CN104498493B - CRISPR/Cas9特异性敲除乙型肝炎病毒的方法以及用于特异性靶向HBV DNA的gRNA - Google Patents

Abstract

本发明涉及分子生物学与生物医药技术领域，具体的说，本发明涉及基于CRISPR***的gRNA序列及其组合在乙型肝炎病毒治疗中的应用。本发明根据CRISPR gRNA的设计原则以及不同基因型HBV序列的保守性区域，设计了8种指导RNA（gRNA），并且将其构建在PX330表达载体上。在细胞模型和小鼠模型中利用这8条gRNA，及其组合指导的CRISPR/Cas9***，可以有效的抑制乙型肝炎病毒基因的表达和复制。多条gRNA联合使用可以得到更好的效果，且可以更好地抑制不同基因型的HBV复制。这一***有易于操作，对HBV复制的抑制效率高，对于多种基因型适用的特点。因此，本发明涉及的gRNA及其组合有望在制备治疗乙型肝炎病毒新型药物中得到应用。

Description

CRISPR/Cas9特异性敲除乙型肝炎病毒的方法以及用于特异 性靶向HBV DNA的gRNA

技术领域

本发明属于基因工程领域，更具体地说涉及CRISPR/Cas9特异性敲除乙型肝炎病毒的方法以及用于特异性靶向HBVDNA的gRNA。

背景技术

乙型肝炎病毒（Hepatitis B virus， HBV）是一种能够引起多种肝脏疾病，包括肝炎，肝纤维化，肝细胞癌的病毒。全世界范围内，慢性乙肝感染者有近3.5亿人，而其中近三分之一感染者在我国。乙型肝炎病毒给全世界，尤其是我国的医疗卫生体系带来了巨大的挑战。目前针对HBV的治疗方法主要包括两种：1. α干扰素（IFN-α）和聚乙二醇化α干扰素（PEG-IFN-α），干扰素治疗HBV感染的机制目前尚未完全明确，普遍认为可能是通过直接抑制病毒复制或者免疫调节作用；2. 核苷类似物，当前临床使用的核苷类似物主要包括拉米夫定，阿德福韦酯，恩替卡韦，替诺夫韦，替比夫定等。这些药物主要作用于乙肝病毒的逆转录酶，通过抑制病毒的逆转录从而抑制病毒的复制。但是，当前的这些药物都只能抑制病毒的复制，并不能直接清除已经存在的病毒基因组DNA，复发率高，且需要长期用药。因此，当前迫切需要新的方法来清除病毒的基因组DNA，达到功能性治愈的效果。

2010 年，Engineered zinc finger nucleases (ZFNs) 被应用于HBV cccDNA 的敲除，效率大约为26%。2013 年，Engineered transcription activator-like effectors(TALEs) 也被用于突变HBV cccDNA，效率大约为35%，相应地，HBsAg 降低了大约20%。很明显，现有的技术还很难满足需要，人们期待找到更高效的HBV cccDNA 的敲除策略。

CRISPR/Cas***是从细菌和古生菌对抗外来病毒或质粒的适应性免疫***发展而来，包括三种不同的类型，其中TypeⅡ型的CRISPR/Cas***的DNA内切酶Cas9只有一个亚基，结构最为简单，所以应用也最广泛。除了Cas9蛋白外，该***还包括两条短的CRISPRRNAs（crRNAs）和trans-activating crRNAs（tracrRNA）。成熟的crRNA-tracrRNA复合体可以通过碱基互补配对指导Cas9蛋白到靶序列上，并在PAM（protospacer adjacent motif）附近特异性剪切DNA双链，形成DSB（double strand break）。DSB可以通过两种途径被修复，一种是非同源重组的末端接合（Non-Homologous End Joining NHEJ）DNA修复方式， 另一种是同源重组修复（ Homology Directed Repair HDR) 方式。NHEJ修复方式可能产生碱基的***或缺失，从而产生移码突变，或者也可能突变成终止密码子，这些突变形式都可以改变目的基因的开放阅读框；HDR方式需要一段与被剪切片段同源的模板片段来修复DSB，这种修复方式可以将被用来作为模板的同源片段的序列复制到目的基因中，所以可以利用这种修复方式将特定的基因片段引入到目的基因中。

由于CRISPR/Cas***具有操作简单，切割效率更高等特点，这一***被认为有更好的应用前景。之前发表的基于CRISPR/Cas9***对抗HBV复制的研究均有局限性（Su-RuLin, et al., Molecular Therapy-Nucleic Acids, 2014, 3, e186; Christoph Seegerand Ji A Sohn, Molecular Therapy-Nucleic Acids, 2014, 3, e216）：首先，都以单一基因型HBV序列为模板设计gRNAs并进行实验，并未选取HBV序列的保守区域（即HBV基因突变率低的区域）。由于HBV病毒基因突变率高，针对单个基因型HBV设计的gRNA有很高的脱靶几率，而且也不能抑制其他基因型HBV的复制，因此其后期的临床转化可能会有较大的局限性；其次，Christoph Seeger仅在细胞水平上检测了HBV部分抗原的表达水平，虽然Su-RuLin的报道中进行了小鼠模型验证，但是抑制效果均不理想（仅抑制了20%左右）。

因此，开发出一些高效、靶向乙型肝炎病毒保守序列、且突变率较低区域的gRNA，对于CRISPR/Cas9 ***充分发挥作用极为重要，并具有较大的应用价值。

发明内容

本发明的目的在于通过设计、构建、筛选，最终提供一些基于CRISPR/Cas9***，同时靶向不同基因型和亚型HBV保守基因序列（突变率较低的区域）的高效gRNA及其靶位点序列，并用其抑制HBV复制，从而特异清除HBV。为实现上述目的，本发明以CRISPR/Cas9***的原理及其gRNA的设计原则为基础，通过序列比对找出26种基因型和亚型HBV基因序列的保守区域，以这些保守区域为靶位点设计了一系列gRNA，并以pX330-U6-Chimeric_BB-CBh -hSpCas9（Addgene ID: 42230）为表达载体，构建了gRNA/Cas9表达体系。通过筛选和一系列分析测试，最终选出8个有效的gRNA及其组合，并在细胞及动物模型上加以应用。

本申请的技术方案如下：

一、靶向不同基因型和亚型HBV保守基因序列的高效gRNA及其靶位点序列的设计及构建。

在Genebank中找到26种不同基因型和亚型的HBV基因序列，通过生物信息学分析和序列比对，找到其相对保守的区域，并且这些保守区域与人基因组无相关性。由于HBV复制过程中突变率较高，这些保守区域本身也是复制过程中基因突变率较低的区域，选取位于HBV基因的保守区域作为潜在靶位点（图1）；

通过Crispr 设计工具及gRNA的设计原则，评估HBV DNA序列上得分较高的靶位点设计gRNA，其评估原则包括：1）序列下游是否有PAM（NGG），2）靶位点不能与人类基因组序列相同或相似，脱靶效率低（图1）；

根据设计的gRNA，在其5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后将合成的序列变性、退火，得到具有BbsI粘性末端的双链DNA片段，如下：

正向：5’-CACCNNNNNNNNNNNNNNNNNNNN

反向：NNNNNNNNNNNNNNNNNNNNCAAA-5’

将双链DNA片段和用BbsI酶切过的pX330-U6-Chimeric_BB-CBh -hSpCas9载体（其序列为序列表SEQ ID NO. 17所示）进行连接；

将连接产物转化到大肠杆菌JM109感受态细胞中，涂布于带有氨苄青霉素抗性的LB平板上，筛选阳性菌落，提取阳性菌落质粒进行分析及测序，确定gRNA表达载体构建成功（命名为PX330-U6-HBVgRNA）。

二、在HepG2细胞系中分析检测gRNA对于HBV复制的抑制作用，筛选到8条有效的gRNA（分别命名为T1-T8），gRNA在HBV cccDNA的靶向位点位于至少2种HBV蛋白编码基因的共有序列区域，其对应的DNA序列如序列表SEQ ID NO. 1-8任意一条序列所示，及其组合（Tmix）。

将构建的PX330-U6-HBVgRNA质粒（其序列为序列表SEQ ID NO. 9-16任意一条所述）分别与pHBV1.3和pSV-β-gal共转染HepG2细胞，并用特异靶向敲除绿色荧光蛋白的gRNA（PX330-U6-T_GFP，其序列为序列表SEQ ID NO. 18所示）与pHBV1.3和pSV-β-gal共转染HepG2细胞作为对照；

共转染细胞培养48、72、96小时，分别用乙型肝炎表面s抗原和e抗原诊断试剂通过酶联免疫法（ELISA）法检测培养基中HBV分泌抗原HBsAg和HBeAg的浓度(图2)。通过实验组与对照组的结果对比，可以看出gRNA T1-T8及其组合Tmix对HBV抗原HBsAg和HBeAg的分泌均有不同程度的抑制效果（图2）；

为了检测设计、构建的gRNA清除HBV的效果，采用实时定量PCR（real-time PCR）检测HBV DNA复制水平。在HepG2细胞中同时转染了PX330-U6-HBV gRNA、pHBV1.3和pSVβ-gal三种质粒，将转染后的HepG2细胞培养4天，然后分别提取细胞中和培养基中的壳体化HBVDNA，用real-time PCR测定提取出来的HBV DNA浓度，其中gRNA T1-T8及其组合Tmix能有效降低细胞内和细胞外的HBV DNA浓度（图3）；

为了进一步检测gRNA***对HBV复制及转录的影响，采用Northern Blot检测细胞内HBV RNA的水平。我们在HepG2细胞中同时转染了PX330-U6-HBV gRNA、pHBV1.3和pSVβ-gal三种质粒，将转染后的HepG2细胞培养4天，然后提取细胞中的总RNA，每个样品取等量的RNA跑胶，转膜，探针杂交，抗体孵育，最后加底物曝光并对比实验组与对照组HBV RNA的强弱。与上一步实验结果一致，其中gRNA T1-T8及其组合Tmix能有效降低HBV RNA浓度（图3）。

三、在HepG2细胞系中分析检测本发明gRNA对于不同基因型HBV的抑制效果。为了检测本发明涉及的gRNA是否对于基因型为A, B, C的HBV具有抑制作用，我们以T1, T7，Tmix为例检测了其对于不同基因型的HBV的抑制效果。首先，我们将质粒pHBV1.3 A（HBV 基因型A （Genebank ID：AF305422.1）），pHBV1.3 B（基因型B（Genebank ID：EU570069.1）），pHBV1.3 C（基因型C （Genebank ID：FJ899793.1））分别与不同的PX330-gRNA质粒以及pSVβ-gal共转染HepG2细胞系，2天后检测HBV病毒e抗原以及s抗原的表达水平（图4）。

四、在水动力注射小鼠模型中研究gRNA对于HBV复制的抑制作用。除在细胞内研究中使用的技术之外，还包括免疫组织化学染色法研究小鼠肝脏切片中HBV抗原的表达水平（图5）；从小鼠肝脏中提取细胞基因组，用T7EN1酶切检测和深度测序确认经过gRNA处理的HBV基因组序列是否产生了突变（图6）。

本发明所构建的gRNA表达载体经测序验证表明构建成功。通过体内、外试验和检测，筛选到全新、高效的gRNA T1-T8及其组合Tmix指导的CRISPR/Cas9***均可以更有效的抑制HBV的复制、病毒RNA和抗原的表达以及子代病毒的产生。这8条gRNA及其组合Tmix能更高效的靶向不同基因及亚基因型HBV的保守性区域（突变率较低的区域），同时不靶向人的基因组，并在小鼠动物模型进行HBV清除治疗的应用。最终筛选到的8条gRNA如下表：

编号	在HBV基因组上的靶位点	靶位点所在HBV蛋白编码区域	靶序列 (N20NGG)
				T1	239-261	S、P	TACCGCAGAGTCTAGACTCGTGG
T2	689-711	S、P	CATTTGTTCAGTGGTTCGTAGGG
				T3	1143-1165	EnhancerⅠ、P	GTTGCCGGGCAACGGGGTAAAGG
T4	1407-1429	X、P	AAACAAAGGACGTCCCGCGCTGG
				T5	1597-1619	EnhancerⅡ、X、P	GGTCTCCATGCGACGTGCAGAGG
T6	1917-1939	C、e	GTAGCTCCAAATTCTTTATAAGG
				T7	2384-2406	C、e、P	GACCTTCGTCTGCGAGGCGAGGG
T8	3124-3146	PreSⅠ、P	CCTTCCTGACTGGCGATTGGTGG

此外，本发明还涉及以上8种不同的gRNA的联合使用，可以是任意两种或多种gRNA（Tmix）的联合使用，更进一步的8种gRNA的联合使用比之前报道的基于CRISPR/Cas9***对抗HBV复制的研究能更有效清除HBV，其抑制效率高于90%，尤其是Tmix组合几乎检测不到细胞内病毒的复制以及细胞外子代病毒的产生，这种策略能够进一步降低病毒突变之后逃逸治疗的可能。

因此，本发明提供在CRISPR-Cas9特异性敲除乙型肝炎病毒cccDNA中用于特异性靶向乙型肝炎病毒cccDNA的gRNA在敲除乙型肝炎病毒cccDNA中的应用，以及PX330-U6-HBVgRNA质粒在敲除乙型肝炎病毒cccDNA中和制备抗乙型肝炎病毒药物中的应用。

附图说明

图1为gRNA与不同基因型、基因亚型的HBV序列的比对。

图2为在转染HBV的细胞系中检测分泌性抗原的表达水平。

结果显示gRNA能够有效的抑制HBsAg，HBeAg的表达。

A：HBsAg表达水平；B：HBeAg表达水平。

图3为使用qPCR在转染HBV的细胞系中检测细胞内的病毒复制中间体DNA，以及分泌到培养基中的子代病毒DNA的复制水平。

结果显示，gRNA能够有效抑制HBV复制。

A：细胞内的HBV复制中间体水平；B：细胞外的HBV-DNA的水平；

C：HBV RNA转录水平。

图4为gRNA对于基因型为A, B, C, D的HBV的抑制效果。使用gRNA T1， T7以及组合Tmix表达质粒与不同基因型HBV质粒共转染HepG2细胞系，2天后检测gRNA对于HBV的抑制效果。

A：不同gRNA及组合Tmix对于HBeAg的抑制效果。

B：不同gRNA及组合Tmix对于HBsAg的抑制效果。

图5为在水动力注射小鼠模型中gRNA对于HBV复制的抑制作用。

A：血清HBsAg水平；B：血清HBeAg水平；C：血清中HBV DNA水平；D：肝组织中HBcAg水平；E：肝组织HBV RNA水平。

图6为在小鼠模型中gRNA诱导HBV基因组产生突变的效果。

A：T7 E1实验检测T1及Tmix诱导突变的能力。

B：T7 E1实验检测T7及Tmix诱导突变的能力。

C：深度测序实验检测gRNA T1诱导产生的突变体的类型。

D：深度测序实验检测gRNA T7诱导产生的突变体的类型。

具体实施方式

下面结合附图和具体实施例对本发明的的技术方案做进一步说明。

【实施例1】靶向26种不同基因型的HBV保守性序列的gRNA序列的设计、合成以及真核表达载体构建

1、靶向HBV DNA的gRNA的选择和设计

以D型HBV（Genebank ID：V01460.1）为参考序列，通过crispr.mit.edu上的Crisprdesign工具找到HBV DNA上打分较高的靶位点，靶位点序列的形式为5’-N（20)-NGG3’ 或者5’-CCN-N(20)-3’。与以往的设计不同，我们将26种不同基因型的HBV DNA序列进行比对，从找到的靶位点中选出位于保守性较高区域的gRNA序列，这些保守区域本身也是复制过程中基因突变率较低的区域。同时也对比了人体基因组序列，排除与人体基因组序列同源性高的gRNA，最终设计出一系列全新的、从未报道的gRNA序列及靶点（图1）。从而保障我们设计的gRNA能有效靶向各种HBV基因型及亚型，降低病毒突变而导致的脱靶，以及提高gRNA的效率。

2、靶向HBV DNA的gRNA寡核苷酸序列的合成和真核表达载体的构建

根据选择的gRNA，在其5’端加上CACC得到正向寡核苷酸序列，在其互补链的5’端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后将合成的序列变性、退火，得到具有BbsI粘性末端的双链DNA片段，如下：

正向：5’-CACCNNNNNNNNNNNNNNNNNNNN

反向：NNNNNNNNNNNNNNNNNNNNCAAA-5’

变性、退火体系为：

2μl正向寡核苷酸链（50μM）

2μl反向寡核苷酸链（50μM）

46μ l1×NEB buffer

在PCR仪中按以下程序运行：90°C，4min；70°C，10min；37°C，20min；25°C，20min。

退火后的双链寡核苷酸链含有BbsI的粘性末端，直接与被BbsI酶切过的pX330-U6-Chimeric_BB-CBh -hSpCas9（以下简称PX330）（SEQ ID NO. 17）载体进行连接，可以得PX330-U6-HBV gRNA重组质粒。

酶切体系：

水 39.3μl

10×FD buffer 5μl

BbsI 2μl

PX330 3.7μl(2μg)

37°C水浴1h

酶切后的质粒直接用胶回收试剂盒回收。

连接体系：

退火产物 0.5μl

线性化的PX330质粒 2μl

5×ligation buffer 2μl

T4DNA Ligase(3units/μl) 1μl

水 4.5μl

16°C水浴1h

将上述步骤得到的连接产物转化JM109感受态细胞，涂布于Amp⁺的LB平板，挑取阳性克隆接菌，37°C摇床摇菌过夜，质粒抽提试剂盒提取质粒并进行测序鉴定，得到PX330-U6-HBV gRNA质粒。

【实施例2】在细胞水平验证本发明gRNA指导的CRISPR/Cas9***对HBV复制的抑制

为了验证本发明中设计的gRNA指导的CRISPR/Cas9***是否具有抗HBV复制的能力，本发明中将构建的gRNA的克隆与乙型肝炎病毒的复制子pHBV1.3（基因型D； GenebankID：V01460.1）共同转染进入肝癌细胞系HepG2之中，另外一个靶向于GFP的gRNA（T_GFP）（SEQID NO. 18）被用作对照组。为了将不同孔之间的转染效率标准化，每个孔都转染了相同量β-半乳糖苷酶的表达质粒pSVβ-gal。分别于转染24、48、72小时之后，取培养基上清用来测定乙型肝炎病毒表面抗原（HBsAg）和乙型肝炎病毒e抗原（HBeAg）的表达水平。结果显示，筛选到8个不同的gRNA T1-T8及其组合Tmix指导的CRISPR/Cas9***均在不同程度上抑制了HBsAg和HBeAg的表达水平（图2A和2B）。另外，Northern Blot结果表明，细胞内病毒的3.5kb，2.4 kb，2.1 kb RNA的表达水平均在不同程度上被抑制（图3C）。转染96小时之后，使用荧光定量PCR法测定了不同组分泌到细胞外的子代病毒和细胞内部的病毒DNA复制中间体的含量。结果显示，筛选到的8个gRNA T1-T8及其组合Tmix（8种gRNA等比例混合）均有效的抑制了病毒的DNA复制水平和子代病毒的产生，其抑制效率高于90%，尤其是Tmix组合几乎检测不到细胞内病毒的复制以及细胞外子代病毒的产生，效果远高于比之前的报道及研究（图3A和图3B）。综上所述，本发明中设计构建并筛选到的8种全新的gRNA T1-T8及其组合Tmix均能够指导CRISPR/Cas9***去更有效的抑制乙型肝炎病毒的基因表达、复制及子代病毒的产生。具体实验步骤如下：

1、细胞培养与转染

a、HepG2细胞接种于DMEM培养基中，其中含10%FBS， penicillin （ 100 U/ml ）和 streptomycin （ 100 µg/ml ） 。

b、在转染前一天将细胞铺在24孔板中，等到密度70%-80%时进行转染。

c、转染前移除细胞上原有的培养基，换为新鲜的无血清培养基。

d、以PX330-U6-HBV gRNA质粒:pHBV3.1质粒:β-gal质粒=8:1:1的比例将质粒DNA溶于opti-MEM中，混匀后加入Neofect^TM，轻柔混匀，室温静置15-30min，转染复合物制备完成。Tmix组合为8种gRNA等比例混合，然后以Tmix质粒：pHBV1.3质粒：β-gal质粒=8:1:1溶于opti-MEM中，混匀后加入Neofect^TM，轻柔混匀，室温静置15-30min，转染复合物制备完成。

e、将转染复合物加入细胞培养基中，轻柔混匀。

f、继续培养24、48、72或96小时后，用ELISA、Northern blot或qPCR检测不同的指标。

2、ELISA法测定细胞表面抗原和e抗原表达的变化

转染后的第二天、第三天及第四天分别收集上清，按照乙型肝炎病毒表面抗原诊断试剂盒及乙型肝炎病毒e抗原诊断试剂盒说明书测量上清中的HBV表面抗原及e抗原。具体步骤如下：

a、每孔加入待测样本50μl，设阴、阳性对照各两孔，每孔加入阴性对照（或阳性对照）各50μl，并设空白对照1孔；

b、每孔加入酶结合物各50μl（空白对照除外），充分混匀，封板，置37°C孵育30min；

c、手工洗板：弃去孔内液体，用洗涤液注满各孔，静置5s，甩干，重复5次后拍干；

d、各孔加入显色剂A液、显色剂B液各50μl，充分混匀，封板，37°C孵育；

e、加入终止液50μl，混匀；

f、用酶标仪读书，取波长450nm。

3、细胞内RNA的提取

a、根据培养皿的面积加入适量的TRIZOL（1ml每10cm²），用枪反复吹打，使细胞充***解；

b、室温放置5min，加入氯仿（每1mlTRIZOL加入0.2ml），剧烈震荡15s，室温放置2-3min，4°C 12000g 15min；

c、将上清转移到一个新的EP管中，加入异丙醇沉淀RNA（每1mlTRIZOL加入0.5mL），室温放置10min，4°C 12000g 10min；

d、去掉上清，用75%乙醇洗RNA沉淀，每1mlTRIZOL至少用1ml乙醇，震荡重悬沉淀，4°C 12000g >5min；

e、去掉上清，在空气中干燥5-10min，用无RNA酶的水溶解RNA，室温下放置5 min，测RNA的浓度。

4、Northern blot

a、加入2×loading，先放65°C变性10min，取出后立刻放冰上，上样进行电泳80V跑2h

RNA琼脂糖凝胶配方：1%琼脂糖，水:甲醛=4:1，10×Mops

b、跑完的胶在紫外下照完相后，将多余的部分裁掉；

c、用milliQ水泡胶3次，每次5min，然后用20×SSC泡两次，每次15min，尼龙膜先用milliQ水泡15 min，再加20×SSC泡15 min；

d、裁20多张与胶一样宽的滤纸，4条一样宽但很长的滤纸，从下到上按干纸巾、干滤纸、湿滤纸、膜、胶，湿滤纸、滤纸桥、湿滤纸的顺序搭好转膜，室温转3h；

e、转好的膜先进行UV交联1.8min；

f、取2ml DIG easy Hyb，55°C与杂交，大于1h；

g、取1μl制备好的单链RNA探针，100°C煮5min，取出后立即放入冰上，再稀释入1mlDIG easy Hyb中，直接加500μl到之前封闭的杂交袋中，55°C过夜；

h、以milliQ水配制2×SSC和0.1×SSC，并加入20%SDS，终浓度为0.1%，先用2×SSC泡5min，两次，常温摇，再用0.1×SSC泡胶15min，两次，55°C，中途摇；

i、用washing buffer 洗膜两次，加封闭液，常温摇30min；

j、以block buffer 稀释DIG抗体（1:15000~1:20000），常温，30min；

k、washing buffer 洗膜两遍，每遍15min，再以Detection buffer 涮一遍；

l、将显色底物加在膜上，用胶片曝光。

5、壳体化HBV DNA的提取

a、用冰预冷的PBS洗细胞两次；

b、加400μl Lysis buffer，室温摇10min；

c、裂解后的细胞转入EP管中，13200rpm离心1min；

d、吸取上清，加20μl DNase1和20μlDNase1 buffer 以及5μlRNase A，37°C温浴2h左右；

e、加20μl 0.5M的EDTA，沸水煮5min；

f、加10μl proteinase K(20mg/ml)和20μl 10%SDS，混匀，55°C过夜；

g、按1:1体积添加酚/氯仿(1:1)，猛烈摇匀后静置2min，13200rpm离心5min，去上清；

h、加入2倍体积的乙醇和0.1倍体积的NaAc(3M，PH5.2），室温放置5min，13200rpm离心5min，弃上清；

i、加1ml70%乙醇，重悬沉淀，13200rpm离心3min，弃上清，干燥；

j、加15μl TE buffer 将DNA溶解。

6、qPCR体系

提取的HBV壳体化DNA 2μl

2×SYBR Green Master 10μl

HBV DNA primers 1.2μl

水 6.8μl

qPCR中HBV DNA使用的引物序列（如SEQ ID NO. 19-20所示）为：

HBVF：5`-CTCGTGGTGGACTTCTCTC-3`

HBVR：5`-CTGCAGGATGAAGAGGAA-3`

【实施例3】任意两种或多种gRNA联合使用对HBV复制的抑制

当我们把多种gRNA联合使用时，CRISPR/Cas9***可以同时剪切HBV DNA上的两个或多个位点，使两个或多个位点同时产生突变，从而提高HBV的抑制效率。尤其是当Tmix组合使用时，产生多个HBV DNA断裂点可能因为修复不及时，而使HBV DNA被进一步降解，降低了HBV的存活率，极大提高CRISPR/Cas9***清除HBV的效率。本发明中将8种gRNA的组合Tmix和pHBV1.3、pSVβ-gal共转染HepG2细胞，培养2天或4天后，用ELISA、Northern blot及qPCR等检测其对HBV的抑制效率。从实验结果可以看到，Tmix对于乙型肝炎病毒的抑制效果明显优于单独使用任意一种gRNA，几乎检测不到细胞内HBV病毒复制和细胞外子代病毒的产生（图2和图3）。同样，在小鼠模型中使用Tmix对HBV进行治疗应用，通过各项检测发现，小鼠的血清中检测不到病毒HBeAg、HBsAg和HBV DNA，此外在肝脏组织中也检测不到HBV基因组（图5）。通过突变位点分析实验和深度测序发现，Tmix几乎可以将HBV DNA切成小片段，以至于在血清及肝脏组织中检测不到HBV DNA（图5和图6），从而大大提高HBV的清除效率，并且这种联合使用，可以有效克服乙型肝炎病毒容易突变的问题。具体实施步骤详见实施例二、四和五。

【实施例4】检测本发明gRNA对于不同基因型HBV的抑制效果

为了检测本发明涉及的gRNA是否对于基因型为A, B, C的HBV具有抑制作用， 我们以T1, T7，Tmix为例检测了其对于不同基因型的HBV的抑制效果。首先，我们将质粒pHBV1.3 A（HBV 基因型A （Genebank ID：AF305422.1）），pHBV1.3 B（基因型B（GenebankID：EU570069.1）），pHBV1.3 C（基因型C （Genebank ID：FJ899793.1））和pHBV1.3 D（基因型D（Genebank ID：V01460.1））分别与不同的PX330-gRNA质粒共转染HepG2细胞系，2天后检测病毒e抗原以及S抗原的表达水平。结果如图4所示，T1和T7对于HBsAg以及HBeAg均有不同程度的抑制。尤为显著的是，如结果所示，Tmix对于四种基因型均有4至5倍的抑制效果（图4）。这是由于8条gRNA分别针对HBV的不同保守性位点，克服了HBV序列易突变的问题，所以对于不同的基因型的HBV均有明显的抑制效果。这也体现了8条gRNA组合的优越性和更好的应用前景。

【实施例5】在小鼠模型中应用本发明的gRNA及其组合Tmix清除HBV

我们选用HBV CRISPR gRNA T1和T7以及8种gRNA联合使用的Tmix组合所指导的CRISPR/Cas9***来验证其是否可以在小鼠模型中抑制乙型肝炎病毒的复制及子代病毒的产生，从而清除HBV。通过动力学注射（hydrodynamic injection，HDI）将乙型肝炎病毒的复制子pHBV1.3通过高压尾静脉注射的方法注射到小鼠体内，从而构建乙型肝炎病毒的小鼠模型，同时将表达T_GFP，T1，T7，Tmix的质粒也分别注射到小鼠体内，四天之后，处死小鼠并检测病毒的复制指标。具体实施步骤如下：

1、将5μg pHBV1.3质粒、5μg PSVβ-gal质粒和25μg PX330-U6-HBV gRNA质粒溶于2ml PBS中；

2、用2ml的医用注射针将溶有质粒的PBS快速（<5s)通过尾静脉注射到小鼠体内；

3、注射后的小鼠养四天后，摘除眼球取血，处死后剖开取肝脏，最大的一叶肝脏用甲醛固定，其它的分两部分，一份冻在液氮中，一份保存在RNA保存液中；

4、小鼠的血液在4°C冰箱放置过夜后，3000rpm离心15 min，取上清，即小鼠血清；

5、取少量小鼠血清稀释后用EILSA测其中的乙型肝炎病毒表面抗原和e抗原；

6、从小鼠血清中提取HBV DNA，并用qPCR检测其中的HBV DNA含量；

7、从保存于RNA保存液中的小鼠肝脏中提取RNA，通过Northern blot检测HBV转录产生的RNA量；

8、甲醛中保存的肝脏小叶用来做病理切片，并通过免疫组化的方法检测其中的Core蛋白含量。

如图5所示，小鼠血清中HBeAg、HBsAg的水平均受到明显的抑制（图5A、B）。T7与Tmix组对于HBV核心抗原的表达有明显的抑制作用（图5D）。T1对于HBV核心抗原表达的抑制作用较为微弱，是由于其作用位点不位于核心抗原编码区或者其启动子区域之上（图5D）。而直接检测小鼠肝脏内的病毒RNA水平也显示病毒复制受到了明显的抑制（图5E）。尤其明显的Tmix组，均已检测不到明显病毒RNA阳性的肝细胞（图5D）。综上，本发明中设计的gRNA，尤其是Tmix所指导的CRISPR/Cas9***可以明显的抑制病毒复制，具有清除病毒感染的能力。

理论上，Cas9将基因组DNA切断之后，细胞会通过非同源重组的末端接合的方式将基因组修复。由于这一修复模式易于产生突变，我们用T7 E1实验以及深度测序实验检测了经过gRNA处理的HBV基因组序列是否产生了这一突变。具体实施方式如下：

1、从小鼠肝脏中提取细胞基因组，所用试剂盒为天根细胞血液组织基因组提取试剂盒（DP304），方法按厂家说明书进行。

2、PCR获得包括gRNA靶位点的片段。

PCR体系如下：

10×buffer 5μl

2mM dNTP mix 5 μl

25mM MgSO4 2 μl

50μM Primer F 0.5 μl

50μM Primer R 0.5 μl

模板 10 ng

KOD-Plus-neo 1μl

ddH2O up to 50 μl

PCR 反应循环

95°C 5 min

95°C 30 S

55°C 30 S

68°C 30 S

35 cycles

68°C 10 min

3、使用OMEGA公司胶回收试剂盒（D2500）回收PCR得到的目的片段。具体方法按照公司提供的试验方法进行。

4、目的片段退火。

向回收得到的目的片段中加入NEB buffer 2， 按如下方式退火

90°C 5 min

70°C 20 min

55°C 20 min

37°C 20 min

5、T7 E1 酶切

向退火产物中加入0.5 μl T7 E1，37°C水浴30 min。1%琼脂糖凝胶电泳检测。

如T7 E1实验所示，T1 gRNA的切割产生了579 bp的小片段，T7 gRNA的切割产生了1174 bp的小片段，这与预期是一致的，说明T1与T7 gRNA均在其对应的靶位点产生了切割（图6A和6B）。如深度测序实验所示，T1和T7 gRNA均产生了不同类型的缺失或者***突变，再次说明了T1与T7 gRNA均在其对应的靶位点产生了切割（图6C和6D）。Tmix中8条gRNA在同时起作用，HBV的基因组会被切割为200bp到500bp不等的小片段，这些片段并不能被细胞有效的连接和重组，所以这些小片段会被细胞内的核酸酶降解。正如图6A和6B所示，Tmix中并未检测到突变位点。由于单一的gRNA作用之后，病毒有可能被修复而产生突变，由于突变的不确定性，突变病毒有可能产生更强的复制能力，或者改变的宿主嗜性，这一方式带来了很大的隐患。而多条gRNA联用的策略可以有效的避免了这一弊端。

SEQUENCE LISTING

<110> 武汉大学

<120> CRISPR/Cas9特异性敲除乙型肝炎病毒的方法以及用于特异性靶向HBV

DNA的gRNA

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<170> PatentIn version 3.3

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gttgccgggc aacggggtaa agg 23

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aaacaaagga cgtcccgcgc tgg 23

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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

cgaaacacct accgcagagt ctagactcgg ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 10

<211> 8508

<212> DNA

<213> 人工序列

<400> 10

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

cgaaacaccc atttgttcag tggttcgtag ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 11

<211> 8508

<212> DNA

<213> 人工序列

<400> 11

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 ttgccgggca acggggtaag ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 12

<211> 8508

<212> DNA

<213> 人工序列

<400> 12

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

cgaaacacca aacaaaggac gtcccgcgcg ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 13

<211> 8508

<212> DNA

<213> 人工序列

<400> 13

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 gtctccatgc gacgtgcagg ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 14

<211> 8508

<212> DNA

<213> 人工序列

<400> 14

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 tagctccaaa ttctttatag ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 15

<211> 8508

<212> DNA

<213> 人工序列

<400> 15

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 accttcgtct gcgaggcgag ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 16

<211> 8508

<212> DNA

<213> 人工序列

<400> 16

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

cgaaacaccc cttcctgact ggcgattggg ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 17

<211> 8506

<212> DNA

<213> 人工序列

<400> 17

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 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 900

gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 960

ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1020

tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1080

accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1140

ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1200

ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1260

aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1320

tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1380

acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1440

acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1500

agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1560

agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1620

tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1680

tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1740

aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1800

gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1860

ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1920

tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1980

gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2040

tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2100

gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc 2160

agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2220

agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2280

tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat 2340

acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2400

agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2460

gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2520

gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2580

tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2640

ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2700

ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2760

tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg 2820

gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2880

agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2940

ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga 3000

aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3060

aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3120

atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3180

acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3240

tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3300

acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3360

agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3420

ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta 3480

aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3540

ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3600

acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3660

gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3720

aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3780

agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3840

accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3900

gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3960

gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4020

agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4080

gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4140

ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4200

agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4260

atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4320

acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4380

aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4440

gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4500

ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4560

caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4620

aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4680

tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4740

actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4800

tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4860

ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4920

agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4980

agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg 5040

aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5100

tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5160

acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5220

ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5280

aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5340

agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5400

ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5460

tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 5520

aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5580

tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5640

ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5700

tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5760

gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5820

cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5880

gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 5940

tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 6000

gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 6060

ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 6120

cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 6180

gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 6240

gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6300

ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 6360

tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6420

atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 6480

ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 6540

ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 6600

tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 6660

gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 6720

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 6780

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6840

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 6900

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 6960

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 7020

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 7080

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 7140

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 7200

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 7260

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 7320

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 7380

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7440

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 7500

ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 7560

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 7620

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 7680

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7740

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 7800

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 7860

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 7920

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 7980

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 8040

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 8100

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 8160

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 8220

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 8280

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8340

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 8400

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 8460

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt 8506

<210> 18

<211> 8508

<212> DNA

<213> 人工序列

<400> 18

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 agctggacgg cgacgtaaag ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc 480

gcccaacgac ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg 540

tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat 600

gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca 660

gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat 720

taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc 780

acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg 840

gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg 900

gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag 960

gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac 1020

gctgccttcg ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac 1080

tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt 1140

agctgagcaa gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt 1200

acctggagca cctgcctgaa atcacttttt ttcaggttgg accggtgcca ccatggacta 1260

taaggaccac gacggagact acaaggatca tgatattgat tacaaagacg atgacgataa 1320

gatggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa 1380

gtacagcatc ggcctggaca tcggcaccaa ctctgtgggc tgggccgtga tcaccgacga 1440

gtacaaggtg cccagcaaga aattcaaggt gctgggcaac accgaccggc acagcatcaa 1500

gaagaacctg atcggagccc tgctgttcga cagcggcgaa acagccgagg ccacccggct 1560

gaagagaacc gccagaagaa gatacaccag acggaagaac cggatctgct atctgcaaga 1620

gatcttcagc aacgagatgg ccaaggtgga cgacagcttc ttccacagac tggaagagtc 1680

cttcctggtg gaagaggata agaagcacga gcggcacccc atcttcggca acatcgtgga 1740

cgaggtggcc taccacgaga agtaccccac catctaccac ctgagaaaga aactggtgga 1800

cagcaccgac aaggccgacc tgcggctgat ctatctggcc ctggcccaca tgatcaagtt 1860

ccggggccac ttcctgatcg agggcgacct gaaccccgac aacagcgacg tggacaagct 1920

gttcatccag ctggtgcaga cctacaacca gctgttcgag gaaaacccca tcaacgccag 1980

cggcgtggac gccaaggcca tcctgtctgc cagactgagc aagagcagac ggctggaaaa 2040

tctgatcgcc cagctgcccg gcgagaagaa gaatggcctg ttcggaaacc tgattgccct 2100

gagcctgggc ctgaccccca acttcaagag caacttcgac ctggccgagg atgccaaact 2160

gcagctgagc aaggacacct acgacgacga cctggacaac ctgctggccc agatcggcga 2220

ccagtacgcc gacctgtttc tggccgccaa gaacctgtcc gacgccatcc tgctgagcga 2280

catcctgaga gtgaacaccg agatcaccaa ggcccccctg agcgcctcta tgatcaagag 2340

atacgacgag caccaccagg acctgaccct gctgaaagct ctcgtgcggc agcagctgcc 2400

tgagaagtac aaagagattt tcttcgacca gagcaagaac ggctacgccg gctacattga 2460

cggcggagcc agccaggaag agttctacaa gttcatcaag cccatcctgg aaaagatgga 2520

cggcaccgag gaactgctcg tgaagctgaa cagagaggac ctgctgcgga agcagcggac 2580

cttcgacaac ggcagcatcc cccaccagat ccacctggga gagctgcacg ccattctgcg 2640

gcggcaggaa gatttttacc cattcctgaa ggacaaccgg gaaaagatcg agaagatcct 2700

gaccttccgc atcccctact acgtgggccc tctggccagg ggaaacagca gattcgcctg 2760

gatgaccaga aagagcgagg aaaccatcac cccctggaac ttcgaggaag tggtggacaa 2820

gggcgcttcc gcccagagct tcatcgagcg gatgaccaac ttcgataaga acctgcccaa 2880

cgagaaggtg ctgcccaagc acagcctgct gtacgagtac ttcaccgtgt ataacgagct 2940

gaccaaagtg aaatacgtga ccgagggaat gagaaagccc gccttcctga gcggcgagca 3000

gaaaaaggcc atcgtggacc tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct 3060

gaaagaggac tacttcaaga aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga 3120

agatcggttc aacgcctccc tgggcacata ccacgatctg ctgaaaatta tcaaggacaa 3180

ggacttcctg gacaatgagg aaaacgagga cattctggaa gatatcgtgc tgaccctgac 3240

actgtttgag gacagagaga tgatcgagga acggctgaaa acctatgccc acctgttcga 3300

cgacaaagtg atgaagcagc tgaagcggcg gagatacacc ggctggggca ggctgagccg 3360

gaagctgatc aacggcatcc gggacaagca gtccggcaag acaatcctgg atttcctgaa 3420

gtccgacggc ttcgccaaca gaaacttcat gcagctgatc cacgacgaca gcctgacctt 3480

taaagaggac atccagaaag cccaggtgtc cggccagggc gatagcctgc acgagcacat 3540

tgccaatctg gccggcagcc ccgccattaa gaagggcatc ctgcagacag tgaaggtggt 3600

ggacgagctc gtgaaagtga tgggccggca caagcccgag aacatcgtga tcgaaatggc 3660

cagagagaac cagaccaccc agaagggaca gaagaacagc cgcgagagaa tgaagcggat 3720

cgaagagggc atcaaagagc tgggcagcca gatcctgaaa gaacaccccg tggaaaacac 3780

ccagctgcag aacgagaagc tgtacctgta ctacctgcag aatgggcggg atatgtacgt 3840

ggaccaggaa ctggacatca accggctgtc cgactacgat gtggaccata tcgtgcctca 3900

gagctttctg aaggacgact ccatcgacaa caaggtgctg accagaagcg acaagaaccg 3960

gggcaagagc gacaacgtgc cctccgaaga ggtcgtgaag aagatgaaga actactggcg 4020

gcagctgctg aacgccaagc tgattaccca gagaaagttc gacaatctga ccaaggccga 4080

gagaggcggc ctgagcgaac tggataaggc cggcttcatc aagagacagc tggtggaaac 4140

ccggcagatc acaaagcacg tggcacagat cctggactcc cggatgaaca ctaagtacga 4200

cgagaatgac aagctgatcc gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc 4260

cgatttccgg aaggatttcc agttttacaa agtgcgcgag atcaacaact accaccacgc 4320

ccacgacgcc tacctgaacg ccgtcgtggg aaccgccctg atcaaaaagt accctaagct 4380

ggaaagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa 4440

gagcgagcag gaaatcggca aggctaccgc caagtacttc ttctacagca acatcatgaa 4500

ctttttcaag accgagatta ccctggccaa cggcgagatc cggaagcggc ctctgatcga 4560

gacaaacggc gaaaccgggg agatcgtgtg ggataagggc cgggattttg ccaccgtgcg 4620

gaaagtgctg agcatgcccc aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg 4680

cttcagcaaa gagtctatcc tgcccaagag gaacagcgat aagctgatcg ccagaaagaa 4740

ggactgggac cctaagaagt acggcggctt cgacagcccc accgtggcct attctgtgct 4800

ggtggtggcc aaagtggaaa agggcaagtc caagaaactg aagagtgtga aagagctgct 4860

ggggatcacc atcatggaaa gaagcagctt cgagaagaat cccatcgact ttctggaagc 4920

caagggctac aaagaagtga aaaaggacct gatcatcaag ctgcctaagt actccctgtt 4980

cgagctggaa aacggccgga agagaatgct ggcctctgcc ggcgaactgc agaagggaaa 5040

cgaactggcc ctgccctcca aatatgtgaa cttcctgtac ctggccagcc actatgagaa 5100

gctgaagggc tcccccgagg ataatgagca gaaacagctg tttgtggaac agcacaagca 5160

ctacctggac gagatcatcg agcagatcag cgagttctcc aagagagtga tcctggccga 5220

cgctaatctg gacaaagtgc tgtccgccta caacaagcac cgggataagc ccatcagaga 5280

gcaggccgag aatatcatcc acctgtttac cctgaccaat ctgggagccc ctgccgcctt 5340

caagtacttt gacaccacca tcgaccggaa gaggtacacc agcaccaaag aggtgctgga 5400

cgccaccctg atccaccaga gcatcaccgg cctgtacgag acacggatcg acctgtctca 5460

gctgggaggc gacaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa 5520

gtaagaattc ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 5580

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 5640

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 5700

ggtggggtgg ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg 5760

gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 5820

ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 5880

gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg 5940

catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg 6000

gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6060

ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6120

cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6180

tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga 6240

cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6300

ctggaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga 6360

tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6420

aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat 6480

agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 6540

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 6600

tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 6660

aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 6720

tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 6780

gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 6840

atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 6900

cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6960

tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 7020

caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 7080

ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 7140

cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 7200

taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 7260

agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 7320

cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 7380

caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 7440

taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 7500

ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg 7560

cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 7620

aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 7680

attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 7740

tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 7800

aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 7860

gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 7920

cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 7980

gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 8040

agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8100

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8160

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8220

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8280

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8340

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8400

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8460

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt 8508

<210> 19

<211> 19

<212> DNA

<213> 人工序列

<400> 19

ctcgtggtgg acttctctc 19

<210> 20

<211> 18

<212> DNA

<213> 人工序列

<400> 20

ctgcaggatg aagaggaa 18

<210> 21

<211> 25

<212> DNA

<213> 人工序列

<400> 21

ttccacaacc ttccaccaaa ctctg 25

<210> 22

<211> 25

<212> DNA

<213> 人工序列

<400> 22

gacaaaagaa aattggtaac agcgg 25

<210> 23

<211> 25

<212> DNA

<213> 人工序列

<400> 23

agtctcctga gcattgttca cctca 25

<210> 24

<211> 25

<212> DNA

<213> 人工序列

<400> 24

atccagcgat aaccaggaca agttg 25

Claims (7)

1.在CRISPR-Cas9特异性敲除乙型肝炎病毒cccDNA中用于特异性靶向乙型肝炎病毒cccDNA的gRNA，所述gRNA在乙型肝炎病毒cccDNA上的靶序列符合5’-N(20)-NGG3’或者5’-CCN-N(20)-3’的序列排列规则，在HBV cccDNA上的靶序列是唯一的，所述gRNA在HBVcccDNA的靶向位点位于至少2种HBV蛋白编码基因的共有序列区域,而且是在不同基因型和亚型HBV的基因序列中都保守的区域，其特征在于：gRNA对应的DNA序列如序列表SEQ IDNO.1-8任意一条序列所示。

2.CRISPR-Cas9特异性敲除乙型肝炎病毒cccDNA的方法，包括如下步骤：

(1)权利要求1所述的gRNA，在其对应的DNA序列SEQ ID NO.1-8的5’末端分别加上CACC得到正向寡核苷酸序列，在其互补链的5’末端分别加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后将合成的序列变性、退火，得到具有BbsI粘性末端的双链DNA片段；

(2)将双链DNA片段和用Bbs1酶切过的pX330-U6-Chimeric_BB-CBh-hSpCas9载体进行连接，将连接产物转化到大肠杆菌JM109感受态细胞中，涂布于带有氨苄青霉素抗性的LB平板上，筛选阳性菌落，提取阳性菌落质粒进行分析及测序，确定gRNA表达载体构建成功，命名为PX330-U6-HBVgRNA；

(3)将步骤(2)构建的八种PX330-U6-HBVgRNA质粒等量混合，与pHBV1.3和pSV-β-gal共转染HepG2细胞，并用表达特异靶向敲除绿色荧光蛋白的gRNA的载体PX330-U6-T_GFP与pHBV1.3和pSV-β-gal共转染HepG2细胞作为对照，PX330-U6-T_GFP的核苷酸序列如SEQ IDNO.18所示；

或者，通过动力学注射(hydrodynamic injection，HDI)将乙型肝炎病毒的复制子pHBV1.3通过高压尾静脉注射的方法注射到小鼠体内，从而构建乙型肝炎病毒的小鼠模型，同时将PX330-U6-T_GFP或步骤(2)构建的八种PX330-U6-HBVgRNA质粒等量混合注射到小鼠体内。

3.如权利要求2所述的CRISPR-Cas9特异性敲除乙型肝炎病毒cccDNA的方法，其特征为：8种PX330-U6-HBVgRNA质粒等比例混合成Tmix，然后以Tmix质粒：pHBV1.3质粒：pSV-β-gal质粒＝8:1:1的比例混合，共转染HepG2细胞。

4.如权利要求2所述的CRISPR-Cas9特异性敲除乙型肝炎病毒cccDNA的方法，其特征为：所述的8种PX330-U6-HBVgRNA质粒的序列为SEQ ID NO.9-16。

5.如权利要求1所述的在CRISPR-Cas9特异性敲除乙型肝炎病毒cccDNA中用于特异性靶向乙型肝炎病毒cccDNA的gRNA在非治疗性的敲除乙型肝炎病毒cccDNA中的应用。

6.如权利要求4中所述的PX330-U6-HBVgRNA质粒在非治疗性的敲除乙型肝炎病毒cccDNA中的应用。

7.如权利要求4中所述的PX330-U6-HBVgRNA质粒在制备抗乙型肝炎病毒药物中的应用。