酸编码序列。需要说明的是，SEQ ID NO.8～11的氨基酸序列与SEQ ID NO.12～15的核苷酸序列是一一对应的。
在第三个方面，本发明提供了一种基因编辑工具，包括上述的腺病毒包装系统、sgRNA以及sgRNA包装载体，所述载体的核苷酸序列如SEQ ID NO.16所示。
在第四个方面，本发明提供了一种细胞表达系统，其中含有上述的基因编辑工具，所述细胞为宿主细胞，宿主细胞优选真核细胞或原核细胞，
更优选小鼠细胞或人细胞；
更优选为小鼠脑神经瘤细胞、人胚胎肾细胞或人宫颈癌细胞；
更优选为N2a细胞、HEK293FT细胞或Hela细胞。
在第五个方面，本发明提供了上述的融合蛋白、腺病毒包装系统、基因编辑工具或细胞表达系统在基因编辑中的应用。
在第六个方面，本发明提供了一种基于腺病毒的基因编辑方法，包括步骤：基于上述融合蛋白、腺病毒包装系统、基因编辑工具或细胞表达系统进行体外或体内基因编辑。
综上所述，本发明的有益效果为：
本发明提供了一种融合蛋白，所述融合蛋白是一种新的组合型胞嘧啶/腺嘌呤碱基编辑工具，可以识别NG作为PAM，拓宽了碱基编辑的靶向范围，并且本发明的碱基编辑工具可适用于腺病毒的包装要求，可获得高滴度的腺病毒，并在此基础上完成了本发明。
附图说明
图1为构建获得的N-ancBE4max-NG和C-ancBE4max-NG所示质粒结构示意图；
图2为构建获得的N-ABEmax-NG和C-ABEmax-NG所示质粒结构示意图；
图3为sgRNA的腺病毒(AAV)包装载体结构示意图；
图4为本发明实施例3实验结果示意图，其中，a为
Figure GDA0002935979890000061
Figure GDA0002935979890000062
共同转染293T细胞以后的Sanger测序结果，第一列为靶向DNA序列示意图；第二列实验结果为未转染的负对照，第三列为靶向基因编辑的实验结果，箭头指示为C-to-T编辑位置；b为
Figure GDA0002935979890000063
Figure GDA0002935979890000064
共同转染293T细胞以后的Sanger测序结果，第一列为靶向DNA序列示意图；第二列实验结果为未转染的负对照，第三列为靶向基因编辑的实验结果，箭头指示为A-to-G编辑位置；
图5为本发明实施例4实验结果示意图，其中，a为N-ABEmax-NG病毒滴度测试扩增曲线和三种稀释浓度滴度测试结果；b为C-ABEmax-NG病毒滴度测试扩增曲线和三种稀释浓度滴度测试结果；c为AAV-sgRNA病毒滴度测试扩增曲线和三种稀释浓度滴度测试结果。
具体实施方式
本发明通过将识别NGG的SpCas9或NG PAM的SpCas9-NG与ancBE4max或ABEmax相结合，得到ancBE4max-NG和ABEmax-NG四种编辑工具，并且利用
Figure GDA0002935979890000071
的可在蛋白质水平进行剪切和拼接的特性，将全长的碱基编辑器在以Cas9573-574氨基酸中间位置分开，表达于两个腺病毒载体。经检测，分开表达的基因编辑工具，可获得良好的基因编辑效果，并且可获得高滴度的腺病毒。
本发明涉及生物技术领域，特别是涉及碱基编辑工具的体内基因编辑和基因突变校正用途。本发明提供两种融合蛋白，包括蛋白质内含子intein片段和碱基编辑器的N端或C端片段，所述intein片段包括N端和C端序列，所述碱基编辑器包括ancBE4max-NG和ABEmax-NG，共获得N-ancBE4max-NG、C-ancBE4max-NG、N-ABEmax-NG、C-ABEmax-NG四种AAV表达质粒。本发明提供的融合蛋白可以缩小碱基编辑工具的大小，使其适用于腺病毒(AAV)的包装范围，从而获得高滴度的碱基编辑工具的腺病毒，可以扩展碱基编辑的体内应用和治疗，具有良好的基因治疗前景和产业化前景。
为实现上述目的及其他相关目的，本发明一方面提供一种融合蛋白，包括包括蛋白质内含子intein N片段或C片段和碱基编辑器的N端或C端片段，所述碱基编辑器包括ancBE4max-NG和ABEmax-NG，所述ancBE4max-NG包括APOBEC1 和SpCas9-NG D10A nickase片段，所述ABEmax-NG包括ecTad-ecTadA*二聚体片段和SpCas9-NG D10A nickase片段。
在本发明一些实施方式中，所述intein片段的氨基酸序列包括：
a)如SEQ ID NO.1所示的intein-N氨基酸序列和SEQ ID NO.2的intein-C氨基酸序列；或，
b)与SEQ ID NO.1或SEQ ID NO.2具有80％以上序列相似性的氨基酸序列、且具有a)所限定的氨基酸序列的功能，优选为能够作为内含子进行氨基酸序列剪切和拼接的特征。
在本发明一些实施方式中，所述N-ancBE4max-NG片段由APOBEC1片段和SpCas9-NGD10A nickase片段N端(2-573)融合而成，其氨基酸序列包括：
c)如SEQ ID NO.3所示的氨基酸序列；或，
d)与SEQ ID NO.3具有80％以上序列相似性的氨基酸序列、且具有c)所限定的氨基酸序列的功能。
在本发明一些实施方式中，所述C-ancBE4max-NG片段由SpCas9-NG D10Anickase片段C端(574-1368)与2*UGI、3*FLAG、BPNLS依次融合而成，其氨基酸序列包括：
e)如SEQ ID NO.4所示的氨基酸序列；或，
f)与SEQ ID NO.4具有80％以上序列相似性的氨基酸序列、且具有e)所限定的氨基酸序列的功能，优选的具有e)的蛋白质水平剪切融合所得全长蛋白的胞嘧啶脱氨酶功能，优选为能够识别NG作为PAM。
在本发明一些实施方式中，所述N-ABEmax-NG片段由ecTad-ecTadA*二聚体片段和SpCas9-NG D10A nickase片段N端(2-573)融合而成，其氨基酸序列包括：
g)如SEQ ID NO.5所示的氨基酸序列；或，
h)与SEQ ID NO.5具有80％以上序列相似性的氨基酸序列、且具有g)所限定的氨基酸序列的功能。
在本发明一些实施方式中，所述C-ABEmax-NG片段由SpCas9-NG D10Anickase片段C端(574-1368)与3*FLAG、BPNLS依次融合而成融合而成，其氨基酸序列包括：
i)如SEQ ID NO.6所示的氨基酸序列；或，
j)与SEQ ID NO.6具有80％以上序列相似性的氨基酸序列、且具有i)所限定的氨基酸序列的功能，优选的具有g)和i)相应的蛋白质水平剪切融合所得全长蛋白的腺嘌呤脱氨酶功能，优选为能够识别NG作为PAM。
在本发明一些实施方式中，所述融合蛋白N-ancBE4max-NG自5’端至3’端依次包括APOBEC1片段、SpCas9-NG D10A nickase的N端片段(2-573)、N-intein。
在本发明一些实施方式中，所述融合蛋白C-ancBE4max-NG自5’端至3’端依次包括C-intein、SpCas9-NG D10A nickase的C端片段(574-1368)、2*UGI、3*FLAG、BPNLS。其中，2*UGI指2个UGI肽段先后连接，3*FLAG表示3个FLAG肽段先后连接。
在本发明一些实施方式中，所述融合蛋白N-ABEmax-NG自5’端至3’端依次包括ecTadA-ecTadA*二聚体片段、SpCas9-NG D10A nickase的N端片段(2-573)、N-intein。
在本发明一些实施方式中，所述融合蛋白C-ABEmax-NG自5’端至3’端依次包括C-intein、SpCas9-NG D10A nickase的C端片段(574-1368)3*FLAG、BPNLS。
在本发明一些实施方式中，所述融合蛋白还包括核定位信号片段，优选的，所述核定位信号片段位于功能性元件(即融合蛋白)的N端和/或C端，优选的，所述核定位信号片段的氨基酸序列如SEQ ID NO.7所示。
在本发明一些实施方式中，所述融合蛋白N-ancBE4max-NG的氨基酸序列如SEQ IDNo.8所示；所述融合蛋白C-ancBE4max-NG的氨基酸序列如SEQ ID No.9所示；所述融合蛋白N-ABEmax-NG的氨基酸序列如SEQ ID No.10所示；所述融合蛋白C-ABEmax-NG的氨基酸序列如SEQ ID No.11所示。
在本发明一些实施方式中，所述融合蛋白N-ancBE4max-NG的腺病毒包装系统所包含的DNA序列如SEQ ID No.12所示；所述融合蛋白C-ancBE4max-NG的腺病毒包装系统所包含的DNA序列如SEQ ID No.13所示；所述融合蛋白N-ABEmax-NG的腺病毒包装系统所包含的DNA序列如SEQ ID No.14所示；所述融合蛋白C-ABEmax-NG的腺病毒包装系统所包含的DNA序列如SEQ ID No.15所示。
在本发明一些实施方式中，所述基因编辑工具还包括sgRNA腺病毒包装系统，所述sgRNA包装载体的DNA载体序列如SEQ ID No.16所示。
本发明另一方面提供一种构建体，所述构建体含有所述的分离的多核苷酸。
本发明另一方面提供一种表达系统，所述表达系统含有所述的构建体或基因组中整合有所述的多核苷酸。
在本发明一些实施方式中，所述表达系统的宿主细胞选自真核细胞或原核细胞，优选选自小鼠细胞、人细胞，更优选选自小鼠脑神经瘤细胞、人胚胎肾细胞、或人宫颈癌细胞，更优选选自N2a细胞、HEK293FT细胞、或Hela细胞等。
本发明另一方面提供所述的融合蛋白、所述的分离的多核苷酸、所述的构建体或所述的表达系统在基因编辑中的用途。
在本发明一些实施方式中，所述用途具体为在真核生物的基因编辑中的用途。
本发明另一方面提供一种碱基编辑体系，包括所述的融合蛋白，所述碱基编辑体系还包括sgRNA。
本发明另一方面提供一种基因编辑方法，包括：通过所述的融合蛋白、或所述的碱基编辑体系进行体外或者在体基因编辑。
本发明第一方面提供四种融合蛋白，包括蛋白质内含子intein片段和碱基编辑器的N端或C端片段，所述碱基编辑器包括ancBE4max-NG和ABEmax-NG。具体四种融合蛋白包括：
Figure GDA0002935979890000101
Figure GDA0002935979890000102
Figure GDA0002935979890000103
Figure GDA0002935979890000104
所述融合蛋白BPNLS-3*HA-ancBE4max-NG-N-intein和
C-intein-C-ancBE4max-NG-2*UGF-3*FLAG*BPNLS可以在蛋白质水平通过intein识别剪切，形成全长的ancBE4max-NG，并以NG为PAM序列，与靶向靶点区域的sgRNA相配合，实现对靶点区域内sgRNA 5’端4-8位的
Figure GDA0002935979890000105
的高效碱基编辑，且突变的精准性高，邻近脱靶低；
所述融合蛋白BPNLS-3*HA-N-ABEmax-NG-N-intein和
C-intein-C-ABEmax-NG-3*FLAG*BPNLS可以在蛋白质水平通过intein识别剪切，形成全长的ABEmax-NG，并以NG为PAM序列，与靶向靶点区域的sgRNA相配合，实现对靶点区域内sgRNA 5’端4-7位的A-to-G的高效碱基编辑，且突变的精准性高，邻近脱靶低。
本发明所提供的融合蛋白中，所述的取代、缺失或者添加可以是保守氨基酸取代。所述“保守氨基酸取代”具体可以是指氨基酸残基被其他具有相似侧链的氨基酸残基取代的情况。
本发明所提供的融合蛋白中，还可以包括核定位信号片段(NLS)，所述核定位信号片段可以位于SEQ ID NO.3/4/5/6的N端或C端。所述核定位信号片段可以包括如SEQ IDNO.7所示的氨基酸序列。
本发明第二方面提供一种分离的多核苷酸，编码本发明第一方面所提供的融合蛋白。
本发明第三方面提供一种构建体，所述构建体含有本发明第二方面所提供的分离的多核苷酸。所述构建体通常可以通过将所述分离的多核苷酸插入合适的表达载体中构建获得，本领域技术人员可选择合适的表达载体，例如，所述表达载体可以是包括但不限于pCMV表达载体、pSV2表达载体、pGL3表达载体及其它慢病毒包装载体、腺病毒包装载体等。
本发明第四方面提供一种表达系统，所述表达系统含有本发明第三方面所提供的构建体或基因组中整合有外源的本发明第二方面所提供的分离的多核苷酸。所述表达系统可以是宿主细胞，所述宿主细胞可以表达如上所述的融合蛋白，所述融合蛋白可以与sgRNA相配合，从而可以将所述融合蛋白定位到目标区域，实现目标区域的碱基编辑。在本发明另一具体实施例中，所述宿主细胞可以是真核细胞和/或原核细胞，更具体可以是小鼠细胞、人细胞等，更具体可以是小鼠脑神经瘤细胞、人胚胎肾细胞、人宫颈癌细胞等，更具体可以是N2a细胞、HEK293FT细胞、Hela细胞等。
本发明第五方面提供本发明第一方面所提供的融合蛋白、或本发明第二方面所提供的分离的多核苷酸、或本发明第三方面所提供的构建体、或本发明第四方面所提供的表达系统在基因编辑中的用途，优选为真核生物的基因编辑中的用途，所述真核生物具体可以是后生动物，具体可以是包括但不限于小鼠等。所述用途具体可以是包括但不限于由A到G或者C到T的碱基编辑、利用本发明的碱基编辑工具进行小鼠疾病模型的构建或人类疾病的治疗等。
本发明第六方面提供一种碱基编辑体系，包括本发明第一方面所提供的融合蛋白，所述碱基编辑体系还包括sgRNA。所述sgRNA的序列通常可以与目标区域至少部分互补，从而可以与所述融合蛋白相配合，将所述融合蛋白定位到目标区域，实现靶点区域内sgRNA5’端4-8位C-to-T的碱基编辑或者4-7位的A-to-G的碱基编辑。本发明所提供的碱基编辑体系极大地拓宽了基因组可靶向的范围，可以将NG序列作为PAM，实现sgRNA靶点区域内的碱基编辑，同时缩小了构建载体的质粒大小，从而适用于慢病毒和腺病毒包装系统，适用于动物疾病模型的构建或疾病的基因突变校正治疗。此外，所述融合蛋白还具有编辑精准性高、邻近脱靶低等优点，具有良好的产业化前景。
本发明第七方面提供一种碱基编辑方法，包括：通过本发明第一方面所提供的融合蛋白、或本发明第六方面所提供的碱基编辑体系进行基因编辑。例如，所述基因编辑方法可以包括：在适当条件下培养本发明第四方面所提供的表达系统，从而表达所述融合蛋白，所述融合蛋白可以在与其配合的靶向目标区域的sgRNA存在的条件下，对靶标区域进行碱基编辑。
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。他人一些非本质替换或改进在本发明的保护范围内。实施例中未注明厂商的试剂或仪器均可通过市场购买获得。未详细注明的实验方法，按照常规条件或试剂厂商推荐的方法实施。
实施例1
首先构建ancBE4max-NG和ABEmax-NG质粒，通过Mut Express II FastMutagenesis Kit V2(Vazyme,C214-02)将7个氨基酸突变(R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R)引入ancBE4max和ABEmax质粒，ancBE4max由商业公司全基因合成，ABEmax质粒购自Addgene(#112095)。产生的pCMV-ancBE4max-NG所包含的DNA序列如SEQ ID No.17所示；pCMV-ABEmax-NG所包含的DNA序列如SEQ ID No.18所示。
实施例2
在实施例1所得ancBE4max-NG和ABEmax-NG的基础上，构建如图1和2所示的pAAV-TRE-ancBE4max-NG 2-573-intein-N、pAAV-TRE-intein C-ancBE4max-NG 574-1368、pAAV-TRE-ABEmax-NG 2-573-intein-N、pAAV-TRE-intein C-ABEmax-NG 574-1368。
2.1 pAAV-TRE-ancBE4max-NG-2-573-intein-N、pAAV-TRE-intein C-ancBE4max-NG-574-1368、pAAV-TRE-ABEmax-NG-2-573-intein-N、pAAV-TRE-intein C-ABEmax-NG574-1368质粒的构建
由金唯智生物科技有限公司合成序列如下表1的PCR引物，稀释至10μM作为PCR引物，使用原始的pAAV-TRE作为模板。
表1
N-AAV-For TGCCTGGCCGGCGACACCCTG
N-AAV-Rev CATaagcttAGCGTAATCTGGAACG
N-ancBE4max-For GATTACGCTaagcttATGagcagtgaaaccggaccagtg
N-ancBE4max-Rev TGTCGCCGGCCAGGCActcgattttcttgaagtagtc
N-ABEmax-For GATTACGCTaagcttATGtctgaagtcgagtttagcca
N-ABEmax-Rev TGTCGCCGGCCAGGCActcgattttcttgaagtagtc
C-AAV-For tctggtggtTCTAGAGACTACAA
C-AAV-Rev GTTGTGGGCGATGATGTCGTTAG
C-ABEmax-For CATCATCGCCCACAACtgcttcgactccgtggaaatct
C-ABEmax-Rev CTCTAGAaccaccagagtcacctcccagctgagacag
C-ABEmax-For CATCATCGCCCACAACtgcttcgactccgtggaaatct
C-ABEmax-Rev CTCTAGAaccaccagatgagccgccagacagcattt
使用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)分别扩增载体序列片段和ABEmax或者ancBEmax的N端或者C端片段。扩增体系(参见表2)和PCR反应条件如所示：
表2
水 20μl
2xbuffer 25μl
dNTP 1μl
For引物 1μl
Rev引物 1μl
10XGCN4模板 1μl
高保真酶 1μl
一共 50μl
PCR程序为：95℃、5min，1 cycle；95℃、30S，62℃、30S，72℃、1.5min，30 cycles；72℃、5min，4℃至∞。
PCR扩增产物经通过AxyPrep PCR Clean-up试剂盒(Axygen,AP-PCR-500G)纯化回收后取30-300ng，利用vazyme重组试剂盒进行重组，重组以后进行转化涂板和挑克隆鉴定。对阳性克隆摇菌提取质粒(Axygene：AP-MN-P-250G)并测定浓度。
2.2构建如图3所示的AAV-sgRNA质粒的构建
设计sgRNA并合成oligos，上游序列为：5’-accg-19-21nt-3’，下游序列为：5’-aaac-19-21nt-3’(可替换序列与上游序列互补配对)，上下游序列通过程序(95℃，5min；95℃-85℃at-2℃/s；85℃-25℃at-0.1℃/s；hold at 4℃)退火，连接到经过BsaI(NEB：R0539L)线性化的pGL3-U6-sgRNA(Addgene#51133)载体上。线性化体系如下所示：pGL3-U6-sgRNA 2μg；buffer (NEB：R0539L)6μL；BsaI 2μL；ddH2O补齐到60μL。37℃酶切过夜。连接体系如下：T4连接buffer(NEB：M0202L)1μL，线性化载体20ng，退火的oligo片段(10μM)5μL，T4连接酶(NEB：M0202L)0.5μL，ddH2O补齐到10μL.16℃连接过夜。连接的载体通过转化，挑菌，鉴定。对阳性克隆摇菌提取质粒(Axygene：AP-MN-P-250G)并测定浓度。
SgRNA构建到pGL3-U6成功以后，利用由金唯智生物科技有限公司合成如下表3所示序列的PCR引物，进行PCR反应扩增载体片段和含有目的sgRNA的片段，扩增成功以后，将两者的PCR纯化片段进行重组，获得含有靶向sgRNA的AAV表达载体。对阳性克隆摇菌提取质粒(Axygene：AP-MN-P-250G)并测定浓度。
表3
Figure GDA0002935979890000141
实施例3
利用上述实施例构建的N-ancBE4max-NG+C-anc-BE4max-NG、N-ABEmax-NG+C-ABEmax-NG系统转染HEK293T细胞，过程如下：
3.1 HEK293T细胞(来自ATCC)复苏，在10cm培养皿(Corning,430167)中培养，培养基为混有10％的胎牛血清(HyClone,SV30087)的DMEM(HyClone,SH30243.01)。培养温度为37℃，二氧化碳浓度为5％。多次传代后当细胞密度为80％时，细胞分盘至12孔板。12孔板使用前用1:10稀释的多聚赖氨酸溶液(Sigma，P4707-50ML)包被处理。
3.2当细胞浓度为80％时，用10％血清的DMEM培养基换液，培养2小时使细胞状态恢复最佳。每孔转染的质粒的量分别是N-ancBE4max-NG 0.5ug、C-anc-BE4max-NG 0.5ug、sgRNA 0.5ug或者N-ABEmax-NG 0.5ug、C-ABEmax-NG 0.5ug、sgRNA 0.5ug共同转染293T细胞,将质粒混在100μl的Opti-MEM(Gibco,11058021)培养基。
3.3将4.5μl的Lipofectamine 2000转染试剂(Thermo,11668019)混入100μl的Opti-MEM培养基，静置5分钟。将混有质粒的Opti-MEM加入混有Lipofectamine 2000的Opti-MEM，慢速吹打混匀，静置20分钟。然后将混有质粒和Lipofectamine 2000的Opti-MEM分别加入12孔板。转染6小时后用10％FBS的DMEM换液。转染24小时后，用终浓度为2ng/ml的Puromycin(InvivoGen,nt-pr-1)做药杀处理。转染72小时后收细胞，酚氯仿法抽取基因组DNA。
3.4以选区的内源基因靶向位点上下游各100bp分别设计并合成PCR引物，加水稀释至10μM。用诺唯赞高保真酶试剂盒(Vazyme,p501-d2)PCR扩增各基因组靶向位点片段。PCR产物样品用AxyPrep DNA凝胶回收试剂盒(Axygen,AP-GX-250G)做割胶回收，去除非特异性条带。
测序结果统计如图4所示，其中A为N-ancBE4max-NG+C-anc-BE4max-NG+sgRNA共同转染293T细胞以后的Sanger测序结果，第一列为靶向DNA序列示意图；第二列实验结果为未转染的负对照，第三列为靶向基因编辑的实验结果，箭头指示为C-to-T编辑位置；b为N-ABEmax-NG+C-ABEmax-NG+sgRNA共同转染293T细胞以后的Sanger测序结果，第一列为靶向DNA序列示意图；第二列实验结果为未转染的负对照，第三列为靶向基因编辑的实验结果，箭头指示为A-to-G编辑位置。本实施例中的sg序列分别为：TGTCACAGTTAGCTCAGCCA(PAM为GGT)。由图4可见，组合得到的基因编辑工具N-ancBE4max-NG+C-anc-BE4max-NG可导致高效的C-to-T转换，而N-ABEmax-NG+C-ABEmax-NG可导致高效的A-to-G转换。
实施例4
利用上述实施例构建的pAAV-TRE-N-ABEmax-NG、pAAV-TRE-C-ABEmax-NG、AAV-sgRNA利用HEK293T细胞包装AAV病毒，过程如下：
4.1重组表达质粒同pHelper(携带腺病毒来源的基因)和pAAV-RC(携带AAV复制和衣壳基因)共转染进AAV-293细胞(提供AAV复制和包装所需的反式作用因子)。转染2到3天后重组AAV在包装细胞中组装完成。
4.2从被感染AAV-293细胞中收集AAV病毒颗粒，一般AAV颗粒会富集在包装细胞中，所以收集细胞而后裂解释放AAV颗粒到上清中可以回收大部分的AAV颗粒。这一步得到的病毒上清液随后用于感染各种哺乳动物类细胞系的感染实验。同时上清中的病毒也可以浓缩保留。
4.3浓缩并纯化第三步的病毒上清液，原上清液里面包含了许多细胞蛋白分子和碎片，通过2次CsCl密度梯度离心和1次超滤可以去除绝大部分的细胞蛋白和残留的CsCl离子。动物实验都需要纯化后的病毒才能够进行，否则会达不到所需剂量并引起副作用。感染宿主细胞后，基因表达前，单链病毒必须变成双链病毒。这个转变是重组基因表达的限制步骤，可以通过腺病毒双重感染或依托泊苷(喜树碱或丁酸钠)来加速。然而，加速基因表达的试剂对目标细胞是有毒的，如果残留到细胞上会杀死目标细胞。因此依托泊苷只能短期使用或为了提高病毒滴度时使用。
4.4用定量PCR法测定所得到病毒的滴度，这种方法可以得到被包装到颗粒中的AAV基因组的物理滴度值。AAV的感染滴度值因为感染细胞、AAV外壳蛋白和测试条件不同有很大差异，并且体外的实验数据不能反映体内的感染情况，所以在比较AAV时，定量PCR得到的物理滴度值是一个更客观的数值。图5显示为本发明实施例4实验结果示意图，其中，a为N-ABEmax-NG病毒滴度测试扩增曲线和三种稀释浓度滴度测试结果；b为C-ABEmax-NG病毒滴度测试扩增曲线和三种稀释浓度滴度测试结果；c为AAV-sgRNA病毒滴度测试扩增曲线和三种稀释浓度滴度测试结果。结果显示，本实施例获得了高于1E13滴度的病毒，证明通过intein将碱基编辑工具改造成两部分，从而获得高效用于内源性基因编辑的腺病毒。
综上所述，本发明有效克服了现有技术中的碱基编辑工具适用范围较窄和不适用于腺病毒包装等缺点，具高度产业利用价值。
本发明中涉及的核苷酸或氨基酸序列如下所示(其中SEQ ID NO.4、6、8、9、10、11序列最后的“*”表示终止密码子)：
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
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Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu
35 40 45
Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys
50 55 60
Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg
65 70 75 80
Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly
85 90 95
Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser
100 105 110
Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser
115 120 125
Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly
130 135 140
Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile
145 150 155 160
Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys
165 170 175
Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg
180 185 190
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met
195 200 205
Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys
210 215 220
Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu
225 230 235 240
Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp
245 250 255
Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser
260 265 270
Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp
275 280 285
Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys
290 295 300
Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr
305 310 315 320
Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser
325 330 335
Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg
340 345 350
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr
355 360 365
Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr
370 375 380
Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr
385 390 395 400
Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu
405 410 415
Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu
420 425 430
Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
435 440 445
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe
450 455 460
Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
465 470 475 480
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
485 490 495
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys
500 505 510
Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln
515 520 525
Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro Lys Arg Asn Ser Asp
530 535 540
Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly
545 550 555 560
Phe Val Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val
565 570 575
Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly
580 585 590
Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe
595 600 605
Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys
610 615 620
Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met
625 630 635 640
Leu Ala Ser Ala Arg Phe Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro
645 650 655
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu
660 665 670
Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
675 680 685
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser
690 695 700
Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
705 710 715 720
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
725 730 735
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Arg Ala Phe Lys
740 745 750
Tyr Phe Asp Thr Thr Ile Asp Arg Lys Val Tyr Arg Ser Thr Lys Glu
755 760 765
Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu
770 775 780
Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Arg
785 790 795 800
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr
805 810 815
Lys Asp Asp Asp Asp Lys Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu
820 825 830
Ser Pro Lys Lys Lys Arg Lys Val Glu
835 840
<210> 7
<211> 19
<212> PRT
<213> 合成
<400> 7
Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg
1 5 10 15
Lys Val Glu
<210> 8
<211> 987
<212> PRT
<213> 合成
<400> 8
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Glu Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly Tyr Pro
20 25 30
Tyr Asp Val Pro Asp Tyr Ala Gly Ser Tyr Pro Tyr Asp Val Pro Asp
35 40 45
Tyr Ala Lys Leu Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro
50 55 60
Thr Leu Arg Arg Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp
65 70 75 80
Pro Arg Glu Leu Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Lys Trp
85 90 95
Gly Thr Ser His Lys Ile Trp Arg His Ser Ser Lys Asn Thr Thr Lys
100 105 110
His Val Glu Val Asn Phe Ile Glu Lys Phe Thr Ser Glu Arg His Phe
115 120 125
Cys Pro Ser Thr Ser Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro
130 135 140
Cys Gly Glu Cys Ser Lys Ala Ile Thr Glu Phe Leu Ser Gln His Pro
145 150 155 160
Asn Val Thr Leu Val Ile Tyr Val Ala Arg Leu Tyr His His Met Asp
165 170 175
Gln Gln Asn Arg Gln Gly Leu Arg Asp Leu Val Asn Ser Gly Val Thr
180 185 190
Ile Gln Ile Met Thr Ala Pro Glu Tyr Asp Tyr Cys Trp Arg Asn Phe
195 200 205
Val Asn Tyr Pro Pro Gly Lys Glu Ala His Trp Pro Arg Tyr Pro Pro
210 215 220
Leu Trp Met Lys Leu Tyr Ala Leu Glu Leu His Ala Gly Ile Leu Gly
225 230 235 240
Leu Pro Pro Cys Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr
245 250 255
Phe Phe Thr Ile Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro
260 265 270
His Ile Leu Trp Ala Thr Gly Leu Lys Ser Gly Gly Ser Ser Gly Gly
275 280 285
Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu
290 295 300
Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly
305 310 315 320
Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu
325 330 335
Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg
340 345 350
His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly
355 360 365
Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr
370 375 380
Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
385 390 395 400
Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser
405 410 415
Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly
420 425 430
Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
435 440 445
His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg
450 455 460
Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe
465 470 475 480
Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu
485 490 495
Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro
500 505 510
Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
515 520 525
Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu
530 535 540
Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu
545 550 555 560
Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu
565 570 575
Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala
580 585 590
Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu
595 600 605
Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile
610 615 620
Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His
625 630 635 640
His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro
645 650 655
Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala
660 665 670
Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
675 680 685
Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys
690 695 700
Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly
705 710 715 720
Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg
725 730 735
Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile
740 745 750
Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
755 760 765
Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr
770 775 780
Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala
785 790 795 800
Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn
805 810 815
Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val
820 825 830
Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys
835 840 845
Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu
850 855 860
Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr
865 870 875 880
Phe Lys Lys Ile Glu Cys Leu Ala Gly Asp Thr Leu Ile Thr Leu Ala
885 890 895
Asp Gly Arg Arg Val Pro Ile Arg Glu Leu Val Ser Gln Gln Asn Phe
900 905 910
Ser Val Trp Ala Leu Asn Pro Gln Thr Tyr Arg Leu Glu Arg Ala Arg
915 920 925
Val Ser Arg Ala Phe Cys Thr Gly Ile Lys Pro Val Tyr Arg Leu Thr
930 935 940
Thr Arg Leu Gly Arg Ser Ile Arg Ala Thr Ala Asn His Arg Phe Leu
945 950 955 960
Thr Pro Gln Gly Trp Lys Arg Val Asp Glu Leu Gln Pro Gly Asp Tyr
965 970 975
Leu Ala Leu Pro Arg Arg Ile Pro Thr Ala Ser
980 985
<210> 9
<211> 1083
<212> PRT
<213> 合成
<400> 9
Met Ala Ala Ala Cys Pro Glu Leu Arg Gln Leu Ala Gln Ser Asp Val
1 5 10 15
Tyr Trp Asp Pro Ile Val Ser Ile Glu Pro Asp Gly Val Glu Glu Val
20 25 30
Phe Asp Leu Thr Val Pro Gly Pro His Asn Phe Val Ala Asn Asp Ile
35 40 45
Ile Ala His Asn Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
50 55 60
Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile
65 70 75 80
Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu
85 90 95
Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu
100 105 110
Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys
115 120 125
Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
130 135 140
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp
145 150 155 160
Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
165 170 175
His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val
180 185 190
Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
195 200 205
Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp
210 215 220
Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile
225 230 235 240
Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
245 250 255
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
260 265 270
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
275 280 285
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
290 295 300
Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
305 310 315 320
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu
325 330 335
Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
340 345 350
Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala
355 360 365
Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
370 375 380
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu
385 390 395 400
Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
405 410 415
Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val
420 425 430
Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp
435 440 445
Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
450 455 460
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr
465 470 475 480
Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
485 490 495
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr
500 505 510
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
515 520 525
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr
530 535 540
Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
545 550 555 560
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
565 570 575
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro Lys
580 585 590
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
595 600 605
Lys Tyr Gly Gly Phe Val Ser Pro Thr Val Ala Tyr Ser Val Leu Val
610 615 620
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys
625 630 635 640
Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
645 650 655
Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp
660 665 670
Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
675 680 685
Arg Lys Arg Met Leu Ala Ser Ala Arg Phe Leu Gln Lys Gly Asn Glu
690 695 700
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His
705 710 715 720
Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
725 730 735
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
740 745 750
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
755 760 765
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
770 775 780
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
785 790 795 800
Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Val Tyr Arg
805 810 815
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
820 825 830
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser
835 840 845
Gly Gly Ser Gly Gly Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile
850 855 860
Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met
865 870 875 880
Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp
885 890 895
Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met
900 905 910
Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile
915 920 925
Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser
930 935 940
Gly Gly Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu
945 950 955 960
Thr Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu
965 970 975
Glu Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val
980 985 990
His Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr
995 1000 1005
Ser Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp
1010 1015 1020
Ser Asn Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Ser
1025 1030 1035
Gly Gly Ser Arg Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp
1040 1045 1050
His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Lys Arg Thr Ala
1055 1060 1065
Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val Glu
1070 1075 1080
<210> 10
<211> 1123
<212> PRT
<213> 合成
<400> 10
Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys
1 5 10 15
Arg Lys Val Glu Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly Tyr Pro
20 25 30
Tyr Asp Val Pro Asp Tyr Ala Gly Ser Tyr Pro Tyr Asp Val Pro Asp
35 40 45
Tyr Ala Lys Leu Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met
50 55 60
Arg His Ala Leu Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val
65 70 75 80
Pro Val Gly Ala Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly
85 90 95
Trp Asn Arg Pro Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile
100 105 110
Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile
115 120 125
Asp Ala Thr Leu Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly
130 135 140
Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp
145 150 155 160
Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro
165 170 175
Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu
180 185 190
Cys Ala Ala Leu Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile
195 200 205
Lys Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser
210 215 220
Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr
225 230 235 240
Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe
245 250 255
Ser His Glu Tyr Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala
260 265 270
Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn
275 280 285
Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro
290 295 300
Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met
305 310 315 320
Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro
325 330 335
Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val
340 345 350
Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met
355 360 365
Asp Val Leu His Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu
370 375 380
Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg
385 390 395 400
Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr
405 410 415
Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly
420 425 430
Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly
435 440 445
Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
450 455 460
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
465 470 475 480
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
485 490 495
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
500 505 510
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
515 520 525
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
530 535 540
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
545 550 555 560
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
565 570 575
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
580 585 590
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
595 600 605
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
610 615 620
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
625 630 635 640
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
645 650 655
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
660 665 670
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
675 680 685
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
690 695 700
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
705 710 715 720
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
725 730 735
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
740 745 750
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
755 760 765
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
770 775 780
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
785 790 795 800
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
805 810 815
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
820 825 830
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
835 840 845
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
850 855 860
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
865 870 875 880
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
885 890 895
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
900 905 910
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
915 920 925
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
930 935 940
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
945 950 955 960
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
965 970 975
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
980 985 990
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
995 1000 1005
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Leu
1010 1015 1020
Ala Gly Asp Thr Leu Ile Thr Leu Ala Asp Gly Arg Arg Val Pro
1025 1030 1035
Ile Arg Glu Leu Val Ser Gln Gln Asn Phe Ser Val Trp Ala Leu
1040 1045 1050
Asn Pro Gln Thr Tyr Arg Leu Glu Arg Ala Arg Val Ser Arg Ala
1055 1060 1065
Phe Cys Thr Gly Ile Lys Pro Val Tyr Arg Leu Thr Thr Arg Leu
1070 1075 1080
Gly Arg Ser Ile Arg Ala Thr Ala Asn His Arg Phe Leu Thr Pro
1085 1090 1095
Gln Gly Trp Lys Arg Val Asp Glu Leu Gln Pro Gly Asp Tyr Leu
1100 1105 1110
Ala Leu Pro Arg Arg Ile Pro Thr Ala Ser
1115 1120
<210> 11
<211> 893
<212> PRT
<213> 合成
<400> 11
Met Ala Ala Ala Cys Pro Glu Leu Arg Gln Leu Ala Gln Ser Asp Val
1 5 10 15
Tyr Trp Asp Pro Ile Val Ser Ile Glu Pro Asp Gly Val Glu Glu Val
20 25 30
Phe Asp Leu Thr Val Pro Gly Pro His Asn Phe Val Ala Asn Asp Ile
35 40 45
Ile Ala His Asn Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp
50 55 60
Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile
65 70 75 80
Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu
85 90 95
Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu
100 105 110
Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys
115 120 125
Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys
130 135 140
Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp
145 150 155 160
Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile
165 170 175
His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val
180 185 190
Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
195 200 205
Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp
210 215 220
Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile
225 230 235 240
Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser
245 250 255
Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser
260 265 270
Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
275 280 285
Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp
290 295 300
Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile
305 310 315 320
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu
325 330 335
Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu
340 345 350
Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala
355 360 365
Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
370 375 380
Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu
385 390 395 400
Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser
405 410 415
Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val
420 425 430
Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp
435 440 445
Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His
450 455 460
Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr
465 470 475 480
Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp
485 490 495
Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr
500 505 510
Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu
515 520 525
Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr
530 535 540
Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
545 550 555 560
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys
565 570 575
Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro Lys
580 585 590
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
595 600 605
Lys Tyr Gly Gly Phe Val Ser Pro Thr Val Ala Tyr Ser Val Leu Val
610 615 620
Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys
625 630 635 640
Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn
645 650 655
Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp
660 665 670
Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
675 680 685
Arg Lys Arg Met Leu Ala Ser Ala Arg Phe Leu Gln Lys Gly Asn Glu
690 695 700
Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His
705 710 715 720
Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu
725 730 735
Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile
740 745 750
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys
755 760 765
Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln
770 775 780
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
785 790 795 800
Arg Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Val Tyr Arg
805 810 815
Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
820 825 830
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser
835 840 845
Gly Gly Ser Arg Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His
850 855 860
Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Lys Arg Thr Ala Asp Gly
865 870 875 880
Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val Glu
885 890
<210> 12
<211> 6491
<212> DNA
<213> 合成
<400> 12
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtctcga gtttaccact ccctatcagt 180
gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag 240
tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct 300
atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag 360
tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca 420
ctccctatca gtgatagaga aaagtgaaag tcgagctcgg tacccgggtc gagtaggcgt 480
gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540
cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc 600
cccgaattcg ccaccatgaa gagaacagca gacggaagtg aatttgagtc tccaaagaag 660
aagcgaaaag tggaataccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc 720
ccggactatg caggttccta tccatatgac gttccagatt acgctaagct tatgagcagt 780
gaaaccggac cagtggcagt ggacccaacc ctgaggagac ggattgagcc ccatgaattt 840
gaagtgttct ttgacccaag ggagctgagg aaggagacat gcctgctgta cgagatcaag 900
tggggcacaa gccacaagat ctggcgccac agctccaaga acaccacaaa gcacgtggaa 960
gtgaatttca tcgagaagtt tacctccgag cggcacttct gcccctctac cagctgttcc 1020
atcacatggt ttctgtcttg gagcccttgc ggcgagtgtt ccaaggccat caccgagttc 1080
ctgtctcagc accctaacgt gaccctggtc atctacgtgg cccggctgta tcaccacatg 1140
gaccagcaga acaggcaggg cctgcgcgat ctggtgaatt ctggcgtgac catccagatc 1200
atgacagccc cagagtacga ctattgctgg cggaacttcg tgaattatcc acctggcaag 1260
gaggcacact ggccaagata cccacccctg tggatgaagc tgtatgcact ggagctgcac 1320
gcaggaatcc tgggcctgcc tccatgtctg aatatcctgc ggagaaagca gccccagctg 1380
acatttttca ccattgctct gcagtcttgt cactatcagc ggctgcctcc tcatattctg 1440
tgggctacag gcctgaagtc tggaggatct agcggaggat cctctggcag cgagacacca 1500
ggaacaagcg agtcagcaac accagagagc agtggcggca gcagcggcgg cagcgacaag 1560
aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt gatcaccgac 1620
gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 1680
aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 1740
ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 1800
gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 1860
tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 1920
gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 1980
gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 2040
ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 2100
ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 2160
agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 2220
aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 2280
ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 2340
ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 2400
gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 2460
gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 2520
agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 2580
cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 2640
gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 2700
gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 2760
accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 2820
cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 2880
ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 2940
tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 3000
aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 3060
aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 3120
ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 3180
cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 3240
ctgaaagagg actacttcaa gaaaatcgag tgcctggccg gcgacaccct gatcacactg 3300
gctgatggaa ggagagtgcc tatcagagag ctggtgagcc agcagaactt ctccgtgtgg 3360
gccctgaacc cacagaccta cagactggag agggccagag tgtctcgggc tttttgtaca 3420
ggcatcaagc ccgtgtaccg gctgaccaca cggctgggac gcagcatcag ggctaccgct 3480
aaccaccgct tcctgacacc acagggctgg aagagggtgg acgagctgca gccaggagat 3540
tacctggccc tgccaaggcg catccctacc gcaagctaat ctagataaag atctaacttg 3600
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 3660
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 3720
gtctggctag acacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca 3780
ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc 3840
cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc 3900
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca 3960
accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 4020
cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 4080
tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 4140
ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 4200
tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 4260
taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt 4320
tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 4380
aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag 4440
tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga 4500
cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 4560
cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg 4620
cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 4680
aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 4740
ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 4800
aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 4860
ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 4920
gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 4980
ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 5040
ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 5100
gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 5160
aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 5220
gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 5280
aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 5340
caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 5400
tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 5460
acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 5520
gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 5580
agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 5640
gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 5700
ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 5760
taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 5820
agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 5880
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 5940
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 6000
gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 6060
aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 6120
aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 6180
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 6240
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 6300
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 6360
cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6420
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 6480
tgctcacatg t 6491
<210> 13
<211> 6780
<212> DNA
<213> 合成
<400> 13
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtctcga gtttaccact ccctatcagt 180
gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag 240
tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct 300
atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag 360
tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca 420
ctccctatca gtgatagaga aaagtgaaag tcgagctcgg tacccgggtc gagtaggcgt 480
gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540
cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc 600
cccgaattcg ccaccatggc tgctgcttgc ccagagctga ggcagctggc tcagagcgac 660
gtgtactggg accccatcgt gtccatcgag cccgacggcg tggaggaggt gttcgatctg 720
accgtgcccg gacctcacaa ctttgtggct aacgacatca tcgcccacaa ctgcttcgac 780
tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat 840
ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg 900
gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg 960
aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac 1020
accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc 1080
aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg 1140
atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag 1200
ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc 1260
atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc 1320
gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac 1380
agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg 1440
aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg 1500
cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac 1560
gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg 1620
ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg 1680
aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag 1740
ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc 1800
atcaagagac agctggtgga aacccggcag attacaaagc acgtggcaca gatcctggac 1860
tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc 1920
accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc 1980
gagatcaaca actaccacca cgcccacgac gcctacctaa acgccgtcgt gggaaccgca 2040
ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac 2100
gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac 2160
ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag 2220
atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag 2280
ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa 2340
aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcagacccaa gaggaacagc 2400
gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgtgagc 2460
cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa 2520
ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag 2580
aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc 2640
aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct 2700
gccagattcc tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg 2760
tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag 2820
ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc 2880
tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag 2940
caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc 3000
aatctgggag cccctagagc cttcaagtac tttgacacca ccatcgaccg gaaggtgtac 3060
agaagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac 3120
gagacacgga tcgacctgtc tcagctggga ggtgacagcg gcgggagcgg cgggagcggg 3180
gggagcacta atctgagcga catcattgag aaggagactg ggaaacagct ggtcattcag 3240
gagtccatcc tgatgctgcc tgaggaggtg gaggaagtga tcggcaacaa gccagagtct 3300
gacatcctgg tgcacaccgc ctacgacgag tccacagatg agaatgtgat gctgctgacc 3360
tctgacgccc ccgagtataa gccttgggcc ctggtcatcc aggattctaa cggcgagaat 3420
aagatcaaga tgctgagcgg aggatccgga ggatctggag gcagcaccaa cctgtctgac 3480
atcatcgaga aggagacagg caagcagctg gtcatccagg agagcatcct gatgctgccc 3540
gaagaagtcg aagaagtgat cggaaacaag cctgagagcg atatcctggt ccataccgcc 3600
tacgacgaga gtaccgacga aaatgtgatg ctgctgacat ccgacgcccc agagtataag 3660
ccctgggctc tggtcatcca ggattccaac ggagagaaca aaatcaaaat gctgtctggc 3720
ggctcatctg gtggttctag agactacaag gaccacgatg gcgactacaa ggatcacgac 3780
atcgattaca aggacgatga cgataagaag cggacagctg atggcagcga gttcgagtcc 3840
cccaagaaga agaggaaggt ggagtgattc tagataaaga tctaacttgt ttattgcagc 3900
ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 3960
actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggctaga 4020
cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg 4080
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc 4140
cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat 4200
tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg 4260
cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta 4320
cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt 4380
tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg 4440
ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat 4500
cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac 4560
tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag 4620
ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg 4680
cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct 4740
ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 4800
gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 4860
tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 4920
gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 4980
ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 5040
atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 5100
tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 5160
tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 5220
gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 5280
aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 5340
gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 5400
ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 5460
gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 5520
gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 5580
atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 5640
ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 5700
cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 5760
cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 5820
gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 5880
cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 5940
cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 6000
taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 6060
ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 6120
aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 6180
caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 6240
taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 6300
gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 6360
cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 6420
taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 6480
agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 6540
ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 6600
gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 6660
acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 6720
acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 6780
<210> 14
<211> 6899
<212> DNA
<213> 合成
<400> 14
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtctcga gtttaccact ccctatcagt 180
gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag 240
tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct 300
atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag 360
tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca 420
ctccctatca gtgatagaga aaagtgaaag tcgagctcgg tacccgggtc gagtaggcgt 480
gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540
cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc 600
cccgaattcg ccaccatgaa gagaacagca gacggaagtg aatttgagtc tccaaagaag 660
aagcgaaaag tggaataccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc 720
ccggactatg caggttccta tccatatgac gttccagatt acgctaagct tatgtctgaa 780
gtcgagttta gccacgagta ttggatgagg cacgcactga ccctggcaaa gcgagcatgg 840
gatgaaagag aagtccccgt gggcgccgtg ctggtgcaca acaatagagt gatcggagag 900
ggatggaaca ggccaatcgg ccgccacgac cctaccgcac acgcagagat catggcactg 960
aggcagggag gcctggtcat gcagaattac cgcctgatcg atgccaccct gtatgtgaca 1020
ctggagccat gcgtgatgtg cgcaggagca atgatccaca gcaggatcgg aagagtggtg 1080
ttcggagcac gggacgccaa gaccggcgca gcaggctccc tgatggatgt gctgcaccac 1140
cccggcatga accaccgggt ggagatcaca gagggaatcc tggcagacga gtgcgccgcc 1200
ctgctgagcg atttctttag aatgcggaga caggagatca aggcccagaa gaaggcacag 1260
agctccaccg actctggagg atctagcgga ggatcctctg gaagcgagac accaggcaca 1320
agcgagtccg ccacaccaga gagctccggc ggctcctccg gaggatcctc tgaggtggag 1380
ttttcccacg agtactggat gagacatgcc ctgaccctgg ccaagagggc acgcgatgag 1440
agggaggtgc ctgtgggagc cgtgctggtg ctgaacaata gagtgatcgg cgagggctgg 1500
aacagagcca tcggcctgca cgacccaaca gcccatgccg aaattatggc cctgagacag 1560
ggcggcctgg tcatgcagaa ctacagactg attgacgcca ccctgtacgt gacattcgag 1620
ccttgcgtga tgtgcgccgg cgccatgatc cactctagga tcggccgcgt ggtgtttggc 1680
gtgaggaacg caaaaaccgg cgccgcaggc tccctgatgg acgtgctgca ctaccccggc 1740
atgaatcacc gcgtcgaaat taccgaggga atcctggcag atgaatgtgc cgccctgctg 1800
tgctatttct ttcggatgcc tagacaggtg ttcaatgctc agaagaaggc ccagagctcc 1860
accgactccg gaggatctag cggaggctcc tctggctctg agacacctgg cacaagcgag 1920
agcgcaacac ctgaaagcag cgggggcagc agcggggggt cagacaagaa gtacagcatc 1980
ggcctggcca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg 2040
cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa gaagaacctg 2100
atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct gaagagaacc 2160
gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga gatcttcagc 2220
aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc cttcctggtg 2280
gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga cgaggtggcc 2340
taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga cagcaccgac 2400
aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt ccggggccac 2460
ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct gttcatccag 2520
ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag cggcgtggac 2580
gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa tctgatcgcc 2640
cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct gagcctgggc 2700
ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact gcagctgagc 2760
aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga ccagtacgcc 2820
gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga catcctgaga 2880
gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag atacgacgag 2940
caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac 3000
aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga cggcggagcc 3060
agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga cggcaccgag 3120
gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac cttcgacaac 3180
ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg gcggcaggaa 3240
gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct gaccttccgc 3300
atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg gatgaccaga 3360
aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa gggcgcttcc 3420
gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa cgagaaggtg 3480
ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg 3540
aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca gaaaaaggcc 3600
atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac 3660
tacttcaaga aaatcgagtg cctggccggc gacaccctga tcacactggc tgatggaagg 3720
agagtgccta tcagagagct ggtgagccag cagaacttct ccgtgtgggc cctgaaccca 3780
cagacctaca gactggagag ggccagagtg tctcgggctt tttgtacagg catcaagccc 3840
gtgtaccggc tgaccacacg gctgggacgc agcatcaggg ctaccgctaa ccaccgcttc 3900
ctgacaccac agggctggaa gagggtggac gagctgcagc caggagatta cctggccctg 3960
ccaaggcgca tccctaccgc aagctaatct agataaagat ctaacttgtt tattgcagct 4020
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 4080
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctagac 4140
acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc 4200
gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc 4260
gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt 4320
ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc 4380
gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 4440
acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 4500
cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 4560
tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc 4620
gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 4680
cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg 4740
gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 4800
gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc 4860
tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg 4920
ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 4980
gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg 5040
cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 5100
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 5160
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 5220
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 5280
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 5340
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 5400
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 5460
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 5520
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 5580
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 5640
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 5700
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 5760
tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 5820
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 5880
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 5940
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 6000
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 6060
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 6120
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 6180
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 6240
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 6300
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 6360
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 6420
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 6480
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 6540
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 6600
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 6660
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 6720
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 6780
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 6840
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt 6899
<210> 15
<211> 6210
<212> DNA
<213> 合成
<400> 15
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtctcga gtttaccact ccctatcagt 180
gatagagaaa agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag 240
tcgagtttac cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct 300
atcagtgata gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag 360
tgaaagtcga gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca 420
ctccctatca gtgatagaga aaagtgaaag tcgagctcgg tacccgggtc gagtaggcgt 480
gtacggtggg aggcctatat aagcagagct cgtttagtga accgtcagat cgcctggaga 540
cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc 600
cccgaattcg ccaccatggc tgctgcttgc ccagagctga ggcagctggc tcagagcgac 660
gtgtactggg accccatcgt gtccatcgag cccgacggcg tggaggaggt gttcgatctg 720
accgtgcccg gacctcacaa ctttgtggct aacgacatca tcgcccacaa ctgcttcgac 780
tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat 840
ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg 900
gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg 960
aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac 1020
accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa gcagtccggc 1080
aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt catgcagctg 1140
atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag 1200
ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat taagaagggc 1260
atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc 1320
gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac 1380
agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg 1440
aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct gtactacctg 1500
cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac 1560
gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga caacaaggtg 1620
ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg 1680
aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag 1740
ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc 1800
atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca gatcctggac 1860
tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc 1920
accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc 1980
gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt gggaaccgcc 2040
ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta caaggtgtac 2100
gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac cgccaagtac 2160
ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc caacggcgag 2220
atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt gtgggataag 2280
ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa 2340
aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcagacccaa gaggaacagc 2400
gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg cttcgtgagc 2460
cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa 2520
ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag cttcgagaag 2580
aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga cctgatcatc 2640
aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat gctggcctct 2700
gccagattcc tgcagaaggg aaacgaactg gccctgccct ccaaatatgt gaacttcctg 2760
tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga gcagaaacag 2820
ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat cagcgagttc 2880
tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc ctacaacaag 2940
caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt taccctgacc 3000
aatctgggag cccctagagc cttcaagtac tttgacacca ccatcgaccg gaaggtgtac 3060
agaagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac cggcctgtac 3120
gagacacgga tcgacctgtc tcagctggga ggtgactctg gtggttctag agactacaag 3180
gaccacgatg gcgactacaa ggatcacgac atcgattaca aggacgatga cgataagaag 3240
cggacagctg atggcagcga gttcgagtcc cccaagaaga agaggaaggt ggagtgattc 3300
tagataaaga tctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat 3360
cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 3420
catcaatgta tcttatcatg tctggctaga cacgtgcgga ccgagcggcc gcaggaaccc 3480
ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 3540
ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 3600
agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 3660
caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 3720
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 3780
tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 3840
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 3900
atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 3960
cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 4020
ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 4080
aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 4140
ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 4200
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 4260
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 4320
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 4380
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4440
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 4500
tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 4560
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 4620
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 4680
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 4740
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 4800
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 4860
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 4920
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 4980
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 5040
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 5100
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 5160
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 5220
ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 5280
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 5340
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 5400
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 5460
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 5520
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 5580
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5640
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5700
atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5760
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5820
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5880
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5940
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 6000
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 6060
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 6120
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 6180
tggccttttg ctggcctttt gctcacatgt 6210
<210> 16
<211> 5958
<212> DNA
<213> 合成
<400> 16
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt 180
ttaaacagag aggaatctct cgaggagggc ctatttccca tgattccttc atatttgcat 240
atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata 300
ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa 360
ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg 420
gctttatata tcttgtggaa aggacgaaac accgtactta ggttggaagg ccacgtttta 480
gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc 540
gagtcggtgc ttttttggtc gactttttta gagctagagc gcgtgcgcca attctgcatc 600
gagccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 660
tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 720
catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 780
gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 840
gctattacca tggtcgaggt gagccccacg ttctgcttca ctctccccat ctcccccccc 900
tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc gatgggggcg 960
gggggggggg gggggcgcgc gccgggcggg gcggggcggg gcgaggggcg gggcggggcg 1020
aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt tccttttatg 1080
gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc gggagtcgct 1140
gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 1200
tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 1260
aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag 1320
gggctccggg agggcccttt gtgcgggggg agcggctcgg ggctgtccgc ggggggacgg 1380
ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc 1440
tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt 1500
gctggttatt gtgctgtctc atcattttgg caaagaattg gatcgaattc gccaccatgt 1560
caagactgga caagagcaaa gtcataaact ctgctctgga attactcaat gaagtcggta 1620
tcgaaggcct gacgacaagg aaactcgctc aaaagctggg agttgagcag cctaccctgt 1680
actggcacgt caagaacaag cgggccctgc tcgatgccct ggcaatcgag atgctggaca 1740
ggcatcatac ccacttctgc cccctggaag gcgagtcatg gcaagacttt ctgcggaaca 1800
acgccaagtc attccgctgt gctctcctct cacatcgcga cggggctaaa gtgcatctcg 1860
gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg ttcctgtgtc 1920
agcaaggctt ctccctggag aacgcactgt acgctctgtc cgccgtgggc cactttacac 1980
tgggctgcgt attggaggat caggagcatc aagtagcaaa agaggaaaga gagacaccta 2040
ccaccgattc tatgccccca cttctgagac aagcaattga gctgttcgac catcagggag 2100
ccgaacctgc cttccttttc ggcctggaac taatcatatg tggcctggag aaacagctaa 2160
agtgcgaaag cggcgggccg gccgacgccc ttgacgattt tgacttagac atgctcccag 2220
ccgatgccct tgacgacttt gaccttgata tgctgcctgc tgacgctctt gacgattttg 2280
accttgacat gctccccggg tgaggatcca atcaacctct ggattacaaa atttgtgaaa 2340
gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 2400
tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 2460
cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 2520
gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 2580
tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 2640
ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 2700
ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 2760
cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 2820
tgccggctct gcggcctctt ccgcgacttc gccttcgccc tcagacgagt cggatctccc 2880
tttgggccgc ctccccgcag atctaacttg tttattgcag cttataatgg ttacaaataa 2940
agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 3000
ttgtccaaac tcatcaatgt atcttatcat gtctggctag acacgtggcc gctaccccga 3060
ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg 3120
caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg 3180
cgacaccctg gtgaaccgca cgtgcggacc gagcggccgc aggaacccct agtgatggag 3240
ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc 3300
cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag 3360
gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt 3420
caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 3480
cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 3540
cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 3600
tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg 3660
gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 3720
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct 3780
attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 3840
tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca 3900
ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac 3960
ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 4020
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac 4080
gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt 4140
agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 4200
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4260
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4320
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 4380
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 4440
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 4500
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4560
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 4620
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 4680
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 4740
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 4800
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 4860
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 4920
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 4980
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 5040
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 5100
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 5160
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 5220
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5280
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 5340
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 5400
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc 5460
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 5520
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 5580
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 5640
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 5700
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 5760
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 5820
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 5880
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 5940
ggccttttgc tcacatgt 5958
<210> 17
<211> 8961
<212> DNA
<213> 合成
<400> 17
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420
gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcagcag tgaaaccgga 480
ccagtggcag tggacccaac cctgaggaga cggattgagc cccatgaatt tgaagtgttc 540
tttgacccaa gggagctgag gaaggagaca tgcctgctgt acgagatcaa gtggggcaca 600
agccacaaga tctggcgcca cagctccaag aacaccacaa agcacgtgga agtgaatttc 660
atcgagaagt ttacctccga gcggcacttc tgcccctcta ccagctgttc catcacatgg 720
tttctgtctt ggagcccttg cggcgagtgt tccaaggcca tcaccgagtt cctgtctcag 780
caccctaacg tgaccctggt catctacgtg gcccggctgt atcaccacat ggaccagcag 840
aacaggcagg gcctgcgcga tctggtgaat tctggcgtga ccatccagat catgacagcc 900
ccagagtacg actattgctg gcggaacttc gtgaattatc cacctggcaa ggaggcacac 960
tggccaagat acccacccct gtggatgaag ctgtatgcac tggagctgca cgcaggaatc 1020
ctgggcctgc ctccatgtct gaatatcctg cggagaaagc agccccagct gacatttttc 1080
accattgctc tgcagtcttg tcactatcag cggctgcctc ctcatattct gtgggctaca 1140
ggcctgaagt ctggaggatc tagcggagga tcctctggca gcgagacacc aggaacaagc 1200
gagtcagcaa caccagagag cagtggcggc agcagcggcg gcagcgacaa gaagtacagc 1260
atcggcctgg ccatcggcac caactctgtg ggctgggccg tgatcaccga cgagtacaag 1320
gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat caagaagaac 1380
ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg gctgaagaga 1440
accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca agagatcttc 1500
agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga gtccttcctg 1560
gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg 1620
gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt ggacagcacc 1680
gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa gttccggggc 1740
cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa gctgttcatc 1800
cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg 1860
gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga aaatctgatc 1920
gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc cctgagcctg 1980
ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa actgcagctg 2040
agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg cgaccagtac 2100
gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag cgacatcctg 2160
agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa gagatacgac 2220
gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag 2280
tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat tgacggcgga 2340
gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat ggacggcacc 2400
gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg gaccttcgac 2460
aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct gcggcggcag 2520
gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat cctgaccttc 2580
cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc ctggatgacc 2640
agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga caagggcgct 2700
tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc caacgagaag 2760
gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga gctgaccaaa 2820
gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga gcagaaaaag 2880
gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca gctgaaagag 2940
gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg 3000
ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga caaggacttc 3060
ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct gacactgttt 3120
gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt cgacgacaaa 3180
gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag ccggaagctg 3240
atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct gaagtccgac 3300
ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac ctttaaagag 3360
gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca cattgccaat 3420
ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt ggtggacgag 3480
ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat ggccagagag 3540
aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg gatcgaagag 3600
ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa cacccagctg 3660
cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta cgtggaccag 3720
gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc tcagagcttt 3780
ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag 3840
agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg gcggcagctg 3900
ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc 3960
ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga aacccggcag 4020
attacaaagc acgtggcaca gatcctggac tcccggatga acactaagta cgacgagaat 4080
gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt gtccgatttc 4140
cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca cgcccacgac 4200
gcctacctaa acgccgtcgt gggaaccgca ctgatcaaaa agtaccctaa gctggaaagc 4260
gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc caagagcgag 4320
caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat gaactttttc 4380
aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat cgagacaaac 4440
ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt gcggaaagtg 4500
ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc 4560
aaagagtcta tcagacccaa gaggaacagc gataagctga tcgccagaaa gaaggactgg 4620
gaccctaaga agtacggcgg cttcgtgagc cccaccgtgg cctattctgt gctggtggtg 4680
gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct gctggggatc 4740
accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga agccaagggc 4800
tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct gttcgagctg 4860
gaaaacggcc ggaagagaat gctggcctct gccagattcc tgcagaaggg aaacgaactg 4920
gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga gaagctgaag 4980
ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa gcactacctg 5040
gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc cgacgctaat 5100
ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag agagcaggcc 5160
gagaatatca tccacctgtt taccctgacc aatctgggag cccctagagc cttcaagtac 5220
tttgacacca ccatcgaccg gaaggtgtac agaagcacca aagaggtgct ggacgccacc 5280
ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc tcagctggga 5340
ggtgacagcg gcgggagcgg cgggagcggg gggagcacta atctgagcga catcattgag 5400
aaggagactg ggaaacagct ggtcattcag gagtccatcc tgatgctgcc tgaggaggtg 5460
gaggaagtga tcggcaacaa gccagagtct gacatcctgg tgcacaccgc ctacgacgag 5520
tccacagatg agaatgtgat gctgctgacc tctgacgccc ccgagtataa gccttgggcc 5580
ctggtcatcc aggattctaa cggcgagaat aagatcaaga tgctgagcgg aggatccgga 5640
ggatctggag gcagcaccaa cctgtctgac atcatcgaga aggagacagg caagcagctg 5700
gtcatccagg agagcatcct gatgctgccc gaagaagtcg aagaagtgat cggaaacaag 5760
cctgagagcg atatcctggt ccataccgcc tacgacgaga gtaccgacga aaatgtgatg 5820
ctgctgacat ccgacgcccc agagtataag ccctgggctc tggtcatcca ggattccaac 5880
ggagagaaca aaatcaaaat gctgtctggc ggctcaaaaa gaaccgccga cggcagcgaa 5940
ttcgagccca agaagaagag gaaagtctaa ccggtcatca tcaccatcac cattgagttt 6000
aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 6060
cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 6120
aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 6180
aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 6240
ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga 6300
gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 6360
cacacaacat acgagccgga agcataaagt gtaaagccta ggatgcctaa tgagtgagct 6420
aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 6480
agctgcatta atgaatcggc caacgcgcgg gaagaggcgg tttgcgtatt gggcgctctt 6540
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 6600
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 6660
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 6720
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 6780
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 6840
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 6900
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 6960
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 7020
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 7080
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 7140
actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct 7200
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 7260
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 7320
tcttttctac ggggtctgac actcagtgga acgaaaactc acgttaaggg attttggtca 7380
tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 7440
caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg 7500
cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 7560
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag 7620
acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc 7680
gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag 7740
ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca 7800
tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa 7860
ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 7920
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata 7980
attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca 8040
agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg 8100
ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg 8160
ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 8220
cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 8280
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac 8340
tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca 8400
tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 8460
tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca 8520
gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg 8580
aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca 8640
attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca 8700
gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 8760
tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 8820
gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 8880
cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 8940
tggcagtaca tcaagtgtat c 8961
<210> 18
<211> 8811
<212> DNA
<213> 合成
<400> 18
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg 60
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 120
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact 180
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa 240
atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta 300
ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct 360
agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca 420
gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga agtcgagttt 480
agccacgagt attggatgag gcacgcactg accctggcaa agcgagcatg ggatgaaaga 540
gaagtccccg tgggcgccgt gctggtgcac aacaatagag tgatcggaga gggatggaac 600
aggccaatcg gccgccacga ccctaccgca cacgcagaga tcatggcact gaggcaggga 660
ggcctggtca tgcagaatta ccgcctgatc gatgccaccc tgtatgtgac actggagcca 720
tgcgtgatgt gcgcaggagc aatgatccac agcaggatcg gaagagtggt gttcggagca 780
cgggacgcca agaccggcgc agcaggctcc ctgatggatg tgctgcacca ccccggcatg 840
aaccaccggg tggagatcac agagggaatc ctggcagacg agtgcgccgc cctgctgagc 900
gatttcttta gaatgcggag acaggagatc aaggcccaga agaaggcaca gagctccacc 960
gactctggag gatctagcgg aggatcctct ggaagcgaga caccaggcac aagcgagtcc 1020
gccacaccag agagctccgg cggctcctcc ggaggatcct ctgaggtgga gttttcccac 1080
gagtactgga tgagacatgc cctgaccctg gccaagaggg cacgcgatga gagggaggtg 1140
cctgtgggag ccgtgctggt gctgaacaat agagtgatcg gcgagggctg gaacagagcc 1200
atcggcctgc acgacccaac agcccatgcc gaaattatgg ccctgagaca gggcggcctg 1260
gtcatgcaga actacagact gattgacgcc accctgtacg tgacattcga gccttgcgtg 1320
atgtgcgccg gcgccatgat ccactctagg atcggccgcg tggtgtttgg cgtgaggaac 1380
gcaaaaaccg gcgccgcagg ctccctgatg gacgtgctgc actaccccgg catgaatcac 1440
cgcgtcgaaa ttaccgaggg aatcctggca gatgaatgtg ccgccctgct gtgctatttc 1500
tttcggatgc ctagacaggt gttcaatgct cagaagaagg cccagagctc caccgactcc 1560
ggaggatcta gcggaggctc ctctggctct gagacacctg gcacaagcga gagcgcaaca 1620
cctgaaagca gcgggggcag cagcgggggg tcagacaaga agtacagcat cggcctggcc 1680
atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag 1740
aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc 1800
ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga 1860
agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg 1920
gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat 1980
aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag 2040
aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac 2100
ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc 2160
gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag 2220
acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc 2280
atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc 2340
ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc 2400
aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc 2460
tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt 2520
ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc 2580
gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag 2640
gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt 2700
ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa 2760
gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc 2820
gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc 2880
ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac 2940
ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac 3000
tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag 3060
gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc 3120
ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag 3180
cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg 3240
accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac 3300
ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag 3360
aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc 3420
ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag 3480
gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag 3540
atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag 3600
ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc 3660
cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac 3720
agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa 3780
gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc 3840
cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg 3900
atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc 3960
cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag 4020
ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag 4080
ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc 4140
aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac 4200
tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg 4260
ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag 4320
ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa 4380
ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac 4440
gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc 4500
cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc 4560
cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac 4620
gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac 4680
ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc 4740
aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt 4800
accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg 4860
gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc 4920
caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc 4980
agacccaaga ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag 5040
tacggcggct tcgtgagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa 5100
aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa 5160
agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg 5220
aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg 5280
aagagaatgc tggcctctgc cagattcctg cagaagggaa acgaactggc cctgccctcc 5340
aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag 5400
gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc 5460
gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg 5520
ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc 5580
cacctgttta ccctgaccaa tctgggagcc cctagagcct tcaagtactt tgacaccacc 5640
atcgaccgga aggtgtacag aagcaccaaa gaggtgctgg acgccaccct gatccaccag 5700
agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg tgactctggc 5760
ggctcaaaaa gaaccgccga cggcagcgaa ttcgagccca agaagaagag gaaagtctaa 5820
ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt 5880
ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg 5940
ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt 6000
gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca 6060
atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct 6120
ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc 6180
ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 6240
gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 6300
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 6360
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 6420
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 6480
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 6540
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 6600
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 6660
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 6720
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 6780
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 6840
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 6900
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 6960
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 7020
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 7080
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 7140
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga 7200
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 7260
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 7320
ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 7380
catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 7440
ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 7500
caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 7560
ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 7620
tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 7680
cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 7740
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 7800
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 7860
gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 7920
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 7980
aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 8040
tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 8100
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 8160
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 8220
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 8280
taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc 8340
gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa 8400
gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt 8460
aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc 8520
gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta 8580
gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg 8640
ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga 8700
cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat 8760
gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat c 8811

Claims (7)

1.一种基因编辑工具，其特征在于，所述编辑工具为将碱基A转换为G的N-ABEmax-NG+C-ABEmax-NG编辑系统，所述编辑系统包括融合蛋白、sgRNA和sgRNA包装载体，以及腺病毒包装系统；所述N-ABEmax-NG+C-ABEmax-NG编辑系统的融合蛋白，由N-ABEmax-NG氨基酸片段和C-ABEmax-NG氨基酸片段组成；所述N-ABEmax-NG氨基酸片段自N端至C端依次由BPNLS多肽、3*HA多肽、SpCas9-NG D10A nickase片段N端第2～573个氨基酸组成的多肽和inteinN片段组成，其氨基酸序列如SEQ ID NO:10所示；所述C-ABEmax-NG氨基酸片段自N端至C端依次由intein C片段、SpCas9-NG D10A nickase多肽C端第574～1368个氨基酸组成的多肽、3*FLAG多肽和BPNLS多肽组成，其氨基酸序列如SEQ ID NO:11所示。
2.如权利要求1所述的基因编辑工具，其特征在于，所述如SEQ ID NO:10所示的氨基酸序列其编码的核苷酸序列如SEQ ID NO:14所示；所述如SEQ ID NO:11所示的氨基酸序列其编码的核苷酸序列如SEQ ID NO:15所示。
3.如权利要求1或2所述的基因编辑工具，其特征在于，所述融合蛋白还包括核定位信号多肽片段，所述核定位信号多肽片段位于所述融合蛋白的N端和/或C端。
4.如权利要求3所述的基因编辑工具，其特征在于，所述sgRNA包装载体的核苷酸序列如SEQ ID NO:16所示。
5.如权利要求1~4任一项所述的基因编辑工具在单碱基编辑中的应用。
6.如权利要求5所述的应用，其特征在于，所述单碱基编辑为将碱基A转换为G。
7.一种细胞表达系统，其特征在于，含有如权利要求1~4任一项所述的基因编辑工具，所述细胞为真核宿主细胞或原核宿主细胞。
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