CN112522260B - Crispr系统及其在制备ttn基因突变的扩张型心肌病克隆猪核供体细胞中的应用 - Google Patents

Crispr系统及其在制备ttn基因突变的扩张型心肌病克隆猪核供体细胞中的应用 Download PDF

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CN112522260B
CN112522260B CN202011137413.6A CN202011137413A CN112522260B CN 112522260 B CN112522260 B CN 112522260B CN 202011137413 A CN202011137413 A CN 202011137413A CN 112522260 B CN112522260 B CN 112522260B
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牛冬
汪滔
陶裴裴
王磊
曾为俊
程锐
马翔
赵泽英
刘璐
黄彩云
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Nanjing Qizhen Genetic Engineering Co Ltd
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Abstract

本发明公开了一种CRISPR/Cas9系统及其在制备TTN基因突变的扩张型心肌病克隆猪核供体细胞中的应用。本发明提供了一种sgRNA，命名为sgRNATTN‑E278‑g2，其靶序列结合区如SEQ ID NO：7中第1‑20位核苷酸所示。本发明还提供了一种质粒，命名为质粒pKG‑U6gRNA(TTN‑E278‑g2)，其转录得到sgRNATTN‑E278‑g2。本发明还保护sgRNATTN‑E278‑g2或质粒pKG‑U6gRNA(TTN‑E278‑g2)在制备扩张型心肌病猪模型或扩张型心肌病猪细胞模型中的应用。本发明为扩张型心肌病猪模型的制备奠定了坚实的基础，对于扩张型心肌病药物的研发具有重大应用价值。

Description

CRISPR系统及其在制备TTN基因突变的扩张型心肌病克隆猪 核供体细胞中的应用
技术领域
本发明属于生物技术领域，具体涉及基因编辑领域，更具体涉及一种CRISPR/Cas9系统及其在制备TTN基因突变的扩张型心肌病克隆猪核供体细胞中的应用。
背景技术
扩张型心肌病(DCM，dilated cardiomyopathy，又称充血性心肌病)是一种原因未明的原发性心肌疾病。临床表现患者以中年人居多。起病多缓慢，有时可达10年以上。症状为左或右心室或双侧心室扩大，并伴有心室收缩功能减退，伴或不伴充血性心力衰竭，以室性或房性心律失常、气短和水肿最为常见。此外，尚可有脑、肾、肺等处的栓塞，病情呈进行性加重，死亡可发生于疾病的任何阶段。已经在30％～40％的DCM患者中发现了遗传病因，但其中只有50％与已知的致病基因变异相关。
18％的零散发生DCM和25％的家族性DCM是由TTN基因突变引起的。TTN基因编码人体最大蛋白-肌巨蛋白(titin)，定位于人2q31(CMD1G；MIM 604145)。家族性DCM中多含有TTN截短突变(TTNtv)，包含点突变、删除、插入等各种形式，最终导致TTN编码的titin蛋白提前终止而“截短”，每个家族不尽相同，但是确切的发病机制仍在研究。因此，迫切需要开发出基于TTNtv导致的DCM动物模型以尽快解开发病机制谜团。猪作为大动物，是人类长期以来主要的肉食供应动物，易于大规模繁殖饲养，而且在伦理道德及动物保护等方面要求较低，且猪体型大小和生理功能与人类近似，是理想的人类疾病模型动物。
基因编辑是近年来不断取得重大发展的一种生物技术，其包括从基于同源重组的胚胎干细胞注射到基于核酸酶的ZFN、TALEN、CRISPR/Cas9等，其中CRISPR/Cas9技术是当前最先进的基因编辑技术。目前，基因编辑技术被越来越多地应用到动物模型的制作上。
发明内容
本发明的目的是提供一种CRISPR/Cas9系统及其在制备TTN基因突变的扩张型心肌病克隆猪核供体细胞中的应用。
本发明提供了一种sgRNA，命名为sgRNATTN-E278-g2，其靶序列结合区如SEQ ID NO：7中第1-20位核苷酸所示。具体的，sgRNATTN-E278-g2如SEQ ID NO：7所示。
本发明还提供了一种质粒，命名为质粒pKG-U6gRNA(TTN-E278-g2)，其转录得到sgRNATTN-E278-g2。
本发明还提供了一种试剂盒，包括sgRNATTN-E278-g2。所述试剂盒还包括质粒pKG-GE3。
本发明还提供了一种试剂盒，包括质粒pKG-U6gRNA(TTN-E278-g2)。所述试剂盒还包括质粒pKG-GE3。
本发明还保护sgRNATTN-E278-g2在制备试剂盒中的应用。
本发明还保护质粒pKG-U6gRNA(TTN-E278-g2)在制备试剂盒中的应用。
本发明还保护sgRNATTN-E278-g2和质粒pKG-GE3在制备试剂盒中的应用。
本发明还保护质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3在制备试剂盒中的应用。
以上任一所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备扩张型心肌病猪模型；(c)制备扩张型心肌病猪细胞模型。所述重组细胞为猪重组细胞。所述重组细胞的转化受体细胞为猪细胞。所述重组细胞，即将质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3共转染受体细胞得到的重组细胞。质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的摩尔配比具体可为3：1。质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的质量配比具体可为0.92μg：1.08μg。受体细胞、质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的配比依次为约20万个受体细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g2)：1.08μg质粒pKG-GE3。所述猪细胞可为猪成纤维细胞。所述猪细胞具体可为猪原代成纤维细胞。所述猪具体可为从江香猪。制备扩张型心肌病猪模型时，先制备所述重组细胞，然后将所述重组细胞作为核移植供体细胞采用体细胞克隆技术得到克隆猪，即为扩张型心肌病猪模型。还可以用扩张型心肌病猪模型制备扩张型心肌病猪细胞模型，即分离扩张型心肌病猪模型的相应细胞，即为扩张型心肌病猪细胞模型。
本发明还保护sgRNATTN-E278-g2或质粒pKG-U6gRNA(TTN-E278-g2)或所述试剂盒在制备重组细胞中的应用。所述重组细胞为猪重组细胞。所述重组细胞的转化受体细胞为猪细胞。所述重组细胞，即将质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3共转染受体细胞得到的重组细胞。质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的摩尔配比具体可为3：1。质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的质量配比具体可为0.92μg：1.08μg。受体细胞、质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的配比依次为约20万个受体细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g2)：1.08μg质粒pKG-GE3。所述猪细胞可为猪成纤维细胞。所述猪细胞具体可为猪原代成纤维细胞。所述猪具体可为从江香猪。
本发明还保护sgRNATTN-E278-g2或质粒pKG-U6gRNA(TTN-E278-g2)或所述试剂盒在制备扩张型心肌病猪模型中的应用。本发明还保护sgRNATTN-E278-g2或质粒pKG-U6gRNA(TTN-E278-g2)或所述试剂盒在制备扩张型心肌病猪细胞模型中的应用。制备扩张型心肌病猪模型时，先制备所述重组细胞，然后将所述重组细胞作为核移植供体细胞采用体细胞克隆技术得到克隆猪，即为扩张型心肌病猪模型。还可以用扩张型心肌病猪模型制备扩张型心肌病猪细胞模型，即分离扩张型心肌病猪模型的相应细胞，即为扩张型心肌病猪细胞模型。
本发明还保护一种制备重组细胞的方法，包括如下步骤：将质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3共转染猪细胞，得到重组细胞。质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的摩尔配比具体可为3：1。质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的质量配比具体可为0.92μg：1.08μg。猪细胞、质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3的配比依次为约20万个猪细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g2)：1.08μg质粒pKG-GE3。所述猪细胞可为猪成纤维细胞。所述猪细胞具体可为猪原代成纤维细胞。所述猪具体可为从江香猪。
本发明还保护所述方法制备得到的重组细胞。
本发明还保护所述重组细胞在制备扩张型心肌病猪模型中的应用。本发明还保护所述重组细胞在制备扩张型心肌病猪细胞模型中的应用。制备扩张型心肌病猪模型时，将所述重组细胞作为核移植供体细胞采用体细胞克隆技术得到克隆猪，即为扩张型心肌病猪模型。还可以用扩张型心肌病猪模型制备扩张型心肌病猪细胞模型，即分离扩张型心肌病猪模型的相应细胞，即为扩张型心肌病猪细胞模型。
以上任一所述重组细胞为TTN基因发生基因编辑的重组细胞。
以上任一所述重组细胞为TTN基因缺陷的重组细胞。
以上任一所述重组细胞为TTN基因发生突变的重组细胞。所述突变可为杂合突变(对应基因型为杂合突变型)或纯合突变(对应的基因型为双等位基因相同突变型或双等位基因不同突变型)。
具体来说，所述重组细胞可为如下任一：表1中编号为2、3、4、5、6、7、11、12、14、16、17、18、22、24、26、27、28、29、32、34、35、36、37、38、40的单克隆细胞株。
sgRNATTN-E278-g2靶点：5’-CCTAAACCCAAACACGATGG-3’。
具体来说，所述质粒pKG-U6gRNA(TTN-E278-g2)是借助限制性内切酶BbsI将sgRNATTN-E278-g2的靶序列结合区的编码序列插入pKG-U6gRNA载体得到的。
质粒pKG-GE3中，具有特异融合基因；所述特异融合基因编码特异融合蛋白；
所述特异融合蛋白自N端至C端依次包括如下元件：两个核定位信号(NLS)、Cas9蛋白、两个核定位信号、自剪切多肽P2A、荧光报告蛋白、自裂解多肽T2A、抗性筛选标记蛋白；
质粒pKG-GE3中，由EF1a启动子启动所述特异融合基因的表达；
质粒pKG-GE3中，所述特异融合基因下游具有WPRE序列元件、3’LTR序列元件和bGHpoly(A)signal序列元件。
质粒pKG-GE3中，依次具有如下元件：CMV增强子、EF1a启动子、所述特异融合基因、WPRE序列元件、3’LTR序列元件、bGH poly(A)signal序列元件。
所述特异融合蛋白中，Cas9蛋白上游的两个核定位信号为SV40核定位信号，Cas9蛋白下游的两个核定位信号为nucleoplasmin核定位信号。
所述特异融合蛋白中，荧光报告蛋白具体可为EGFP蛋白。
所述特异融合蛋白中，抗性筛选标记蛋白具体可为Puromycin蛋白。
自剪切多肽P2A的氨基酸序列为“ATNFSLLKQAGDVEENPGP”(发生自剪切的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)。
自裂解多肽T2A的氨基酸序列为“EGRGSLLTCGDVEENPGP”(发生自裂解的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)。
特异融合基因具体如SEQ ID NO：2中第911-6706位核苷酸所示。
CMV增强子如SEQ ID NO：2中第395-680位核苷酸所示。
EF1a启动子如SEQ ID NO：2中第682-890位核苷酸所示。
WPRE序列元件如SEQ ID NO：2中第6722-7310位核苷酸所示。
3’LTR序列元件如SEQ ID NO：2中第7382-7615位核苷酸所示。
bGH poly(A)signal序列元件如SEQ ID NO：2中第7647-7871位核苷酸所示。
质粒pKG-GE3具体如SEQ ID NO：2所示。
质粒pKG-U6gRNA中，具有SEQ ID NO：3中第2280-2637位核苷酸所示的DNA分子。
质粒pKG-U6gRNA具体如SEQ ID NO：3所示。
猪TTN基因信息：编码titin蛋白；位于15号染色体；
GeneID为100519519，Sus scrofa。猪TTN基因编码的蛋白质如GENBANK ACCESSIONNO.XP_020931560.1(linear MAM 13-MAY-2017)所示。基因组DNA中，猪TTN基因具有315个外显子，其中第278外显子如SEQ ID NO：5所示，其编码的蛋白质片段如SEQ ID NO：4所示。
以上任一所述TTN基因具体可为具有编码SEQ ID NO：4所示蛋白质片段的DNA分子的基因。以上任一所述TTN基因具体可为具有SEQ ID NO：5所示DNA分子的基因。以上任一所述TTN基因为猪TTN基因。
与现有技术相比，本发明至少具有如下有益效果：
(1)本发明研究对象(猪)比其他动物(大小鼠、灵长类)具有更好的应用性。
大小鼠等啮齿类动物不论从体型、器官大小、生理、病理等方面都与人相差巨大，无法真实地模拟人类正常的生理、病理状态。研究表明，95％以上在大小鼠中验证有效的药物在人类临床试验中是无效的。就大动物而言，灵长类是与人亲缘关系最近的动物，但其体型小、性成熟晚(6-7岁开始交配)，且为单胎动物，群体扩繁速度极慢，饲养成本也高。另外，灵长类动物克隆效率低、难度大、成本高。
而猪作为模型动物就没有上述缺点，猪是除灵长类外与人亲缘关系最近的动物，其体型、体重、器官大小等与人相近，在解剖学、生理学、营养代谢、疾病发病机制方面等方面与人类极为相似。同时，猪的性成熟早(4-6个月)，繁殖力高，一胎多仔，在2-3年内即可形成一个较大群体。另外，猪的克隆技术非常成熟，克隆及饲养成本较灵长类低得多；而且猪作为人类长期以来的肉食性动物，用猪作为疾病模型动物在动物保护和伦理等方面的要求较低。
(2)采用本发明改造的Cas9高效表达载体进行基因编辑,编辑效率比原载体提高100％以上。
(3)采用本发明改造的Cas9高效表达载体进行基因编辑，通过靶标基因PCR产物测序结果可分析出所获细胞的基因型[纯合突变(包括双等位基因相同突变和双等位基因不同突变)、杂合突变或野生型]，获得纯合突变的概率为30％～50％，大大优于使用胚胎注射技术的模型制备方法(即受精卵注射基因编辑材料)中获得纯合突变的概率(低于5％)。
(4)利用本发明所得到的纯合突变单克隆细胞株进行体细胞核移植动物克隆可直接得到含靶标基因纯合突变的克隆猪，并且该纯合突变可稳定遗传。
在小鼠模型制作中采用的受精卵显微注射基因编辑材料后再进行胚胎移植的方法，因其直接获得纯合突变后代的概率非常低(低于5％)，需要进行后代的杂交选育，这不太适用于妊娠期较长的大动物(如猪)模型制作。因此，本发明采用技术难度大、挑战性高的原代细胞体外编辑并筛选阳性编辑单克隆细胞的方法，后期再通过体细胞核移植动物克隆技术直接获得相应疾病模型猪，可大大缩短模型猪制作周期并节省人力、物力、财力。
本发明为通过基因编辑手段获得扩张型心肌病猪模型奠定了坚实的基础，将有助于研究并揭示由TTNtv引起的DCM发病机制，进一步的可用于进行药物筛选、药效检测、疾病病理、基因治疗及细胞治疗等研究，能够为进一步的临床应用提供有效的实验数据，进而为成功治疗人类因TTNtv导致的DCM提供有力的实验手段。本发明对于扩张型心肌病药物的研发及揭示该病的发病机制具有重大应用价值。
附图说明
图1为质粒pX330的结构示意图。
图2为质粒pKG-GE3的结构示意图。
图3为质粒pKG-U6gRNA的结构示意图。
图4为将20bp左右的DNA分子(用于转录形成gRNA的靶序列结合区)插入质粒pKG-U6gRNA的示意图。
图5为质粒配比优化时的测序结果。
图6为质粒pX330和质粒pKG-GE3的效果比较时的测序结果。
图7为实施例3中以3只猪的基因组DNA为模板进行PCR扩增后的电泳图。
图8为实施例3中以18只猪的基因组DNA为模板进行PCR扩增后的电泳图。
图9为实施例3的步骤四中的测序峰图。
图10为实施例4中得到的单克隆细胞的靶基因PCR产物电泳图。
图11为编号TTN-1的单克隆细胞的正向测序与野生型比对的结果。
图12为编号为TTN-2的单克隆细胞的正向测序与野生型比对的结果。
图13为编号为TTN-3的单克隆细胞的正向测序与野生型比对的结果。
图14为编号为TTN-12的单克隆细胞的正向测序和反向测序同时与野生型比对的结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。实施例中构建的重组质粒，均已进行测序验证。完全培养液(％为体积比)：15％胎牛血清(Gibco)+83％DMEM培养基(Gibco)+1％Penicillin-Streptomycin(Gibco)+1％HEPES(Solarbio)。细胞培养条件：37℃，5％CO2、5％O2的恒温培养箱。
制备猪原代成纤维细胞的方法：①取猪耳组织0.5g，除毛，然后用75﹪酒精浸泡30-40s，然后用含5％(体积比)Penicillin-Streptomycin(Gibco)的PBS缓冲液洗涤5次，然后用PBS缓冲液洗涤一次；②用剪刀将组织剪碎，采用5mL 1％胶原酶溶液(Sigma)，37℃消化1h，然后500g离心5min，弃上清；③将沉淀用1mL完全培养液重悬，然后铺入含10mL完全培养基并已用0.2％明胶(VWR)封盘的直径为10的细胞培养皿中，培养至细胞长满皿底60％左右；④完成步骤③后，采用胰蛋白酶消化并收集细胞，然后重悬于完全培养液。
实施例1、质粒的制备
制备质粒pX330-U6-Chimeric_BB-CBh-hSpCas9，如SEQ ID NO：1所示。质粒pX330-U6-Chimeric_BB-CBh-hSpCas9，简称质粒pX330。
制备质粒pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，如SEQ ID NO：2所示。质粒pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，简称质粒pKG-GE3。
制备质粒pKG-U6gRNA，如SEQ ID NO：3所示。
质粒pX330、质粒pKG-GE3、质粒pKG-U6gRNA均为环形质粒。
质粒pX330的结构示意图见图1。SEQ ID NO：1中，第440-725位核苷酸组成CMV增强子，第727-1208位核苷酸组成chickenβ-actin启动子，第1304-1324位核苷酸编码SV40核定位信号(NLS)，第1325-5449位核苷酸编码Cas9蛋白，第5450-5497位核苷酸编码nucleoplasmin核定位信号(NLS)。
质粒pKG-GE3的结构示意图见图2。SEQ ID NO：2中，第395-680位核苷酸组成CMV增强子，第682-890位核苷酸组成EF1a启动子，第986-1006位核苷酸编码核定位信号(NLS)，第1016-1036位核苷酸编码核定位信号(NLS)，第1037-5161位核苷酸编码Cas9蛋白，第5162-5209位核苷酸编码核定位信号(NLS)，第5219-5266位核苷酸编码核定位信号(NLS)，第5276-5332位核苷酸编码自剪切多肽P2A(自剪切多肽P2A的氨基酸序列为“ATNFSLLKQAGDVEENPGP”，发生自剪切的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)，第5333-6046位核苷酸编码EGFP蛋白，第6056-6109位核苷酸编码自裂解多肽T2A(自裂解多肽T2A的氨基酸序列为“EGRGSLLTCGDVEENPGP”，发生自裂解的断裂位置为C端开始第一个氨基酸残基和第二个氨基酸残基之间)，第6110-6703位核苷酸编码Puromycin蛋白(简称Puro蛋白)，第6722-7310位核苷酸组成WPRE序列元件，第7382-7615位核苷酸组成3’LTR序列元件，第7647-7871位核苷酸组成bGH poly(A)signal序列元件。SEQID NO：2中，第911-6706形成融合基因，表达融合蛋白。由于自剪切多肽P2A和自裂解多肽T2A的存在，融合蛋白自发形成如下三个蛋白：具有Cas9蛋白的蛋白、具有EGFP蛋白的蛋白和具有Puro蛋白的蛋白。
与质粒pX330相比，质粒pKG-GE3主要进行了如下改造：①去除残留的gRNA骨架序列(GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTT)，降低干扰；②将原有chickenβ-actin启动子改造为具更高表达活性的EF1a启动子，增加Cas9基因的蛋白表达能力；③在Cas9基因的上游和下游均增加核定位信号编码基因(NLS)，增加Cas9蛋白的核定位能力；④原质粒无任何真核细胞筛选标记，不利于阳性转化细胞的筛选和富集，依次在Cas9基因的下游插入P2A-EGFP-T2A-PURO编码基因，赋予载体荧光和真核细胞抗性筛选能力；⑤插入WPRE元件和3’LTR序列元件，增强Cas9基因的蛋白翻译能力。
质粒pKG-U6gRNA的结构示意图见图3。SEQ ID NO：3中，第2280-2539位核苷酸组成hU6启动子，第2558-2637位核苷酸用于转录形成gRNA骨架。使用时，将20bp左右的DNA分子(用于转录形成gRNA的靶序列结合区)插入质粒pKG-U6gRNA，形成重组质粒，示意图见图4,在细胞中重组质粒转录得到gRNA。
实施例2、质粒pX330和质粒pKG-GE3的效果比较
选择位于RAG1基因的高效gRNA靶点：
RAG1-gRNA4的靶点：5’-AGTTATGGCAGAACTCAGTG-3’。
用于扩增包含靶点的片段的引物如下：
RAG1-nF126：5’-CCCCATCCAAAGTTTTTAAAGGA-3’；
RAG1-nR525：5’-TGTGGCAGATGTCACAGTTTAGG-3’。
初生从江香猪(雌性，血型AO)，制备猪原代成纤维细胞。
一、制备重组质粒
取质粒pKG-U6gRNA，用限制性内切酶BbsI进行酶切，回收载体骨架(约3kb的线性大片段)。分别合成RAG1-4S和RAG1-4A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(RAG1-gRNA4)。
RAG1-4S：5’-caccgAGTTATGGCAGAACTCAGTG-3’；
RAG1-4A：5’-aaacCACTGAGTTCTGCCATAACTc-3’。
RAG1-4S和RAG1-4A均为单链DNA分子。
二、质粒配比优化
第一组：将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.44μg质粒pKG-U6gRNA(RAG1-gRNA4)：1.56μg质粒pKG-GE3。即质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3的摩尔配比为：1：1。
第二组：将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.72μg质粒pKG-U6gRNA(RAG1-gRNA4)：1.28μg质粒pKG-GE3。即质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3的摩尔配比为：2：1。
第三组：将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(RAG1-gRNA4)：1.08μg质粒pKG-GE3。即质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3的摩尔配比为：3：1。
第四组：将质粒pKG-U6gRNA(RAG1-gRNA4)转染致猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：1μg质粒pKG-U6gRNA(RAG1-gRNA4)。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，提取基因组DNA，采用RAG1-nF126和RAG1-nR525组成的引物对进行PCR扩增，然后进行电泳。
电泳后回收目的条带并进行测序，测序结果见图5。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。第一组至第三组的基因编辑效率依次为9％、53％、66％。第四组不发生基因编辑。结果表明，第三组编辑效率最高，确定单gRNA质粒与Cas9质粒最适用量为摩尔比3：1，质粒实际用量为0.92μg：1.08μg。
三、质粒pX330和质粒pKG-GE3的效果比较
1、共转染
RAG1-B组：将质粒pKG-U6gRNA(RAG1-gRNA4)转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(RAG1-gRNA4)。
RAG1-330组：将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pX330共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(RAG1-gRNA4)：1.08μg质粒pX330。
RAG1-KG组：将质粒pKG-U6gRNA(RAG1-gRNA4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(RAG1-gRNA4)：1.08μg质粒pKG-GE3。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，提取基因组DNA，采用RAG1-nF126和RAG1-nR525组成的引物对进行PCR扩增，将产物进行测序。
通过利用Synthego ICE工具分析测序峰图得出不同靶点的编辑效率。RAG1-B组不发生基因编辑。RAG1-330组、RAG1-KG组的编辑效率依次为28％、68％。测序结果示例性峰图见图6。结果表明，与采用质粒pX330相比，采用质粒pKG-GE3使得基因编辑效率显著提高。
实施例3、TTN基因敲除的靶点的筛选
猪TTN基因信息：编码titin蛋白；位于15号染色体；
GeneID为100519519，Sus scrofa。猪TTN基因编码的蛋白质如GENBANK ACCESSIONNO.XP_020931560.1(linear MAM 13-MAY-2017)所示。基因组DNA中，猪TTN基因具有315个外显子，其中第278外显子如SEQ ID NO：5所示，其编码的蛋白质片段如SEQ ID NO：4所示。
一、TTN基因敲除预设靶点及邻近基因组序列保守性分析
18只初生从江香猪，其中雌性10只(分别命名1、2、3、4、5、6、7、8、9、10)、雄性8只(分别命名为A、B、C、D、E、F、G、H)。
分别以3只猪(雌性，1、2、3)的基因组DNA为模板，分别采用TTN-E278-gRNA-JDF1和TTN-E278-gRNA-JDR1组成的引物对或TTN-E278-gRNA-JDF2和TTN-E278-gRNA-JDR2组成的引物对进行PCR扩增，然后进行电泳，见图7。TTN-E278-gRNA-JDF2和TTN-E278-gRNA-JDR2组成的引物对效果更好。
分别以18只猪的基因组DNA为模板，采用TTN-E278-gRNA-JDF2和TTN-E278-gRNA-JDR2组成的引物对进行PCR扩增，然后进行电泳，见图8。回收PCR扩增产物并进行测序，将测序结果与公共数据库中的TTN基因序列进行比对分析。
TTN-E278-gRNA-JDF1：5’-ATTGTGGAGAAACGAGAAGCG-3’；
TTN-E278-gRNA-JDR1：5’-CAAAGTTAACTCTCTGTGTCT-3’。
TTN-E278-gRNA-JDF2：5’-CACTGGGACCTCCCTCTGATA-3’；
TTN-E278-gRNA-JDR2：5’-TAATGGGTAGGGCCCACTATC-3’。
二、筛选靶点
通过筛选NGG(避开可能的突变位点)初步筛选到若干靶点，经过预实验进一步从中筛选到4个靶点。
4个靶点分别如下：
sgRNATTN-E278-g1靶点：5’-GATGGGGGCAGTAAGATCAC-3’；
sgRNATTN-E278-g2靶点：5’-CCTAAACCCAAACACGATGG-3’；
sgRNATTN-E278-g3靶点：5’-AAAGAAAAGGCTCAGACCAG-3’；
sgRNATTN-E278-g4靶点：5’-GTTGTGAAGAATCTAACTGA-3’。
三、制备重组质粒
取质粒pKG-U6gRNA，用限制性内切酶BbsI进行酶切，回收载体骨架(约3kb的线性大片段)。
分别合成TTN-E278-gRNA1-S和TTN-E278-gRNA1-A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(TTN-E278-g1)。质粒pKG-U6gRNA(TTN-E278-g1)表达SEQ ID NO：6所示的sgRNATTN-E278-g1。
SEQ ID NO：6：
GAUGGGGGCAGUAAGAUCACguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
分别合成TTN-E278-gRNA2-S和TTN-E278-gRNA2-A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(TTN-E278-g2)。质粒pKG-U6gRNA(TTN-E278-g2)表达SEQ ID NO：7所示的sgRNATTN-E278-g2。
SEQ ID NO：7：
CCUAAACCCAAACACGAUGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
分别合成TTN-E278-gRNA3-S和TTN-E278-gRNA3-A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(TTN-E278-g3)。质粒pKG-U6gRNA(TTN-E278-g3)表达SEQ ID NO：8所示的sgRNATTN-E278-g3。
SEQ ID NO：8：
AAAGAAAAGGCUCAGACCAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
分别合成TTN-E278-gRNA4-S和TTN-E278-gRNA4-A，然后混合并进行退火，得到具有粘性末端的双链DNA分子。将具有粘性末端的双链DNA分子和载体骨架连接，得到质粒pKG-U6gRNA(TTN-E278-g4)。质粒pKG-U6gRNA(TTN-E278-g4)表达SEQ ID NO：9所示的sgRNATTN-E278-g4。
SEQ ID NO：9：
GUUGUGAAGAAUCUAACUGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu
TTN-E278-gRNA1-S：5’-caccGATGGGGGCAGTAAGATCAC-3’；
TTN-E278-gRNA1-A：5’-aaacGTGATCTTACTGCCCCCATC-3’；
TTN-E278-gRNA2-S：5’-caccgCCTAAACCCAAACACGATGG-3’；
TTN-E278-gRNA2-A：5’-aaacCCATCGTGTTTGGGTTTAGGc-3’；
TTN-E278-gRNA3-S：5’-caccgAAAGAAAAGGCTCAGACCAG-3’；
TTN-E278-gRNA3-A：5’-aaacCTGGTCTGAGCCTTTTCTTTc-3’；
TTN-E278-gRNA4-S：5’-caccGTTGTGAAGAATCTAACTGA-3’；
TTN-E278-gRNA4-A：5’-aaacTCAGTTAGATTCTTCACAAC-3’。
TTN-E278-gRNA1-S、TTN-E278-gRNA1-A、TTN-E278-gRNA2-S、TTN-E278-gRNA2-A、TTN-E278-gRNA3-S、TTN-E278-gRNA3-A、TTN-E278-gRNA4-S、TTN-E278-gRNA4-A均为单链DNA分子。
四、不同靶点的编辑效率比较
初生从江香猪(雌性，血型AO)，制备猪原代成纤维细胞。
1、共转染
第一组：将质粒pKG-U6gRNA(TTN-E278-g1)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g1)：1.08μg质粒pKG-GE3。
第二组：将质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g2)：1.08μg质粒pKG-GE3。
第三组：将质粒pKG-U6gRNA(TTN-E278-g3)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g3)：1.08μg质粒pKG-GE3。
第四组：将质粒pKG-U6gRNA(TTN-E278-g4)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g4)：1.08μg质粒pKG-GE3。
第五组：猪原代成纤维细胞，未进行任何转染操作。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，然后裂解细胞并提取基因组DNA，采用TTN-E278-gRNA-JDF2和TTN-E278-gRNA-JDR2组成的引物对进行PCR扩增，然后进行电泳，电泳图见图9。回收目标片段并进行测序，利用Synthego ICE工具分析测序峰图得出不同靶点的基因编辑效率。第一组至第四组的基因编辑效率依次为0％、35％、23％、16％。第五组不发生基因编辑。结果表明，第二组编辑效率最高，sgRNATTN-E278-g2的靶点为最优靶点。
实施例4、利用体细胞克隆的方法制备TTN基因编辑单克隆细胞
初生从江香猪(雌性，血型AO)，制备猪原代成纤维细胞。
1、共转染
将质粒pKG-U6gRNA(TTN-E278-g2)和质粒pKG-GE3共转染猪原代成纤维细胞。配比：约20万个猪原代成纤维细胞：0.92μg质粒pKG-U6gRNA(TTN-E278-g2)：1.08μg质粒pKG-GE3。
共转染采用电击转染的方式，采用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪(参数设置为：1450V、10ms、3pulse)。
2、完成步骤1后，采用完全培养液培养16-18小时，然后更换新的完全培养液进行培养。培养总时间为48小时。
3、完成步骤2后，采用胰蛋白酶消化并收集细胞，然后用完全培养液洗涤，然后用完全培养液重悬，然后分别挑取各个单克隆转移到96孔板中(每个孔1个细胞，每个孔中装有100μl完全培养液)，培养2周(每2-3天更换新的完全培养液)。
4、完成步骤3后，采用胰蛋白酶消化并收集细胞(每孔得到的细胞，约2/3接种到装有完全培养液的6孔板中，剩余的1/3收集在1.5mL离心管中)。
5、取步骤4的6孔板，培养直至细胞长至80％汇合度，采用胰蛋白酶消化并收集细胞，使用细胞冻存液(90％完全培养基+10％DMSO，体积比)将细胞冻存。
6、取步骤4的离心管，取细胞，提取基因组DNA，采用TTN-E278-gRNA-JDF2和TTN-E278-gRNA-JDR2组成的引物对进行PCR扩增，然后进行电泳。将猪原代成纤维细胞作为野生型对照。电泳图见图10。图10中的泳道编号与表1中的细胞编号一致。
7、完成步骤6后，回收PCR扩增产物并测序。
猪原代成纤维细胞的测序结果只有一种，其基因型为纯合野生型。如果某一单克隆细胞的测序结果有两种，一种与猪原代成纤维细胞的测序结果一致，另一种与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换)，该单克隆细胞的基因型为杂合型；如果某一单克隆细胞的测序结果为两种，均与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换)，该单克隆细胞的基因型为双等位基因不同突变型；如果某一单克隆细胞的测序结果为一种，且与猪原代成纤维细胞的测序结果相比发生了突变(突变包括一个或多个核苷酸的缺失、插入或替换)，该单克隆细胞的基因型为双等位基因相同突变型；如果某一单克隆细胞的测序结果为一种，且与猪原代成纤维细胞的测序结果一致，该单克隆细胞的基因型为纯合野生型。
结果见表1。编号为2、4、7、11、14、17、18、22、26、27、32、35、37、38、40的单克隆细胞的基因型为双等位基因相同突变型。编号为12、24、29的单克隆细胞的基因型为双等位基因不同突变型。编号为3、5、6、16、28、34、36的单克隆细胞的基因型为杂合型。得到基因编辑单克隆细胞的比率为25/40。
示例性的测序比对结果见图11至图14。图11是编号为TTN-1的单克隆细胞的正向测序与野生型比对的结果，判定为纯合野生型。图12是编号为TTN-2的单克隆细胞的正向测序与野生型比对的结果，判定为双等位基因相同突变型。图13是编号为TTN-3的单克隆细胞的正向测序与野生型比对的结果，判定为杂合型。图14是编号为TTN-12的单克隆细胞的正向测序和反向测序同时与野生型比对的结果，判定为双等位基因不同突变型。
表1
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以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
SEQUENCE LISTING
<110> 南京启真基因工程有限公司
<120> CRISPR系统及其在制备TTN基因突变的扩张型心肌病克隆猪核供体细胞中的应用
<130> GNCYX202174
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 8484
<212> DNA
<213> Artificial sequence
<400> 1
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360
agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420
aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480
ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540
aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660
acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720
ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780
ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840
ggggggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 900
agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 960
aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct 1020
ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga 1080
gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta 1140
agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca 1200
ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa 1260
ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa 1320
ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc tggacatcgg 1380
caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt 1440
caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct 1500
gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca gaagaagata 1560
caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa 1620
ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa 1680
gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta 1740
ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg 1800
gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg 1860
cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta 1920
caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct 1980
gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga 2040
gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt 2100
caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga 2160
cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc 2220
cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat 2280
caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct 2340
gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt 2400
cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt 2460
ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa 2520
gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca 2580
ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt 2640
cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt 2700
gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac 2760
catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat 2820
cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag 2880
cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga 2940
gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct 3000
gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat 3060
cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg 3120
cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa 3180
cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat 3240
cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa 3300
gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga 3360
caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa 3420
cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca 3480
ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc 3540
cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg 3600
ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa 3660
gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg 3720
cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta 3780
cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg 3840
gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat 3900
cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc 3960
cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat 4020
tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga 4080
taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc 4140
acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga 4200
agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt 4260
ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt 4320
cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga 4380
ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc 4440
taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct 4500
ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat 4560
cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt 4620
gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcctgcc 4680
caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg 4740
cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg 4800
caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag 4860
cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa 4920
ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag 4980
aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc cctccaaata 5040
tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa 5100
tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca 5160
gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc 5220
cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct 5280
gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca ccaccatcga 5340
ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc accagagcat 5400
caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc 5460
ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag agctcgctga 5520
tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 5580
tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 5640
tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 5700
ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga acccctagtg 5760
atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 5820
gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc 5880
ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 5940
atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6000
tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt 6060
tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6120
tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg 6180
gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 6240
agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct 6300
cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg 6360
agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat 6420
ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 6480
caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 6540
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 6600
cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 6660
tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 6720
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 6780
aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 6840
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 6900
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 6960
agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 7020
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 7080
tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 7140
atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 7200
ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 7260
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 7320
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 7380
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 7440
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 7500
ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca gatggtaagc 7560
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 7620
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 7680
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 7740
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 7800
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 7860
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 7920
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 7980
ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 8040
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 8100
ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 8160
gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 8220
gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 8280
gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 8340
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 8400
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 8460
tttgctggcc ttttgctcac atgt 8484
<210> 2
<211> 10476
<212> DNA
<213> Artificial sequence
<400> 2
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc tagcgcgtgc 360
gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc 420
cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga 480
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 540
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 600
ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 660
tagtcatcgc tattaccatg ggggcagagc gcacatcgcc cacagtcccc gagaagttgg 720
ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa 780
gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 840
cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggttggac 900
cggtgccacc atggactata aggaccacga cggagactac aaggatcatg atattgatta 960
caaagacgat gacgataaga tggcccccaa aaagaaacga aaggtgggtg ggtccccaaa 1020
gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg 1080
cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc 1140
cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat 1200
cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc 1260
cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa 1320
cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga 1380
agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta 1440
ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa 1500
ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt 1560
cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct 1620
ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc 1680
caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca 1740
gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct 1800
gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa 1860
ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga 1920
cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt 1980
gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca 2040
ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa 2100
agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag 2160
ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga 2220
actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg 2280
cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga 2340
tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat 2400
cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa 2460
gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc 2520
ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct 2580
gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa 2640
atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat 2700
cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta 2760
cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa 2820
cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga 2880
caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga 2940
cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat 3000
gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa 3060
cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt 3120
cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat 3180
ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc 3240
cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt 3300
gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca 3360
gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat 3420
caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa 3480
cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact 3540
ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa 3600
ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga 3660
caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa 3720
cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct 3780
gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac 3840
aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa 3900
gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa 3960
ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta 4020
cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt 4080
cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga 4140
aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac 4200
cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga 4260
aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag 4320
catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga 4380
gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc 4440
taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa 4500
agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat 4560
catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa 4620
agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa 4680
cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct 4740
gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc 4800
ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga 4860
gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga 4920
caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa 4980
tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga 5040
caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat 5100
ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga 5160
caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gcggctccaa 5220
gcggcctgcc gcgacgaaga aagcgggaca ggccaagaaa aagaaaggat ccggcgcaac 5280
aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa 5340
gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 5400
cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 5460
cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 5520
cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 5580
cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 5640
cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 5700
cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 5760
caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 5820
gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 5880
gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 5940
ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 6000
cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggct ccggcgaggg 6060
caggggaagt cttctaacat gcggggacgt ggaggaaaat cccggcccaa ccgagtacaa 6120
gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc 6180
cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg 6240
ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg 6300
ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg 6360
ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc 6420
gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct 6480
ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct 6540
ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc 6600
ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc 6660
cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga 6720
caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 6780
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 6840
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 6900
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 6960
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 7020
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 7080
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 7140
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 7200
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 7260
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta 7320
agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 7380
ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 7440
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 7500
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 7560
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc 7620
cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 7680
cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 7740
aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 7800
ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 7860
gggctctatg gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 7920
atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 7980
cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg 8040
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 8100
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 8160
aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 8220
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 8280
tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt 8340
ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 8400
ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 8460
cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 8520
cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 8580
caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 8640
tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 8700
ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 8760
gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 8820
cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 8880
tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 8940
tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 9000
cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 9060
tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 9120
agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 9180
ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 9240
ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 9300
aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 9360
gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 9420
tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 9480
ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc 9540
cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 9600
atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 9660
cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 9720
ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 9780
cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 9840
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 9900
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 9960
taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 10020
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 10080
agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 10140
gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 10200
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 10260
ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 10320
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 10380
tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 10440
ggttcctggc cttttgctgg ccttttgctc acatgt 10476
<210> 3
<211> 3120
<212> DNA
<213> Artificial sequence
<400> 3
gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60
cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180
aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240
ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300
ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360
tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420
tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540
gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600
acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660
gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720
acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780
gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900
gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020
agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920
ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980
cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040
cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100
acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160
cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220
accatgatta cgccaagctt gcatgcaggc ctctgcagtc gacgggcccg ggatccgatg 2280
ataaacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 2340
tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 2400
gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 2460
ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 2520
gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag 2580
ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc 2640
tagcgcgtgc gccaattctg cagacaaatg gctctagagg tacccataga tctagatgca 2700
ttcgcgaggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 2760
accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 2820
atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 2880
ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 2940
gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 3000
cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3060
tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3120
<210> 4
<211> 5701
<212> PRT
<213> Sus scrofa
<400> 4
Pro Pro Gly Pro Pro Ser Asn Ala Arg Val Thr Asp Thr Thr Lys Lys
1 5 10 15
Ser Ala Ser Leu Ala Trp Gly Lys Pro His Tyr Asp Gly Gly Leu Glu
20 25 30
Ile Thr Gly Tyr Val Val Glu His Gln Lys Val Gly Glu Asp Ala Trp
35 40 45
Ile Lys Asp Thr Thr Gly Thr Ala Leu Arg Ile Thr Glu Phe Val Val
50 55 60
Pro Asp Leu Gln Thr Lys Glu Lys Tyr Asn Phe Arg Ile Ser Ala Ile
65 70 75 80
Asn Asp Ala Gly Val Gly Glu Pro Ala Val Ile Pro Asn Val Glu Ile
85 90 95
Val Glu Arg Glu Met Ala Pro Asp Phe Glu Leu Asp Ala Glu Leu Arg
100 105 110
Arg Thr Leu Val Val Arg Ala Gly Leu Ser Ile Arg Ile Phe Val Pro
115 120 125
Ile Lys Gly Arg Pro Ala Pro Glu Val Thr Trp Thr Lys Asp Asp Ile
130 135 140
Asn Leu Lys Asn Arg Ala Asn Ile Glu Asn Thr Glu Ser Phe Thr Leu
145 150 155 160
Leu Ile Ile Pro Glu Cys Asn Arg Tyr Asp Thr Gly Lys Phe Val Met
165 170 175
Thr Ile Glu Asn Pro Ala Gly Lys Lys Ser Gly Phe Val Asn Val Arg
180 185 190
Val Leu Asp Thr Pro Gly Pro Val Leu Asn Leu Arg Pro Thr Asp Ile
195 200 205
Thr Lys Glu Ser Val Thr Leu His Trp Asp Leu Pro Leu Ile Asp Gly
210 215 220
Gly Ser Arg Ile Thr Asn Tyr Ile Val Glu Lys Arg Glu Ala Thr Arg
225 230 235 240
Lys Ser Tyr Ser Thr Val Thr Thr Lys Cys His Lys Cys Thr Tyr Lys
245 250 255
Val Thr Gly Leu Ser Glu Gly Cys Glu Tyr Phe Phe Arg Val Met Ala
260 265 270
Glu Asn Glu Tyr Gly Ile Gly Glu Pro Ala Glu Thr Thr Glu Pro Val
275 280 285
Arg Ala Ser Glu Ala Pro Ser Pro Pro Asp Ser Leu Asn Ile Met Asp
290 295 300
Ile Thr Lys Ser Thr Val Ser Leu Ala Trp Pro Lys Pro Lys His Asp
305 310 315 320
Gly Gly Ser Lys Ile Thr Gly Tyr Val Ile Glu Ala Gln Arg Lys Gly
325 330 335
Ser Asp Gln Trp Thr His Ile Thr Thr Val Lys Gly Leu Glu Cys Val
340 345 350
Val Lys Asn Leu Thr Glu Gly Glu Glu Tyr Thr Phe Gln Val Met Ala
355 360 365
Val Asn Ser Ala Gly Arg Ser Ala Pro Arg Glu Ser Arg Pro Val Ile
370 375 380
Val Lys Glu Gln Thr Met Leu Pro Glu Leu Asp Leu Arg Gly Ile Tyr
385 390 395 400
Gln Lys Leu Val Ile Ala Lys Ala Gly Asp Asn Ile Lys Val Glu Ile
405 410 415
Pro Val Leu Gly Arg Pro Lys Pro Thr Val Thr Trp Lys Lys Gly Asp
420 425 430
Gln Ile Leu Lys Gln Thr Gln Arg Val Asn Phe Glu Asn Thr Ala Thr
435 440 445
Ser Thr Ile Leu Asn Ile Asn Glu Cys Val Arg Ser Asp Ser Gly Pro
450 455 460
Tyr Pro Leu Thr Ala Arg Asn Ile Val Gly Glu Val Gly Asp Val Ile
465 470 475 480
Thr Ile Gln Val His Asp Ile Pro Gly Pro Pro Thr Gly Pro Ile Lys
485 490 495
Phe Asp Glu Val Ser Ser Asp Phe Val Thr Phe Ser Trp Glu Pro Pro
500 505 510
Glu Asn Asp Gly Gly Val Pro Ile Ser Asn Tyr Val Val Glu Met Arg
515 520 525
Gln Thr Asp Ser Thr Thr Trp Val Glu Leu Ala Thr Thr Val Ile Arg
530 535 540
Thr Thr Tyr Lys Ala Thr Arg Leu Thr Thr Gly Val Glu Tyr Gln Phe
545 550 555 560
Arg Val Lys Ala Gln Asn Arg Tyr Gly Val Gly Pro Gly Ile Thr Ser
565 570 575
Ala Pro Val Val Ala Asn Tyr Pro Phe Lys Val Pro Gly Pro Pro Gly
580 585 590
Thr Pro Gln Val Thr Ala Val Thr Lys Asp Ser Ile Thr Ile Ser Trp
595 600 605
His Glu Pro Leu Ser Asp Gly Gly Ser Pro Ile Leu Gly Tyr His Ile
610 615 620
Glu Arg Lys Glu Arg Asn Gly Ile Leu Trp Gln Thr Val Asn Lys Thr
625 630 635 640
Ile Val Pro Gly Asn Ile Phe Lys Ser Ser Gly Leu Thr Asp Gly Ile
645 650 655
Ala Tyr Glu Phe Arg Val Ile Ala Glu Asn Met Ala Gly Lys Ser Lys
660 665 670
Pro Ser Lys Pro Ser Glu Pro Met Leu Ala Leu Asp Pro Ile Asp Pro
675 680 685
Pro Gly Lys Pro Ile Pro Leu Asn Ile Thr Arg Asn Thr Val Thr Leu
690 695 700
Lys Trp Ala Lys Pro Glu Tyr Thr Gly Gly Phe Lys Ile Thr Ser Tyr
705 710 715 720
Ile Val Glu Lys Arg Asp Leu Pro Asn Gly Arg Trp Leu Lys Ala Asn
725 730 735
Phe Ser Asn Ile Leu Glu Asn Glu Phe Thr Val Ser Gly Leu Thr Glu
740 745 750
Asp Ala Ala Tyr Glu Phe Arg Val Ile Ala Lys Asn Ala Ala Gly Ala
755 760 765
Ile Ser Pro Pro Ser Glu Pro Ser Asp Ala Ile Thr Cys Lys Asp Asp
770 775 780
Ile Glu Ala Pro Arg Ile Met Val Asp Val Lys Phe Lys Asp Thr Ile
785 790 795 800
Ile Leu Lys Ala Gly Glu Thr Phe Lys Leu Glu Ala Asp Val Ser Gly
805 810 815
Arg Pro Pro Pro Thr Met Glu Trp Thr Lys Asp Gly Lys Glu Leu Glu
820 825 830
Asn Thr Ala Lys Leu Glu Ile Lys Ile Ala Asp Phe Ser Thr Asn Leu
835 840 845
Val Asn Lys Asp Ser Leu Arg Arg Asp Gly Gly Ala Tyr Val Leu Thr
850 855 860
Ala Thr Asn Pro Gly Gly Phe Ala Lys His Ile Phe Asn Val Lys Val
865 870 875 880
Leu Asp Arg Pro Gly Pro Pro Glu Gly Pro Leu Ala Val Ser Glu Val
885 890 895
Thr Ser Glu Lys Cys Val Leu Ser Trp Leu Pro Pro Leu Asp Asp Gly
900 905 910
Gly Ala Lys Ile Asp Tyr Tyr Val Val Gln Lys Arg Glu Thr Ser Arg
915 920 925
Leu Ala Trp Thr Asn Val Ala Ser Glu Val Gln Val Thr Lys Leu Lys
930 935 940
Val Thr Lys Leu Leu Lys Gly Asn Glu Tyr Ile Phe Arg Val Met Ala
945 950 955 960
Val Asn Lys Tyr Gly Val Gly Glu Pro Leu Glu Ser Glu Pro Val Leu
965 970 975
Ala Val Asp Pro Tyr Gly Pro Pro Asp Pro Pro Lys Asn Pro Glu Val
980 985 990
Thr Thr Ile Thr Lys Asp Ser Met Val Val Cys Trp Gly His Pro Asp
995 1000 1005
Ser Asp Gly Gly Ser Glu Ile Ile Asn Tyr Ile Val Glu Arg Arg
1010 1015 1020
Asp Lys Ala Gly Gln Arg Trp Val Lys Cys Asn Lys Lys Thr Leu
1025 1030 1035
Thr Asp Leu Arg Tyr Lys Val Ser Gly Leu Thr Glu Gly His Glu
1040 1045 1050
Tyr Glu Phe Arg Ile Met Ala Glu Asn Ala Ala Gly Ile Ser Ala
1055 1060 1065
Pro Ser Ala Thr Ser Pro Phe Tyr Lys Ala Cys Asp Ala Val Phe
1070 1075 1080
Lys Pro Gly Pro Pro Gly Asn Pro Arg Val Leu Asp Thr Ser Arg
1085 1090 1095
Ser Ser Ile Ser Ile Ala Trp Asn Lys Pro Ile Tyr Asp Gly Gly
1100 1105 1110
Ser Glu Ile Thr Gly Tyr Met Val Glu Ile Ala Leu Pro Glu Glu
1115 1120 1125
Asp Glu Trp Lys Ile Val Thr Pro Pro Ala Gly Leu Lys Ala Thr
1130 1135 1140
Ser Tyr Thr Ile Thr Asn Leu Val Glu Asn Gln Glu Tyr Lys Ile
1145 1150 1155
Arg Ile Tyr Ala Met Asn Ser Glu Gly Ile Gly Glu Pro Ala Leu
1160 1165 1170
Val Pro Gly Thr Pro Lys Ala Glu Asp Arg Met Leu Pro Pro Glu
1175 1180 1185
Ile Glu Leu Asp Ala Asp Leu Arg Lys Val Val Ile Ile Arg Ala
1190 1195 1200
Cys Cys Thr Leu Arg Leu Phe Val Pro Ile Lys Gly Arg Pro Ala
1205 1210 1215
Pro Glu Val Lys Trp Thr Arg Glu His Gly Glu Ser Leu Asp Lys
1220 1225 1230
Ala Ser Ile Glu Ser Thr Ser Ser Tyr Thr Leu Leu Ile Val Gly
1235 1240 1245
Asn Val Asn Arg Phe Asp Ser Gly Lys Tyr Ile Leu Thr Ile Glu
1250 1255 1260
Asn Ser Ser Gly Ser Lys Ser Ala Phe Val Asn Val Arg Val Leu
1265 1270 1275
Asp Thr Pro Gly Pro Pro Gln Asp Leu Lys Val Lys Glu Val Thr
1280 1285 1290
Lys Thr Ser Ala Thr Leu Thr Trp Glu Pro Pro Leu Leu Asp Gly
1295 1300 1305
Gly Ser Lys Ile Lys Asn Tyr Ile Val Glu Lys Arg Glu Ser Thr
1310 1315 1320
Arg Lys Ala Tyr Ser Thr Val Ala Thr Asn Cys His Lys Thr Ser
1325 1330 1335
Trp Lys Val Asp Gln Leu Gln Glu Gly Cys Ser Tyr Tyr Phe Arg
1340 1345 1350
Val Leu Ala Glu Asn Glu Tyr Gly Ile Gly Leu Pro Ala Glu Thr
1355 1360 1365
Ala Glu Ser Val Lys Ala Ser Glu Arg Pro Leu Pro Pro Gly Lys
1370 1375 1380
Ile Thr Leu Val Asp Val Thr Arg Asn Ser Val Ser Leu Ser Trp
1385 1390 1395
Glu Lys Pro Glu His Asp Gly Gly Ser Arg Ile Leu Gly Tyr Ile
1400 1405 1410
Val Glu Met Gln Ser Lys Gly Ser Asp Lys Trp Val Thr Cys Ala
1415 1420 1425
Thr Val Lys Val Thr Glu Ala Thr Ile Thr Gly Leu Ile Gln Gly
1430 1435 1440
Glu Glu Tyr Ser Phe Arg Val Ser Ala Gln Asn Glu Lys Gly Ile
1445 1450 1455
Ser Asp Pro Arg Gln Leu Ser Val Pro Val Ile Ala Lys Asp Leu
1460 1465 1470
Val Ile Pro Pro Ala Phe Lys Leu Leu Phe Asn Thr Tyr Thr Val
1475 1480 1485
Leu Ala Gly Glu Asp Leu Lys Val Asp Val Pro Phe Ile Gly Arg
1490 1495 1500
Pro Ile Pro Ala Val Thr Trp His Lys Asp Asp Val Pro Leu Lys
1505 1510 1515
Gln Thr Thr Arg Val Asn Ala Glu Ser Thr Glu Asn Asn Ser Leu
1520 1525 1530
Leu Thr Ile Lys Glu Ala Cys Arg Glu Asp Val Gly His Tyr Val
1535 1540 1545
Val Lys Leu Thr Asn Ser Ala Gly Glu Ala Thr Glu Thr Leu Asn
1550 1555 1560
Ile Ile Val Leu Asp Lys Pro Gly Pro Pro Thr Gly Pro Val Lys
1565 1570 1575
Met Glu Glu Val Thr Ala Asp Ser Ile Thr Leu Ser Trp Gly Pro
1580 1585 1590
Pro Lys Tyr Asp Gly Gly Ser Ser Ile Asn Asn Tyr Ile Val Glu
1595 1600 1605
Lys Arg Asp Thr Ser Thr Thr Thr Trp His Ile Val Ser Ala Thr
1610 1615 1620
Val Ala Arg Thr Thr Ile Lys Ala Cys Arg Leu Lys Thr Gly Cys
1625 1630 1635
Glu Tyr Gln Phe Arg Ile Ala Ala Glu Asn Arg Tyr Gly Lys Ser
1640 1645 1650
Thr Tyr Leu Thr Ser Glu Pro Ile Val Ala Gln Tyr Pro Phe Lys
1655 1660 1665
Val Pro Gly Pro Pro Gly Thr Pro Phe Val Thr His Ser Ser Arg
1670 1675 1680
Asp Ser Met Glu Val Gln Trp Asn Glu Pro Val Ser Asp Gly Gly
1685 1690 1695
Ser Lys Val Ile Gly Tyr His Leu Glu Arg Lys Glu Arg Asn Asn
1700 1705 1710
Ile Leu Trp Val Lys Leu Asn Lys Thr Pro Ile Pro Gln Thr Lys
1715 1720 1725
Phe Lys Thr Thr Gly Leu Asp Glu Gly Ile Glu Tyr Glu Phe Arg
1730 1735 1740
Val Ser Ala Glu Asn Ile Val Gly Ile Gly Lys Pro Ser Lys Val
1745 1750 1755
Ser Glu Cys Tyr Thr Ala Arg Asp Pro Cys Asp Pro Pro Gly Lys
1760 1765 1770
Pro Glu Ala Ile Ile Val Thr Arg Asn Ser Val Thr Leu Gln Trp
1775 1780 1785
Lys Lys Pro Thr Tyr Asp Gly Gly Ser Lys Ile Thr Gly Tyr Ile
1790 1795 1800
Val Glu Lys Lys Glu Leu Pro Glu Gly Arg Trp Met Lys Ala Ser
1805 1810 1815
Phe Thr Asn Val Ile Asp Thr Gln Phe Glu Val Thr Gly Leu Val
1820 1825 1830
Glu Asp Asn Arg Tyr Glu Phe Arg Val Ile Ala Arg Asn Ala Ala
1835 1840 1845
Gly Val Phe Ser Glu Pro Ser Glu Ser Thr Gly Ala Ile Thr Val
1850 1855 1860
Arg Asp Glu Val Glu Pro Pro Arg Ile Arg Met Asp Pro Lys Tyr
1865 1870 1875
Lys Asp Thr Ile Val Val His Ala Gly Glu Leu Phe Lys Ile Asp
1880 1885 1890
Ala Asp Ile Tyr Gly Lys Pro Ile Pro Thr Thr Gln Trp Ile Lys
1895 1900 1905
Gly Asp Gln Glu Leu Ser Asn Thr Ala Arg Leu Glu Ile Lys Ser
1910 1915 1920
Thr Asp Phe Ala Thr Ser Leu Ser Val Lys Asp Ala Val Arg Val
1925 1930 1935
Asp Ser Gly Asn Tyr Ile Leu Lys Ala Lys Asn Val Ala Gly Glu
1940 1945 1950
Lys Ser Val Thr Val Asn Val Lys Val Leu Asp Arg Pro Gly Pro
1955 1960 1965
Pro Glu Gly Pro Ile Val Ile Ser Gly Val Thr Ala Glu Lys Cys
1970 1975 1980
Ala Leu Ala Trp Lys Pro Pro Leu Gln Asp Gly Gly Ser Asp Ile
1985 1990 1995
Ile Asn Tyr Ile Val Glu Arg Arg Glu Thr Ser Arg Leu Val Trp
2000 2005 2010
Thr Val Val Asp Ala Asn Val Gln Thr Leu Ser Cys Lys Val Thr
2015 2020 2025
Lys Leu Leu Glu Gly Asn Glu Tyr Ile Phe Arg Ile Met Ala Val
2030 2035 2040
Asn Lys Tyr Gly Val Gly Glu Pro Leu Glu Ser Glu Pro Val Ile
2045 2050 2055
Ala Lys Asn Pro Phe Val Val Pro Asp Ala Pro Lys Ala Pro Glu
2060 2065 2070
Ile Thr Ala Val Thr Lys Asp Ser Met Ile Val Val Trp Glu Arg
2075 2080 2085
Pro Ala Ser Asp Gly Gly Ser Glu Ile Leu Gly Tyr Val Leu Glu
2090 2095 2100
Lys Arg Asp Lys Glu Gly Ile Arg Trp Thr Arg Cys His Lys Arg
2105 2110 2115
Leu Ile Gly Glu Leu Arg Leu Arg Val Thr Gly Leu Leu Glu Asn
2120 2125 2130
His Asn Tyr Glu Phe Arg Val Ser Ala Glu Asn Ala Ala Gly Leu
2135 2140 2145
Ser Glu Pro Ser Pro Pro Ser Ala Tyr Gln Lys Ala Cys Asp Pro
2150 2155 2160
Ile Tyr Lys Pro Gly Pro Pro Asn Asn Pro Lys Val Ile Asp Ile
2165 2170 2175
Thr Arg Ser Ser Val Phe Leu Ser Trp Gly Lys Pro Ile Tyr Asp
2180 2185 2190
Gly Gly Cys Glu Ile Gln Gly Tyr Ile Val Glu Lys Cys Asp Val
2195 2200 2205
Ser Val Gly Glu Trp Thr Met Cys Thr Pro Pro Thr Gly Ile Asn
2210 2215 2220
Lys Thr Asn Ile Glu Val Glu Lys Leu Leu Glu Lys His Glu Tyr
2225 2230 2235
Asn Phe Arg Ile Cys Ala Ile Asn Lys Ala Gly Val Gly Glu His
2240 2245 2250
Ala Asp Val Pro Gly Pro Val Ile Val Glu Glu Lys Met Glu Ala
2255 2260 2265
Pro Asp Leu Asp Leu Asp Met Glu Leu Arg Lys Ile Ile Asn Ile
2270 2275 2280
Arg Ala Gly Gly Ser Leu Arg Leu Phe Val Pro Ile Lys Gly Arg
2285 2290 2295
Pro Thr Pro Glu Val Lys Trp Gly Lys Val Asp Gly Glu Ile Arg
2300 2305 2310
Asp Ala Ala Ile Ile Asp Ser Thr Ser Ser Phe Thr Ser Leu Val
2315 2320 2325
Leu Asp Asn Val Asn Arg Tyr Asp Ser Gly Lys Tyr Thr Leu Thr
2330 2335 2340
Leu Glu Asn Ser Ser Gly Thr Lys Ser Ala Phe Val Thr Val Arg
2345 2350 2355
Val Leu Asp Thr Pro Ser Pro Pro Val Asn Leu Lys Val Thr Glu
2360 2365 2370
Ile Thr Lys Asp Ser Val Ser Ile Thr Trp Glu Pro Pro Leu Leu
2375 2380 2385
Asp Gly Gly Ser Lys Ile Lys Asn Tyr Ile Val Glu Lys Arg Glu
2390 2395 2400
Ala Thr Arg Lys Ser Tyr Ala Ala Val Val Thr Asn Cys His Lys
2405 2410 2415
Asn Ser Trp Lys Ile Asp Gln Leu Gln Glu Gly Cys Ser Tyr Tyr
2420 2425 2430
Phe Arg Val Thr Ala Glu Asn Glu Tyr Gly Ile Gly Leu Pro Ala
2435 2440 2445
His Thr Asp Asp Pro Val Lys Val Ala Glu Val Pro Gln Pro Pro
2450 2455 2460
Gly Lys Ile Thr Val Asp Asp Val Thr Arg Asn Ser Val Ser Leu
2465 2470 2475
Ser Trp Thr Lys Pro Glu His Asp Gly Gly Ser Lys Ile Ile Gln
2480 2485 2490
Tyr Ile Val Glu Met Gln Ala Lys His Ser Glu Lys Trp Ser Glu
2495 2500 2505
Cys Ala Arg Val Lys Ser Leu Glu Ala Val Ile Thr Asn Leu Thr
2510 2515 2520
Gln Gly Glu Glu Tyr Leu Phe Arg Val Val Ala Val Asn Glu Lys
2525 2530 2535
Gly Arg Ser Asp Pro Arg Ser Leu Ala Val Pro Ile Val Ala Lys
2540 2545 2550
Asp Leu Val Ile Glu Pro Asp Val Arg Pro Gly Phe Asn Thr Tyr
2555 2560 2565
Ser Val Gln Val Gly Gln Asp Leu Lys Ile Glu Val Pro Val Ser
2570 2575 2580
Gly Arg Pro Lys Pro Thr Ile Thr Trp Thr Lys Asp Gly Leu Pro
2585 2590 2595
Leu Lys Gln Thr Thr Arg Ile Asn Val Thr Asp Ser Leu Asp Leu
2600 2605 2610
Thr Val Leu Ser Ile Lys Glu Thr His Lys Asp Asp Gly Gly His
2615 2620 2625
Tyr Gly Ile Thr Val Ala Asn Val Val Gly Gln Lys Thr Ala Ser
2630 2635 2640
Ile Glu Ile Ile Thr Leu Asp Lys Pro Asp Pro Pro Lys Gly Pro
2645 2650 2655
Val Lys Phe Asp Glu Val Ser Ala Glu Ser Ile Thr Leu Ser Trp
2660 2665 2670
Glu Pro Pro Leu Tyr Thr Gly Gly Cys Gln Ile Thr Asn Tyr Ile
2675 2680 2685
Val Gln Lys Arg Asp Thr Thr Thr Thr Val Trp Asp Val Val Ser
2690 2695 2700
Ala Thr Val Ala Arg Thr Thr Leu Lys Val Thr Lys Leu Lys Thr
2705 2710 2715
Gly Thr Glu Tyr Gln Phe Arg Ile Phe Ala Glu Asn Arg Tyr Gly
2720 2725 2730
Gln Ser Phe Ala Leu Glu Ser Glu Pro Ile Val Ala Gln Tyr Pro
2735 2740 2745
Tyr Lys Glu Pro Gly Pro Pro Gly Thr Pro Phe Ala Thr Ala Ile
2750 2755 2760
Ser Lys Asp Ser Met Val Ile Gln Trp His Glu Pro Val Asn Asn
2765 2770 2775
Gly Gly Ser Pro Ile Ile Gly Tyr His Leu Glu Arg Lys Glu Arg
2780 2785 2790
Asn Ser Ile Leu Trp Thr Lys Val Asn Lys Thr Ile Ile His Asp
2795 2800 2805
Thr Gln Phe Lys Ala Val Asn Leu Glu Glu Gly Ile Glu Tyr Glu
2810 2815 2820
Phe Arg Val Tyr Ala Glu Asn Ile Val Gly Val Gly Lys Ala Ser
2825 2830 2835
Lys Asn Ser Glu Cys Tyr Val Ala Arg Asp Pro Cys Asp Pro Pro
2840 2845 2850
Gly Thr Pro Glu Ala Ile Ile Val Lys Arg Asn Glu Ile Thr Leu
2855 2860 2865
Gln Trp Thr Lys Pro Ala Tyr Asp Gly Gly Ser Ile Ile Thr Gly
2870 2875 2880
Tyr Ile Val Glu Lys Arg Asp Leu Pro Glu Gly Arg Trp Met Lys
2885 2890 2895
Ala Ser Phe Thr Asn Val Ile Glu Thr Gln Phe Thr Val Ser Gly
2900 2905 2910
Leu Thr Glu Asp Gln Arg Tyr Glu Phe Arg Val Ile Ala Lys Asn
2915 2920 2925
Ala Ala Gly Thr Ile Ser Lys Pro Ser Asp Ser Thr Gly Pro Ile
2930 2935 2940
Thr Ala Lys Asp Glu Val Glu Leu Pro Arg Ile Ser Met Asp Pro
2945 2950 2955
Lys Phe Arg Asp Thr Ile Val Val Asn Ala Gly Glu Thr Phe Arg
2960 2965 2970
Leu Glu Ala Asp Val His Gly Lys Pro Leu Pro Thr Ile Glu Trp
2975 2980 2985
Leu Arg Gly Asp Lys Glu Ile Glu Glu Ser Ala Arg Phe Glu Ile
2990 2995 3000
Lys Asn Thr Asp Phe Lys Ala Leu Leu Ile Val Lys Asp Ala Ile
3005 3010 3015
Arg Ile Asp Gly Gly Gln Tyr Ile Leu Arg Ala Ser Asn Val Ala
3020 3025 3030
Gly Ser Lys Ser Phe Pro Val Asn Val Lys Val Leu Asp Arg Pro
3035 3040 3045
Gly Pro Pro Glu Gly Pro Val Gln Val Thr Gly Val Thr Ala Glu
3050 3055 3060
Lys Cys Thr Leu Ser Trp Ser Pro Pro Leu Gln Asp Gly Gly Ser
3065 3070 3075
Asp Ile Ser His Tyr Val Val Glu Lys Arg Glu Thr Ser Arg Leu
3080 3085 3090
Ala Trp Thr Val Val Ala Ser Glu Val Val Thr Asn Ser Leu Lys
3095 3100 3105
Val Thr Lys Leu Leu Glu Gly Asn Glu Tyr Ile Phe Arg Ile Met
3110 3115 3120
Ala Val Asn Lys Tyr Gly Val Gly Glu Pro Leu Glu Ser Ala Pro
3125 3130 3135
Val Leu Met Lys Asn Pro Phe Val Pro Pro Gly Pro Pro Lys Ser
3140 3145 3150
Leu Glu Ile Thr Asn Val Ala Lys Asp Ser Met Thr Val Cys Trp
3155 3160 3165
Asn Arg Pro Asp Ser Asp Gly Gly Ser Glu Ile Ile Gly Tyr Ile
3170 3175 3180
Val Glu Lys Arg Asp Arg Ser Gly Ile Arg Trp Ile Lys Cys Asn
3185 3190 3195
Lys Arg Arg Val Thr Asp Leu Arg Leu Arg Val Thr Gly Leu Thr
3200 3205 3210
Glu Asp His Glu Tyr Glu Phe Arg Val Ser Ala Glu Asn Ala Ala
3215 3220 3225
Gly Val Gly Glu Pro Ser Ser Pro Thr Val Tyr Tyr Lys Ala Cys
3230 3235 3240
Asp Pro Val Phe Lys Pro Gly Pro Pro Thr Asn Ala His Ile Val
3245 3250 3255
Asp Thr Thr Lys Asn Ser Ile Thr Leu Ala Trp Gly Lys Pro Ile
3260 3265 3270
Tyr Asp Gly Gly Ser Glu Ile Leu Gly Tyr Ile Val Glu Ile Cys
3275 3280 3285
Lys Ala Asp Glu Glu Glu Trp Gln Ile Val Thr Pro Gln Thr Gly
3290 3295 3300
Leu Lys Ala Thr Arg Phe Glu Ile Ser Lys Leu Thr Glu His Gln
3305 3310 3315
Glu Tyr Lys Ile Arg Val Cys Ala Leu Asn Lys Val Gly Leu Gly
3320 3325 3330
Glu Ala Thr Ser Val Pro Gly Thr Val Lys Pro Glu Asp Lys Leu
3335 3340 3345
Glu Ala Pro Glu Leu Asp Leu Asp Ser Glu Leu Arg Lys Gly Ile
3350 3355 3360
Val Val Arg Ala Gly Gly Ser Val Arg Ile His Ile Pro Phe Lys
3365 3370 3375
Gly Arg Pro Thr Pro Glu Ile Thr Trp Ser Arg Glu Glu Gly Glu
3380 3385 3390
Phe Thr Asp Lys Val Gln Ile Glu Lys Gly Ala Asn Ser Thr Gln
3395 3400 3405
Leu Ser Ile Asp Asn Cys Asp Arg Asn Asp Ala Gly Lys Tyr Ile
3410 3415 3420
Leu Lys Leu Glu Asn Ser Ser Gly Ser Lys Ser Ala Phe Val Thr
3425 3430 3435
Val Lys Val Leu Asp Thr Pro Gly Pro Pro Gln Asn Leu Ala Val
3440 3445 3450
Lys Glu Val Lys Lys Asp Ser Val Leu Leu Thr Trp Glu Pro Pro
3455 3460 3465
Ile Ile Asp Gly Gly Ala Lys Ile Lys Asn Tyr Val Ile Asp Lys
3470 3475 3480
Arg Glu Ser Thr Arg Lys Ala Tyr Ala Asn Val Ser Asn Lys Cys
3485 3490 3495
Ser Lys Thr Ser Phe Lys Val Glu Asn Leu Thr Glu Gly Ala Thr
3500 3505 3510
Tyr Tyr Phe Arg Val Met Ala Glu Asn Glu Phe Gly Ile Gly Val
3515 3520 3525
Pro Val Glu Thr Val Asp Ala Val Lys Ala Ala Glu Pro Pro Ser
3530 3535 3540
Pro Pro Gly Lys Val Thr Leu Thr Asp Val Ser Gln Thr Ser Ala
3545 3550 3555
Ser Leu Met Trp Glu Lys Pro Glu His Asp Gly Gly Ser Arg Ile
3560 3565 3570
Leu Gly Tyr Val Val Glu Met Gln Pro Lys Gly Thr Glu Lys Trp
3575 3580 3585
Ser Val Val Ala Glu Ser Lys Val Cys Asn Ala Ser Val Thr Gly
3590 3595 3600
Leu Ser Ser Gly Gln Glu Tyr Gln Phe Arg Val Lys Ala Tyr Asn
3605 3610 3615
Glu Lys Gly Arg Ser Asp Pro Arg Val Leu Gly Val Pro Val Ile
3620 3625 3630
Ala Lys Asp Leu Thr Ile Gln Pro Ser Phe Gln Leu Pro Phe Asn
3635 3640 3645
Thr Tyr Thr Val Gln Ala Gly Glu Asp Leu Lys Ile Glu Ile Pro
3650 3655 3660
Val Ile Gly Arg Pro Arg Pro Gln Ile Ser Trp Val Lys Asp Gly
3665 3670 3675
Glu Leu Leu Lys Gln Thr Thr Arg Val Asn Val Glu Glu Thr Ala
3680 3685 3690
Thr Ser Thr Ile Leu His Ile Lys Glu Ser Asn Lys Asp Asp Phe
3695 3700 3705
Gly Lys Tyr Thr Ile Thr Ala Thr Asn Ser Ala Gly Thr Ala Thr
3710 3715 3720
Glu Asn Leu Ser Ile Ile Val Leu Glu Lys Pro Gly Pro Pro Val
3725 3730 3735
Gly Pro Val Arg Phe Asp Glu Val Ser Ala Asp Phe Val Val Ile
3740 3745 3750
Ser Trp Glu Pro Pro Ala Tyr Thr Gly Gly Cys Gln Ile Ser Asn
3755 3760 3765
Tyr Ile Val Glu Lys Arg Asp Thr Thr Thr Thr Thr Trp His Met
3770 3775 3780
Val Ser Ala Thr Val Ala Arg Thr Thr Ile Lys Ile Thr Lys Leu
3785 3790 3795
Lys Thr Gly Ser Glu Tyr Gln Phe Arg Ile Phe Ala Glu Asn Arg
3800 3805 3810
Tyr Gly Lys Ser Ala Ser Leu Asp Ser Lys Pro Val Ile Val Gln
3815 3820 3825
Tyr Pro Phe Lys Glu Pro Gly Pro Pro Gly Thr Pro Phe Val Thr
3830 3835 3840
Ser Ile Ser Lys Asp Gln Met Leu Val Gln Trp His Glu Pro Val
3845 3850 3855
Asn Asp Gly Gly Ser Lys Val Ile Gly Tyr His Leu Glu Gln Lys
3860 3865 3870
Glu Lys Asn Ser Ile Leu Trp Ile Lys Leu Asn Lys Thr Pro Ile
3875 3880 3885
Gln Asp Thr Lys Phe Lys Thr Thr Gly Leu Asp Glu Gly Leu Glu
3890 3895 3900
Tyr Glu Phe Lys Val Ser Ala Glu Asn Ile Val Gly Ile Gly Lys
3905 3910 3915
Pro Ser Lys Val Ser Glu Cys Tyr Val Ala Arg Asp Pro Cys Asp
3920 3925 3930
Pro Pro Gly Arg Pro Glu Ala Ile Val Ile Thr Arg Asn Asn Val
3935 3940 3945
Thr Leu Lys Trp Lys Lys Pro Ala Tyr Asp Gly Gly Ser Lys Ile
3950 3955 3960
Thr Gly Tyr Ile Val Glu Lys Lys Asp Leu Pro Asp Gly Arg Trp
3965 3970 3975
Met Lys Ala Ser Phe Thr Asn Val Leu Glu Thr Glu Phe Thr Val
3980 3985 3990
Ser Gly Leu Val Glu Asp Gln Arg Tyr Glu Phe Arg Val Ile Ala
3995 4000 4005
Arg Asn Ala Ala Gly Asn Phe Ser Glu Pro Ser Glu Ser Thr Gly
4010 4015 4020
Ala Ile Thr Ala Arg Asp Glu Ile Asp Ala Pro Asn Ala Ser Leu
4025 4030 4035
Asp Pro Lys Tyr Lys Asp Val Ile Val Val His Ala Gly Glu Thr
4040 4045 4050
Phe Val Leu Glu Ala Asp Ile Arg Gly Lys Pro Ile Pro Asp Ile
4055 4060 4065
Val Trp Leu Lys Asp Gly Lys Glu Leu Glu Glu Thr Thr Ala Arg
4070 4075 4080
Met Glu Ile Lys Ser Thr Ile Gln Lys Thr Thr Leu Val Val Lys
4085 4090 4095
Asp Cys Ile Arg Ser Asp Gly Gly Gln Tyr Ile Leu Lys Leu Ser
4100 4105 4110
Asn Val Gly Gly Thr Lys Ser Ile Pro Ile Thr Val Lys Val Leu
4115 4120 4125
Asp Lys Pro Gly Pro Pro Glu Gly Pro Leu Lys Val Ser Gly Val
4130 4135 4140
Thr Ala Glu Lys Cys Tyr Leu Ala Trp Asn Pro Pro Leu His Asp
4145 4150 4155
Gly Gly Ala Asn Ile Ser His Tyr Ile Ile Glu Lys Arg Glu Thr
4160 4165 4170
Ser Arg Leu Ser Trp Thr Gln Val Ser Thr Glu Val Gln Ala Leu
4175 4180 4185
Asn Tyr Lys Val Thr Lys Leu Leu Pro Gly Asn Glu Tyr Ile Phe
4190 4195 4200
Arg Val Met Ala Val Asn Lys Tyr Gly Ile Gly Glu Pro Leu Glu
4205 4210 4215
Ser Glu Pro Ile Ile Ala Arg Asn Pro Tyr Lys Pro Pro Gly Pro
4220 4225 4230
Pro Ser Pro Pro Glu Val Ser Ala Ile Thr Lys Asp Ser Met Val
4235 4240 4245
Val Thr Trp Ala Arg Pro Ala Asp Asp Gly Gly Ala Glu Ile Glu
4250 4255 4260
Gly Tyr Ile Leu Glu Lys Arg Asp Lys Glu Gly Ile Arg Trp Thr
4265 4270 4275
Lys Cys Asn Lys Lys Thr Leu Thr Asp Leu Arg Phe Arg Val Thr
4280 4285 4290
Gly Leu Thr Glu Gly His Ser Tyr Glu Phe Arg Val Ala Ala Glu
4295 4300 4305
Asn Ala Ala Gly Val Gly Glu Pro Ser Glu Pro Ser Val Phe Tyr
4310 4315 4320
Arg Ala Cys Asp Ala Leu Tyr Pro Pro Gly Pro Pro Ser Asn Pro
4325 4330 4335
Lys Val Thr Asp Thr Ser Arg Ser Ser Val Ser Leu Ala Trp Asn
4340 4345 4350
Lys Pro Ile Tyr Asp Gly Gly Ala Pro Val Lys Gly Tyr Val Val
4355 4360 4365
Glu Val Lys Glu Ala Thr Ala Asp Glu Trp Thr Thr Cys Thr Pro
4370 4375 4380
Pro Thr Gly Leu Gln Gly Lys Gln Phe Thr Val Thr Glu Leu Lys
4385 4390 4395
Glu Asn Thr Glu Tyr Asn Phe Arg Ile Cys Ala Ile Asn Ser Glu
4400 4405 4410
Gly Val Gly Glu Pro Ala Thr Ile Pro Gly Ser Val Val Ala Lys
4415 4420 4425
Glu Arg Gln Glu Pro Pro Glu Ile Glu Leu Asp Ala Asp Leu Arg
4430 4435 4440
Lys Val Val Ile Leu Arg Ala Ser Ala Thr Leu Arg Leu Phe Val
4445 4450 4455
Thr Ile Lys Gly Arg Pro Glu Pro Glu Val Lys Trp Glu Lys Ala
4460 4465 4470
Glu Gly Thr Leu Thr Asp Arg Ala Gln Ile Glu Val Thr Ser Ser
4475 4480 4485
Tyr Thr Met Leu Val Ile Asp Asn Val Thr Arg Phe Asp Ser Gly
4490 4495 4500
Arg Tyr Asn Leu Thr Leu Glu Asn Asn Ser Gly Ser Lys Thr Ala
4505 4510 4515
Phe Val Asn Val Arg Val Leu Asp Ser Pro Ser Ala Pro Val Asn
4520 4525 4530
Leu Asn Val Arg Glu Val Lys Lys Asp Ser Val Thr Leu Ala Trp
4535 4540 4545
Glu Pro Pro Leu Ile Asp Gly Gly Ala Lys Ile Thr Asn Tyr Ile
4550 4555 4560
Val Glu Lys Arg Glu Thr Thr Arg Lys Ala Tyr Ala Thr Val Thr
4565 4570 4575
Asn Asn Cys Thr Lys Asn Thr Phe Lys Ile Glu Asn Leu Gln Glu
4580 4585 4590
Gly Cys Ser Tyr Tyr Phe Arg Val Leu Ala Ala Asn Glu Tyr Gly
4595 4600 4605
Ile Gly Leu Pro Ala Glu Thr Thr Glu Pro Val Lys Val Ser Glu
4610 4615 4620
Pro Pro Leu Pro Pro Gly Arg Val Thr Leu Val Asp Val Thr Arg
4625 4630 4635
Ser Thr Ala Thr Ile Lys Trp Glu Lys Pro Glu Ser Asp Gly Gly
4640 4645 4650
Ser Lys Ile Ile Gly Tyr Val Val Glu Met Gln Thr Lys Gly Ser
4655 4660 4665
Glu Lys Trp Ser Thr Cys Thr Gln Val Lys Thr Leu Glu Ala Thr
4670 4675 4680
Ile Ser Gly Leu Thr Ala Gly Glu Glu Tyr Ile Phe Arg Val Ala
4685 4690 4695
Ala Ile Asn Glu Lys Gly Lys Ser Asp Pro Arg Gln Leu Gly Val
4700 4705 4710
Pro Val Ile Ala Arg Asp Ile Glu Ile Lys Pro Ser Val Glu Leu
4715 4720 4725
Pro Phe Asn Thr Phe Asn Val Lys Ala Arg Asp Gln Leu Lys Ile
4730 4735 4740
Asp Val Pro Phe Lys Gly Arg Pro Gln Ala Thr Val Ser Trp Lys
4745 4750 4755
Lys Asp Gly Gln Thr Leu Lys Glu Thr Thr Arg Val Asn Val Ser
4760 4765 4770
Ser Ser Lys Thr Val Thr Ser Leu Ile Ile Lys Glu Ala Ser Arg
4775 4780 4785
Glu Asp Val Gly Thr Tyr Glu Leu Cys Val Ser Asn Ser Ala Gly
4790 4795 4800
Ser Ile Thr Val Pro Ile Thr Val Ile Val Leu Asp Arg Pro Gly
4805 4810 4815
Pro Pro Gly Pro Ile His Ile Asp Glu Val Ser Cys Asp Asn Ile
4820 4825 4830
Thr Ile Ser Trp Asn Pro Pro Glu Tyr Asp Gly Gly Cys Gln Ile
4835 4840 4845
Ser Asn Tyr Ile Val Glu Lys Arg Glu Thr Thr Ser Thr Thr Trp
4850 4855 4860
His Val Val Ser Gln Ala Val Ala Arg Thr Ser Ile Lys Ile Val
4865 4870 4875
Arg Leu Val Thr Gly Ser Glu Tyr Gln Phe Arg Val Cys Ala Glu
4880 4885 4890
Asn Arg Tyr Gly Lys Ser Ser Tyr Ser Asp Ser Pro Ala Val Val
4895 4900 4905
Ala Glu Tyr Pro Phe Ser Pro Pro Gly Pro Pro Gly Thr Pro Arg
4910 4915 4920
Val Val His Ala Thr Lys Ser Thr Met Val Val Thr Trp Gln Val
4925 4930 4935
Pro Val Asn Asp Gly Gly Ser Gln Val Leu Gly Tyr His Leu Glu
4940 4945 4950
Tyr Lys Glu Arg Ser Ser Ile Leu Trp Ser Lys Ala Asn Lys Ile
4955 4960 4965
Leu Ile Thr Asp Thr Gln Met Lys Val Ser Gly Leu Asp Glu Gly
4970 4975 4980
Leu Met Tyr Glu Tyr Arg Val Tyr Ala Glu Asn Ile Ala Gly Ile
4985 4990 4995
Gly Lys Cys Ser Lys Ser Cys Glu Pro Val Pro Ala Arg Asp Pro
5000 5005 5010
Cys Asp Pro Pro Gly Gln Pro Glu Val Thr Asn Ile Thr Arg Lys
5015 5020 5025
Ser Val Ser Leu Lys Trp Ser Lys Pro Arg Tyr Asp Gly Gly Ala
5030 5035 5040
Lys Ile Thr Gly Tyr Ile Ile Glu Arg Arg Glu Leu Pro Asp Gly
5045 5050 5055
Arg Trp Leu Lys Cys Asn Phe Thr Asn Val Gln Glu Thr Tyr Phe
5060 5065 5070
Glu Val Thr Glu Leu Thr Glu Asp Gln Arg Tyr Glu Phe Arg Val
5075 5080 5085
Phe Ala Lys Asn Ala Ala Asp Ser Val Ser Glu Pro Ser Glu Ser
5090 5095 5100
Thr Gly Pro Ile Thr Val Lys Asp Asp Val Glu Ala Pro Arg Ile
5105 5110 5115
Met Met Asp Val Lys Phe Arg Asp Val Ile Val Val Lys Ala Gly
5120 5125 5130
Glu Val Leu Lys Ile Asn Ala Asp Val Ala Gly Arg Pro Leu Pro
5135 5140 5145
Val Ile Ser Trp Ala Lys Asp Gly Val Glu Ile Glu Glu Arg Ala
5150 5155 5160
Arg Thr Glu Ile Val Ser Thr Asp Tyr Asn Thr Leu Leu Thr Val
5165 5170 5175
Lys Asp Cys Val Arg Arg Asp Ser Gly Gln Tyr Val Leu Thr Val
5180 5185 5190
Lys Asn Val Ala Gly Thr Arg Ser Met Ala Val Asn Cys Lys Val
5195 5200 5205
Leu Asp Lys Pro Gly Pro Pro Ala Gly Pro Leu Gln Ile Thr Gly
5210 5215 5220
Leu Thr Ala Glu Lys Cys Ser Leu Ser Trp Gly Pro Pro Gln Glu
5225 5230 5235
Asp Gly Gly Ala Ala Ile Asp Tyr Tyr Ile Val Glu Lys Arg Glu
5240 5245 5250
Thr Ser His Leu Ala Trp Thr Ile Cys Glu Gly Glu Leu Arg Thr
5255 5260 5265
Thr Ser Cys Lys Val Thr Lys Leu Leu Lys Gly Asn Glu Tyr Ile
5270 5275 5280
Phe Arg Val Thr Gly Val Asn Lys Tyr Gly Val Gly Glu Pro Leu
5285 5290 5295
Glu Ser Val Ala Val Lys Ala Leu Asp Pro Phe Thr Val Pro Ser
5300 5305 5310
Pro Pro Thr Ser Leu Glu Ile Thr Ser Val Thr Lys Glu Phe Met
5315 5320 5325
Thr Leu Cys Trp Ser Arg Pro Glu Ser Asp Gly Gly Ser Glu Ile
5330 5335 5340
Ser Gly Tyr Ile Ile Glu Arg Arg Glu Lys Asn Ser Leu Arg Trp
5345 5350 5355
Val Arg Val Asn Lys Arg Pro Val Tyr Asp Leu Arg Val Lys Ser
5360 5365 5370
Thr Gly Leu Arg Glu Gly Cys Glu Tyr Glu Tyr Arg Val Tyr Ala
5375 5380 5385
Glu Asn Ala Ala Gly Leu Ser Leu Pro Ser Glu Thr Ser Pro Leu
5390 5395 5400
Val Arg Ala Glu Asp Pro Val Phe Leu Pro Ser Pro Pro Ser Lys
5405 5410 5415
Pro Gln Ile Val Asp Ser Gly Lys Thr Asn Ile Thr Ile Ser Trp
5420 5425 5430
Val Lys Pro Leu Phe Asp Gly Gly Thr Pro Ile Thr Gly Tyr Thr
5435 5440 5445
Val Glu Tyr Lys Lys Ser Ser Glu Thr Asp Trp His Thr Ala Ile
5450 5455 5460
Gln Asn Phe Arg Gly Thr Glu Tyr Thr Ala Ser Gly Leu Thr Thr
5465 5470 5475
Gly Thr Glu Tyr Val Phe Arg Val Arg Ser Ile Asn Lys Val Gly
5480 5485 5490
Ala Ser Asp Pro Ser Asp Ser Ser Asp Pro Gln Ile Ala Lys Glu
5495 5500 5505
Arg Glu Glu Glu Pro Val Phe Asp Leu Asp Ser Glu Met Lys Lys
5510 5515 5520
Thr Leu Ile Val Lys Ala Gly Ser Ser Phe Thr Met Thr Val Pro
5525 5530 5535
Phe Arg Gly Arg Pro Val Pro Ser Val Ser Trp Ser Lys Pro Asp
5540 5545 5550
Thr Asp Leu Arg Thr Arg Ala Tyr Ile Asp Ser Thr Asp Ser Arg
5555 5560 5565
Thr Ser Leu Thr Ile Glu Asn Ala Asn Arg Asn Asp Ser Gly Lys
5570 5575 5580
Tyr Thr Leu Thr Ile Gln Asn Ile Leu Asn Ala Ala Ser Leu Thr
5585 5590 5595
Leu Val Val Lys Val Leu Asp Ser Pro Gly Pro Pro Ala Gly Val
5600 5605 5610
Thr Val Arg Asp Ile Thr Lys Glu Ser Ala Val Leu Ala Trp Asp
5615 5620 5625
Val Pro Glu Asn Asp Gly Gly Ala Pro Val Lys Asn Tyr His Ile
5630 5635 5640
Glu Lys Arg Glu Ala Ser Lys Lys Ala Trp Val Ser Val Thr Asn
5645 5650 5655
Asn Cys Asn Arg Leu Ser Tyr Lys Ile Thr Asn Leu Gln Glu Gly
5660 5665 5670
Ala Ile Tyr Tyr Phe Arg Val Ser Gly Glu Asn Glu Phe Gly Val
5675 5680 5685
Gly Val Pro Ala Glu Thr Lys Glu Gly Val Lys Ile Thr
5690 5695 5700
<210> 5
<211> 17106
<212> DNA
<213> Sus scrofa
<400> 5
atcccccagg accaccttca aacgcacgtg tcactgatac taccaagaaa tctgcttctt 60
tggcatgggg caagcctcac tatgatggtg gccttgaaat cactggctat gtcgtagagc 120
atcaaaaagt cggagaggat gcctggataa aggatacaac aggaactgcc ctcagaatca 180
ctgagtttgt tgtccctgac cttcagacta aagaaaaata caactttaga atcagtgcca 240
tcaatgacgc aggtgttggg gagccagccg tgatcccaaa tgttgaaatt gtagaacgag 300
agatggctcc tgattttgaa ctagatgccg agcttcgaag aacgcttgtt gtgagagcgg 360
gtctcagtat tagaatattt gtgcccatta aaggtcgtcc tgctcctgaa gtgacatgga 420
ccaaagacga tatcaacctt aaaaaccgag ctaacatcga gaatacagaa tcattcactc 480
tcctgattat cccagaatgt aacagatatg acaccggtaa atttgtgatg accattgaaa 540
accctgctgg aaagaaaagt ggctttgtta atgttagagt cttggacacc ccaggacccg 600
tccttaacct gaggcctact gacatcacaa aggagagtgt caccctgcac tgggacctcc 660
ctctgataga tgggggctca cgtataacaa actacattgt ggagaaacga gaagcgacac 720
ggaaatctta ctccacagtc accactaagt gccataagtg tacatataaa gttactggct 780
tatcggaagg atgtgaatac ttcttcagag tgatggcaga aaatgaatac ggaattgggg 840
agccagcgga aacaacagag cctgtaagag cctccgaagc gccgtcacca ccagacagtc 900
ttaacattat ggacataact aagagcacag tcagcctggc gtggcctaaa cccaaacacg 960
atgggggcag taagatcact ggctatgtga ttgaagccca aagaaaaggc tcagaccagt 1020
ggacccacat cacaaccgtg aaggggttag aatgtgttgt gaagaatcta actgaagggg 1080
aagaatatac cttccaggtg atggcagtga acagtgcagg gagaagtgcc ccgagagaaa 1140
gcagacccgt cattgtcaag gagcagacaa tgcttccaga attggatctc cgtggcattt 1200
atcagaaatt agtcattgcc aaagctggtg acaacatcaa agttgaaatt ccagtgcttg 1260
gtcgaccaaa gcccacggtg acatggaaaa aaggagacca gattcttaaa cagacacaga 1320
gagttaactt tgaaaataca gcaacctcaa ccatcttaaa tatcaatgag tgtgtaagaa 1380
gtgatagtgg gccctaccca ttaacagcaa ggaacatcgt gggagaggtt ggggacgtca 1440
tcaccattca agtccatgat atcccagggc cgccgactgg accaatcaaa tttgatgaag 1500
tttcatctga ttttgtcacc ttctcttggg agccacctga gaacgacggg ggtgtgccaa 1560
taagcaacta cgtggtagaa atgcgacaga ccgacagtac cacatgggtt gagttagcaa 1620
ccaccgtcat acgcactacc tataaagcca ctcgcctcac tactggggtg gaatatcaat 1680
tccgagtaaa agctcaaaac agatacggag ttggaccagg catcacatca gcacctgtag 1740
tcgccaacta tccatttaag gttcctgggc ctcctggtac ccctcaggta actgcagtta 1800
ccaaagattc cataacaatt agctggcacg agcctctttc tgatggtgga agccccattt 1860
taggatatca catcgaaaga aaagaacgaa atggtattct ctggcagact gtgaacaaaa 1920
caatagtacc aggcaacatt ttcaaatcaa gtggacttac ggatggcatt gcttatgaat 1980
tccgggtgat cgcagaaaac atggcaggca aaagcaagcc gagcaaacca tctgaaccta 2040
tgttagctct ggatcccatt gacccacctg ggaaaccaat tcctctaaat attactagaa 2100
acacagtgac acttaaatgg gctaagcctg agtatactgg aggctttaaa attaccagtt 2160
acatagtcga aaagagagat cttcctaatg gacggtggct aaaggccaac ttcagcaaca 2220
tcttggagaa tgaatttaca gtcagcggcc taacagaaga tgccgcatat gaattccgcg 2280
tgattgccaa aaatgctgct ggtgctatta gcccaccatc tgaaccatca gatgctatca 2340
catgcaagga tgacattgag gcaccaagga taatggtaga cgtcaaattt aaggacacaa 2400
tcatattaaa agcaggtgaa acattcaagc tggaagctga cgtttcaggt cgcccacctc 2460
caacaatgga atggaccaaa gatggaaagg agcttgaaaa cacagccaaa ttagaaatta 2520
aaattgcaga tttctctact aatctggtaa acaaggattc actaagaagg gatggtggcg 2580
cctatgtcct tacagcaact aatcccggtg gttttgccaa acacattttc aatgttaaag 2640
ttcttgatag accaggccca cctgaaggac ctttggctgt atctgaagtg acatcagaaa 2700
aatgtgtatt gtcatggttg cctccactgg atgatggagg cgccaaaatt gattattacg 2760
tggttcagaa acgtgaaacc agcagattgg catggacgaa cgtagcctcc gaagtgcaag 2820
tcacaaagct aaaggtcact aaactgttga aaggcaatga atacatattc cgtgtcatgg 2880
ctgtaaataa atatggggtt ggagagccac tggaatcaga gcctgtgctt gcagtagatc 2940
cttatggacc tcctgatcca cccaaaaacc ctgaagttac aactattact aaagattcca 3000
tggttgtctg ctggggacat cctgactctg atggtggaag tgaaatcatc aattacattg 3060
tggaacggcg cgacaaagct ggccaacggt gggtgaaatg caacaaaaaa acccttactg 3120
atttaagata taaagtgtct ggactgacag agggacatga atacgagttt cggatcatgg 3180
ctgagaacgc tgctggaatc agtgcaccaa gtgccaccag tccattttat aaagcttgtg 3240
atgctgtgtt taaaccgggc ccaccaggta acccacgtgt cttagataca agcagatcat 3300
ccatttcaat tgcttggaac aaacctatct atgatggtgg ttccgagatc actgggtata 3360
tggttgagat cgccctgccg gaagaagacg aatggaagat tgtcacacca ccagccgggc 3420
tcaaggcaac ctcttacacc atcaccaacc ttgtagagaa tcaggaatat aaaatccgca 3480
tctatgccat gaattctgaa ggcatcgggg aaccagcgct tgttcctgga actccaaagg 3540
ctgaggacag gatgctgcct ccagagattg aactggatgc tgacctgcgc aaagttgtta 3600
ttataagagc ttgctgtacc ctgagactct ttgttccaat caaaggaaga cctgcaccag 3660
aggtgaagtg gacccgggaa catggggaat ctttagataa agctagcatt gaatccacaa 3720
gctcttatac cctccttatc gttggaaacg taaacagatt tgacagtggc aaatacatac 3780
taactataga gaacagctca ggcagcaagt ctgcatttgt caacgtcaga gtgctagata 3840
caccaggccc tccacaggat ctgaaggtaa aggaggtcac taagacatcg gctacactca 3900
catgggagcc tcctctgctt gatggaggtt caaaaataaa gaactatatt gttgaaaagc 3960
gggaatccac aaggaaagca tattcaactg ttgcaacaaa ttgccataag acttcctgga 4020
aggtagacca gctccaagaa ggctgtagtt actacttcag ggttcttgct gaaaatgaat 4080
atggcatcgg gctgccagct gaaaccgcag aatctgtaaa agcatcagaa agacctctcc 4140
ctccaggaaa aataactttg gtggatgtca caagaaatag tgtgtcactc tcttgggaga 4200
aacctgagca tgatggaggc agccgaattc taggctacat tgtagagatg cagagcaaag 4260
gcagtgacaa atgggtcaca tgtgccacag tcaaagtcac tgaagccact attactggat 4320
taattcaggg tgaagagtac tctttccgtg tttcagctca gaatgaaaag ggcatcagcg 4380
atccaagaca actgagtgtg ccagtgattg ccaaagatct tgtcattcca ccagccttca 4440
aactcctgtt caatacttac accgtactgg caggtgaaga cctaaaagtt gacgttccat 4500
tcattggacg ccctatacca gctgtaacct ggcataaaga tgatgtgcca ctgaagcaga 4560
caaccagagt aaatgcagag agcacagaaa ataactcact gctgacgata aaggaggcct 4620
gccgagaaga tgttggacat tatgtagtta agctgactaa ctcggcaggt gaagctactg 4680
aaacccttaa tattatcgtt cttgacaaac cagggcctcc aacaggacca gttaaaatgg 4740
aggaagtgac tgctgacagc atcactctct cctggggccc acctaagtat gatggtggaa 4800
gttctatcaa taactacatt gtagaaaaac gggacacatc cacaaccacc tggcatattg 4860
tgtcagctac tgttgcaagg actacaataa aagcttgcag attaaagact ggatgtgaat 4920
atcagttcag gattgccgct gaaaacagat atggaaagag cacctatctc acttcagagc 4980
ctattgtagc ccagtatcca ttcaaagttc ctggtccccc tggcacaccg ttcgtgacac 5040
actcctccag ggacagcatg gaagtacagt ggaacgagcc agtcagtgat ggaggcagca 5100
aagtcattgg ctatcatcta gaacgcaagg aaagaaacaa catcctctgg gtcaagttga 5160
acaagacacc tattcctcaa accaagttta aaacaactgg cctagatgaa ggcattgaat 5220
atgaattcag agtctccgca gagaacattg tgggcatcgg caagccaagt aaagtgtcag 5280
aatgctacac agcccgtgat ccatgcgacc caccaggaaa gcccgaggca atcattgtca 5340
caaggaattc tgtgactctt cagtggaaga aacccactta tgatggtgga agcaagatca 5400
ctggttatat tgttgagaag aaagaactgc ctgagggccg ctggatgaaa gccagtttta 5460
ccaatgtcat tgacactcag tttgaagtaa ctggcctagt tgaagataac agatatgagt 5520
tccgggttat agctcgaaat gctgcaggag tgtttagtga gccttcagaa agtacagggg 5580
caataacagt aagagatgag gtagaaccac cacgaattcg tatggatcca aaatacaaag 5640
acacaattgt ggttcatgct ggtgagttat tcaagattga cgcagatatc tatggcaaac 5700
caataccaac cactcagtgg ataaaaggtg atcaggagct ttcaaataca gctcgattag 5760
aaataaagag cactgatttt gccaccagtc tcagtgttaa agatgcagtg cgtgttgaca 5820
gtggaaatta catactgaag gccaaaaatg ttgcgggaga gaaatcagtt actgtaaacg 5880
tcaaagttct cgatagacca gggccacctg aaggacctat tgttatatca ggagttacag 5940
cagaaaaatg cgcactagct tggaaacccc cactgcaaga tggtggcagt gatatcataa 6000
attatatcgt ggaaaggaga gaaaccagcc gcttggtttg gactgtggtt gatgccaatg 6060
tgcagaccct cagctgcaag gttactaagc ttcttgaagg caatgaatat attttccgta 6120
taatggctgt aaacaaatat ggtgttggtg aacctcttga atccgagccg gtaattgcca 6180
aaaatccatt cgtagtgcca gatgcaccaa aggctccaga aatcacagca gtgacaaagg 6240
actcaatgat cgttgtatgg gaaaggccag catctgatgg tggcagtgaa attctcggat 6300
atgtgcttga aaagcgggat aaagagggca tcagatggac aagatgccac aaacgtctta 6360
taggggaatt gcgcttgaga gtaactggac tcttagaaaa tcacaactat gaattcagag 6420
tctcagccga gaatgctgct ggacttagcg aaccaagccc gccttctgct taccaaaagg 6480
cttgtgatcc tatttataaa ccaggacctc caaacaatcc caaagtcatt gatattacca 6540
gatcttccgt cttcctttct tggggcaaac caatctatga cggtggctgt gaaatccaag 6600
gatacattgt tgaaaaatgt gacgtgagtg ttggtgaatg gacaatgtgc actccaccaa 6660
caggaatcaa caaaacaaac atagaagtag agaagctgct agaaaagcat gaatacaact 6720
tccgtatctg tgctattaac aaagctggag ttggagaaca tgctgatgtc cctggacctg 6780
ttatagttga agaaaaaatg gaagcaccgg accttgatct tgacatggaa ctgaggaaaa 6840
ttataaatat aagggcaggt ggctccttaa ggttatttgt tcctattaaa ggtcgtccta 6900
caccagaagt caaatggggc aaggtagatg gtgaaatccg cgatgcagct ataatcgaca 6960
gcactagcag cttcacctct cttgttcttg acaatgttaa ccgatatgac agtggaaaat 7020
atactctcac gttagaaaac agcagtggaa caaagtcagc ctttgttact gtgagagtcc 7080
tggacacacc aagtccacct gttaatctga aagtcacaga aatcactaaa gactcggtat 7140
caattacatg ggaacctcct ttgctggatg gaggatccaa aataaaaaat tacattgttg 7200
agaaacgtga agccacaaga aaatcatatg cagcagttgt aacaaattgc cataagaatt 7260
cttggaaaat tgatcaactc caagaaggtt gcagttacta ctttagagtc acagctgaga 7320
atgagtatgg tattggcctt cccgcccaca cagatgatcc agttaaggtt gcagaagtcc 7380
cacaacctcc aggaaaaata actgtggatg atgtcaccag aaacagtgtc tctttgagtt 7440
ggacaaagcc tgaacatgat ggaggcagta aaatcattca gtatattgtg gaaatgcaag 7500
ctaaacacag tgagaaatgg tcagagtgtg ctcgagtcaa gtcccttgaa gcagtaatca 7560
ccaacctaac tcagggagaa gaatatcttt ttagagttgt ggctgtaaat gaaaagggga 7620
gaagtgatcc aaggtctctt gcagttccaa tagttgccaa agatctggtc attgaaccag 7680
atgtaagacc tggatttaac acttacagtg tacaggttgg ccaagatttg aaaatagaag 7740
tgccagtttc tggacgtcct aagccaacta ttacctggac aaaagatggt cttccactga 7800
agcagactac aagaatcaat gttactgact cgcttgacct cactgtactc agtattaaag 7860
aaactcataa ggatgatggt ggacattatg gaatcacagt tgctaatgtt gttggtcaaa 7920
agacagcatc cattgaaatt ataactctag ataaacctga tcctccaaaa ggacctgtta 7980
aatttgatga agtcagtgct gaaagtatta cattatcttg ggaacctcca ttatatacag 8040
gaggctgcca gatcactaat tacatcgttc agaaaagaga tacaaccacc acagtatggg 8100
atgttgtttc tgctactgtt gccagaacta cactcaaagt gaccaaactg aaaactggaa 8160
cagaatacca attcagaata tttgctgaaa acagatatgg acaaagcttt gccttggaat 8220
ctgagccaat tgtagctcaa tatccctaca aagaaccagg ccctccaggc acaccatttg 8280
ccacagccat ttccaaagac tccatggtta tacagtggca cgaaccagtc aataatggtg 8340
gaagcccaat cataggttac caccttgaga gaaaggaaag aaacagtatt ttgtggacaa 8400
aagtcaacaa aactatcatc catgacaccc agtttaaagc agtcaatctt gaagaaggca 8460
ttgaatatga gttcagagtt tatgctgaaa atattgtggg tgtaggcaaa gcaagcaaga 8520
attccgagtg ctatgtagcc agagatccct gtgacccacc aggaacccca gaagcaataa 8580
tagttaaaag aaatgagatc accttacaat ggaccaaacc tgcttatgat ggtggaagta 8640
taattactgg ttacattgta gagaaacgtg atttgcctga gggtcgctgg atgaaagcca 8700
gctttacaaa tgtcattgaa actcagttta ctgtatcagg tcttactgaa gatcaaagat 8760
atgaattcag agtcattgca aagaatgcgg ctggtacaat aagtaaacct tctgacagta 8820
ctggaccaat tactgccaag gatgaagttg aactcccaag aatttcaatg gatccaaaat 8880
tcagagacac aattgtggta aatgcaggag aaacattcag gcttgaagct gatgtccatg 8940
gaaagcctct acctaccatt gaatggttaa gaggagataa ggaaattgaa gaatctgcta 9000
gatttgaaat aaagaacacg gatttcaagg ctttacttat tgtaaaggat gccattagaa 9060
ttgacggagg acagtatatt ttacgagctt ccaatgtggc aggttctaag tcattcccag 9120
taaatgtaaa agtgttagat agaccaggcc ctccagaagg gccagtccag gttactggag 9180
tcactgctga aaaatgtact ttatcatggt ccccaccact tcaagatggt ggcagtgaca 9240
tttctcacta tgttgttgaa aagagagaaa caagtcggct tgcctggact gttgttgctt 9300
cagaggttgt caccaattct ctgaaagtta ccaaactctt agaggggaat gaatatattt 9360
tccgtataat ggctgtcaac aaatacggtg tcggagagcc tttggaatct gcaccggtac 9420
taatgaaaaa tccgtttgtg cctcctggac caccaaaaag cttggaaatc acaaatgttg 9480
ccaaggactc catgactgtc tgctggaacc gtccagatag tgatggtgga agcgagatta 9540
ttggttacat tgtagagaaa agagacagaa gtggcattag gtggataaaa tgtaataaac 9600
gccgcgttac agacttgcgt ctcagagtaa caggattaac agaagatcat gagtacgaat 9660
tcagagtctc tgcagaaaat gctgctgggg ttggagaacc aagttcacct acagtttatt 9720
ataaggcctg tgatcctgtg ttcaaacctg gccctcccac caatgcacac attgtagaca 9780
ccactaaaaa ctcaattaca cttgcctggg gtaaacccat ctacgatggt ggcagtgaga 9840
tcctgggata tatagtagaa atctgtaaag cagatgaaga agaatggcaa atagttactc 9900
cacagactgg cctgaaagcc actcgatttg aaatttcaaa actcactgaa caccaagagt 9960
ataaaatacg agtctgtgcc ctcaataaag tcggtctagg cgaggctaca tcggttcctg 10020
gtactgtgaa accagaagat aagcttgaag cacctgaact tgaccttgac tctgaattaa 10080
ggaaaggaat cgttgtgaga gctggtggat ctgtcagaat tcacattcca ttcaaaggtc 10140
gcccaacacc tgagatcacc tggtctcgag aggaaggtga attcacagat aaggtccaaa 10200
ttgaaaaagg agcaaactct acccaactat caatagataa ctgtgataga aatgatgctg 10260
gaaaatatat tctcaagctg gaaaacagca gtggatctaa atctgctttt gtaactgtga 10320
aagttcttga caccccagga ccaccacaga acttggcagt taaagaagtg aagaaagatt 10380
ctgtccttct cacatgggaa ccacccatca ttgatggggg tgcaaagatc aagaactatg 10440
ttattgacaa acgtgagtcg accagaaaag cttatgccaa tgtgagtaat aaatgcagca 10500
aaacaagctt taaagttgag aatcttacag aaggagccac ttactacttt agagtaatgg 10560
ctgaaaacga atttggaatt ggtgttccag tggaaactgt tgatgctgtg aaagctgccg 10620
aacctccttc cccaccagga aaggttacac tcactgacgt atcccagacc agtgcatcac 10680
ttatgtggga gaaacctgag catgatggtg gtagcaggat cctggggtat gttgttgaaa 10740
tgcaacccaa aggaactgaa aaatggagtg ttgtggctga atctaaagtc tgcaatgcat 10800
ctgttaccgg tttgagttct ggacaagagt atcagttccg tgtcaaggct tacaatgaga 10860
aaggaagaag tgatccaaga gtgcttggtg ttcccgtcat agccaaggac ttgactatac 10920
agcccagttt tcagttacca ttcaacacat acactgttca agctggagaa gatcttaaaa 10980
tcgaaattcc ggttattggc cgaccaagac ctcaaatttc atgggtcaaa gatggtgagc 11040
ttcttaaaca gacaacaaga gtaaatgttg aggaaacagc tacttcaact attttgcaca 11100
ttaaagaaag taacaaagat gactttggaa aatacaccat aacagcaaca aacagtgcgg 11160
gtacggcaac cgaaaatctc agtattatcg ttttggaaaa gcctggacct ccagttgggc 11220
cagttcggtt tgatgaagtt agtgcagact ttgtggtcat atcttgggaa cccccagcct 11280
acactggtgg ctgccaaata agcaactaca ttgtagagaa gcgagataca acgaccacca 11340
cttggcacat ggtatcggca acagttgcaa ggacaacaat taaaataacc aagctgaaaa 11400
caggcagtga gtaccagttt agaatttttg cagaaaacag gtatggaaag agtgcctccc 11460
tggattctaa accagttatt gtacaatatc catttaaaga accaggacca cctggcactc 11520
cttttgtgac atcaatctca aaagatcaga tgcttgtgca atggcatgag cccgttaatg 11580
atggcggtag caaagttatt ggctaccatc ttgaacagaa agaaaagaac agcattttat 11640
ggatcaaatt aaataagact cccattcaag acaccaaatt caaaacaact gggcttgacg 11700
aaggcctcga gtatgagttc aaagtttctg ctgaaaatat tgttggcatc ggcaagccga 11760
gcaaagtgtc agaatgttat gttgctcgtg atccatgtga tccaccaggt cgccccgaag 11820
ccattgttat tacaagaaac aacgtcacac tgaaatggaa gaaacctgcc tatgacggtg 11880
gcagcaaaat aacaggttat attgtagaga agaaagatct acctgatggc cgttggatga 11940
aagccagctt caccaatgta ttggaaactg aatttacagt gagtggactt gtagaagacc 12000
aaagatatga atttagagta attgcaagaa atgcagctgg caactttagt gaaccatctg 12060
aaagcactgg tgccattact gcacgagatg aaattgatgc acccaatgcc tcactggatc 12120
ccaaatataa agatgtcatt gttgtccatg caggagagac ttttgtcctt gaagctgata 12180
ttcgtggcaa acctatacct gatatcgtct ggttaaaaga tggaaaagaa cttgaagaaa 12240
caaccgccag gatggaaatt aagtctacta ttcagaaaac gacgcttgtt gtcaaagact 12300
gtatacgtag tgatggagga caatacatcc tgaaactcag caatgttggt ggtacaaagt 12360
ctattcccat cactgtaaag gtacttgaca agccagggcc tcctgaaggg cctctgaaag 12420
tttccggagt tactgctgaa aaatgttacc tggcatggaa cccaccttta cacgatggtg 12480
gcgctaatat ttcacattac atcattgaga agagagagac aagcagactg tcctggaccc 12540
aggtttcaac tgaagtacag gcccttaact acaaagtcac taaacttctt cctggcaatg 12600
agtacatttt ccgtgtcatg gccgtgaata aatatggaat tggagagcct ctggaatctg 12660
agcccatcat agcccgtaac ccatataaac caccaggtcc tccttcacca cctgaagtct 12720
cagcaatcac caaagattct atggttgtaa cgtgggcacg cccagcagat gatggaggtg 12780
cggaaatcga gggctacatt cttgaaaaac gagataagga aggcattcga tggaccaagt 12840
gcaacaagaa aacactaacg gatcttcgat tcagggtaac tggcctgacc gaaggtcatt 12900
cctatgaatt cagagttgct gctgaaaatg cagctggcgt gggtgaacct agcgagccat 12960
ctgttttcta tcgtgcgtgt gacgctttgt atccaccagg accaccaagt aatccaaaag 13020
taacagacac ctccagatct tctgtctccc tggcatggaa taaaccaatt tatgacgggg 13080
gtgcacctgt taagggctat gttgttgagg tcaaagaagc cactgctgat gaatggacaa 13140
catgcactcc accaacagga ttacaaggaa agcaattcac agtgactgag cttaaagaaa 13200
acactgaata taacttccgt atttgtgcca tcaattctga aggcgtgggt gaacctgcaa 13260
ctatacctgg ttcggttgtt gctaaagaga ggcaagagcc accagaaata gaacttgatg 13320
ctgatctcag aaaggtggtc attctgcgtg cgagtgctac tctacgctta tttgtcacta 13380
tcaaaggtcg accagaacct gaagttaaat gggaaaaggc cgaaggcacg ctcactgaca 13440
gggcccagat tgaggtgacc agctcatata caatgttggt aatagataat gttaccagat 13500
ttgacagtgg tcggtacaat ctgacactgg aaaataacag tggctctaaa actgctttcg 13560
ttaacgtcag agttcttgac tcaccaagtg cccctgtgaa tctgaatgta agagaagtga 13620
agaaagactc ggtaacattg gcttgggaac caccacttat tgatggcgga gctaagatta 13680
caaactacat cgttgaaaaa cgagaaacta caagaaaagc atatgctact gttacaaaca 13740
actgcacaaa gaacactttt aaaattgaaa atcttcaaga aggatgttct tactacttcc 13800
gagtcttggc tgccaatgaa tatgggattg gtttgccagc agaaacaacg gaacccgtta 13860
aagtttctga accacccctc ccacctggaa gagtaactct tgttgacgtg acccgtagca 13920
cagctacgat taagtgggag aaaccagaaa gtgatggtgg cagcaaaatt ataggttatg 13980
tagttgaaat gcagactaaa gggagtgaaa aatggagcac atgcacacaa gttaagactc 14040
tagaagcaac aatatctggc ctgactgctg gagaggagta tatcttcagg gtagctgcaa 14100
ttaatgaaaa aggaaaaagt gatccaagac aactgggagt acctgtaatt gcaagagaca 14160
ttgaaataaa gccttcggtt gagcttcctt tcaatacttt caatgtaaag gccagagacc 14220
aactgaagat tgatgttcca tttaaaggaa gacctcaggc gactgtaagc tggaaaaaag 14280
atggtcagac tcttaaagag acaactagag tcaatgtttc ttcttcaaag actgtaacat 14340
cactaattat taaggaagcg tcaagggaag atgttggaac atatgaatta tgtgtttcaa 14400
acagtgctgg atctataaca gttcctatta ccgtaattgt ccttgacaga ccaggacctc 14460
ctggacctat tcatattgat gaggttagtt gtgacaatat aaccatttct tggaatcctc 14520
cagaatatga tggcggctgc cagattagca attacattgt tgaaaagaga gaaaccacct 14580
ctacaacgtg gcatgtggtt tcacaagcag ttgcaagaac atccatcaag atagttcgtc 14640
tggtaacagg aagtgagtat cagttccgtg tctgtgcaga aaaccgctat ggaaagagct 14700
cctacagtga ctctccagct gttgttgcag agtatccatt cagcccccct ggtcctcctg 14760
gtactcctag agttgtgcat gccacaaaat ccaccatggt ggtaacctgg caagtgccag 14820
ttaatgatgg aggaagtcaa gtacttggct atcaccttga gtataaagaa agaagcagca 14880
ttctttggtc aaaagcaaat aaaatcctca tcactgatac tcaaatgaaa gtttccggcc 14940
ttgatgaagg actcatgtat gagtatcgtg tatatgctga gaatattgct ggaatcggta 15000
aatgcagtaa atcttgtgag ccagtccctg caagagatcc ttgtgaccct cctggacaac 15060
ctgaagtcac aaatatcacg agaaaatcag tgtcacttaa atggtctaag ccacgttatg 15120
acggtggagc taagatcacg ggatacatta ttgaacgtag agaactacca gatggccgat 15180
ggctgaagtg caattttact aacgtacaag aaacatactt tgaagtaacc gaacttacgg 15240
aagatcagcg ttatgaattc cgtgtttttg caaagaatgc tgccgactca gttagtgagc 15300
cctctgaatc tactggacct attacagtta aagacgacgt tgaggctcca agaattatga 15360
tggatgtcaa gttccgagat gttattgttg tcaaagctgg agaggtcctt aagataaatg 15420
cagacgttgc agggcgacca ctaccagtaa tttcctgggc caaggatggt gtagaaatag 15480
aagagagggc aagaacagaa atcgtctcaa cagactataa tactttgctg acagtgaaag 15540
actgtgtccg acgagactct gggcagtatg tgctaacagt gaagaacgtt gcaggaactc 15600
ggtctatggc agttaattgc aaggtacttg ataagcctgg cccaccagca ggaccacttc 15660
aaataactgg cctcaccgct gagaaatgct ctctttcctg gggaccaccc caagaagatg 15720
gtggcgcagc tattgactat tacattgtag aaaaacgcga gacaagccac cttgcatgga 15780
caatatgtga aggagagtta aggacaacat cctgtaaagt aactaagtta ctcaaaggca 15840
atgaatacat ctttagagtg acaggtgtta ataaatatgg tgttggtgag cccctggaga 15900
gtgtggctgt aaaggcatta gatccattta cagttccaag tccacccaca tctttggaaa 15960
ttacatctgt gaccaaagag ttcatgacac tttgctggtc aaggccagag agtgatggag 16020
gcagtgaaat ttctggatac ataattgaga ggcgagagaa gaacagcctg cgctgggtgc 16080
gtgtaaacaa gaggccagtt tacgatctga gagtgaaatc aacaggactt cgggaaggat 16140
gtgaatatga gtaccgggtg tatgcagaaa atgctgcagg cctaagcctt ccgagtgaaa 16200
cctctccctt agttcgggca gaagatccag tattcttacc atctcctcca tccaaacccc 16260
aaatcgtgga ctctggcaag accaacataa caattagctg ggttaagccc ttatttgatg 16320
gtggcacccc aataactgga tacactgtag aatacaaaaa atccagtgaa actgactggc 16380
acactgccat tcagaacttc aggggcacag agtacacagc aagtggacta accacaggaa 16440
ccgaatatgt tttcagagta agatctatca ataaggttgg agctagtgat cccagtgata 16500
gctccgatcc ccagatagca aaggaaagag aagaagagcc tgtatttgat cttgacagtg 16560
aaatgaagaa gaccttgatt gtcaaggccg gctcctcatt taccatgact gtgcctttcc 16620
gaggaagacc agtacccagc gtctcctgga gtaaaccaga cactgacctc cgcactagag 16680
cttatattga ctccacagac tctcgcacat cactgactat tgaaaatgcc aacagaaatg 16740
attctggaaa atacacatta acaattcaga atattttgaa cgctgcttca ctgaccttag 16800
tcgtcaaggt cttagattct cctggtcctc cagccggtgt taccgtacgc gatataacaa 16860
aagagtctgc agtgttagcc tgggatgttc ctgaaaacga tggtggagca ccagtgaaga 16920
attaccacat agaaaaacgg gaggccagta agaaagcatg ggtctcggta accaacaact 16980
gtaaccgcct ctcctacaaa atcaccaact tacaagaagg agcaatatac tacttcagag 17040
tctctggaga aaatgagttt ggtgttggtg taccagctga aacgaaggaa ggagtaaaaa 17100
taacag 17106
<210> 6
<211> 100
<212> RNA
<213> Artificial sequence
<400> 6
gaugggggca guaagaucac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 7
<211> 100
<212> RNA
<213> Artificial sequence
<400> 7
ccuaaaccca aacacgaugg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 8
<211> 100
<212> RNA
<213> Artificial sequence
<400> 8
aaagaaaagg cucagaccag guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 9
<211> 100
<212> RNA
<213> Artificial sequence
<400> 9
guugugaaga aucuaacuga guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100

Claims (8)

1.一种sgRNA，其靶序列结合区如SEQ ID NO：7中第1-20位核苷酸所示。
2.一种质粒，其转录得到权利要求1所述sgRNA。
3.一种试剂盒，包括权利要求1所述sgRNA或权利要求2所述质粒；所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备扩张型心肌病猪模型；(c)制备扩张型心肌病猪细胞模型。
4.如权利要求3所述的试剂盒，其特征在于：所述试剂盒还包括质粒pKG-GE3；质粒pKG-GE3如SEQ ID NO：2所示。
5.权利要求1所述sgRNA或权利要求2所述质粒在制备试剂盒中的应用；所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备扩张型心肌病猪模型；(c)制备扩张型心肌病猪细胞模型。
6.两种质粒在制备试剂盒中的应用；
所述两种质粒为权利要求2所述的质粒和SEQ ID NO：2所示的质粒pKG-GE3；
所述试剂盒的用途为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备扩张型心肌病猪模型；(c)制备扩张型心肌病猪细胞模型。
7.权利要求1所述sgRNA或权利要求2所述质粒或权利要求3所述试剂盒或权利要求4所述试剂盒的应用，为如下(a)或(b)或(c)：(a)制备重组细胞；(b)制备扩张型心肌病猪模型；(c)制备扩张型心肌病猪细胞模型。
8.一种制备重组细胞的方法，包括如下步骤：将权利要求2所述质粒和权利要求4中所述的质粒pKG-GE3共转染猪细胞，得到TTN基因发生突变的重组细胞。
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