CN1942585A - 含人丙型肝炎病毒全长基因组的核酸构建物、核酸构建物转入其中的重组全长病毒基因组复制型细胞和生产丙型肝炎病毒颗粒的方法 - Google Patents

含人丙型肝炎病毒全长基因组的核酸构建物、核酸构建物转入其中的重组全长病毒基因组复制型细胞和生产丙型肝炎病毒颗粒的方法 Download PDF

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CN1942585A
CN1942585A CNA2005800115150A CN200580011515A CN1942585A CN 1942585 A CN1942585 A CN 1942585A CN A2005800115150 A CNA2005800115150 A CN A2005800115150A CN 200580011515 A CN200580011515 A CN 200580011515A CN 1942585 A CN1942585 A CN 1942585A
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胁田隆字
加藤孝宣
伊达朋子
宫本道子
田边纯一
曾根三郎
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Toray Industries Inc
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Abstract

本发明提供有效复制含全长HCV基因组序列的RNA的方法和使用细胞培养系统生产含全长HCV复制子RNA或全长HCV基因组RNA的HCV病毒颗粒的方法。此外，本发明涉及生产丙型肝炎病毒颗粒的方法，其包括培养其中导入复制子RNA的细胞，并在培养基中产生病毒颗粒，其中所述复制子RNA包含的核苷酸序列含有基因型2a丙型肝炎病毒的全长基因组RNA序列、至少一种选择标记基因和/或至少一种报告基因以及至少一种IRES序列，或含有基因型2a丙型肝炎病毒的全长基因组RNA。再进一步，本发明还涉及丙型肝炎疫苗和抗丙型肝炎病毒颗粒的抗体。

Description

含人丙型肝炎病毒全长基因组的核酸构建物、核酸构建物转入其中 的重组全长病毒基因组复制型细胞和生产丙型肝炎病毒颗粒的方法
技术领域
本发明涉及含丙型肝炎病毒全长基因组的核酸构建物、生产丙型肝炎病毒颗粒的体外方法和所生产的丙型肝炎病毒颗粒的用途。
背景技术
丙型肝炎病毒(HCV)属于黄病毒科家族，是一种具有单链正义RNA基因组的病毒，已知引起丙型肝炎。
HCV通过持续感染引起慢性肝炎。目前，在世界范围内观察到的慢性肝炎的重要病因是持续HCV感染。实际上，大约50％的持续感染个体发展成慢性肝炎。这些患者约有20％在10-20年间由慢性肝炎转变成肝硬化，这些患者中有一些进一步发展成晚期致死性病症，例如肝癌。
丙型肝炎目前主要通过使用干扰素-α或干扰素-β的疗法或使用干扰素-α和利巴韦林(一种嘌呤-核苷衍生物)组合的疗法治疗。但是，即使在实施这些疗法时，在所有受治疗患者中也仅有约60％观察到疗效。当观察到疗效后终止治疗时，一半以上的患者疾病复发。
一个重要目标是开发有效对抗丙型肝炎的治疗剂或预防剂。在工业化国家丙型肝炎发病率高，最终引发严重后果，目前还没有可用的病因疗法。因此，需要开发HCV-特异性化学疗法和疫苗疗法。开发抗HCV药物的目标可为抑制HCV复制或抑制HCV感染细胞。
最近，已制备了HCV亚基因组RNA复制子系统，作为HCV衍生的自主复制RNA(参见特许文献1、2和3，非特许文献1-4)。在HCV亚基因组RNA复制子系统中，制备其中HCV基因组结构基因被去除并被药物选择性标记基因取代的HCV复制子RNA，并将其导入培养细胞中，由此使复制子RNA在细胞中自主复制。使用这些系统使得有可能分析HCV的复制机制。但是，这是一个其中仅可在HCV病毒增殖和复制过程中评价病毒RNA复制的实验系统，不能分析被感染细胞中HCV病毒颗粒形成和胞外释放或感染另一细胞的过程。
此时，只能在使用动物如黑猩猩的实验系统中评价HCV病毒颗粒形成和胞外释放以及感染另一细胞的过程(非特许文献5)。但是，使用活生物(例如动物)的实验系统很复杂，非常难分析。因此，为了分析HCV病毒颗粒形成和胞外释放以及感染另一细胞的过程，为了生产具有抑制该过程的作用机制的抗HCV药物，必须建立重现该过程的高度简单化的实验系统，即在细胞培养实验系统中的HCV病毒颗粒生产系统。
此外，一旦可使用细胞培养系统稳定提供HCV病毒颗粒，就有可能使用分子生物学技术对病毒减毒或生产非感染性HCV病毒，这可用于疫苗。
特许文献1：日本特许公开(公报)第2001-17187号
特许文献2：国际专利申请PCT/JP03/15038
特许文献3：JP特许申请第2003-329082号
非特许文献1：Lohmann等，Science，(1999)285，110-113页
非特许文献2：Blight等，Science，(2000)290，1972-1974页
非特许文献3：Friebe等，J.Virol.，(2001)75(24)：12047-12057页
非特许文献4：Ikeda等，J.Virol.，(2002)76(6)：2997-3006页
非特许文献5：Kolykhalov等，Science，(1997)277，570-574页
非特许文献6：Kato等，Gastroenterology，(2003)125，1808-1817页
非特许文献7：Yanagi等，Proc.Natl.Acad.Sci.，(1997)96(16)：8738-8743页
非特许文献8：Okamoto等，J.Gen.Virol.，(1991)73，2697-26704页
非特许文献9：Aoyagi等，J.Clin.Microbiol.，(1999)37(6)：1802-1808页
发明公开
本发明的目标是提供含全长HCV基因组序列的RNA的有效复制方法和在细胞培养系统中生产含全长HCV复制子RNA或全长HCV基因组RNA的HCV病毒颗粒的方法。本发明的目标迄今为止还从未实现过。
为实现以上目标，本发明的发明人深入研究，结果开发出一种在细胞培养系统中生产HCV病毒颗粒的方法。即，本发明如下所述。
[1]一种复制子RNA，其包含的核苷酸序列含有基因型2a丙型肝炎病毒基因组RNA的5′非翻译区、核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列、NS5B蛋白编码序列和3′非翻译区，至少一种选择标记基因和/或至少一种报告基因，以及至少一种IRES序列。
在该复制子RNA中，优选核苷酸序列含在5′-3′方向为以下顺序的5′非翻译区、至少一种选择标记基因和/或至少一种报告基因，和至少一种IRES序列，以及核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列、NS5B蛋白编码序列和3′非翻译区。
在此复制子RNA的更优选实施方案中，基因型2a丙型肝炎病毒基因组RNA是含SEQ ID NO：12所示核苷酸序列的RNA。
在此复制子RNA的再更优选实施方案中，5′非翻译区含SEQ IDNO：1所示核苷酸序列，核心蛋白编码序列含SEQ ID NO：2所示核苷酸序列，E1蛋白编码序列含SEQ ID NO：3所示核苷酸序列，E2蛋白编码序列含SEQ ID NO：4所示核苷酸序列，NS2蛋白编码序列含SEQ ID NO：5所示核苷酸序列，NS3蛋白编码序列含SEQ ID NO：6所示核苷酸序列，NS4A蛋白编码序列含SEQ ID NO：7所示核苷酸序列，NS4B蛋白编码序列含SEQ ID NO：8所示核苷酸序列，NS5A蛋白编码序列含SEQ ID NO：9所示核苷酸序列，NS5B蛋白编码序列含SEQ ID NO：10所示核苷酸序列，而3′非翻译区含SEQ ID NO：11所示核苷酸序列。
[2]含以下(a)或(b)RNA的复制子RNA：
(a)含SEQ ID NO：13所示核苷酸序列的RNA；或
(b)含通过缺失、置换或添加1-100个核苷酸而衍生自SEQ ID NO：13所示核苷酸序列的核苷酸序列、并具有自主复制能力和病毒颗粒生产能力的RNA。
[3]一种生产细胞的方法，其中所述细胞复制复制子RNA并生产病毒颗粒，该方法包括将上述[1]或[2]中任一项的复制子RNA导入到细胞中。
用于该方法的细胞优选为增殖性细胞。同样或否则，用于该方法的细胞优选为真核细胞。
用于该方法的真核细胞优选为人肝源细胞、人子宫颈源细胞或人胎肾源细胞。所述真核细胞更优选为Huh7细胞、HepG2细胞、IMY-N9细胞、HeLa细胞或293细胞。
[4]利用上述[3]的方法可获得的细胞，其复制复制子RNA并生产病毒颗粒。
[5]生产丙型肝炎病毒颗粒的方法，包括培养上述[4]的细胞，以使该细胞生产病毒颗粒。
[6]利用上述[5]的方法可获得的丙型肝炎病毒颗粒。
[7]生产丙型肝炎病毒感染细胞的方法，包括培养上述[4]的细胞，并用培养物中的病毒颗粒感染其它细胞。
[8]通过上述[7]的方法可获得的丙型肝炎病毒感染细胞。
[9]筛选抗丙型肝炎病毒物质的方法，包括在测试物质存在下培养选自以下(a)、(b)和(c)中的至少一种：
(a)上述[4]的细胞，
(b)上述[8]的丙型肝炎病毒感染细胞，和
(c)上述[6]的丙型肝炎病毒颗粒和丙型肝炎病毒允许细胞；
并检测所获培养物中的复制子RNA或病毒颗粒。
[10]丙型肝炎疫苗，包含上述[6]的丙型肝炎病毒颗粒或其部分。
[11]使用上述[6]的丙型肝炎病毒颗粒或其部分作为抗原生产丙型肝炎疫苗的方法。
[12]通过使用上述[1]或[2]的复制子RNA生产用于基因治疗的嗜肝病毒载体的方法。
[13]利用上述[12]的方法可获得的嗜肝病毒载体。
[14]在细胞中复制和/或表达外源基因的方法，包括将编码外源基因的RNA插入到上述[1]或[2]中任一项的复制子RNA中，并将其导入到所述细胞中。
[15]一种细胞生产方法，其中所述细胞复制RNA并生产病毒颗粒，该方法包括将含SEQ ID NO：12所示核苷酸序列的RNA导入到该细胞中。
[16]生产丙型肝炎病毒颗粒的方法，包括将含SEQ ID NO：12所示核苷酸序列的RNA导入到细胞中，并培养细胞，使该细胞生产病毒颗粒。
[17]上述[15]或[16]的方法，其中所述细胞为增殖性细胞。
[18]生产含外源基因的病毒载体的方法，包括将编码外源基因的RNA插入到含SEQ ID NO：12所示核苷酸序列的RNA中，将其导入到细胞中，并培养细胞，使该细胞生产病毒颗粒。
[19]抗上述[6]的丙型肝炎病毒颗粒的抗体。
在本申请要求其优先权的日本特许申请第2004-045489号的说明书和附图中的内容结合到本文中。
附图简述
图1是一幅示意图，显示了构建模板DNA的方法，该模板DNA用于制备本发明的全长HCV复制子HCV或全长HCV基因组RNA。图1的上部显示了质粒克隆pJFH1的结构，其通过将全长HCV基因组插入到T7启动子下游产生。图1的下部显示了含全长HCV基因组序列的质粒克隆pFGREP-JFH1的结构，其中含新霉素抗性基因和EMCV IRES的DNA片段插入到pJFH1的T7启动子和5′非翻译区下游。该图中显示的术语如下。T7：T7RNA启动子，5′UTR：5′非翻译区，C：核心蛋白，E1、E2：包膜蛋白。NS2、NS3、NS4A、NS4B、4A、4B：非结构蛋白。3′UTR：3′非翻译区。AgeI、PmeI、XbaI：限制酶AgeI、PmeI和XbaI的限制性位点。GDD：对应于NS5B蛋白活性中心的氨基酸基序GDD的位点。neo：新霉素抗性基因。EMCVIRES：脑心肌炎病毒内部核糖体进入位点；
图2是一幅照片，显示了RNA印迹分析的结果，其表明rJFH-1在其中已导入全长HCV基因组RNA rJFH-1的Huh7细胞中复制；
图3显示了培养基中HCV核心蛋白定量的结果。空心圆代表其中已导入rJFH1的细胞，实心圆代表其中已导入rJFH1/GND的细胞；
图4图示了HCV核心蛋白和全长HCV基因组RNA的量，以及通过蔗糖密度梯度分级分离已导入rJFH-1的Huh7细胞的培养上清液所收集的各个级分的比重。实心圆、空心圆和阴影圆分别代表HCV核心蛋白、全长HCV基因组RNA和比重。
图5是一幅照片，显示了其中已转染入全长HCV复制子RNArFGREP-JFH1的Huh7细胞的集落形成；
图6是一幅照片，显示了全长HCV复制子RNA在全长HCV复制子RNA复制型细胞克隆中的复制，其中所述克隆已通过将rFGREP-JFH1转染入Huh7细胞中建立；
图7是一幅照片，显示了使用宿主细胞的基因组DNA作为模板和新霉素抗性基因特异性引物的PCR扩增的结果，用于证实新霉素抗性基因整合入基因组DNA中。M：DNA大小标记物，P：阳性对照，N：Huh7细胞；
图8是一幅照片，显示了蛋白质印迹分析的结果，其表明核心蛋白在其中已导入全长HCV复制子RNA rFGREP-JFH1的Huh7细胞中表达；
图9是一幅照片，显示了蛋白质印迹分析的结果，其表明NS3蛋白在其中已导入全长HCV复制子RNA rFGREP-JFH1的Huh7细胞中表达；
图10是一幅照片，显示了蛋白质印迹分析的结果，其表明NS5A蛋白在其中已导入全长HCV复制子RNA rFGREP-JFH1的Huh7细胞中表达；
图11图示了HCV核心蛋白和全长HCV复制子RNA的量，以及通过蔗糖密度梯度分级分离已导入rFGREP-JFH1的Huh7细胞的培养上清液所收集的各个级分的比重。实心圆、空心圆和阴影圆分别代表HCV核心蛋白、全长HCV复制子RNA和比重；
图12是一幅照片，显示了Huh7细胞的集落形成，其中已加入全长HCV复制子RNA复制型细胞培养上清液中的病毒颗粒。
实施本发明的最佳方式
本发明如下详细阐述。
1.全长HCV复制子RNA
丙型肝炎病毒(HCV)基因组是单股正链RNA，含约9600个核苷酸。此基因组RNA含5′非翻译区(也称为5′NTR或5′UTR)、由结构区和非结构区组成的翻译区以及3′非翻译区(也称为3′NTR或3′UTR)。HCV结构蛋白在结构区中编码，多种非结构蛋白在非结构区中编码。
这样的HCV结构蛋白(core、E1蛋白和E2蛋白)和非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A和NS5B)如下产生：首先将翻译区翻译成单个连续的多蛋白，然后通过蛋白酶限制性切割多蛋白释放。在这些结构蛋白和非结构蛋白(即HCV的病毒蛋白)中，core是核心蛋白，E1和E2是包膜蛋白。非结构蛋白是参与病毒自身复制的蛋白，已知NS2具有金属蛋白酶活性，已知NS3具有丝氨酸蛋白酶活性(在N端侧的1/3)和解旋酶活性(在C端侧的2/3)。此外，NS4A是NS3蛋白酶活性的辅因子，已报道NS5B具有RNA依赖性RNA聚合酶活性。
本发明的发明人使用HVC基因组RNA构建了具有自主复制能力和病毒颗粒生产能力的复制子RNA。
已通过修饰HCV基因组RNA产生的具有自主复制能力的RNA在本文中叫做“复制子RNA”或“RNA复制子”。在本说明书中，源自HCV的复制子RNA还可叫做HCV-RNA复制子。含全长HCV基因组RNA的本发明复制子RNA在本文叫做“全长HCV复制子RNA”。本发明的全长HCV复制子RNA具有生产病毒颗粒的能力。
在本发明的全长HCV复制子RNA的优选实施方案中，丙型肝炎病毒优选为但不限于基因型2a丙型肝炎病毒。在本发明中，“基因型2a丙型肝炎病毒”或“基因型2a HCV”指按照Simmonds等(参见Simmonds，P.等，Hepatology，(1994)10，1321-1324页)的国际分类法鉴别为基因型2a的肝炎病毒。在本发明中，“基因型2a丙型肝炎病毒”或“基因型2a HCV”不仅包括具有天然HCV基因组RNA的病毒，而且包括具有其中天然HCV基因组序列已被人工修饰的基因组RNA的病毒。基因型2a HCV的具体实例包括JFH-1毒株(参见日本特许公开(公告)第2002-171978号)。
在本说明书中，“丙型肝炎病毒基因组RNA”指含丙型肝炎病毒单链正义RNA基因组整个区域的核苷酸序列的RNA。基因型2a丙型肝炎病毒的基因组RNA优选为但不限于含SEQ ID NO：12所示核苷酸序列的RNA。
本发明全长HCV复制子的一个实施方案是包含核苷酸序列的复制子RNA，该核苷酸序列包含5′非翻译区、核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列、NS5B蛋白编码序列和3′非翻译区、至少一种选择标记基因或报告基因以及至少一种IRES序列。
本发明的全长HCV复制子RNA不限于但优选包含：在5′-3′方向为以下顺序的5′非翻译区、至少一种选择标记基因或报告基因、至少一种IRES序列、核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列、NS5B蛋白编码序列和3′非翻译区。
在本申请的说明书中，“5′非翻译区”(5′NTR或5′UTR)、“核心蛋白编码序列”(核心区或C区)、“E1蛋白编码序列”(E1区)、“E2蛋白编码序列”(E2区)、“NS2蛋白编码序列”(NS2区)、“NS3蛋白编码序列”(NS3区)、“NS4A蛋白编码序列”(NS4A区)、“NS4B蛋白编码序列”(NS4B区)、“NS5A蛋白编码序列”(NS5A区)、“NS5B蛋白编码序列”(NS5B区)和“3′非翻译区”(3′NTR或3′UTR)，以及其它特定区域或位点，以包含基因型2a HCV病毒JFH-1毒株(日本特许公开(公报)第2002-171978号)基因组完整区域的全长基因组RNA(SEQ ID NO：12)为基准定义。
另外，本发明丙型肝炎病毒(HCV)基因组中的部分区域或位点可以SEQ ID NO：1-11所示序列为基准定义，SEQ ID NO：1-11是JFH-1毒株基因组RNA(SEQ ID NO：12)的部分核苷酸序列。JFH-1毒株全长基因组RNA(源自JFH-1克隆；SEQ ID NO：12)的“5′非翻译区”包含SEQ ID NO：1所示核苷酸序列。“核心蛋白编码序列”包含SEQ IDNO：2所示核苷酸序列。“E1蛋白编码序列”包含SEQ ID NO：3所示核苷酸序列。“E2蛋白编码序列”包含SEQ ID NO：4所示核苷酸序列。“NS2蛋白编码序列”包含SEQ ID NO：5所示核苷酸序列。“NS3蛋白编码序列”包含SEQ ID NO：6所示核苷酸序列。“NS4A蛋白编码序列”包含SEQ ID NO：7所示核苷酸序列。“NS4B蛋白编码序列”包含SEQ ID NO：8所示核苷酸序列。“NS5A蛋白编码序列”包含SEQ IDNO：9所示核苷酸序列。“NS5B蛋白编码序列”包含SEQ ID NO：10所示核苷酸序列。“3′非翻译区”包含SEQ ID NO：11所示核苷酸序列。
例如，HCV源RNA序列中的区域或位点可由SEQ ID NO：1-12的核苷酸序列中的核苷酸编号限定，核苷酸编号通过比对RNA序列和SEQ ID NO：1-12所示核苷酸序列来确定。在比对中，可存在空位、添加、缺失、置换等。
在本发明的更优选实施方案中，包含在全长HCV复制子RNA中的5′非翻译区、核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列、NS5B蛋白编码序列和3′非翻译区优选分别包含SEQ ID NO：1-11所示核苷酸序列。
本发明的全长HCV复制子RNA的优选实施方案为含SEQ ID NO：1-11所示核苷酸序列、至少一种标记基因和/或报告基因和至少一种IRES序列的复制子RNA。
本发明中的“选择标记基因”指赋予细胞选择能力的基因，使得只有表达该基因的细胞才可被选择。选择标记基因的一般性实例包括抗生素抗性基因。本发明中优选的选择标记基因实例包括新霉素抗性基因、胸苷激酶基因、卡那霉素抗性基因、抗硫胺素抗性基因、腺苷酰转移酶基因、Zeocin抗性基因和嘌呤霉素抗性基因。优选新霉素抗性基因和胸苷激酶基因，更优选新霉素抗性基因。但是，本发明中的选择标记基因不限于这些基因。
此外，在本发明中，“报告基因”指其编码基因产物可作为该基因表达指示剂的标记基因。报告基因的一般性实例包括催化光发射反应或显色反应的酶的结构基因。本发明中的报告基因的优选实例包括源自转座子Tn9的氯霉素乙酰转移酶基因、大肠杆菌源的β-葡糖醛酸酶基因或β-半乳糖苷酶基因、荧光素酶基因、绿色荧光蛋白基因、得自水母的水母发光蛋白基因和分泌型胎盘碱性磷酸酶(SEAP)基因。但是，本发明中的报告基因不限于这些基因。
在全长复制子RNA中可包含以上选择标记基因和报告基因中的仅一种，或者可同时包含二者。一种或多种选择标记基因或报告基因可存在于一个全长HCV复制子RNA中。
在本发明中，“IRES序列”指内部核糖体进入位点，其允许核糖体通过结合RNA内部启动翻译。本发明中的IRES序列的优选实例包括但不限于EMCV IRES(脑心肌炎病毒的内部核糖体进入位点)、FMDV IRES和HCV IRES。更优选EMCV IRES和HCV IRES，EMCVIRES是最优选的序列。
本发明的全长HCV复制子RNA的再更优选实施方案是含SEQID NO：13所示核苷酸序列的RNA。此外，含在SEQ ID NO：13所示核苷酸序列中通过缺失、置换或添加1-100个核苷酸(优选1-30个，更优选1-10个，再更优选1-6个，最优选1至若干(2-5)个核苷酸)而衍生自SEQ ID NO：13所示核苷酸序列的核苷酸序列、具有自主复制能力和病毒颗粒生产能力的复制子RNA是全长HCV复制子RNA的优选实施方案，也包括在本发明范围中。
本发明的全长HCV复制子RNA还可包含编码任选外源基因的RNA，其中所述外源基因要在全长复制子RNA已导入其中的细胞中表达。编码外源基因的RNA还可连接在5′非翻译区下游，或连接在选择标记基因或报告基因的上游或下游，或连接在3′非翻译区上游。编码外源基因的RNA可插入在核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列和NS5B蛋白编码序列之间的任意位点。
当含外源基因编码RNA的全长HCV复制子RNA在已导入其的细胞中翻译时，其可表达外源基因编码的基因产物。因此，含外源基因编码RNA的全长HCV复制子RNA还可适用于在细胞中生产外源基因的基因产物。
本发明的全长HCV复制子RNA可进一步包含核酶。核酶连接在选择标记基因和/或报告基因下游，使得可通过核酶的自切割活性将选择标记基因和/或报告基因由IRES序列、核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列和NS5B蛋白编码序列以及3′非翻译区上切下。
在本发明的全长HCV复制子RNA中，上述选择标记基因和/或报告基因、病毒蛋白编码序列和外源基因、核酶等被连接在一起，使得它们由全长HCV复制子RNA以正确的读框翻译。在这些序列中，全长复制子RNA编码的蛋白优选通过蛋白酶切割位点等互相连接，使得蛋白被翻译或表达为多蛋白，接着由蛋白酶切割成各个蛋白。
本发明还涉及编码本发明复制子RNA的DNA载体，优选表达载体。
在本发明中，RNA的“自主复制能力”指RNA在导入细胞中时能够自主生长。RNA的自主复制能力可通过以下方法证实，但该方法不是限制性的。用目的RNA转染Huh7细胞并培养。由所获得的培养细胞提取RNA，并使用能够特异性检测导入RNA的探针对RNA进行RNA印迹杂交。目的RNA检测证实自主复制。在本说明书实施例中有关集落形成能力实验、HCV蛋白表达验证、复制子RNA检测等的描述中阐述了证实自主复制能力的具体方法的实例。
此外，在本发明中，RNA的“病毒颗粒生产能力”指当RNA导入到细胞(例如培养细胞，如Huh7细胞)中时在细胞中产生病毒颗粒。病毒颗粒生产能力可通过例如将使用RNA特异性引物的RT-PCR法用于检测导入该RNA的细胞的培养上清液来证实。其还可通过对培养上清液进行蔗糖密度梯度法以分离病毒颗粒和通过检测HCV蛋白来证实。在本说明书实施例中有关集落形成能力实验、HCV蛋白表达验证、复制子RNA检测等的描述中阐述了具体方法的实例。
2.全长HCV复制子RNA的制备
可使用本领域技术人员已知的基因工程技术制备本发明的全长HCV复制子RNA。可使用例如但不限于JFH-1毒株作为基因型2a丙型肝炎病毒，利用以下的方法制备全长HCV复制子RNA。
首先，常规地再构建对应于JFH-1毒株基因组RNA完整区域(SEQ ID NO：12；该序列在国际DNA数据库中在检索号AB047639下)的DNA，并将其插入到RNA启动子下游，以便制备DNA克隆。本文使用的“对应于RNA的DNA”指具有通过用T(胸腺嘧啶)取代U(尿嘧啶)而来源于RNA核苷酸序列的核苷酸序列的DNA。以上RNA启动子优选包含在质粒克隆中。优选RNA启动子的实例不限于但包括T7RNA启动子、SP6 RNA启动子和SP3 RNA启动子，特别优选T7 RNA启动子。
接着，将选择标记基因和/或报告基因以及IRES序列编码DNA插入到上述DNA克隆中。优选将选择标记基因和/或报告基因插入到5′非翻译区下游，将IRES序列插入到更下游。
随后，使用如上制备的DNA克隆作为模板，使用RNA聚合酶合成RNA。RNA合成可通过标准方法由5′非翻译区开始。当DNA克隆为质粒克隆时，可用使用限制酶由质粒克隆切下的DNA片段作为模板合成RNA。另外，优选要合成的RNA的3′端与病毒基因组RNA的3′非翻译区的末端具有相同序列，未添加或缺失其它序列。由此合成的RNA是本发明的全长HCV复制子RNA。
3.HCV颗粒的制备
可通过将如上所述制备的全长HCV复制子RNA导入细胞中，获得可复制全长HCV复制子RNA(优选持续复制(即具有复制子RNA复制能力))的重组细胞。在本说明书中，复制全长HCV复制子RNA的重组细胞被称为“全长HCV复制子RNA复制型细胞”。
全长HCV复制子RNA复制型细胞可生产病毒颗粒。生产的病毒颗粒在由HCV病毒蛋白组成的外壳中含全长HCV复制子RNA。因此，本发明的全长HCV复制子RNA复制型细胞生产的病毒颗粒是HCV颗粒。也就是说，在本发明中，可通过培养全长HCV复制子RNA复制型细胞，在细胞培养系统中制备HCV颗粒。优选地，可通过培养全长HCV复制子RNA复制型细胞，并收集在培养物(优选培养上清液)中产生的病毒颗粒，获得HCV颗粒。
或者，可利用通过将全长HCV基因组RNA导入到细胞中获得的重组细胞生产HCV颗粒。全长HCV基因组RNA在其中导入本发明的全长HCV基因组RNA(优选来源于JFH-1克隆的全长HCV基因组RNA，更优选具有SEQ ID NO：12所示核苷酸序列的RNA)的细胞中高效复制。在本说明书中，复制全长HCV基因组RNA的细胞称为“全长HCV基因组RNA复制型细胞”。全长HCV基因组RNA复制型细胞可生产病毒颗粒。全长HCV基因组RNA复制型细胞生产的病毒颗粒在由HCV病毒蛋白组成的壳中包含全长HCV基因组RNA。因此，由其中导入本发明的全长HCV基因组RNA的细胞生产的病毒颗粒是HCV颗粒。不限于但优选地可通过培养其中导入来源于JFH-1克隆的全长HCV基因组RNA(例如具有SEQ ID NO：12所示核苷酸序列的RNA)的细胞，在细胞培养系统中制备HCV颗粒。例如，可通过培养其中导入全长HCV基因组RNA(例如具有SEQ IDNO：12所示核苷酸序列的RNA)的细胞，并收集在培养物(优选培养上清液)中产生的病毒颗粒，获得HCV颗粒。
对于要导入上述全长HCV复制子RNA或全长HCV基因组RNA的细胞，可使用任何细胞，只要其可被传代培养。这样的细胞优选为真核细胞，更优选为人细胞，再更优选为人肝源细胞、人子宫颈源细胞或人胎肾源细胞。优选可使用增殖性细胞，包括癌细胞系、干细胞系等细胞，更优选使用Huh7细胞、HepG2细胞、IMY-N9细胞、HeLa细胞和293细胞等。对于这些细胞，可使用市售的细胞，这些细胞可得自细胞保藏机构，或者也可使用由任意细胞(例如癌细胞或干细胞)建立的细胞系。
可使用本领域技术人员已知的任何技术，实现将全长HCV复制子RNA或全长HCV基因组RNA导入到细胞中。这样的导入方法的实例包括电穿孔、粒子枪法、脂转染法、磷酸钙法、微注射法、DEAESepharose法等。特别优选使用电穿孔的方法。
全长HCV复制子RNA或全长HCV基因组RNA可单独导入，或者可在与其它核酸混合后导入。为改变全长HCV复制子RNA或全长HCV基因组RNA的量，同时将要导入的RNA量保持在一定水平，将需要量的要导入的全长HCV复制子RNA或全长HCV基因组RNA与由要导入RNA的细胞提取的总细胞RNA混合，以使总RNA量达到一定水平，然后混合物用于导入到细胞中。可按照使用的导入方法确定用于导入到细胞中的复制子RNA的量，其优选为1pg-100μg，更优选为10pg-10μg。
可利用在全长HCV复制子RNA中的选择标记基因或报告基因的表达，选择全长HCV复制子RNA复制型细胞。具体地说，例如，可在培养基中培养这种进行了全长HCV复制子RNA细胞导入处理的细胞，其中细胞可由于表达选择标记基因而被选择。或者，在进行全长HCV复制子RNA细胞导入处理的细胞培养后，可检测报告基因(例如荧光蛋白)的表达。
作为实例，当全长HCV复制子RNA包含新霉素抗性基因作为选择标记基因时，将用全长HCV复制子RNA进行电穿孔法的细胞接种入培养皿中。在培养12-72小时(优选16-48小时)后，以0.05mg/ml-3.0mg/ml的浓度将G418(新霉素)加入到培养皿中。优选在接种后将细胞连续培养10-40天，更优选14-28天，同时一周更换培养基两次，可通过用结晶紫染色活细胞，选择复制导入的全长HCV复制子RNA的细胞的集落。
可通过标准方法由形成的集落克隆细胞。由此获得的复制全长HCV复制子RNA的细胞克隆在本说明书中被称为“全长HCV复制子RNA复制型细胞克隆”。本发明的全长HCV复制子RNA复制型细胞包括全长HCV复制子RNA复制型细胞克隆。
对于全长HCV复制子RNA复制型细胞，可通过检测复制的全长HCV复制子RNA，确认全长HCV复制子RNA的选择标记基因或报告基因没有整合在宿主基因组DNA中，并进一步检测HCV蛋白，证实全长HCV复制子RNA在细胞或细胞克隆中的实际复制。
可按照本领域技术人员已知的任意RNA检测方法，检测已复制的全长HCV复制子RNA。例如，可通过使用全长HCV复制子RNA特异性DNA片段作为探针的RNA杂交法，检测由细胞提取的总RNA中的全长HCV复制子RNA。
而且，可通过例如但不限于对由细胞提取出的基因组DNA进行PCR，以扩增至少一部分选择标记基因或报告基因，然后确认没有扩增产物，来证实全长HCV复制子RNA中的选择标记基因或报告基因没有整合在宿主基因组DNA中。因为一般认为在证实扩增产物的细胞中选择标记基因或报告基因可能已整合入宿主基因组中，所以有可能全长HCV复制子RNA自身不复制。在此情况下，可如下所述通过检测HCV蛋白进一步证实全长HCV复制子RNA的复制。
可通过例如使抗HCV蛋白(其由导入的全长HCV复制子RNA表达)的抗体与提取的细胞蛋白反应，检测HCV蛋白。该方法可通过本领域技术人员已知的任意蛋白检测方法进行。具体地说，例如，可通过将由细胞提取出的蛋白样品印迹在硝酸纤维素膜上，使抗HCV蛋白抗体(例如抗-NS3特异性抗体或由丙型肝炎患者收集的抗血清)与硝酸纤维素膜反应，并检测抗HCV蛋白抗体，检测HCV蛋白。如果在提取的细胞蛋白中检测出HCV蛋白，则可推断该细胞复制全长HCV复制子RNA，并表达HCV蛋白。
可通过本领域技术人员已知的任意病毒检测方法，证实全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞的病毒颗粒生产能力。例如，通过蔗糖密度梯度分级分离怀疑生产病毒颗粒的细胞的培养上清液，并测定各个级分的级分密度、HCV核心蛋白浓度以及全长HCV复制子RNA或全长HCV基因组RNA的量。结果，如果核心蛋白的峰与全长HCV复制子RNA或全长HCV基因组RNA的峰一致，显示检测峰的级分的密度(例如1.18-1.20mg)小于分级分离25％NP40(聚氧乙烯(9)辛基苯基醚)处理的培养上清液所获得的等同级分的密度，则可认为细胞具有病毒颗粒生产能力。
例如可使用核心蛋白、E1蛋白或E2蛋白的抗体检测培养上清液中释放的HCV病毒颗粒。另外，可通过使用特异性引物的RT-PCR法扩增和检测培养上清液中的全长HCV复制子RNA，间接检测HCV病毒颗粒的存在。
4.用本发明的HCV颗粒感染另一细胞
本发明的HCV病毒颗粒具有感染细胞(优选HCV允许细胞)的能力。本发明还涉及生产丙型肝炎病毒感染细胞的方法，包括培养全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞，并用在由此获得的培养物(优选培养上清液)中的病毒颗粒感染另一细胞(优选HCV允许细胞)。在本发明中，HCV允许细胞指对HCV敏感的细胞，优选但不限于肝细胞或淋巴系细胞。具体地说，肝细胞包括原代肝细胞、Huh7细胞、HepG2细胞、IMY-N9细胞、HeLa细胞、203细胞等。淋巴系细胞包括但不限于Molt4细胞、HPB-Ma细胞、Daudi细胞等。
当用本发明的全长HCV复制子RNA复制型细胞生产的HCV颗粒感染细胞(例如HCV允许细胞)时，全长HCV复制子RNA在感染细胞中复制，病毒颗粒也在感染细胞中形成。因为用在全长HCV复制子RNA复制型细胞中产生的病毒颗粒感染的细胞表达选择标记基因和/或报告基因，所以可利用表达选择和/或检测感染细胞。通过用在本发明的全长HCV复制子RNA复制型细胞中产生的病毒颗粒感染细胞，全长HCV复制子RNA在细胞中复制，可进一步生产病毒颗粒。
更进一步，通过用在本发明的全长HCV基因组RNA复制型细胞中产生的HCV颗粒感染细胞(例如HCV允许细胞)，全长HCV基因组RNA在感染细胞中复制，病毒颗粒也在感染细胞中形成。通过用在本发明的全长HCV基因组RNA复制型细胞中产生的病毒颗粒感染细胞，全长HCV基因组RNA在细胞中复制，而且可生产病毒颗粒。
在全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞中产生的HCV病毒颗粒可感染HCV允许动物，例如黑猩猩等，并在其中诱发由HCV引起的肝炎。
5.本发明的其它实施方案
全长HCV复制子RNA在本发明的全长HCV复制子RNA复制型细胞中高效复制。另外，全长HCV基因组RNA在本发明的全长HCV基因组RNA复制型细胞中高效复制。因此，可使用本发明的全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞高效生产全长HCV复制子RNA或全长HCV基因组RNA。
在本发明中，可通过培养全长HCV复制子RNA复制型细胞，由培养物(培养细胞和/或培养基)中提取RNA，对RNA进行电泳法，并分离和纯化全长HCV复制子RNA，生产全长HCV复制子RNA。还可通过相似的方法，使用全长HCV基因组RNA复制型细胞生产全长HCV基因组RNA。通过这样的方法生产的RNA包含丙型肝炎病毒的全长基因组序列。在此情况下，可通过选择标记基因和/或报告基因和IRES序列中断丙型肝炎病毒的全长基因组序列。借助于包含所提供的丙型肝炎病毒全长基因组序列的RNA的生产方法，使得有可能更详细地分析丙型肝炎病毒基因组。
再者，本发明的全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞可适用于生产HCV蛋白。HCV蛋白可利用本领域技术人员已知的任何方法生产。例如，可通过将全长HCV复制子RNA或全长HCV基因组RNA导入到细胞中，培养重组细胞，并利用已知方法由如此获得的培养物(培养细胞和/或培养基)中收集蛋白，生产HCV蛋白。
再者，本发明的HCV病毒颗粒可具有嗜肝性。因此，可使用本发明的全长HCV复制子RNA生产嗜肝病毒载体。该病毒载体适用于基因治疗。在本发明中，通过将外源基因编码RNA整合入全长HCV复制子RNA或全长HCV基因组RNA中并将整合的RNA导入细胞中表达，外源基因可被导入到细胞中，并在细胞中复制。此外，通过制备其中全长HCV复制子RNA或全长HCV基因组RNA的E1蛋白编码序列和/或E2蛋白编码序列被其它生物物种来源病毒的外壳蛋白编码序列置换的RNA，使该RNA有可能感染不同的生物物种。还是在此情况下，外源基因整合入全长HCV复制子RNA或全长HCV基因组RNA中，这可用作在肝细胞中表达外源基因的嗜肝病毒载体。
本发明还涉及生产携带外源基因的病毒载体的方法，包括将外源基因编码RNA插入到含SEQ ID NO：12所示核苷酸序列的RNA中，将其导入到细胞中，并培养细胞，以生产病毒颗粒。
本发明提供含本发明的HCV颗粒或其部分的丙型肝炎疫苗，生产含本发明的HCV颗粒或其部分的丙型肝炎疫苗的方法。
具体地说，如上制备的HCV颗粒可直接用作疫苗，或者可在利用本领域已知方法减毒或灭活后使用。例如，可通过使用柱层析、过滤、离心等纯化HCV颗粒，获得HCV疫苗母液。可由此HCV疫苗母液制备减毒活HCV疫苗或灭活HCV疫苗。可通过例如将灭活剂(如福尔马林、β-丙内酯、戊二醛等)加入到病毒悬浮液中并混合，使其与病毒反应(Appaiahgari等，Vaccine，(2004)22(27-28)，3669-3675页)，进行病毒灭活。
为生产本发明的疫苗，有可能使用其中通过使用公开已知的技术引入突变减弱或消除病原性的HCV复制子RNA。
本发明的疫苗制备成溶液或悬浮液给予。还有可能制备为适于溶解或悬浮在液体中的固体物质形式。制品可在脂质体中乳化或囊化。活性免疫原性组分如HCV颗粒经常与药物可接受的并适于活性成分的赋形剂混合。合适的赋形剂包括例如水、生理盐水、葡萄糖、甘油、乙醇及其混合物。此外，如果有需要，疫苗可包含少量的助剂(例如增湿剂或乳化剂)、pH缓冲剂和/或用于增强疫苗效力的佐剂。有效佐剂的实例包括但不限于以下物质：氢氧化铝、N-乙酰-胞壁酰-L-苏氨酰-D-异谷氨酰胺(thr-MDP)、N-乙酰-正-胞壁酰-L-丙氨酰-D-异谷氨酰胺(CGP 11637，nor-MDP)、N-乙酰胞壁酰-L-丙氨酰-D-异谷氨酰胺-L-丙氨酸-2-(1′-2′-二棕榈酰-sn-甘油-3-羟基磷酰氧基)-乙胺(CGP19835A，称为MTP-PE)和RIBI。RIBI在2％鲨烯/Tween 80乳浊液中包含三种提取自细菌的组分，即单磷酰脂质A、二分枝菌酸海藻糖和细胞壁骨架(HPL+TDM+CWS)。可通过检测抗免疫原性HCV颗粒的抗体的量测定佐剂效力，其中所述抗体通过给予由HCV颗粒组成的疫苗产生。
本发明的疫苗一般胃肠外给予，例如通过注射，如皮下或肌内注射。适于其它给予途径的其它剂型包括栓剂，在某些情况下包括口服制剂。
如果有需要，可将一种或多种具有佐剂活性的上述化合物加入到HCV疫苗中。佐剂为此免疫系统的非特异性刺激因子，在宿主中增强对HCV疫苗的免疫应答。该技术领域已知佐剂的具体实例包括完全弗氏佐剂、不完全弗氏佐剂、维生素E、非离子型嵌段共聚物、胞壁酰二肽、皂苷、矿物油、植物油和Carbopol。特别适用于粘膜的佐剂包括例如大肠杆菌不耐热毒素(LT)和霍乱毒素(CT)。其它合适的佐剂包括例如氢氧化铝、磷酸铝或氧化铝、油乳浊液(例如Bayol(注册商标)或Marcol 52(注册商标))、皂苷或维生素E增溶物。在优选的实施方案中，本发明的疫苗包含佐剂。
例如，对于皮下、皮内、肌内和静脉内给予的注射，本发明的HCV疫苗中可包含的药物可接受的载体和稀释剂的具体实例包括稳定剂、糖类(例如山梨醇、甘露醇、淀粉、蔗糖、葡萄糖、葡聚糖)、蛋白(例如白蛋白或酪蛋白)、含蛋白的物质(例如牛血清白蛋白或脱脂奶)和缓冲液(例如磷酸盐缓冲液)。
用于栓剂的常规粘合剂和载体包括例如聚亚烷基二醇或甘油三酯。栓剂可由含0.5％-50％(优选1％-20％)活性成分的混合物配制。口服制剂可包含通常使用的赋形剂。这样的赋形剂包括例如药用级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。
本发明的疫苗可以溶液、悬浮液、片剂、丸剂、胶囊、延迟释放制剂或粉末剂型生产，并可包含10-95％(优选25-70％)的活性成分(病毒颗粒或其部分)。
本发明的疫苗通过适于剂型的方法并以预防和/或治疗有效量给予。剂量为0.01μg-100,000μg，这取决于要治疗的患者、患者免疫系统中的抗体形成能力和需要的保护水平。其还依赖于给予途径，例如口服、皮下、皮内、肌内、静脉内等。
该疫苗可通过单次给予程序给予，或优选通过复合给予程序给予。在复合给予程序中，在给予开始时进行1-10次单独给予，接着以维持和/或增强免疫反应所需要的间隔给予。例如，可在1-4个月后进行另一类给予，作为第二次给予。必要时，可在几个月后持续给予。给予方案至少部分地按照个体患者的需要来确定，取决于主治医师的判断。
此外，含免疫原性HCV颗粒的疫苗可与其它免疫控制剂(例如免疫球蛋白)共同给予。
HCV颗粒疫苗可通过给予健康个体，以诱发对HCV的免疫应答，用于预防性地对抗可能的新HCV感染。HCV颗粒疫苗还可通过给予感染HCV的患者并在体内诱发针对HCV的强免疫应答，用作治疗性疫苗，以清除HCV。
全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞或用在这些细胞中产生的病毒颗粒感染的丙型肝炎病毒感染细胞，可用作测试系统，用于筛选促进或抑制例如丙型肝炎病毒复制、病毒颗粒重构和病毒颗粒释放的物质(抗丙型肝炎病毒的物质)。具体地说，例如，可通过确定测试物是否促进或抑制全长HCV复制子RNA或全长HCV基因组RNA的复制或者病毒颗粒的形成或释放，在测试物存在下培养这些细胞，并检测所获培养物中的全长HCV复制子RNA或全长HCV基因组RNA或病毒颗粒，筛选促进或抑制丙型肝炎病毒生长的物质。在此情况下，可通过测定由上述细胞提取的RNA制品中的全长HCV复制子RNA或全长HCV基因组RNA的量、比率或存在情况，对培养物中的全长HCV复制子RNA或全长HCV基因组RNA进行检测。可通过检测培养上清液中HCV蛋白的量、比率或存在情况，对培养物(主要是培养上清液)中的病毒颗粒进行检测。
而且，可通过检测该培养物中的病毒颗粒，研究由HCV感染患者的血清纯化的免疫球蛋白是否可预防本发明HCV颗粒的感染。在此测试中，可使用已用本发明的HCV病毒颗粒免疫的小鼠、大鼠、兔等的血清。可使用采用部分HCV蛋白、HCV基因等进行的免疫。可以相似的方式对其它感染预防性物质进行该测试。
产生的抗本发明HCV病毒颗粒的本发明抗体包括多克隆抗体和单克隆抗体。当优选多克隆抗体时，第一步用本发明的HCV颗粒免疫选定的哺乳动物(例如小鼠、兔、山羊、绵羊、马等)。由被免疫动物收集血清，并用已知方法加工。如果含针对HCV表位的多克隆抗体的血清包含针对其它抗原的抗体，则可通过免疫亲和层析纯化这些血清。生产多克隆抗血清的方法及其治疗方法是本领域已知的。可由已感染HCV的哺乳动物分离多克隆抗体。
本领域技术人员可容易地生产针对HCV表位的单克隆抗体。已知的常用方法是生产产生单克隆抗体的杂交瘤。例如，可使用在Current Protocols in Immunology(John Wiley & Sons，Inc.)中描述的方法。
可通过细胞融合或其它方法，例如用肿瘤基因DNA直接转化B淋巴细胞或用EB病毒转导，生产产生单克隆抗体的细胞系。
通过这些方法获得的单克隆抗体和多克隆抗体用于诊断、治疗和预防HCV。
使用本发明的HCV颗粒生产的抗体可与药物可接受的增溶剂、添加剂、稳定剂、缓冲剂等一起给予。可选择任何给予途径，但优选皮下、皮内和肌内给予，更优选静脉内给予。
在本发明的全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞中产生的HCV颗粒和HCV允许细胞可作为测试系统，用于筛选可刺激或抑制HCV与细胞结合的物质。具体地说，例如，可通过在测试物存在下培养本发明的全长HCV复制子RNA复制型细胞中产生的HCV颗粒和HCV允许细胞，检测所获培养物中的全长HCV复制子RNA或病毒颗粒，并确定测试物是否促进或抑制复制子RNA复制或病毒颗粒形成，筛选可促进或抑制丙型肝炎病毒生长的物质。
可按照上述技术或以下实施例对全长HCV复制子RNA或全长HCV基因组RNA进行这样的检测。上述测试系统可用于生产和评价丙型肝炎病毒感染的预防剂、治疗剂或诊断剂。
具体地说，上述本发明测试系统的应用实例包括以下几点：
(1)筛选抑制HCV生长和感染的物质
抑制HCV生长和感染的物质包括例如直接或间接影响HCV生长和感染的有机化合物、通过与HCV基因组或其互补链中的靶序列杂交而直接或间接影响HCV生长或HCV蛋白翻译的反义寡核苷酸等。
(2)评价在细胞培养物中具有抗病毒活性的各种物质
前述各种物质包括通过合理药物设计或高通量筛选获得的物质(例如纯化和分离的酶)。
(3)鉴别治疗HCV感染患者的新靶标
例如，本发明的全长HCV复制子RNA复制型细胞或全长HCV基因组RNA复制型细胞可用于鉴别可在HCV生长中起重要作用的宿主细胞蛋白。
(4)评价HCV对药物等的抗性获得能力，并鉴别与抗性相关的突变
(5)生产病毒蛋白作为抗原，用于开发、生产和评价丙型肝炎病毒感染的诊断剂和治疗剂
(6)生产病毒蛋白作为抗原，用于开发、生产和评价用于丙型肝炎病毒感染的疫苗和生产减毒HCV
(7)生产用于诊断和治疗丙型肝炎病毒感染的单克隆或多克隆抗体。
基于以下的实施例和附图将更具体地描述本发明。但本发明的技术范围不限于这些实施例。
实施例1：得自全长HCV基因组RNA的全长HCV复制子RNA的制备
(A)构建表达载体
构建质粒DNA，其中DNA(JFH-1克隆)插入到pUC19质粒中T7RNA启动子序列的下游，此DNA(JFH-1克隆)含已由暴发性肝衰竭患者中分离出的丙型肝炎病毒JFH-1毒株(基因型2a)的全长基因组cDNA。
具体地说，将通过扩增JFH-1毒株的病毒RNA所获得的RT-PCR片段克隆入pGEM-T EASY载体(Promega)中，以获得质粒pGEM1-258、pGEM44-486、pGEM317-849、pGEM617-1323、pGEM1141-2367、pGEM2285-3509、pGEM3471-4665、pGEM4547-5970、pGEM5883-7003、pGEM6950-8035、pGEM7984-8892、pGEM8680-9283、pGEM9231-9634和pGEM9594-9678(参见非特许文献6)。通过使用PCR法和限制酶，将包含在这些质粒中的病毒基因组RNA来源的cDNA连接在一起，以克隆全长病毒基因组cDNA。将T7R RNA启动子序列插入到全长病毒基因组cDNA的上游。在下文中，以此方式构建的质粒DNA称为pJFH1(图1的上部)。上述JFH-1克隆的制备已描述于特许文献1和非特许文献3。此外，JFH-1克隆的全长cDNA的核苷酸序列以检索号AB047639登记在国际DNA数据库(DDBJ/EMBL/GenBank)中。
接着，通过将EMCV-IRES(脑心肌炎病毒的内部核糖体进入位点)和新霉素抗性基因(neo；也称为新霉素磷酸转移酶基因)插入到pJFH1质粒DNA(图1的下部)的5′非翻译区和核心蛋白区之间，构建质粒DNA pFGREP-JFH1。该构建方法与先前的公开内容(非特许文献4)一致。此外，通过导入突变制备突变质粒克隆pJFH1/GND和pFGREP-JFH1/GND，其中所述突变将对应于RNA聚合酶(由pJFH1和pFGREP-JFH1中的NS5B区编码)活性中心的氨基酸基序GDD变为GND。因为由突变克隆pJFH1/GND和pFGREP-JFH1/GND编码的NS5B蛋白活性位点的氨基酸序列被改变，所以不能由突变克隆表达复制复制子RNA所需的活性NS5B蛋白。
此外，通过将荧光素酶基因插入到pFGREP-JFH1的第415-420的MluI位点和第2075-2082的PmeI位点之间，以用荧光素酶基因替代pFGREP-JFH1的新霉素抗性基因，制备pFGREP-JFH1/Luc，作为导入报告基因的表达载体。另外，通过将pFGREP-JFH1/Luc第10933位的G突变为A，制备突变体pFGREP-JFH1/Luc/GND，其中NS5bRNA聚合酶活性中心的GDD基序被变为GND。
通过将绿色荧光蛋白基因插入到pFGREP-JFH1的第415-420的MluI位点和第1142-1149的PmeI位点之间，制备pFGREP-JFH1/EGFP，其中pFGREP-JFH1的新霉素抗性基因被绿色荧光蛋白基因取代。另外，通过将pFGREP-JFH1/EGFP第10000位的G突变为A，制备突变体pFGREP-JFH1/EGFP/GND，其中NS5b RNA聚合酶活性中心的GDD基序被变为GND。
通过将分泌型胎盘碱性磷酸酶基因插入到pFGREP-JFH1的第415-420的MluI位点和第1982-1989的PmeI位点之间，以用分泌型胎盘碱性磷酸酶基因取代pFGREP-JFH1的新霉素抗性基因，制备pFGREP-JFH1/SEAP。另外，通过将pFGREP-JFH1/SEAP第10840位的G突变为A，制备突变体pFGREP-JFH1/SEAP/GND，其中NS5bRNA聚合酶活性中心的GDD基序被变为GND。
(B)全长HCV基因组RNA和全长HCV复制子RNA的制备
如上构建表达载体，用限制酶XbaI消化pJFH1、pJFH1/GND、pFGREP-JFH1和pFGREP-JFH1/GND，以制备模板DNA，用于合成全长HCV基因组RNA和全长HCV复制子RNA。随后，通过于30℃温育30分钟，用20U绿豆核酸酶的50μl反应溶液处理10-20μg各XbaI片段。绿豆核酸酶是这样一种酶：其催化涉及选择性消化双链DNA的单链部分的反应。通常，如果使用以上XbaI片段原样作为模板合成RNA，则合成在3′端具有4个额外碱基CUGA的复制子RNA，其为XbaI识别位点的一部分。因此，在该实施例中，通过用绿豆核酸酶处理XbaI片段，由XbaI片段去除4碱基CUGA。随后，对绿豆核酸酶处理后的含XbaI片段的溶液进行标准蛋白去除处理，以获得无4碱基CUGA的纯化XbaI片段，作为以下使用的模板DNA。
接着，使用T7 RNA聚合酶由该模板体外合成RNA。使用MEGAscript(Ambion Co.)进行RNA合成。按照生产商的说明书使20μl含0.5-1.0μg模板DNA的反应混合物反应。
在RNA合成后，将DNA酶(2U)加入到反应混合物中，于37℃反应15分钟，然后用酸-苯酚处理提取RNA，以去除模板DNA。以此方式由源自pJFH1、pJFH1/GND、pFGREP-JFH1和pFGREP-JFH1/GND的模板DNA合成的RNA分别称为rJFH1、rJFH1/GND、rFGREP-JFH1和rFGREP-JFH1/GND。这些RNA的核苷酸序列分别示于SEQ ID NO：12、13、14和15的rJFH-1、rFGREP-JFH1、rJFH1/GND和rFGREP-JFH1/GND。rJFH1是本发明全长HCV基因组RNA的实例，与JFH-1毒株的全长HCV基因组具有相同的序列结构。rFGREP-JFH1是本发明全长HCV复制子RNA的实例。
随后，通过分别使用如上制备的表达载体pFGREP-JFH1/Luc、pFGREP-JFH1/Luc/GND、pFGREP-JFH1/EGFP、pFGREP-JFH1/EGFP/GND、pFGREP-JFH1/S EAP和pFGREP-JFH1/SEAP/GND作为模板，生产为HCV复制子RNA的rFGR-JFH1/Luc(SEQ ID NO：21)、rFGR-JFH1/Luc/GND((SEQ ID NO：22)、rFGR-JFH1/EGFP(SEQID NO：23)、rFGR-JFH1/EGFP/GND(SEQ ID NO：24)、rFGR-JFH1/SEAP(SEQ ID NO：25)和rFGR-JFH1/SEAP/GND(SEQ ID NO：26)。
实施例2：全长HCV基因组RNA在细胞中的复制和病毒颗粒的产生
(C)全长HCV基因组RNA在细胞中的复制和病毒颗粒的产生
将如上合成的各种量的全长HCV基因组RNA(rJFH1或rJFH1/GND)与由Huh7细胞提取的总RNA混合，使RNA量达到10μg。随后，利用电穿孔法将混合的RNA导入到Huh7细胞中。将进行电穿孔处理的Huh7细胞接种入培养皿中。在温育12、24、48和72小时后，收集细胞，提取RNA，并通过RNA印迹法分析。按照Molecular Cloning，A laboratory Manual，第2版，J.Sambrook，E.F.Fritsch，T.Maniatis，Cold Spring Harbor Laboratory Press(1989)进行RNA印迹分析。具体地说，对由温育后细胞提取的RNA进行变性琼脂糖凝胶电泳，并在电泳后将RNA转移至带正电荷的尼龙膜上。使由pJFH1制备的32P标记DNA或RNA探针与前述转移至膜上的RNA杂交。漂洗膜，并使膜对胶片曝光，以检测对JFH-1克隆的全长HCV基因组RNA特异性的RNA条带。
如图2所示，当rJFH1/GND转染至细胞中时，在转染后4小时导入RNA的条带被证实为弱信号，但信号随着传代次数变得更弱，转染后24小时的条带信号几乎不可检测。相比之下，当转染rJFH1时，导入RNA条带的信号强度首先变弱，和rJFH1/GND转染后4-12小时的情况一样，但在转染后24小时证实了清晰的RNA信号。证实的信号是HCV基因组RNA特异性的。也就是说，一般认为某些导入的全长HCV基因组RNA复制并生长。对于其中为RNA复制酶的NS5B活性基序突变的rJFH1/GND，未观察到复制，这表明NS5B活性对全长HCV基因组RNA复制很重要。此外，对得自丙型肝炎病毒，例如H77毒株(非特许文献7)、J6毒株(非特许文献8)和本发明发明人由慢性肝炎患者分离的JCH1毒株(非特许文献6)的全长基因组RNA(先前已全部被分离)进行相同的实验，但证实这些毒株的全长HCV基因组RNA未复制。
(D)转染细胞培养物的培养基中HCV病毒颗粒的检测
将如上所述的电穿孔处理的Huh7细胞接种在培养皿中，培养12、24、48和72小时，然后检测培养上清液中的HCV核心蛋白。按照Ortho HCV抗原IRMA检验(非特许文献9)进行检测。如图3所示，在用rJFH1转染后48和72小时检测培养上清液中的核心蛋白。为检验此核心蛋白是否作为病毒颗粒被分泌，通过蔗糖密度梯度分级分离rJFH1转染后72小时的培养基。在离心管中，2ml 60％(重量/重量)蔗糖溶液(溶解在50mM Tris pH 7.5/0.1M NaCl/1mM EDTA中)、1ml 50％蔗糖溶液、1ml 40％蔗糖溶液、1ml 30％蔗糖溶液、1mlof 20％蔗糖溶液和1ml 10％蔗糖溶液分层，4ml样品培养上清液覆盖于其上。在Beckman转子SW41 Ti中以400,000RPM于4℃离心16小时。在离心后，由管底部收集各0.5ml的级分。测定各个级分中的密度、HCV核心蛋白浓度和全长HCV基因组RNA的量。按照Takeuchi T，Katsume A，Tanaka T，Abe A，Inoue K，Tsukiyama-KoharaK，Kawaguchi R，Tanaka S，Kohara M，“Real-Time detection system forquantification of Hepatitis C virus genome”，Gastroenterology 116：636-642(1999)，通过检测全长HCV基因组RNA的5′非翻译区的RNA，用定量RT-PCR法进行全长HCV基因组RNA的检测。具体地说，使用合成引物R6-130-S17：5′-CGGGAGAGCCATAGTGG-3′(SEQ IDNO：16)、R6-290-R19：5′-AGTACCACAAGGCCTTTCG-3′(SEQ ID NO：17)和TaqMan探针：R6-148-S21FT，5′-CTGCGGAACCGGTGAGTACAC-3′(SEQ ID NO：18)以及EZ rTthRNA PCR试剂盒，PCR扩增由细胞提取的RNA中包含的全长HCV基因组RNA，然后通过ABI Prism 7700序列检测系统检测。
如图4所示，在级分11中核心蛋白的峰与全长HCV基因组RNA的峰一致。该级分的密度约为1.18mg/ml，其表明比重比迄今为止所报道的核心蛋白和核酸缀合物的比重低。此外，当在用0.25％NP40处理培养上清液后进行相似的分级分离时，核心蛋白和全长HCV基因组RNA的峰迁移至约1.28mg/ml的比重。也就是说，一般认为NP40处理剥落了病毒颗粒的表面膜(其含脂质，于是具有较低比重)，产生仅由核酸和核心蛋白组成的核心颗粒，因此比重增加。以上结果表明，通过将rJFH1转染至Huh7细胞中，全长HCV基因组RNA在细胞中复制，结果形成病毒颗粒，并分泌入培养上清液中。
实施例3
(E)全长HCV复制子RNA复制型细胞的制备和细胞克隆的建立
通过如实施例2所述，将实施例1中制备的rFGREP-JFH1和rFGREP-JFH1/GND转染入Huh7细胞，制备全长HCV复制子RNA复制型细胞，然后尝试建立全长HCV复制子RNA复制型细胞克隆。
首先，在分别将rFGREP-JFH1和rFGREP-JFH1/GND转染入Huh7细胞中后，将细胞接种在培养皿中。在培养16-24小时后，加入不同浓度的G418。继续培养，同时每周更换两次培养基。在培养21天后，用结晶紫染色存活细胞。计数染色的集落，计算获得的集落数/用于转染的RNA重量。对于某些培养皿又继续培养，以克隆存活细胞的集落。分别由克隆细胞中提取RNA、基因组DNA和蛋白，然后检测全长HCV复制子RNA，研究新霉素抗性基因在基因组DNA中的整合以及HCV蛋白的表达。这些结果在下文详细给出。
(F)集落形成能力
以上转染的结果表明，对于在1.0mg/ml G418浓度下用rFGREP-JFH1转染的Huh7细胞(图5的左部)，集落形成能力/1μg用于转染的复制子RNA是368CFU(集落形成单位)/μg RNA。相反，对于用rFGREP-JFH1/GND转染的Huh7细胞(图5的右部)，未观测到集落形成。这表明，用rFGREP-JFH1复制子RNA转染的Huh7细胞的集落形成能力依赖于由rFGREP-JFH1表达的NS5B(RNA聚合酶)的活性。也就是说，一般认为在集落形成细胞中，借助于由rFGREP-JFH1表达的NS5B的作用，rFGREP-JFH1复制子RNA自主复制，引起新霉素抗性基因持续表达，因此保持了G418抗性，结果使细胞生长成为可能。
(G)在已建立细胞克隆中的全长HCV复制子RNA的检测
利用酸-苯酚提取法，由全长HCV复制子RNA复制型细胞克隆中提取总RNA，该细胞按照以上章节(E)通过将rFGREP-JFH1转染入Huh7细胞中而建立。随后，利用RNA印迹法检测此总RNA。在该方法中，使用pFGREP-JFH1特异性探针。以相似的方式由未转染的Huh7细胞提取的总RNA(在图6中显示为“Huh7”)，一个除由Huh7细胞提取的总RNA以外还含体外合成的107个拷贝的复制子RNA的样品(在图6中显示为“107”)和一个除由Huh7细胞提取的总RNA以外还含体外合成的108个拷贝的复制子RNA的样品(在图6中显示为“108”)用作对照。在图6中，1-4表示细胞克隆号。
结果，用rFGREP-JFH1特异性探针检测与rFGREP-JFH1具有相似大小的RNA(图6)。由该结果证实转染的rFGREP-JFH1复制子RNA在细胞克隆中复制和生长。还表明细胞克隆中的复制子RNA的量有差异。如图6所示，例如，克隆2中的复制子RNA的量少于其它克隆。
(H)验证新霉素抗性基因有无整合入基因组DNA中
对于按照(E)所获得的细胞克隆1-8(如图7中的FGR-JFH1/2-1至FGR-JFH1/2-8所示)，使用新霉素抗性基因特异性引物(有义引物NEO-S3：5′-AACAAGATGGATTGCACGCA-3′(SEQ ID NO：19)，反义引物NEO-R：5′-CGTCAAGAAGGCGATAGAAG-3′(SEQ ID NO：20))，并使用由每个细胞克隆提取的宿主基因组DNA作为模板，进行PCR扩增，以便证实各个细胞克隆抗G418的抗性并非是由于新霉素抗性基因整合入宿主细胞基因组中。结果，如图7所示，未观测到显示扩增新霉素抗性基因的阳性克隆。
(H)的结果证实，全长HCV复制子RNA在通过本发明的全长HCV复制子RNA转染建立的细胞克隆中复制。
(I)HCV蛋白的检测
通过标准方法由通过rFGREP-JFH1转染建立的细胞克隆提取蛋白，然后通过SDS-PAGE和蛋白质印迹法分析。在此情况下检测的细胞克隆与以上章节(G)中使用的克隆相同。通过将制备的全长HCV基因组RNA瞬时转染入Huh7细胞中获得细胞提取物，将其用作阳性对照(在图8、9和10中显示为JFH-1)。通过转染HCV亚基因组RNA复制子(SGR-JFH1)获得的克隆的细胞提取物用作核心蛋白的阴性对照，并用作NS3和NS5a蛋白的阳性对照(在图8、9和10中显示为SGR-JFH1)。未转染的Huh7细胞的细胞提取物用作所有蛋白的阴性对照(在图8、9和10中显示为Huh7)。将由各个细胞克隆提取的蛋白样品印迹在PVDF膜(Immobilon-P，Millipore)上，然后使用抗核心蛋白特异性抗体和抗NS3特异性抗体(由Dr.Moradpour惠赠；Wolk B等，J.Virology，2000，74：2293-2304)检测由其中的全长HCV复制子RNA编码的核心蛋白和NS3蛋白。如图8和9所示，对于通过rFGREP-JFH1转染建立的细胞克隆1-4，检测各个蛋白中与阳性对照大小相同的蛋白。因为在未转染Huh7细胞中既没有检测到核心蛋白，也没有检测到NS3蛋白，所以证实在细胞克隆1-4中已转染的全长HCV复制子RNA自主复制，表达核心蛋白和NS3蛋白。
此外，对于每个细胞克隆，已如上所述检验NS3蛋白的表达，还使用丙型肝炎患者的血清作为抗体检验由全长HCV复制子RNA表达的NS5A蛋白(图10)。
由上述(H)和(I)的结果证实，在已通过全长HCV复制子RNA转染建立的细胞克隆中，全长HCV复制子RNA复制，病毒蛋白也表达。
(J)全长HCV复制子RNA复制型细胞中的病毒颗粒生产
按照以上章节(E)将rFGREP-JFH1转染入Huh7细胞中，建立全长HCV复制子RNA复制型细胞克隆2和3(FGR-JFH1/2-3)，然后回收其培养上清液。按照与上述(D)相似的方法检测培养上清液中的HCV病毒颗粒。结果示于图11。在图11中，阴影圆代表各个级分的比重(g/ml)。实心圆代表核心蛋白的量(fmol/L)。空心圆代表全长HCV复制子RNA的滴度(×0.1拷贝/mL)。
如图11所示，核心蛋白峰与比重约1.18-1.20mg/ml的级分中的全长HCV复制子RNA的峰一致。还在较轻级分中发现小峰。以上结果表明，全长HCV复制子RNA在用rFGREP-JFH11转染的Huh7细胞中复制，形成病毒颗粒并分泌入其培养上清液中。
实施例4
(K)采用培养上清液中的病毒颗粒的感染实验
通过将在(H)中使用的细胞克隆1-8(即FGR-JFH1/2-1、FGR-JFH1/2-2、FGR-JFH1/2-3、FGR-JFH1/2-4、FGR-JFH1/2-5、FGR-JGF1/2-6、FGR-JFH1/2-7、FGR-JFH1/2-8)的各个培养上清液加入Huh7细胞中，用在培养上清液中的病毒颗粒感染Huh7细胞。在第2天，以0.3mg/ml向感染的Huh7细胞的培养基中加入G418，再培养Huh7细胞21天。在结束培养后，固定细胞，并用结晶紫染色。对于分别用FGR-JFH1/2-3、FGR-JFH1/2-5和FGR-JFH1/2-6的培养上清液感染的细胞，观测到集落形成。另一方面，对于用作对照的亚基因组复制子细胞SGR-JFH1/4-1(描述于非特许文献6)的培养上清液感染的细胞，未观测到集落形成。图12显示了与加入的4ml或8mlFGR-JFH1/2-3或SGR-JFH1/4-1培养上清液培养21天后染色培养皿的照片。在其中接种与4ml或8ml FGR-JFH1/2-3培养上清液混合的细胞的培养皿中，分别发现3个和9个集落。但是，在其中接种与SGR-JFH1/4-1的培养上清液混合的细胞的培养皿中，未观测到集落。
随后，克隆分别使用FGR-JFH1/2-3和FGR-JFH1/2-5的培养上清液以丙型肝炎病毒感染形成的集落。由用FGR-JFH1/2-3的培养上清液感染的培养皿建立3个克隆FGR-JFH1/C2-3-11、FGR-JFH1/C2-3-12和FGR-JFH1/C2-3-13，并由用FGR-JFH1/C2-5的培养上清液感染的培养皿建立2个克隆FGR-JFH1/C2-5-11和FGR-JFH1/C2-5-12。
当用FGR-JFH1/C2-3-11、FGR-JFH1/C2-3-12、FGR-JFH1/C2-3-13、FGR-JFH1/C2-5-11和FGR-JFH1/C2-5-12各个细胞克隆的培养上清液感染Huh7细胞时，在分别用FGR-JFH1/C2-3-12和FGR-JFH1/C2-5-12的培养上清液感染的培养皿中观测到集落形成。由用FGR-JFH1/C2-3-12的培养上清液感染的细胞建立另两个克隆FGR-JFH1/C2-3-12-1和FGR-JFH1/C2-3-12-2。由用FGR-JFH1/C2-5-12的培养上清液感染的细胞建立另两个克隆FGR-JFH1/C2-5-12-1和FGR-JFH1/C2-5-12-2。
已用全长HCV复制子RNA复制型细胞的培养上清液感染的细胞建立细胞，由这些细胞克隆提取RNA、蛋白和基因组DNA。通过使用基因组DNA作为模板的PCR，检验新霉素抗性基因在这些细胞克隆的基因组DNA中的整合，结果全为阴性。而且，通过使用RNA作为模板的定量PCR可检测细胞中复制的全长HCV复制子RNA。再进一步，可检测培养上清液中的核心蛋白。这些结果表明，由本发明的全长HCV复制子RNA复制型细胞生产的含全长HCV复制子RNA的病毒颗粒可感染另一细胞。
工业实用性
按照本发明的方法，可在细胞培养系统中制备HCV病毒颗粒。使用本发明的复制子RNA，可在细胞培养系统中有效生产含全长HCV基因组RNA的RNA。而且，通过使用其中导入本发明的全长HCV复制子RNA或全长HCV基因组RNA的细胞，全长HCV复制子RNA或全长HCV基因组RNA可在细胞培养系统中复制，本发明的HCV病毒颗粒可在细胞培养系统中连续生产。其中导入本发明的全长HCV复制子RNA或全长HCV基因组RNA的细胞还可用作测试系统，用于筛选影响HCV复制过程、病毒颗粒形成过程和病毒颗粒胞外释放过程的各种物质。本发明的全长HCV复制子RNA和全长HCV基因组RNA以及病毒颗粒还用作外源基因的病毒载体。本发明的病毒颗粒或其部分可包含在疫苗中，作为抗丙型肝炎病毒的疫苗抗原。此外，其中本发明的病毒颗粒和其它细胞一起培养的系统可用作测试系统，用于筛选对病毒颗粒感染细胞有影响的各种物质。本发明的全长HCV复制子RNA或全长HCV基因组RNA用作模板，该模板能使HCV全长基因组序列简单再生。
本文提及的所有出版物、特许和特许申请都全文在此引入作为参考。
无内容序列表
SEQ ID NO：1代表来自JFH-1克隆的HCV基因组RNA的5′非翻译区序列。
SEQ ID NO：2代表来自JFH-1克隆的HCV基因组RNA的核心蛋白编码序列。
SEQ ID NO：3代表来自JFH-1克隆的HCV基因组RNA的E1蛋白编码序列。
SEQ ID NO：4代表来自JFH-1克隆的HCV基因组RNA的E2蛋白编码序列。
SEQ ID NO：5代表来自JFH-1克隆的HCV基因组RNA的NS2蛋白编码序列。
SEQ ID NO：6代表来自JFH-1克隆的HCV基因组RNA的NS3蛋白编码序列。
SEQ ID NO：7代表来自JFH-1克隆的HCV基因组RNA的NS4A蛋白编码序列。
SEQ ID NO：8代表来自JFH-1克隆的HCV基因组RNA的NS4B蛋白编码序列。
SEQ ID NO：9代表来自JFH-1克隆的HCV基因组RNA的NS5A蛋白编码序列。
SEQ ID NO：10代表来自JFH-1克隆的HCV基因组RNA的NS5B蛋白编码序列。
SEQ ID NO：11代表来自JFH-1克隆的HCV基因组RNA的3′非翻译区序列。
SEQ ID NO：12代表来自JFH-1克隆的全长HCV基因组RNA序列。
SEQ ID NO：13代表包含来自JFH-1克隆的全长HCV基因组RNA的复制子RNA序列。
SEQ ID NO：14代表来自JFH-1克隆的全长HCV基因组RNA序列，其中氨基酸基序GDD已突变为GND。
SEQ ID NO：15代表包含来自JFH-1克隆的全长HCV基因组RNA的复制子RNA序列，其中氨基酸基序GDD已突变为GND。
SEQ ID NO：16-20代表引物序列。
SEQ ID NO：21代表来源于表达载体pFGREP-JFH1/Luc的复制子RNA的序列。
SEQ ID NO：22代表来源于表达载体pFGREP-JFH1/Luc/GND的复制子RNA的序列。
SEQ ID NO：23代表来源于表达载体pFGREP-JFH1/EGFP的复制子RNA的序列。
SEQ ID NO：24代表来源于表达载体pFGREP-JFH1/EGFP/GND的复制子RNA的序列。
SEQ ID NO：25代表来源于表达载体pFGREP-JFH1/SEAP的复制子RNA的序列。
SEQ ID NO：26代表来源于表达载体pFGREP-JFH1/SEAP/GND的复制子RNA的序列。
                                序列表
<110>东京都医学研究机构
     日本东丽工业株式会社
<120>含人丙型肝炎病毒全长基因组的核酸构建物、核酸构建物转入其中的重组全长病毒基因组复制型细胞和生产人丙型肝炎病毒颗粒的方法
<130>PH-2372-PCT
<140>PCT/JP2005/003232
<141>2005-02-21
<150>JP 2004-045489
<151>2004-02-20
<160>26
<170>PatentIn Ver.2.1
<210>1
<211>340
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的5′非翻译区
<220>
<223>发明人：Wakita，Takaji
     发明人：Kato，Takanobu
     发明人：Date，Tomoko
     发明人：Miyamoto，Michiko
     发明人：Tanabe，Junichi
     发明人：Sone，Saburo
<400>1
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cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc                             340
<210>2
<211>573
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的核心蛋白编码序列
<400>2
augagcacaa auccuaaacc ucaaagaaaa accaaaagaa acaccaaccg ucgcccagaa       60
gacguuaagu ucccgggcgg cggccagauc guuggcggag uauacuuguu gccgcgcagg       120
ggccccaggu ugggugugcg cacgacaagg aaaacuucgg agcgguccca gccacguggg       180
agacgccagc ccauccccaa agaucggcgc uccacuggca aggccugggg aaaaccaggu       240
cgccccuggc cccuauaugg gaaugaggga cucggcuggg caggauggcu ccuguccccc       300
cgaggcucuc gccccuccug gggccccacu gacccccggc auaggucgcg caacgugggu       360
aaagucaucg acacccuaac guguggcuuu gccgaccuca ugggguacau ccccgucgua       420
ggcgccccgc uuaguggcgc cgccagagcu gucgcgcacg gcgugagagu ccuggaggac       480
gggguuaauu augcaacagg gaaccuaccc gguuuccccu uuucuaucuu cuugcuggcc       540
cuguuguccu gcaucaccgu uccggucucu gcu                                    573
<210>3
<211>576
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<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的E1蛋白编码序列
<400>3
gcccagguga agaauaccag uagcagcuac auggugacca augacugcuc caaugacagc       60
aucacuuggc agcucgaggc ugcgguucuc cacguccccg ggugcguccc gugcgagaga       120
guggggaaua cgucacggug uugggugcca gucucgccaa acauggcugu gcggcagccc       180
ggugcccuca cgcagggucu gcggacgcac aucgauaugg uugugauguc cgccaccuuc       240
ugcucugcuc ucuacguggg ggaccucugu ggcgggguga ugcucgcggc ccagguguuc       300
aucgucucgc cgcaguacca cugguuugug caagaaugca auugcuccau cuacccuggc       360
accaucacug gacaccgcau ggcaugggac augaugauga acuggucgcc cacggccacc       420
augauccugg cguacgugau gcgcgucccc gaggucauca uagacaucgu uagcggggcu       480
cacuggggcg ucauguucgg cuuggccuac uucucuaugc agggagcgug ggcgaagguc       540
auugucaucc uucugcuggc cgcuggggug gacgcg                                 576
<210>4
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<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的E2蛋白编码序列
<400>4
ggcaccacca ccguuggagg cgcuguugca cguuccacca acgugauugc cggcguguuc       60
agccauggcc cucagcagaa cauucagcuc auuaacacca acggcaguug gcacaucaac       120
cguacugccu ugaauugcaa ugacuccuug aacaccggcu uucucgcggc cuuguucuac       180
accaaccgcu uuaacucguc agggugucca gggcgccugu ccgccugccg caacaucgag       240
gcuuuccgga uagggugggg cacccuacag uacgaggaua augucaccaa uccagaggau       300
augaggccgu acugcuggca cuacccccca aagccgugug gcguaguccc cgcgaggucu       360
guguguggcc caguguacug uuucaccccc agcccgguag uagugggcac gaccgacaga       420
cguggagugc ccaccuacac auggggagag aaugagacag augucuuccu acugaacagc       480
acccgaccgc cgcagggcuc augguucggc ugcacgugga ugaacuccac ugguuucacc       540
aagacuugug gcgcgccacc uugccgcacc agagcugacu ucaacgccag cacggacuug       600
uugugcccua cggauuguuu uaggaagcau ccugaugcca cuuauauuaa gugugguucu       660
gggcccuggc ucacaccaaa gugccugguc cacuacccuu acagacucug gcauuacccc       720
ugcacaguca auuuuaccau cuucaagaua agaauguaug uagggggggu ugagcacagg       780
cucacggccg caugcaacuu cacucguggg gaucgcugcg acuuggagga cagggacagg       840
agucagcugu cuccucuguu gcacucuacc acggaauggg ccauccugcc cugcaccuac       900
ucagacuuac ccgcuuuguc aacuggucuu cuccaccuuc accagaacau cguggacgua       960
caauacaugu auggccucuc accugcuauc acaaaauacg ucguucgaug ggagugggug       1020
guacucuuau uccugcucuu agcggacgcc agagucugcg ccugcuugug gaugcucauc       1080
uuguugggcc aggccgaagc agcauuggag aaguuggucg ucuugcacgc ugcgagugcg       1140
gcuaacugcc auggccuccu auauuuugcc aucuucuucg uggcagcuug gcacaucagg       1200
ggucgggugg uccccuugac caccuauugc cucacuggcc uauggcccuu cugccuacug       1260
cucauggcac ugccccggca ggcuuaugcc                                        1290
<210>5
<211>651
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的NS2蛋白编码序列
<400>5
uaugacgcac cugugcacgg acagauaggc guggguuugu ugauauugau cacccucuuc       60
acacucaccc cgggguauaa gacccuccuc ggccaguguc uguggugguu gugcuaucuc       120
cugacccugg gggaagccau gauucaggag uggguaccac ccaugcaggu gcgcggcggc       180
cgcgauggca ucgcgugggc cgucacuaua uucugcccgg gugugguguu ugacauuacc       240
aaauggcuuu uggcguugcu ugggccugcu uaccucuuaa gggccgcuuu gacacaugug       300
ccguacuucg ucagagcuca cgcucugaua aggguaugcg cuuuggugaa gcagcucgcg       360
ggggguaggu auguucaggu ggcgcuauug gcccuuggca gguggacugg caccuacauc       420
uaugaccacc ucacaccuau gucggacugg gccgcuagcg gccugcgcga cuuagcgguc       480
gccguggaac ccaucaucuu caguccgaug gagaagaagg ucaucgucug gggagcggag       540
acggcugcau guggggacau ucuacaugga cuucccgugu ccgcccgacu cggccaggag       600
auccuccucg gcccagcuga uggcuacacc uccaaggggu ggaagcuccu u                651
<210>6
<211>1893
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的NS3蛋白编码序列
<400>6
gcucccauca cugcuuaugc ccagcaaaca cgaggccucc ugggcgccau aguggugagu       60
augacggggc gugacaggac agaacaggcc ggggaagucc aaauccuguc cacagucucu       120
caguccuucc ucggaacaac caucucgggg guuuugugga cuguuuacca cggagcuggc       180
aacaagacuc uagccggcuu acgggguccg gucacgcaga uguacucgag ugcugagggg       240
gacuugguag gcuggcccag ccccccuggg accaagucuu uggagccgug caagugugga       300
gccgucgacc uauaucuggu cacgcggaac gcugauguca ucccggcucg gagacgcggg       360
gacaagcggg gagcauugcu cuccccgaga cccauuucga ccuugaaggg guccucgggg       420
gggccggugc ucugcccuag gggccacguc guugggcucu uccgagcagc ugugugcucu       480
cggggcgugg ccaaauccau cgauuucauc cccguugaga cacucgacgu uguuacaagg       540
ucucccacuu ucagugacaa cagcacgcca ccggcugugc cccagaccua ucaggucggg       600
uacuugcaug cuccaacugg caguggaaag agcaccaagg ucccugucgc guaugccgcc       660
cagggguaca aaguacuagu gcuuaacccc ucgguagcug ccacccuggg guuuggggcg       720
uaccuaucca aggcacaugg caucaauccc aacauuagga cuggagucag gaccgugaug       780
accggggagg ccaucacgua cuccacauau ggcaaauuuc ucgccgaugg gggcugcgcu       840
agcggcgccu augacaucau cauaugcgau gaaugccacg cuguggaugc uaccuccauu       900
cucggcaucg gaacgguccu ugaucaagca gagacagccg gggucagacu aacugugcug       960
gcuacggcca caccccccgg gucagugaca accccccauc ccgauauaga agagguaggc       1020
cucgggcggg agggugagau ccccuucuau gggagggcga uuccccuauc cugcaucaag       1080
ggagggagac accugauuuu cugccacuca aagaaaaagu gugacgagcu cgcggcggcc       1140
cuucggggca ugggcuugaa ugccguggca uacuauagag gguuggacgu cuccauaaua       1200
ccagcucagg gagauguggu ggucgucgcc accgacgccc ucaugacggg guacacugga       1260
gacuuugacu ccgugaucga cugcaaugua gcggucaccc aagcugucga cuucagccug       1320
gaccccaccu ucacuauaac cacacagacu gucccacaag acgcugucuc acgcagucag       1380
cgccgcgggc gcacagguag aggaagacag ggcacuuaua gguauguuuc cacuggugaa       1440
cgagccucag gaauguuuga caguguagug cuuugugagu gcuacgacgc aggggcugcg       1500
ugguacgauc ucacaccagc ggagaccacc gucaggcuua gagcguauuu caacacgccc       1560
ggccuacccg ugugucaaga ccaucuugaa uuuugggagg caguuuucac cggccucaca       1620
cacauagacg cccacuuccu cucccaaaca aagcaagcgg gggagaacuu cgcguaccua       1680
guagccuacc aagcuacggu gugcgccaga gccaaggccc cucccccguc cugggacgcc       1740
auguggaagu gccuggcccg acucaagccu acgcuugcgg gccccacacc ucuccuguac       1800
cguuugggcc cuauuaccaa ugaggucacc cucacacacc cugggacgaa guacaucgcc       1860
acaugcaugc aagcugaccu ugaggucaug acc                                    1893
<210>7
<211>162
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的NS4A蛋白编码序列
<400>7
agcacguggg uccuagcugg aggaguccug gcagccgucg ccgcauauug ccuggcgacu       60
ggaugcguuu ccaucaucgg ccgcuugcac gucaaccagc gagucgucgu ugcgccggau       120
aaggaggucc uguaugaggc uuuugaugag auggaggaau gc                          162
<210>8
<211>783
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的NS4B蛋白编码序列
<400>8
gccucuaggg cggcucucau cgaagagggg cagcggauag ccgagauguu gaaguccaag       60
auccaaggcu ugcugcagca ggccucuaag caggcccagg acauacaacc cgcuaugcag       120
gcuucauggc ccaaagugga acaauuuugg gccagacaca uguggaacuu cauuagcggc       180
auccaauacc ucgcaggauu gucaacacug ccagggaacc ccgcgguggc uuccaugaug       240
gcauucagug ccgcccucac caguccguug ucgaccagua ccaccauccu ucucaacauc       300
augggaggcu gguuagcguc ccagaucgca ccacccgcgg gggccaccgg cuuugucguc       360
aguggccugg ugggggcugc cgugggcagc auaggccugg guaaggugcu gguggacauc       420
cuggcaggau auggugcggg cauuucgggg gcccucgucg cauucaagau caugucuggc       480
gagaagcccu cuauggaaga ugucaucaau cuacugccug ggauccuguc uccgggagcc       540
cugguggugg gggucaucug cgcggccauu cugcgccgcc acgugggacc gggggagggc       600
gcgguccaau ggaugaacag gcuuauugcc uuugcuucca gaggaaacca cgucgccccu       660
acucacuacg ugacggaguc ggaugcgucg cagcguguga cccaacuacu uggcucucuu       720
acuauaacca gccuacucag aagacuccac aauuggauaa cugaggacug ccccauccca       780
ugc                                                                     783
<210>9
<211>1398
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的NS5A蛋白编码序列
<400>9
uccggauccu ggcuccgcga cgugugggac uggguuugca ccaucuugac agacuucaaa       60
aauuggcuga ccucuaaauu guuccccaag cugcccggcc uccccuucau cucuugucaa       120
aagggguaca agggugugug ggccggcacu ggcaucauga ccacgcgcug cccuugcggc       180
gccaacaucu cuggcaaugu ccgccugggc ucuaugagga ucacagggcc uaaaaccugc       240
augaacaccu ggcaggggac cuuuccuauc aauugcuaca cggagggcca gugcgcgccg       300
aaacccccca cgaacuacaa gaccgccauc uggagggugg cggccucgga guacgcggag       360
gugacgcagc augggucgua cuccuaugua acaggacuga ccacugacaa ucugaaaauu       420
ccuugccaac uaccuucucc agaguuuuuc uccugggugg acggugugca gauccauagg       480
uuugcaccca caccaaagcc guuuuuccgg gaugaggucu cguucugcgu ugggcuuaau       540
uccuaugcug ucggguccca gcuucccugu gaaccugagc ccgacgcaga cguauugagg       600
uccaugcuaa cagauccgcc ccacaucacg gcggagacug cggcgcggcg cuuggcacgg       660
ggaucaccuc caucugaggc gagcuccuca gugagccagc uaucagcacc gucgcugcgg       720
gccaccugca ccacccacag caacaccuau gacguggaca uggucgaugc caaccugcuc       780
auggagggcg guguggcuca gacagagccu gaguccaggg ugcccguucu ggacuuucuc       840
gagccaaugg ccgaggaaga gagcgaccuu gagcccucaa uaccaucgga gugcaugcuc       900
cccaggagcg gguuuccacg ggccuuaccg gcuugggcac ggccugacua caacccgccg       960
cucguggaau cguggaggag gccagauuac caaccgccca ccguugcugg uugugcucuc       1020
ccccccccca agaaggcccc gacgccuccc ccaaggagac gccggacagu gggucugagc       1080
gagagcacca uaucagaagc ccuccagcaa cuggccauca agaccuuugg ccagcccccc       1140
ucgagcggug augcaggcuc guccacgggg gcgggcgccg ccgaauccgg cgguccgacg       1200
uccccuggug agccggcccc cucagagaca gguuccgccu ccucuaugcc cccccucgag       1260
ggggagccug gagauccgga ccuggagucu gaucagguag agcuucaacc ucccccccag       1320
gggggggggg uagcucccgg uucgggcucg gggucuuggu cuacuugcuc cgaggaggac       1380
gauaccaccg ugugcugc                                                     1398
<210>10
<211>1773
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的NS5B蛋白编码序列
<400>10
uccaugucau acuccuggac cggggcucua auaacucccu guagccccga agaggaaaag       60
uugccaauca acccuuugag uaacucgcug uugcgauacc auaacaaggu guacuguaca       120
acaucaaaga gcgccucaca gagggcuaaa aagguaacuu uugacaggac gcaagugcuc       180
gacgcccauu augacucagu cuuaaaggac aucaagcuag cggcuuccaa ggucagcgca       240
aggcuccuca ccuuggagga ggcgugccag uugacuccac cccauucugc aagauccaag       300
uauggauucg gggccaagga gguccgcagc uuguccggga gggccguuaa ccacaucaag       360
uccgugugga aggaccuccu ggaagaccca caaacaccaa uucccacaac caucauggcc       420
aaaaaugagg uguucugcgu ggaccccgcc aaggggggua agaaaccagc ucgccucauc       480
guuuacccug accucggcgu ccgggucugc gagaaaaugg cccucuauga cauuacacaa       540
aagcuuccuc aggcgguaau gggagcuucc uauggcuucc aguacucccc ugcccaacgg       600
guggaguauc ucuugaaagc augggcggaa aagaaggacc ccauggguuu uucguaugau       660
acccgaugcu ucgacucaac cgucacugag agagacauca ggaccgagga guccauauac       720
caggccugcu cccugcccga ggaggcccgc acugccauac acucgcugac ugagagacuu       780
uacguaggag ggcccauguu caacagcaag ggucaaaccu gcgguuacag acguugccgc       840
gccagcgggg ugcuaaccac uagcaugggu aacaccauca caugcuaugu gaaagcccua       900
gcggccugca aggcugcggg gauaguugcg cccacaaugc ugguaugcgg cgaugaccua       960
guagucaucu cagaaagcca ggggacugag gaggacgagc ggaaccugag agccuucacg       1020
gaggccauga ccagguacuc ugccccuccu ggugaucccc ccagaccgga auaugaccug       1080
gagcuaauaa cauccuguuc cucaaaugug ucuguggcgu ugggcccgcg gggccgccgc       1140
agauacuacc ugaccagaga cccaaccacu ccacucgccc gggcugccug ggaaacaguu       1200
agacacuccc cuaucaauuc auggcuggga aacaucaucc aguaugcucc aaccauaugg       1260
guucgcaugg uccuaaugac acacuucuuc uccauucuca ugguccaaga cacccuggac       1320
cagaaccuca acuuugagau guauggauca guauacuccg ugaauccuuu ggaccuucca       1380
gccauaauug agagguuaca cgggcuugac gccuuuucua ugcacacaua cucucaccac       1440
gaacugacgc ggguggcuuc agcccucaga aaacuugggg cgccaccccu cagggugugg       1500
aagagucggg cucgcgcagu cagggcgucc cucaucuccc guggagggaa agcggccguu       1560
ugcggccgau aucucuucaa uugggcggug aagaccaagc ucaaacucac uccauugccg       1620
gaggcgcgcc uacuggacuu auccaguugg uucaccgucg gcgccggcgg gggcgacauu       1680
uuucacagcg ugucgcgcgc ccgaccccgc ucauuacucu ucggccuacu ccuacuuuuc       1740
guagggguag gccucuuccu acuccccgcu cgg                                    1773
<210>11
<211>239
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的丙型肝炎病毒基因组RNA的3′非翻译区
<400>11
uagagcggca cacacuaggu acacuccaua gcuaacuguu ccuuuuuuuu uuuuuuuuuu       60
uuuuuuuuuu uuuuuuuuuu uuuuucuuuu uuuuuuuuuu cccucuuucu ucccuucuca       120
ucuuauucua cuuucuuucu ugguggcucc aucuuagccc uagucacggc uagcugugaa       180
agguccguga gccgcaugac ugcagagagu gccguaacug gucucucugc agaucaugu        239
<210>12
<211>9707
<212>RNA
<213>丙型肝炎病毒
<220>
<223>来自JFH-1克隆的全长丙型肝炎病毒基因组RNA
<400>12
gaauucuaau acgacucacu auagaccugc cccuaauagg ggcgacacuc cgccaugaau       60
cacuccccug ugaggaacua cugucuucac gcagaaagcg ccuagccaug gcguuaguau       120
gagugucgua cagccuccag gccccccccu cccgggagag ccauaguggu cugcggaacc       180
ggugaguaca ccggaauugc cgggaagacu ggguccuuuc uuggauaaac ccacucuaug       240
cccggccauu ugggcgugcc cccgcaagac ugcuagccga guagcguugg guugcgaaag       300
gccuuguggu acugccugau agggcgcuug cgagugcccc gggaggucuc guagaccgug       360
caccaugagc acaaauccua aaccucaaag aaaaaccaaa agaaacacca accgucgccc       420
agaagacguu aaguucccgg gcggcggcca gaucguuggc ggaguauacu uguugccgcg       480
caggggcccc agguugggug ugcgcacgac aaggaaaacu ucggagcggu cccagccacg       540
ugggagacgc cagcccaucc ccaaagaucg gcgcuccacu ggcaaggccu ggggaaaacc       600
aggucgcccc uggccccuau augggaauga gggacucggc ugggcaggau ggcuccuguc       660
cccccgaggc ucucgccccu ccuggggccc cacugacccc cggcauaggu cgcgcaacgu       720
ggguaaaguc aucgacaccc uaacgugugg cuuugccgac cucauggggu acauccccgu       780
cguaggcgcc ccgcuuagug gcgccgccag agcugucgcg cacggcguga gaguccugga       840
ggacgggguu aauuaugcaa cagggaaccu acccgguuuc cccuuuucua ucuucuugcu       900
ggcccuguug uccugcauca ccguuccggu cucugcugcc caggugaaga auaccaguag       960
cagcuacaug gugaccaaug acugcuccaa ugacagcauc acuuggcagc ucgaggcugc       1020
gguucuccac guccccgggu gcgucccgug cgagagagug gggaauacgu cacgguguug       1080
ggugccaguc ucgccaaaca uggcugugcg gcagcccggu gcccucacgc agggucugcg       1140
gacgcacauc gauaugguug ugauguccgc caccuucugc ucugcucucu acguggggga       1200
ccucuguggc ggggugaugc ucgcggccca gguguucauc gucucgccgc aguaccacug       1260
guuugugcaa gaaugcaauu gcuccaucua cccuggcacc aucacuggac accgcauggc       1320
augggacaug augaugaacu ggucgcccac ggccaccaug auccuggcgu acgugaugcg       1380
cguccccgag gucaucauag acaucguuag cggggcucac uggggcguca uguucggcuu       1440
ggccuacuuc ucuaugcagg gagcgugggc gaaggucauu gucauccuuc ugcuggccgc       1500
ugggguggac gcgggcacca ccaccguugg aggcgcuguu gcacguucca ccaacgugau       1560
ugccggcgug uucagccaug gcccucagca gaacauucag cucauuaaca ccaacggcag       1620
uuggcacauc aaccguacug ccuugaauug caaugacucc uugaacaccg gcuuucucgc       1680
ggccuuguuc uacaccaacc gcuuuaacuc gucagggugu ccagggcgcc uguccgccug       1740
ccgcaacauc gaggcuuucc ggauagggug gggcacccua caguacgagg auaaugucac       1800
caauccagag gauaugaggc cguacugcug gcacuacccc ccaaagccgu guggcguagu       1860
ccccgcgagg ucugugugug gcccagugua cuguuucacc cccagcccgg uaguaguggg       1920
cacgaccgac agacguggag ugcccaccua cacaugggga gagaaugaga cagaugucuu       1980
ccuacugaac agcacccgac cgccgcaggg cucaugguuc ggcugcacgu ggaugaacuc       2040
cacugguuuc accaagacuu guggcgcgcc accuugccgc accagagcug acuucaacgc       2100
cagcacggac uuguugugcc cuacggauug uuuuaggaag cauccugaug ccacuuauau       2160
uaaguguggu ucugggcccu ggcucacacc aaagugccug guccacuacc cuuacagacu       2220
cuggcauuac cccugcacag ucaauuuuac caucuucaag auaagaaugu auguaggggg       2280
gguugagcac aggcucacgg ccgcaugcaa cuucacucgu ggggaucgcu gcgacuugga       2340
ggacagggac aggagucagc ugucuccucu guugcacucu accacggaau gggccauccu       2400
gcccugcacc uacucagacu uacccgcuuu gucaacuggu cuucuccacc uucaccagaa       2460
caucguggac guacaauaca uguauggccu cucaccugcu aucacaaaau acgucguucg       2520
augggagugg gugguacucu uauuccugcu cuuagcggac gccagagucu gcgccugcuu       2580
guggaugcuc aucuuguugg gccaggccga agcagcauug gagaaguugg ucgucuugca       2640
cgcugcgagu gcggcuaacu gccauggccu ccuauauuuu gccaucuucu ucguggcagc       2700
uuggcacauc aggggucggg ugguccccuu gaccaccuau ugccucacug gccuauggcc       2760
cuucugccua cugcucaugg cacugccccg gcaggcuuau gccuaugacg caccugugca       2820
cggacagaua ggcguggguu uguugauauu gaucacccuc uucacacuca ccccggggua       2880
uaagacccuc cucggccagu gucuguggug guugugcuau cuccugaccc ugggggaagc       2940
caugauucag gaguggguac cacccaugca ggugcgcggc ggccgcgaug gcaucgcgug       3000
ggccgucacu auauucugcc cggguguggu guuugacauu accaaauggc uuuuggcguu       3060
gcuugggccu gcuuaccucu uaagggccgc uuugacacau gugccguacu ucgucagagc       3120
ucacgcucug auaaggguau gcgcuuuggu gaagcagcuc gcggggggua gguauguuca       3180
gguggcgcua uuggcccuug gcagguggac uggcaccuac aucuaugacc accucacacc       3240
uaugucggac ugggccgcua gcggccugcg cgacuuagcg gucgccgugg aacccaucau       3300
cuucaguccg auggagaaga aggucaucgu cuggggagcg gagacggcug caugugggga       3360
cauucuacau ggacuucccg uguccgcccg acucggccag gagauccucc ucggcccagc       3420
ugauggcuac accuccaagg gguggaagcu ccuugcuccc aucacugcuu augcccagca       3480
aacacgaggc cuccugggcg ccauaguggu gaguaugacg gggcgugaca ggacagaaca       3540
ggccggggaa guccaaaucc uguccacagu cucucagucc uuccucggaa caaccaucuc       3600
ggggguuuug uggacuguuu accacggagc uggcaacaag acucuagccg gcuuacgggg       3660
uccggucacg cagauguacu cgagugcuga gggggacuug guaggcuggc ccagcccccc       3720
ugggaccaag ucuuuggagc cgugcaagug uggagccguc gaccuauauc uggucacgcg       3780
gaacgcugau gucaucccgg cucggagacg cggggacaag cggggagcau ugcucucccc       3840
gagacccauu ucgaccuuga agggguccuc gggggggccg gugcucugcc cuaggggcca       3900
cgucguuggg cucuuccgag cagcugugug cucucggggc guggccaaau ccaucgauuu       3960
cauccccguu gagacacucg acguuguuac aaggucuccc acuuucagug acaacagcac       4020
gccaccggcu gugccccaga ccuaucaggu cggguacuug caugcuccaa cuggcagugg       4080
aaagagcacc aaggucccug ucgcguaugc cgcccagggg uacaaaguac uagugcuuaa       4140
ccccucggua gcugccaccc ugggguuugg ggcguaccua uccaaggcac auggcaucaa       4200
ucccaacauu aggacuggag ucaggaccgu gaugaccggg gaggccauca cguacuccac       4260
auauggcaaa uuucucgccg augggggcug cgcuagcggc gccuaugaca ucaucauaug       4320
cgaugaaugc cacgcugugg augcuaccuc cauucucggc aucggaacgg uccuugauca       4380
agcagagaca gccgggguca gacuaacugu gcuggcuacg gccacacccc ccgggucagu       4440
gacaaccccc caucccgaua uagaagaggu aggccucggg cgggagggug agauccccuu       4500
cuaugggagg gcgauucccc uauccugcau caagggaggg agacaccuga uuuucugcca       4560
cucaaagaaa aagugugacg agcucgcggc ggcccuucgg ggcaugggcu ugaaugccgu       4620
ggcauacuau agaggguugg acgucuccau aauaccagcu cagggagaug ugguggucgu       4680
cgccaccgac gcccucauga cgggguacac uggagacuuu gacuccguga ucgacugcaa       4740
uguagcgguc acccaagcug ucgacuucag ccuggacccc accuucacua uaaccacaca       4800
gacuguccca caagacgcug ucucacgcag ucagcgccgc gggcgcacag guagaggaag       4860
acagggcacu uauagguaug uuuccacugg ugaacgagcc ucaggaaugu uugacagugu       4920
agugcuuugu gagugcuacg acgcaggggc ugcgugguac gaucucacac cagcggagac       4980
caccgucagg cuuagagcgu auuucaacac gcccggccua cccguguguc aagaccaucu       5040
ugaauuuugg gaggcaguuu ucaccggccu cacacacaua gacgcccacu uccucuccca       5100
aacaaagcaa gcgggggaga acuucgcgua ccuaguagcc uaccaagcua cggugugcgc       5160
cagagccaag gccccucccc cguccuggga cgccaugugg aagugccugg cccgacucaa       5220
gccuacgcuu gcgggcccca caccucuccu guaccguuug ggcccuauua ccaaugaggu       5280
cacccucaca cacccuggga cgaaguacau cgccacaugc augcaagcug accuugaggu       5340
caugaccagc acgugggucc uagcuggagg aguccuggca gccgucgccg cauauugccu       5400
ggcgacugga ugcguuucca ucaucggccg cuugcacguc aaccagcgag ucgucguugc       5460
gccggauaag gagguccugu augaggcuuu ugaugagaug gaggaaugcg ccucuagggc       5520
ggcucucauc gaagaggggc agcggauagc cgagauguug aaguccaaga uccaaggcuu       5580
gcugcagcag gccucuaagc aggcccagga cauacaaccc gcuaugcagg cuucauggcc       5640
caaaguggaa caauuuuggg ccagacacau guggaacuuc auuagcggca uccaauaccu       5700
cgcaggauug ucaacacugc cagggaaccc cgcgguggcu uccaugaugg cauucagugc       5760
cgcccucacc aguccguugu cgaccaguac caccauccuu cucaacauca ugggaggcug       5820
guuagcgucc cagaucgcac cacccgcggg ggccaccggc uuugucguca guggccuggu       5880
gggggcugcc gugggcagca uaggccuggg uaaggugcug guggacaucc uggcaggaua       5940
uggugcgggc auuucggggg cccucgucgc auucaagauc augucuggcg agaagcccuc       6000
uauggaagau gucaucaauc uacugccugg gauccugucu ccgggagccc uggugguggg       6060
ggucaucugc gcggccauuc ugcgccgcca cgugggaccg ggggagggcg cgguccaaug       6120
gaugaacagg cuuauugccu uugcuuccag aggaaaccac gucgccccua cucacuacgu       6180
gacggagucg gaugcgucgc agcgugugac ccaacuacuu ggcucucuua cuauaaccag       6240
ccuacucaga agacuccaca auuggauaac ugaggacugc cccaucccau gcuccggauc       6300
cuggcuccgc gacguguggg acuggguuug caccaucuug acagacuuca aaaauuggcu       6360
gaccucuaaa uuguucccca agcugcccgg ccuccccuuc aucucuuguc aaaaggggua       6420
caagggugug ugggccggca cuggcaucau gaccacgcgc ugcccuugcg gcgccaacau       6480
cucuggcaau guccgccugg gcucuaugag gaucacaggg ccuaaaaccu gcaugaacac       6540
cuggcagggg accuuuccua ucaauugcua cacggagggc cagugcgcgc cgaaaccccc       6600
cacgaacuac aagaccgcca ucuggagggu ggcggccucg gaguacgcgg aggugacgca       6660
gcaugggucg uacuccuaug uaacaggacu gaccacugac aaucugaaaa uuccuugcca       6720
acuaccuucu ccagaguuuu ucuccugggu ggacggugug cagauccaua gguuugcacc       6780
cacaccaaag ccguuuuucc gggaugaggu cucguucugc guugggcuua auuccuaugc       6840
ugucgggucc cagcuucccu gugaaccuga gcccgacgca gacguauuga gguccaugcu       6900
aacagauccg ccccacauca cggcggagac ugcggcgcgg cgcuuggcac ggggaucacc       6960
uccaucugag gcgagcuccu cagugagcca gcuaucagca ccgucgcugc gggccaccug       7020
caccacccac agcaacaccu augacgugga cauggucgau gccaaccugc ucauggaggg       7080
cgguguggcu cagacagagc cugaguccag ggugcccguu cuggacuuuc ucgagccaau       7140
ggccgaggaa gagagcgacc uugagcccuc aauaccaucg gagugcaugc uccccaggag       7200
cggguuucca cgggccuuac cggcuugggc acggccugac uacaacccgc cgcucgugga       7260
aucguggagg aggccagauu accaaccgcc caccguugcu gguugugcuc uccccccccc       7320
caagaaggcc ccgacgccuc ccccaaggag acgccggaca gugggucuga gcgagagcac       7380
cauaucagaa gcccuccagc aacuggccau caagaccuuu ggccagcccc ccucgagcgg       7440
ugaugcaggc ucguccacgg gggcgggcgc cgccgaaucc ggcgguccga cguccccugg       7500
ugagccggcc cccucagaga cagguuccgc cuccucuaug cccccccucg agggggagcc       7560
uggagauccg gaccuggagu cugaucaggu agagcuucaa ccuccccccc aggggggggg       7620
gguagcuccc gguucgggcu cggggucuug gucuacuugc uccgaggagg acgauaccac       7680
cgugugcugc uccaugucau acuccuggac cggggcucua auaacucccu guagccccga       7740
agaggaaaag uugccaauca acccuuugag uaacucgcug uugcgauacc auaacaaggu       7800
guacuguaca acaucaaaga gcgccucaca gagggcuaaa aagguaacuu uugacaggac       7860
gcaagugcuc gacgcccauu augacucagu cuuaaaggac aucaagcuag cggcuuccaa       7920
ggucagcgca aggcuccuca ccuuggagga ggcgugccag uugacuccac cccauucugc       7980
aagauccaag uauggauucg gggccaagga gguccgcagc uuguccggga gggccguuaa       8040
ccacaucaag uccgugugga aggaccuccu ggaagaccca caaacaccaa uucccacaac       8100
caucauggcc aaaaaugagg uguucugcgu ggaccccgcc aaggggggua agaaaccagc       8160
ucgccucauc guuuacccug accucggcgu ccgggucugc gagaaaaugg cccucuauga       8220
cauuacacaa aagcuuccuc aggcgguaau gggagcuucc uauggcuucc aguacucccc       8280
ugcccaacgg guggaguauc ucuugaaagc augggcggaa aagaaggacc ccauggguuu       8340
uucguaugau acccgaugcu ucgacucaac cgucacugag agagacauca ggaccgagga       8400
guccauauac caggccugcu cccugcccga ggaggcccgc acugccauac acucgcugac       8460
ugagagacuu uacguaggag ggcccauguu caacagcaag ggucaaaccu gcgguuacag       8520
acguugccgc gccagcgggg ugcuaaccac uagcaugggu aacaccauca caugcuaugu       8580
gaaagcccua gcggccugca aggcugcggg gauaguugcg cccacaaugc ugguaugcgg       8640
cgaugaccua guagucaucu cagaaagcca ggggacugag gaggacgagc ggaaccugag       8700
agccuucacg gaggccauga ccagguacuc ugccccuccu ggugaucccc ccagaccgga       8760
auaugaccug gagcuaauaa cauccuguuc cucaaaugug ucuguggcgu ugggcccgcg       8820
gggccgccgc agauacuacc ugaccagaga cccaaccacu ccacucgccc gggcugccug       8880
ggaaacaguu agacacuccc cuaucaauuc auggcuggga aacaucaucc aguaugcucc       8940
aaccauaugg guucgcaugg uccuaaugac acacuucuuc uccauucuca ugguccaaga       9000
cacccuggac cagaaccuca acuuugagau guauggauca guauacuccg ugaauccuuu       9060
ggaccuucca gccauaauug agagguuaca cgggcuugac gccuuuucua ugcacacaua       9120
cucucaccac gaacugacgc ggguggcuuc agcccucaga aaacuugggg cgccaccccu       9180
cagggugugg aagagucggg cucgcgcagu cagggcgucc cucaucuccc guggagggaa       9240
agcggccguu ugcggccgau aucucuucaa uugggcggug aagaccaagc ucaaacucac       9300
uccauugccg gaggcgcgcc uacuggacuu auccaguugg uucaccgucg gcgccggcgg       9360
gggcgacauu uuucacagcg ugucgcgcgc ccgaccccgc ucauuacucu ucggccuacu       9420
ccuacuuuuc guagggguag gccucuuccu acuccccgcu cgguagagcg gcacacacua       9480
gguacacucc auagcuaacu guuccuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu       9540
uuuuuuuucu uuuuuuuuuu uuucccucuu ucuucccuuc ucaucuuauu cuacuuucuu       9600
ucuugguggc uccaucuuag cccuagucac ggcuagcugu gaaagguccg ugagccgcau       9660
gacugcagag agugccguaa cuggucucuc ugcagaucau gucuaga                     9707
<210>13
<211>11111
<212>RNA
<213>人工序列
<220>
<223>人工序列描述：含来自JFH-1克隆的全长丙型肝炎病毒基因组RNA的复制子RNA
<400>13
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca       420
cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac       480
aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu       540
ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc       600
guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg       660
aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc       720
uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc       780
ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau       840
ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc       900
cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca       960
uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga       1020
cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau       1080
ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc       1140
ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac       1200
ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug       1260
cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga       1320
aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa       1380
ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa       1440
caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu       1500
gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg       1560
uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg       1620
ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca       1680
caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg       1740
acgugguuuu ccuuugaaaa acacgaugau accaugagca caaauccuaa accucaaaga       1800
aaaaccaaaa gaaacaccaa ccgucgccca gaagacguua aguucccggg cggcggccag       1860
aucguuggcg gaguauacuu guugccgcgc aggggcccca gguugggugu gcgcacgaca       1920
aggaaaacuu cggagcgguc ccagccacgu gggagacgcc agcccauccc caaagaucgg       1980
cgcuccacug gcaaggccug gggaaaacca ggucgccccu ggccccuaua ugggaaugag       2040
ggacucggcu gggcaggaug gcuccugucc ccccgaggcu cucgccccuc cuggggcccc       2100
acugaccccc ggcauagguc gcgcaacgug gguaaaguca ucgacacccu aacguguggc       2160
uuugccgacc ucauggggua cauccccguc guaggcgccc cgcuuagugg cgccgccaga       2220
gcugucgcgc acggcgugag aguccuggag gacgggguua auuaugcaac agggaaccua       2280
cccgguuucc ccuuuucuau cuucuugcug gcccuguugu ccugcaucac cguuccgguc       2340
ucugcugccc aggugaagaa uaccaguagc agcuacaugg ugaccaauga cugcuccaau       2400
gacagcauca cuuggcagcu cgaggcugcg guucuccacg uccccgggug cgucccgugc       2460
gagagagugg ggaauacguc acgguguugg gugccagucu cgccaaacau ggcugugcgg       2520
cagcccggug cccucacgca gggucugcgg acgcacaucg auaugguugu gauguccgcc       2580
accuucugcu cugcucucua cgugggggac cucuguggcg gggugaugcu cgcggcccag       2640
guguucaucg ucucgccgca guaccacugg uuugugcaag aaugcaauug cuccaucuac       2700
ccuggcacca ucacuggaca ccgcauggca ugggacauga ugaugaacug gucgcccacg       2760
gccaccauga uccuggcgua cgugaugcgc guccccgagg ucaucauaga caucguuagc       2820
ggggcucacu ggggcgucau guucggcuug gccuacuucu cuaugcaggg agcgugggcg       2880
aaggucauug ucauccuucu gcuggccgcu gggguggacg cgggcaccac caccguugga       2940
ggcgcuguug cacguuccac caacgugauu gccggcgugu ucagccaugg cccucagcag       3000
aacauucagc ucauuaacac caacggcagu uggcacauca accguacugc cuugaauugc       3060
aaugacuccu ugaacaccgg cuuucucgcg gccuuguucu acaccaaccg cuuuaacucg       3120
ucaggguguc cagggcgccu guccgccugc cgcaacaucg aggcuuuccg gauagggugg       3180
ggcacccuac aguacgagga uaaugucacc aauccagagg auaugaggcc guacugcugg       3240
cacuaccccc caaagccgug uggcguaguc cccgcgaggu cugugugugg cccaguguac       3300
uguuucaccc ccagcccggu aguagugggc acgaccgaca gacguggagu gcccaccuac       3360
acauggggag agaaugagac agaugucuuc cuacugaaca gcacccgacc gccgcagggc       3420
ucaugguucg gcugcacgug gaugaacucc acugguuuca ccaagacuug uggcgcgcca       3480
ccuugccgca ccagagcuga cuucaacgcc agcacggacu uguugugccc uacggauugu       3540
uuuaggaagc auccugaugc cacuuauauu aagugugguu cugggcccug gcucacacca       3600
aagugccugg uccacuaccc uuacagacuc uggcauuacc ccugcacagu caauuuuacc       3660
aucuucaaga uaagaaugua uguagggggg guugagcaca ggcucacggc cgcaugcaac       3720
uucacucgug gggaucgcug cgacuuggag gacagggaca ggagucagcu gucuccucug       3780
uugcacucua ccacggaaug ggccauccug cccugcaccu acucagacuu acccgcuuug       3840
ucaacugguc uucuccaccu ucaccagaac aucguggacg uacaauacau guauggccuc       3900
ucaccugcua ucacaaaaua cgucguucga ugggaguggg ugguacucuu auuccugcuc       3960
uuagcggacg ccagagucug cgccugcuug uggaugcuca ucuuguuggg ccaggccgaa       4020
gcagcauugg agaaguuggu cgucuugcac gcugcgagug cggcuaacug ccauggccuc       4080
cuauauuuug ccaucuucuu cguggcagcu uggcacauca ggggucgggu gguccccuug       4140
accaccuauu gccucacugg ccuauggccc uucugccuac ugcucauggc acugccccgg       4200
caggcuuaug ccuaugacgc accugugcac ggacagauag gcguggguuu guugauauug       4260
aucacccucu ucacacucac cccgggguau aagacccucc ucggccagug ucuguggugg       4320
uugugcuauc uccugacccu gggggaagcc augauucagg aguggguacc acccaugcag       4380
gugcgcggcg gccgcgaugg caucgcgugg gccgucacua uauucugccc ggguguggug       4440
uuugacauuaccaaauggcu  uuuggcguug cuugggccug cuuaccucuu aagggccgcu       4500
uugacacaug ugccguacuu cgucagagcu cacgcucuga uaaggguaug cgcuuuggug       4560
aagcagcucg cgggggguag guauguucag guggcgcuau uggcccuugg cagguggacu       4620
ggcaccuaca ucuaugacca ccucacaccu augucggacu gggccgcuag cggccugcgc       4680
gacuuagcgg ucgccgugga acccaucauc uucaguccga uggagaagaa ggucaucguc       4740
uggggagcgg agacggcugc auguggggac auucuacaug gacuucccgu guccgcccga       4800
cucggccagg agauccuccu cggcccagcu gauggcuaca ccuccaaggg guggaagcuc       4860
cuugcuccca ucacugcuua ugcccagcaa acacgaggcc uccugggcgc cauaguggug       4920
aguaugacgg ggcgugacag gacagaacag gccggggaag uccaaauccu guccacaguc       4980
ucucaguccu uccucggaac aaccaucucg gggguuuugu ggacuguuua ccacggagcu       5040
ggcaacaaga cucuagccgg cuuacggggu ccggucacgc agauguacuc gagugcugag       5100
ggggacuugg uaggcuggcc cagccccccu gggaccaagu cuuuggagcc gugcaagugu       5160
ggagccgucg accuauaucu ggucacgcgg aacgcugaug ucaucccggc ucggagacgc       5220
ggggacaagc ggggagcauu gcucuccccg agacccauuu cgaccuugaa gggguccucg       5280
ggggggccgg ugcucugccc uaggggccac gucguugggc ucuuccgagc agcugugugc       5340
ucucggggcg uggccaaauc caucgauuuc auccccguug agacacucga cguuguuaca       5400
aggucuccca cuuucaguga caacagcacg ccaccggcug ugccccagac cuaucagguc       5460
ggguacuugc augcuccaac uggcagugga aagagcacca aggucccugu cgcguaugcc       5520
gcccaggggu acaaaguacu agugcuuaac cccucgguag cugccacccu gggguuuggg       5580
gcguaccuau ccaaggcaca uggcaucaau cccaacauua ggacuggagu caggaccgug       5640
augaccgggg aggccaucac guacuccaca uauggcaaau uucucgccga ugggggcugc       5700
gcuagcggcg ccuaugacau caucauaugc gaugaaugcc acgcugugga ugcuaccucc       5760
auucucggca ucggaacggu ccuugaucaa gcagagacag ccggggucag acuaacugug       5820
cuggcuacgg ccacaccccc cgggucagug acaacccccc aucccgauau agaagaggua       5880
ggccucgggc gggaggguga gauccccuuc uaugggaggg cgauuccccu auccugcauc       5940
aagggaggga gacaccugau uuucugccac ucaaagaaaa agugugacga gcucgcggcg       6000
gcccuucggg gcaugggcuu gaaugccgug gcauacuaua gaggguugga cgucuccaua       6060
auaccagcuc agggagaugu gguggucguc gccaccgacg cccucaugac gggguacacu       6120
ggagacuuug acuccgugau cgacugcaau guagcgguca cccaagcugu cgacuucagc       6180
cuggacccca ccuucacuau aaccacacag acugucccac aagacgcugu cucacgcagu       6240
cagcgccgcg ggcgcacagg uagaggaaga cagggcacuu auagguaugu uuccacuggu       6300
gaacgagccu caggaauguu ugacagugua gugcuuugug agugcuacga cgcaggggcu       6360
gcgugguacg aucucacacc agcggagacc accgucaggc uuagagcgua uuucaacacg       6420
cccggccuac ccguguguca agaccaucuu gaauuuuggg aggcaguuuu caccggccuc       6480
acacacauag acgcccacuu ccucucccaa acaaagcaag cgggggagaa cuucgcguac       6540
cuaguagccu accaagcuac ggugugcgcc agagccaagg ccccuccccc guccugggac       6600
gccaugugga agugccuggc ccgacucaag ccuacgcuug cgggccccac accucuccug       6660
uaccguuugg gcccuauuac caaugagguc acccucacac acccugggac gaaguacauc       6720
gccacaugca ugcaagcuga ccuugagguc augaccagca cguggguccu agcuggagga       6780
guccuggcag ccgucgccgc auauugccug gcgacuggau gcguuuccau caucggccgc       6840
uugcacguca accagcgagu cgucguugcg ccggauaagg agguccugua ugaggcuuuu       6900
gaugagaugg aggaaugcgc cucuagggcg gcucucaucg aagaggggca gcggauagcc       6960
gagauguuga aguccaagau ccaaggcuug cugcagcagg ccucuaagca ggcccaggac       7020
auacaacccg cuaugcaggc uucauggccc aaaguggaac aauuuugggc cagacacaug       7080
uggaacuuca uuagcggcau ccaauaccuc gcaggauugu caacacugcc agggaacccc       7140
gcgguggcuu ccaugauggc auucagugcc gcccucacca guccguuguc gaccaguacc       7200
accauccuuc ucaacaucau gggaggcugg uuagcguccc agaucgcacc acccgcgggg       7260
gccaccggcu uugucgucag uggccuggug ggggcugccg ugggcagcau aggccugggu       7320
aaggugcugg uggacauccu ggcaggauau ggugcgggca uuucgggggc ccucgucgca       7380
uucaagauca ugucuggcga gaagcccucu auggaagaug ucaucaaucu acugccuggg       7440
auccugucuc cgggagcccu gguggugggg gucaucugcg cggccauucu gcgccgccac       7500
gugggaccgg gggagggcgc gguccaaugg augaacaggc uuauugccuu ugcuuccaga       7560
ggaaaccacg ucgccccuac ucacuacgug acggagucgg augcgucgca gcgugugacc       7620
caacuacuug gcucucuuac uauaaccagc cuacucagaa gacuccacaa uuggauaacu       7680
gaggacugcc ccaucccaug cuccggaucc uggcuccgcg acguguggga cuggguuugc       7740
accaucuuga cagacuucaa aaauuggcug accucuaaau uguuccccaa gcugcccggc       7800
cuccccuuca ucucuuguca aaagggguac aagggugugu gggccggcac uggcaucaug       7860
accacgcgcu gcccuugcgg cgccaacauc ucuggcaaug uccgccuggg cucuaugagg       7920
aucacagggc cuaaaaccug caugaacacc uggcagggga ccuuuccuau caauugcuac       7980
acggagggcc agugcgcgcc gaaacccccc acgaacuaca agaccgccau cuggagggug       8040
gcggccucgg aguacgcgga ggugacgcag caugggucgu acuccuaugu aacaggacug       8100
accacugaca aucugaaaau uccuugccaa cuaccuucuc cagaguuuuu cuccugggug       8160
gacggugugc agauccauag guuugcaccc acaccaaagc cguuuuuccg ggaugagguc       8220
ucguucugcg uugggcuuaa uuccuaugcu gucggguccc agcuucccug ugaaccugag       8280
cccgacgcag acguauugag guccaugcua acagauccgc cccacaucac ggcggagacu       8340
gcggcgcggc gcuuggcacg gggaucaccu ccaucugagg cgagcuccuc agugagccag       8400
cuaucagcac cgucgcugcg ggccaccugc accacccaca gcaacaccua ugacguggac       8460
auggucgaug ccaaccugcu cauggagggc gguguggcuc agacagagcc ugaguccagg       8520
gugcccguuc uggacuuucu cgagccaaug gccgaggaag agagcgaccu ugagcccuca       8580
auaccaucgg agugcaugcu ccccaggagc ggguuuccac gggccuuacc ggcuugggca       8640
cggccugacu acaacccgcc gcucguggaa ucguggagga ggccagauua ccaaccgccc       8700
accguugcug guugugcucu cccccccccc aagaaggccc cgacgccucc cccaaggaga       8760
cgccggacag ugggucugag cgagagcacc auaucagaag cccuccagca acuggccauc       8820
aagaccuuug gccagccccc cucgagcggu gaugcaggcu cguccacggg ggcgggcgcc       8880
gccgaauccg gcgguccgac guccccuggu gagccggccc ccucagagac agguuccgcc       8940
uccucuaugc ccccccucga gggggagccu ggagauccgg accuggaguc ugaucaggua       9000
gagcuucaac cuccccccca gggggggggg guagcucccg guucgggcuc ggggucuugg       9060
ucuacuugcu ccgaggagga cgauaccacc gugugcugcu ccaugucaua cuccuggacc       9120
ggggcucuaa uaacucccug uagccccgaa gaggaaaagu ugccaaucaa cccuuugagu       9180
aacucgcugu ugcgauacca uaacaaggug uacuguacaa caucaaagag cgccucacag       9240
agggcuaaaa agguaacuuu ugacaggacg caagugcucg acgcccauua ugacucaguc       9300
uuaaaggaca ucaagcuagc ggcuuccaag gucagcgcaa ggcuccucac cuuggaggag       9360
gcgugccagu ugacuccacc ccauucugca agauccaagu auggauucgg ggccaaggag       9420
guccgcagcu uguccgggag ggccguuaac cacaucaagu ccguguggaa ggaccuccug       9480
gaagacccac aaacaccaau ucccacaacc aucauggcca aaaaugaggu guucugcgug       9540
gaccccgcca agggggguaa gaaaccagcu cgccucaucg uuuacccuga ccucggcguc       9600
cgggucugcg agaaaauggc ccucuaugac auuacacaaa agcuuccuca ggcgguaaug       9660
ggagcuuccu auggcuucca guacuccccu gcccaacggg uggaguaucu cuugaaagca       9720
ugggcggaaa agaaggaccc cauggguuuu ucguaugaua cccgaugcuu cgacucaacc       9780
gucacugaga gagacaucag gaccgaggag uccauauacc aggccugcuc ccugcccgag       9840
gaggcccgca cugccauaca cucgcugacu gagagacuuu acguaggagg gcccauguuc       9900
aacagcaagg gucaaaccug cgguuacaga cguugccgcg ccagcggggu gcuaaccacu       9960
agcaugggua acaccaucac augcuaugug aaagcccuag cggccugcaa ggcugcgggg       10020
auaguugcgc ccacaaugcu gguaugcggc gaugaccuag uagucaucuc agaaagccag       10080
gggacugagg aggacgagcg gaaccugaga gccuucacgg aggccaugac cagguacucu       10140
gccccuccug gugauccccc cagaccggaa uaugaccugg agcuaauaac auccuguucc       10200
ucaaaugugu cuguggcguu gggcccgcgg ggccgccgca gauacuaccu gaccagagac       10260
ccaaccacuc cacucgcccg ggcugccugg gaaacaguua gacacucccc uaucaauuca       10320
uggcugggaa acaucaucca guaugcucca accauauggg uucgcauggu ccuaaugaca       10380
cacuucuucu ccauucucau gguccaagac acccuggacc agaaccucaa cuuugagaug       10440
uauggaucag uauacuccgu gaauccuuug gaccuuccag ccauaauuga gagguuacac       10500
gggcuugacg ccuuuucuau gcacacauac ucucaccacg aacugacgcg gguggcuuca       10560
gcccucagaa aacuuggggc gccaccccuc agggugugga agagucgggc ucgcgcaguc       10620
agggcguccc ucaucucccg uggagggaaa gcggccguuu gcggccgaua ucucuucaau       10680
ugggcgguga agaccaagcu caaacucacu ccauugccgg aggcgcgccu acuggacuua       10740
uccaguuggu ucaccgucgg cgccggcggg ggcgacauuu uucacagcgu gucgcgcgcc       10800
cgaccccgcu cauuacucuu cggccuacuc cuacuuuucg uagggguagg ccucuuccua       10860
cuccccgcuc gguagagcgg cacacacuag guacacucca uagcuaacug uuccuuuuuu       10920
uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuucuu uuuuuuuuuu uucccucuuu       10980
cuucccuucu caucuuauuc uacuuucuuu cuugguggcu ccaucuuagc ccuagucacg       11040
gcuagcugug aaagguccgu gagccgcaug acugcagaga gugccguaac uggucucucu       11100
gcagaucaug u                                                            11111
<210>14
<211>11111
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：来自JFH-1克隆的全长丙型肝炎病毒基因组RNA，其中氨基酸基序GDD已突变为GND
<400>14
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg ucgcccaaug auugaacaag auggauugca       420
cgcagguucu ccggccgcuu ggguggagag gcuauucggc uaugacuggg cacaacagac       480
aaucggcugc ucugaugccg ccguguuccg gcugucagcg caggggcgcc cgguucuuuu       540
ugucaagacc gaccuguccg gugcccugaa ugaacugcag gacgaggcag cgcggcuauc       600
guggcuggcc acgacgggcg uuccuugcgc agcugugcuc gacguuguca cugaagcggg       660
aagggacugg cugcuauugg gcgaagugcc ggggcaggau cuccugucau cucaccuugc       720
uccugccgag aaaguaucca ucauggcuga ugcaaugcgg cggcugcaua cgcuugaucc       780
ggcuaccugc ccauucgacc accaagcgaa acaucgcauc gagcgagcac guacucggau       840
ggaagccggu cuugucgauc aggaugaucu ggacgaagag caucaggggc ucgcgccagc       900
cgaacuguuc gccaggcuca aggcgcgcau gcccgacggc gaggaucucg ucgugaccca       960
uggcgaugcc ugcuugccga auaucauggu ggaaaauggc cgcuuuucug gauucaucga       1020
cuguggccgg cugggugugg cggaccgcua ucaggacaua gcguuggcua cccgugauau       1080
ugcugaagag cuuggcggcg aaugggcuga ccgcuuccuc gugcuuuacg guaucgccgc       1140
ucccgauucg cagcgcaucg ccuucuaucg ccuucuugac gaguucuucu gaguuuaaac       1200
ccucucccuc cccccccccu aacguuacug gccgaagccg cuuggaauaa ggccggugug       1260
cguuugucua uauguuauuu uccaccauau ugccgucuuu uggcaaugug agggcccgga       1320
aaccuggccc ugucuucuug acgagcauuc cuaggggucu uuccccucuc gccaaaggaa       1380
ugcaaggucu guugaauguc gugaaggaag caguuccucu ggaagcuucu ugaagacaaa       1440
caacgucugu agcgacccuu ugcaggcagc ggaacccccc accuggcgac aggugccucu       1500
gcggccaaaa gccacgugua uaagauacac cugcaaaggc ggcacaaccc cagugccacg       1560
uugugaguug gauaguugug gaaagaguca aauggcucuc cucaagcgua uucaacaagg       1620
ggcugaagga ugcccagaag guaccccauu guaugggauc ugaucugggg ccucggugca       1680
caugcuuuac auguguuuag ucgagguuaa aaaaacgucu aggccccccg aaccacgggg       1740
acgugguuuu ccuuugaaaa acacgaugau accaugagca caaauccuaa accucaaaga       1800
aaaaccaaaa gaaacaccaa ccgucgccca gaagacguua aguucccggg cggcggccag       1860
aucguuggcg gaguauacuu guugccgcgc aggggcccca gguugggugu gcgcacgaca       1920
aggaaaacuu cggagcgguc ccagccacgu gggagacgcc agcccauccc caaagaucgg       1980
cgcuccacug gcaaggccug gggaaaacca ggucgccccu ggccccuaua ugggaaugag       2040
ggacucggcu gggcaggaug gcuccugucc ccccgaggcu cucgccccuc cuggggcccc       2100
acugaccccc ggcauagguc gcgcaacgug gguaaaguca ucgacacccu aacguguggc       2160
uuugccgacc ucauggggua cauccccguc guaggcgccc cgcuuagugg cgccgccaga       2220
gcugucgcgc acggcgugag aguccuggag gacgggguua auuaugcaac agggaaccua       2280
cccgguuucc ccuuuucuau cuucuugcug gcccuguugu ccugcaucac cguuccgguc       2340
ucugcugccc aggugaagaa uaccaguagc agcuacaugg ugaccaauga cugcuccaau       2400
gacagcauca cuuggcagcu cgaggcugcg guucuccacg uccccgggug cgucccgugc       2460
gagagagugg ggaauacguc acgguguugg gugccagucu cgccaaacau ggcugugcgg       2520
cagcccggug cccucacgca gggucugcgg acgcacaucg auaugguugu gauguccgcc       2580
accuucugcu cugcucucua cgugggggac cucuguggcg gggugaugcu cgcggcccag       2640
guguucaucg ucucgccgca guaccacugg uuugugcaag aaugcaauug cuccaucuac       2700
ccuggcacca ucacuggaca ccgcauggca ugggacauga ugaugaacug gucgcccacg       2760
gccaccauga uccuggcgua cgugaugcgc guccccgagg ucaucauaga caucguuagc       2820
ggggcucacu ggggcgucau guucggcuug gccuacuucu cuaugcaggg agcgugggcg       2880
aaggucauug ucauccuucu gcuggccgcu gggguggacg cgggcaccac caccguugga       2940
ggcgcuguug cacguuccac caacgugauu gccggcgugu ucagccaugg cccucagcag       3000
aacauucagc ucauuaacac caacggcagu uggcacauca accguacugc cuugaauugc       3060
aaugacuccu ugaacaccgg cuuucucgcg gccuuguucu acaccaaccg cuuuaacucg       3120
ucaggguguc cagggcgccu guccgccugc cgcaacaucg aggcuuuccg gauagggugg       3180
ggcacccuac aguacgagga uaaugucacc aauccagagg auaugaggcc guacugcugg       3240
cacuaccccc caaagccgug uggcguaguc cccgcgaggu cugugugugg cccaguguac       3300
uguuucaccc ccagcccggu aguagugggc acgaccgaca gacguggagu gcccaccuac       3360
acauggggag agaaugagac agaugucuuc cuacugaaca gcacccgacc gccgcagggc       3420
ucaugguucg gcugcacgug gaugaacucc acugguuuca ccaagacuug uggcgcgcca       3480
ccuugccgca ccagagcuga cuucaacgcc agcacggacu uguugugccc uacggauugu       3540
uuuaggaagc auccugaugc cacuuauauu aagugugguu cugggcccug gcucacacca       3600
aagugccugg uccacuaccc uuacagacuc uggcauuacc ccugcacagu caauuuuacc       3660
aucuucaaga uaagaaugua uguagggggg guugagcaca ggcucacggc cgcaugcaac       3720
uucacucgug gggaucgcug cgacuuggag gacagggaca ggagucagcu gucuccucug       3780
uugcacucua ccacggaaug ggccauccug cccugcaccu acucagacuu acccgcuuug       3840
ucaacugguc uucuccaccu ucaccagaac aucguggacg uacaauacau guauggccuc       3900
ucaccugcua ucacaaaaua cgucguucga ugggaguggg ugguacucuu auuccugcuc       3960
uuagcggacg ccagagucug cgccugcuug uggaugcuca ucuuguuggg ccaggccgaa       4020
gcagcauugg agaaguuggu cgucuugcac gcugcgagug cggcuaacug ccauggccuc       4080
cuauauuuug ccaucuucuu cguggcagcu uggcacauca ggggucgggu gguccccuug       4140
accaccuauu gccucacugg ccuauggccc uucugccuac ugcucauggc acugccccgg       4200
caggcuuaug ccuaugacgc accugugcac ggacagauag gcguggguuu guugauauug       4260
aucacccucu ucacacucac cccgggguau aagacccucc ucggccagug ucuguggugg       4320
uugugcuauc uccugacccu gggggaagcc augauucagg aguggguacc acccaugcag       4380
gugcgcggcg gccgcgaugg caucgcgugg gccgucacua uauucugccc ggguguggug       4440
uuugacauua ccaaauggcu uuuggcguug cuugggccug cuuaccucuu aagggccgcu       4500
uugacacaug ugccguacuu cgucagagcu cacgcucuga uaaggguaug cgcuuuggug       4560
aagcagcucg cgggggguag guauguucag guggcgcuau uggcccuugg cagguggacu       4620
ggcaccuaca ucuaugacca ccucacaccu augucggacu gggccgcuag cggccugcgc       4680
gacuuagcgg ucgccgugga acccaucauc uucaguccga uggagaagaa ggucaucguc       4740
uggggagcgg agacggcugc auguggggac auucuacaug gacuucccgu guccgcccga       4800
cucggccagg agauccuccu cggcccagcu gauggcuaca ccuccaaggg guggaagcuc       4860
cuugcuccca ucacugcuua ugcccagcaa acacgaggcc uccugggcgc cauaguggug       4920
aguaugacgg ggcgugacag gacagaacag gccggggaag uccaaauccu guccacaguc       4980
ucucaguccu uccucggaac aaccaucucg gggguuuugu ggacuguuua ccacggagcu       5040
ggcaacaaga cucuagccgg cuuacggggu ccggucacgc agauguacuc gagugcugag       5100
ggggacuugg uaggcuggcc cagccccccu gggaccaagu cuuuggagcc gugcaagugu       5160
ggagccgucg accuauaucu ggucacgcgg aacgcugaug ucaucccggc ucggagacgc       5220
ggggacaagc ggggagcauu gcucuccccg agacccauuu cgaccuugaa gggguccucg       5280
ggggggccgg ugcucugccc uaggggccac gucguugggc ucuuccgagc agcugugugc       5340
ucucggggcg uggccaaauc caucgauuuc auccccguug agacacucga cguuguuaca       5400
aggucuccca cuuucaguga caacagcacg ccaccggcug ugccccagac cuaucagguc       5460
ggguacuugc augcuccaac uggcagugga aagagcacca aggucccugu cgcguaugcc       5520
gcccaggggu acaaaguacu agugcuuaac cccucgguag cugccacccu gggguuuggg       5580
gcguaccuau ccaaggcaca uggcaucaau cccaacauua ggacuggagu caggaccgug       5640
augaccgggg aggccaucac guacuccaca uauggcaaau uucucgccga ugggggcugc       5700
gcuagcggcg ccuaugacau caucauaugc gaugaaugcc acgcugugga ugcuaccucc       5760
auucucggca ucggaacggu ccuugaucaa gcagagacag ccggggucag acuaacugug       5820
cuggcuacgg ccacaccccc cgggucagug acaacccccc aucccgauau agaagaggua       5880
ggccucgggc gggaggguga gauccccuuc uaugggaggg cgauuccccu auccugcauc       5940
aagggaggga gacaccugau uuucugccac ucaaagaaaa agugugacga gcucgcggcg       6000
gcccuucggg gcaugggcuu gaaugccgug gcauacuaua gaggguugga cgucuccaua       6060
auaccagcuc agggagaugu gguggucguc gccaccgacg cccucaugac gggguacacu       6120
ggagacuuug acuccgugau cgacugcaau guagcgguca cccaagcugu cgacuucagc       6180
cuggacccca ccuucacuau aaccacacag acugucccac aagacgcugu cucacgcagu       6240
cagcgccgcg ggcgcacagg uagaggaaga cagggcacuu auagguaugu uuccacuggu       6300
gaacgagccu caggaauguu ugacagugua gugcuuugug agugcuacga cgcaggggcu       6360
gcgugguacg aucucacacc agcggagacc accgucaggc uuagagcgua uuucaacacg       6420
cccggccuac ccguguguca agaccaucuu gaauuuuggg aggcaguuuu caccggccuc       6480
acacacauag acgcccacuu ccucucccaa acaaagcaag cgggggagaa cuucgcguac       6540
cuaguagccu accaagcuac ggugugcgcc agagccaagg ccccuccccc guccugggac       6600
gccaugugga agugccuggc ccgacucaag ccuacgcuug cgggccccac accucuccug       6660
uaccguuugg gcccuauuac caaugagguc acccucacac acccugggac gaaguacauc       6720
gccacaugca ugcaagcuga ccuugagguc augaccagca cguggguccu agcuggagga       6780
guccuggcag ccgucgccgc auauugccug gcgacuggau gcguuuccau caucggccgc       6840
uugcacguca accagcgagu cgucguugcg ccggauaagg agguccugua ugaggcuuuu       6900
gaugagaugg aggaaugcgc cucuagggcg gcucucaucg aagaggggca gcggauagcc       6960
gagauguuga aguccaagau ccaaggcuug cugcagcagg ccucuaagca ggcccaggac       7020
auacaacccg cuaugcaggc uucauggccc aaaguggaac aauuuugggc cagacacaug       7080
uggaacuuca uuagcggcau ccaauaccuc gcaggauugu caacacugcc agggaacccc       7140
gcgguggcuu ccaugauggc auucagugcc gcccucacca guccguuguc gaccaguacc       7200
accauccuuc ucaacaucau gggaggcugg uuagcguccc agaucgcacc acccgcgggg       7260
gccaccggcu uugucgucag uggccuggug ggggcugccg ugggcagcau aggccugggu       7320
aaggugcugg uggacauccu ggcaggauau ggugcgggca uuucgggggc ccucgucgca       7380
uucaagauca ugucuggcga gaagcccucu auggaagaug ucaucaaucu acugccuggg       7440
auccugucuc cgggagcccu gguggugggg gucaucugcg cggccauucu gcgccgccac       7500
gugggaccgg gggagggcgc gguccaaugg augaacaggc uuauugccuu ugcuuccaga       7560
ggaaaccacg ucgccccuac ucacuacgug acggagucgg augcgucgca gcgugugacc       7620
caacuacuug gcucucuuac uauaaccagc cuacucagaa gacuccacaa uuggauaacu       7680
gaggacugcc ccaucccaug cuccggaucc uggcuccgcg acguguggga cuggguuugc       7740
accaucuuga cagacuucaa aaauuggcug accucuaaau uguuccccaa gcugcccggc       7800
cuccccuuca ucucuuguca aaagggguac aagggugugu gggccggcac uggcaucaug       7860
accacgcgcu gcccuugcgg cgccaacauc ucuggcaaug uccgccuggg cucuaugagg       7920
aucacagggc cuaaaaccug caugaacacc uggcagggga ccuuuccuau caauugcuac       7980
acggagggcc agugcgcgcc gaaacccccc acgaacuaca agaccgccau cuggagggug       8040
gcggccucgg aguacgcgga ggugacgcag caugggucgu acuccuaugu aacaggacug       8100
accacugaca aucugaaaau uccuugccaa cuaccuucuc cagaguuuuu cuccugggug       8160
gacggugugc agauccauag guuugcaccc acaccaaagc cguuuuuccg ggaugagguc       8220
ucguucugcg uugggcuuaa uuccuaugcu gucggguccc agcuucccug ugaaccugag       8280
cccgacgcag acguauugag guccaugcua acagauccgc cccacaucac ggcggagacu       8340
gcggcgcggc gcuuggcacg gggaucaccu ccaucugagg cgagcuccuc agugagccag       8400
cuaucagcac cgucgcugcg ggccaccugc accacccaca gcaacaccua ugacguggac       8460
auggucgaug ccaaccugcu cauggagggc gguguggcuc agacagagcc ugaguccagg       8520
gugcccguuc uggacuuucu cgagccaaug gccgaggaag agagcgaccu ugagcccuca       8580
auaccaucgg agugcaugcu ccccaggagc ggguuuccac gggccuuacc ggcuugggca       8640
cggccugacu acaacccgcc gcucguggaa ucguggagga ggccagauua ccaaccgccc       8700
accguugcug guugugcucu cccccccccc aagaaggccc cgacgccucc cccaaggaga       8760
cgccggacag ugggucugag cgagagcacc auaucagaag cccuccagca acuggccauc       8820
aagaccuuug gccagccccc cucgagcggu gaugcaggcu cguccacggg ggcgggcgcc       8880
gccgaauccg gcgguccgac guccccuggu gagccggccc ccucagagac agguuccgcc       8940
uccucuaugc ccccccucga gggggagccu ggagauccgg accuggaguc ugaucaggua       9000
gagcuucaac cuccccccca gggggggggg guagcucccg guucgggcuc ggggucuugg       9060
ucuacuugcu ccgaggagga cgauaccacc gugugcugcu ccaugucaua cuccuggacc       9120
ggggcucuaa uaacucccug uagccccgaa gaggaaaagu ugccaaucaa cccuuugagu       9180
aacucgcugu ugcgauacca uaacaaggug uacuguacaa caucaaagag cgccucacag       9240
agggcuaaaa agguaacuuu ugacaggacg caagugcucg acgcccauua ugacucaguc       9300
uuaaaggaca ucaagcuagc ggcuuccaag gucagcgcaa ggcuccucac cuuggaggag       9360
gcgugccagu ugacuccacc ccauucugca agauccaagu auggauucgg ggccaaggag       9420
guccgcagcu uguccgggag ggccguuaac cacaucaagu ccguguggaa ggaccuccug       9480
gaagacccac aaacaccaau ucccacaacc aucauggcca aaaaugaggu guucugcgug       9540
gaccccgcca agggggguaa gaaaccagcu cgccucaucg uuuacccuga ccucggcguc       9600
cgggucugcg agaaaauggc ccucuaugac auuacacaaa agcuuccuca ggcgguaaug       9660
ggagcuuccu auggcuucca guacuccccu gcccaacggg uggaguaucu cuugaaagca       9720
ugggcggaaa agaaggaccc cauggguuuu ucguaugaua cccgaugcuu cgacucaacc       9780
gucacugaga gagacaucag gaccgaggag uccauauacc aggccugcuc ccugcccgag       9840
gaggcccgca cugccauaca cucgcugacu gagagacuuu acguaggagg gcccauguuc       9900
aacagcaagg gucaaaccug cgguuacaga cguugccgcg ccagcggggu gcuaaccacu       9960
agcaugggua acaccaucac augcuaugug aaagcccuag cggccugcaa ggcugcgggg       10020
auaguugcgc ccacaaugcu gguaugcggc aaugaccuag uagucaucuc agaaagccag       10080
gggacugagg aggacgagcg gaaccugaga gccuucacgg aggccaugac cagguacucu       10140
gccccuccug gugauccccc cagaccggaa uaugaccugg agcuaauaac auccuguucc       10200
ucaaaugugu cuguggcguu gggcccgcgg ggccgccgca gauacuaccu gaccagagac       10260
ccaaccacuc cacucgcccg ggcugccugg gaaacaguua gacacucccc uaucaauuca       10320
uggcugggaa acaucaucca guaugcucca accauauggg uucgcauggu ccuaaugaca       10380
cacuucuucu ccauucucau gguccaagac acccuggacc agaaccucaa cuuugagaug       10440
uauggaucag uauacuccgu gaauccuuug gaccuuccag ccauaauuga gagguuacac       10500
gggcuugacg ccuuuucuau gcacacauac ucucaccacg aacugacgcg gguggcuuca       10560
gcccucagaa aacuuggggc gccaccccuc agggugugga agagucgggc ucgcgcaguc       10620
agggcguccc ucaucucccg uggagggaaa gcggccguuu gcggccgaua ucucuucaau       10680
ugggcgguga agaccaagcu caaacucacu ccauugccgg aggcgcgccu acuggacuua       10740
uccaguuggu ucaccgucgg cgccggcggg ggcgacauuu uucacagcgu gucgcgcgcc       10800
cgaccccgcu cauuacucuu cggccuacuc cuacuuuucg uagggguagg ccucuuccua       10860
cuccccgcuc gguagagcgg cacacacuag guacacucca uagcuaacug uuccuuuuuu       10920
uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuucuu uuuuuuuuuu uucccucuuu       10980
cuucccuucu caucuuauuc uacuuucuuu cuugguggcu ccaucuuagc ccuagucacg       11040
gcuagcugug aaagguccgu gagccgcaug acugcagaga gugccguaac uggucucucu       11100
gcagaucaug u                                                            11111
<210>15
<211>9707
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：含来自JFH-1克隆的全长丙型肝炎病毒基因组RNA的复制子RNA，其中氨基酸基序GDD已突变为GND
<400>15
gaauucuaau acgacucacu auagaccugc cccuaauagg ggcgacacuc cgccaugaau       60
cacuccccug ugaggaacua cugucuucac gcagaaagcg ccuagccaug gcguuaguau       120
gagugucgua cagccuccag gccccccccu cccgggagag ccauaguggu cugcggaacc       180
ggugaguaca ccggaauugc cgggaagacu ggguccuuuc uuggauaaac ccacucuaug       240
cccggccauu ugggcgugcc cccgcaagac ugcuagccga guagcguugg guugcgaaag       300
gccuuguggu acugccugau agggcgcuug cgagugcccc gggaggucuc guagaccgug       360
caccaugagc acaaauccu aaaccucaaag aaaaaccaaa agaaacacca accgucgccc       420
agaagacguu aaguucccgg gcggcggcca gaucguuggc ggaguauacu uguugccgcg       480
caggggcccc agguugggug ugcgcacgac aaggaaaacu ucggagcggu cccagccacg       540
ugggagacgc cagcccaucc ccaaagaucg gcgcuccacu ggcaaggccu ggggaaaacc       600
aggucgcccc uggccccuau augggaauga gggacucggc ugggcaggau ggcuccuguc       660
cccccgaggc ucucgccccu ccuggggccc cacugacccc cggcauaggu cgcgcaacgu       720
ggguaaaguc aucgacaccc uaacgugugg cuuugccgac cucauggggu acauccccgu       780
cguaggcgcc ccgcuuagug gcgccgccag agcugucgcg cacggcguga gaguccugga       840
ggacgggguu aauuaugcaa cagggaaccu acccgguuuc cccuuuucua ucuucuugcu       900
ggcccuguug uccugcauca ccguuccggu cucugcugcc caggugaaga auaccaguag       960
cagcuacaug gugaccaaug acugcuccaa ugacagcauc acuuggcagc ucgaggcugc       1020
gguucuccac guccccgggu gcgucccgug cgagagagug gggaauacgu cacgguguug       1080
ggugccaguc ucgccaaaca uggcugugcg gcagcccggu gcccucacgc agggucugcg       1140
gacgcacauc gauaugguug ugauguccgc caccuucugc ucugcucucu acguggggga       1200
ccucuguggc ggggugaugc ucgcggccca gguguucauc gucucgccgc aguaccacug       1260
guuugugcaa gaaugcaauu gcuccaucua cccuggcacc aucacuggac accgcauggc       1320
augggacaug augaugaacu ggucgcccac ggccaccaug auccuggcgu acgugaugcg       1380
cguccccgag gucaucauag acaucguuag cggggcucac uggggcguca uguucggcuu       1440
ggccuacuuc ucuaugcagg gagcgugggc gaaggucauu gucauccuuc ugcuggccgc       1500
ugggguggac gcgggcacca ccaccguugg aggcgcuguu gcacguucca ccaacgugau       1560
ugccggcgug uucagccaug gcccucagca gaacauucag cucauuaaca ccaacggcag       1620
uuggcacauc aaccguacug ccuugaauug caaugacucc uugaacaccg gcuuucucgc       1680
ggccuuguuc uacaccaacc gcuuuaacuc gucagggugu ccagggcgcc uguccgccug       1740
ccgcaacauc gaggcuuucc ggauagggug gggcacccua caguacgagg auaaugucac       1800
caauccagag gauaugaggc cguacugcug gcacuacccc ccaaagccgu guggcguagu       1860
ccccgcgagg ucugugugug gcccagugua cuguuucacc cccagcccgg uaguaguggg       1920
cacgaccgac agacguggag ugcccaccua cacaugggga gagaaugaga cagaugucuu       1980
ccuacugaac agcacccgac cgccgcaggg cucaugguuc ggcugcacgu ggaugaacuc       2040
cacugguuuc accaagacuu guggcgcgcc accuugccgc accagagcug acuucaacgc       2100
cagcacggac uuguugugcc cuacggauug uuuuaggaag cauccugaug ccacuuauau       2160
uaaguguggu ucugggcccu ggcucacacc aaagugccug guccacuacc cuuacagacu       2220
cuggcauuac cccugcacag ucaauuuuac caucuucaag auaagaaugu auguaggggg       2280
gguugagcac aggcucacgg ccgcaugcaa cuucacucgu ggggaucgcu gcgacuugga       2340
ggacagggac aggagucagc ugucuccucu guugcacucu accacggaau gggccauccu       2400
gcccugcacc uacucagacu uacccgcuuu gucaacuggu cuucuccacc uucaccagaa       2460
caucguggac guacaauaca uguauggccu cucaccugcu aucacaaaau acgucguucg       2520
augggagugg gugguacucu uauuccugcu cuuagcggac gccagagucu gcgccugcuu       2580
guggaugcuc aucuuguugg gccaggccga agcagcauug gagaaguugg ucgucuugca       2640
cgcugcgagu gcggcuaacu gccauggccu ccuauauuuu gccaucuucu ucguggcagc       2700
uuggcacauc aggggucggg ugguccccuu gaccaccuau ugccucacug gccuauggcc       2760
cuucugccua cugcucaugg cacugccccg gcaggcuuau gccuaugacg caccugugca       2820
cggacagaua ggcguggguu uguugauauu gaucacccuc uucacacuca ccccggggua       2880
uaagacccuc cucggccagu gucuguggug guugugcuau cuccugaccc ugggggaagc       2940
caugauucag gaguggguac cacccaugca ggugcgcggc ggccgcgaug gcaucgcgug       3000
ggccgucacu auauucugcc cggguguggu guuugacauu accaaauggc uuuuggcguu       3060
gcuugggccu gcuuaccucu uaagggccgc uuugacacau gugccguacu ucgucagagc       3120
ucacgcucug auaaggguau gcgcuuuggu gaagcagcuc gcggggggua gguauguuca       3180
gguggcgcua uuggcccuug gcagguggac uggcaccuac aucuaugacc accucacacc       3240
uaugucggac ugggccgcua gcggccugcg cgacuuagcg gucgccgugg aacccaucau       3300
cuucaguccg auggagaaga aggucaucgu cuggggagcg gagacggcug caugugggga       3360
cauucuacau ggacuucccg uguccgcccg acucggccag gagauccucc ucggcccagc       3420
ugauggcuac accuccaagg gguggaagcu ccuugcuccc aucacugcuu augcccagca       3480
aacacgaggc cuccugggcg ccauaguggu gaguaugacg gggcgugaca ggacagaaca       3540
ggccggggaa guccaaaucc uguccacagu cucucagucc uuccucggaa caaccaucuc       3600
ggggguuuug uggacuguuu accacggagc uggcaacaag acucuagccg gcuuacgggg       3660
uccggucacg cagauguacu cgagugcuga gggggacuug guaggcuggc ccagcccccc       3720
ugggaccaag ucuuuggagc cgugcaagug uggagccguc gaccuauauc uggucacgcg       3780
gaacgcugau gucaucccgg cucggagacg cggggacaag cggggagcau ugcucucccc       3840
gagacccauu ucgaccuuga agggguccuc gggggggccg gugcucugcc cuaggggcca       3900
cgucguuggg cucuuccgag cagcugugug cucucggggc guggccaaau ccaucgauuu       3960
cauccccguu gagacacucg acguuguuac aaggucuccc acuuucagug acaacagcac       4020
gccaccggcu gugccccaga ccuaucaggu cggguacuug caugcuccaa cuggcagugg       4080
aaagagcacc aaggucccug ucgcguaugc cgcccagggg uacaaaguac uagugcuuaa       4140
ccccucggua gcugccaccc ugggguuugg ggcguaccua uccaaggcac auggcaucaa       4200
ucccaacauu aggacuggag ucaggaccgu gaugaccggg gaggccauca cguacuccac       4260
auauggcaaa uuucucgccg augggggcug cgcuagcggc gccuaugaca ucaucauaug       4320
cgaugaaugc cacgcugugg augcuaccuc cauucucggc aucggaacgg uccuugauca       4380
agcagagaca gccgggguca gacuaacugu gcuggcuacg gccacacccc ccgggucagu       4440
gacaaccccc caucccgaua uagaagaggu aggccucggg cgggagggug agauccccuu       4500
cuaugggagg gcgauucccc uauccugcau caagggaggg agacaccuga uuuucugcca       4560
cucaaagaaa aagugugacg agcucgcggc ggcccuucgg ggcaugggcu ugaaugccgu       4620
ggcauacuau agaggguugg acgucuccau aauaccagcu cagggagaug ugguggucgu       4680
cgccaccgac gcccucauga cgggguacac uggagacuuu gacuccguga ucgacugcaa       4740
uguagcgguc acccaagcug ucgacuucag ccuggacccc accuucacua uaaccacaca       4800
gacuguccca caagacgcug ucucacgcag ucagcgccgc gggcgcacag guagaggaag       4860
acagggcacu uauagguaug uuuccacugg ugaacgagcc ucaggaaugu uugacagugu       4920
agugcuuugu gagugcuacg acgcaggggc ugcgugguac gaucucacac cagcggagac       4980
caccgucagg cuuagagcgu auuucaacac gcccggccua cccguguguc aagaccaucu       5040
ugaauuuugg gaggcaguuu ucaccggccu cacacacaua gacgcccacu uccucuccca       5100
aacaaagcaa gcgggggaga acuucgcgua ccuaguagcc uaccaagcua cggugugcgc       5160
cagagccaag gccccucccc cguccuggga cgccaugugg aagugccugg cccgacucaa       5220
gccuacgcuu gcgggcccca caccucuccu guaccguuug ggcccuauua ccaaugaggu       5280
cacccucaca cacccuggga cgaaguacau cgccacaugc augcaagcug accuugaggu       5340
caugaccagc acgugggucc uagcuggagg aguccuggca gccgucgccg cauauugccu       5400
ggcgacugga ugcguuucca ucaucggccg cuugcacguc aaccagcgag ucgucguugc       5460
gccggauaag gagguccugu augaggcuuu ugaugagaug gaggaaugcg ccucuagggc       5520
ggcucucauc gaagaggggc agcggauagc cgagauguug aaguccaaga uccaaggcuu       5580
gcugcagcag gccucuaagc aggcccagga cauacaaccc gcuaugcagg cuucauggcc       5640
caaaguggaa caauuuuggg ccagacacau guggaacuuc auuagcggca uccaauaccu       5700
cgcaggauug ucaacacugc cagggaaccc cgcgguggcu uccaugaugg cauucagugc       5760
cgcccucacc aguccguugu cgaccaguac caccauccuu cucaacauca ugggaggcug       5820
guuagcgucc cagaucgcac cacccgcggg ggccaccggc uuugucguca guggccuggu       5880
gggggcugcc gugggcagca uaggccuggg uaaggugcug guggacaucc uggcaggaua       5940
uggugcgggc auuucggggg cccucgucgc auucaagauc augucuggcg agaagcccuc       6000
uauggaagau gucaucaauc uacugccugg gauccugucu ccgggagccc uggugguggg       6060
ggucaucugc gcggccauuc ugcgccgcca cgugggaccg ggggagggcg cgguccaaug       6120
gaugaacagg cuuauugccu uugcuuccag aggaaaccac gucgccccua cucacuacgu       6180
gacggagucg gaugcgucgc agcgugugac ccaacuacuu ggcucucuua cuauaaccag       6240
ccuacucaga agacuccaca auuggauaac ugaggacugc cccaucccau gcuccggauc       6300
cuggcuccgc gacguguggg acuggguuug caccaucuug acagacuuca aaaauuggcu       6360
gaccucuaaa uuguucccca agcugcccgg ccuccccuuc aucucuuguc aaaaggggua       6420
caagggugug ugggccggca cuggcaucau gaccacgcgc ugcccuugcg gcgccaacau       6480
cucuggcaau guccgccugg gcucuaugag gaucacaggg ccuaaaaccu gcaugaacac       6540
cuggcagggg accuuuccua ucaauugcua cacggagggc cagugcgcgc cgaaaccccc       6600
cacgaacuac aagaccgcca ucuggagggu ggcggccucg gaguacgcgg aggugacgca       6660
gcaugggucg uacuccuaug uaacaggacu gaccacugac aaucugaaaa uuccuugcca       6720
acuaccuucu ccagaguuuu ucuccugggu ggacggugug cagauccaua gguuugcacc       6780
cacaccaaag ccguuuuucc gggaugaggu cucguucugc guugggcuua auuccuaugc       6840
ugucgggucc cagcuucccu gugaaccuga gcccgacgca gacguauuga gguccaugcu       6900
aacagauccg ccccacauca cggcggagac ugcggcgcgg cgcuuggcac ggggaucacc       6960
uccaucugag gcgagcuccu cagugagcca gcuaucagca ccgucgcugc gggccaccug       7020
caccacccac agcaacaccu augacgugga cauggucgau gccaaccugc ucauggaggg       7080
cgguguggcu cagacagagc cugaguccag ggugcccguu cuggacuuuc ucgagccaau       7140
ggccgaggaa gagagcgacc uugagcccuc aauaccaucg gagugcaugc uccccaggag       7200
cggguuucca cgggccuuac cggcuugggc acggccugac uacaacccgc cgcucgugga       7260
aucguggagg aggccagauu accaaccgcc caccguugcu gguugugcuc uccccccccc       7320
caagaaggcc ccgacgccuc ccccaaggag acgccggaca gugggucuga gcgagagcac       7380
cauaucagaa gcccuccagc aacuggccau caagaccuuu ggccagcccc ccucgagcgg       7440
ugaugcaggc ucguccacgg gggcgggcgc cgccgaaucc ggcgguccga cguccccugg       7500
ugagccggcc cccucagaga cagguuccgc cuccucuaug cccccccucg agggggagcc       7560
uggagauccg gaccuggagu cugaucaggu agagcuucaa ccuccccccc aggggggggg       7620
gguagcuccc gguucgggcu cggggucuug gucuacuugc uccgaggagg acgauaccac       7680
cgugugcugc uccaugucau acuccuggac cggggcucua auaacucccu guagccccga       7740
agaggaaaag uugccaauca acccuuugag uaacucgcug uugcgauacc auaacaaggu       7800
guacuguaca acaucaaaga gcgccucaca gagggcuaaa aagguaacuu uugacaggac       7860
gcaagugcuc gacgcccauu augacucagu cuuaaaggac aucaagcuag cggcuuccaa       7920
ggucagcgca aggcuccuca ccuuggagga ggcgugccag uugacuccac cccauucugc       7980
aagauccaag uauggauucg gggccaagga gguccgcagc uuguccggga gggccguuaa       8040
ccacaucaag uccgugugga aggaccuccu ggaagaccca caaacaccaa uucccacaac       8100
caucauggcc aaaaaugagg uguucugcgu ggaccccgcc aaggggggua agaaaccagc       8160
ucgccucauc guuuacccug accucggcgu ccgggucugc gagaaaaugg cccucuauga       8220
cauuacacaa aagcuuccuc aggcgguaau gggagcuucc uauggcuucc aguacucccc       8280
ugcccaacgg guggaguauc ucuugaaagc augggcggaa aagaaggacc ccauggguuu       8340
uucguaugau acccgaugcu ucgacucaac cgucacugag agagacauca ggaccgagga       8400
guccauauac caggccugcu cccugcccga ggaggcccgc acugccauac acucgcugac       8460
ugagagacuu uacguaggag ggcccauguu caacagcaag ggucaaaccu gcgguuacag       8520
acguugccgc gccagcgggg ugcuaaccac uagcaugggu aacaccauca caugcuaugu       8580
gaaagcccua gcggccugca aggcugcggg gauaguugcg cccacaaugc ugguaugcgg       8640
caaugaccua guagucaucu cagaaagcca ggggacugag gaggacgagc ggaaccugag       8700
agccuucacg gaggccauga ccagguacuc ugccccuccu ggugaucccc ccagaccgga       8760
auaugaccug gagcuaauaa cauccuguuc cucaaaugug ucuguggcgu ugggcccgcg       8820
gggccgccgc agauacuacc ugaccagaga cccaaccacu ccacucgccc gggcugccug       8880
ggaaacaguu agacacuccc cuaucaauuc auggcuggga aacaucaucc aguaugcucc       8940
aaccauaugg guucgcaugg uccuaaugac acacuucuuc uccauucuca ugguccaaga       9000
cacccuggac cagaaccuca acuuugagau guauggauca guauacuccg ugaauccuuu       9060
ggaccuucca gccauaauug agagguuaca cgggcuugac gccuuuucua ugcacacaua       9120
cucucaccac gaacugacgc ggguggcuuc agcccucaga aaacuugggg cgccaccccu       9180
cagggugugg aagagucggg cucgcgcagu cagggcgucc cucaucuccc guggagggaa       9240
agcggccguu ugcggccgau aucucuucaa uugggcggug aagaccaagc ucaaacucac       9300
uccauugccg gaggcgcgcc uacuggacuu auccaguugg uucaccgucg gcgccggcgg       9360
gggcgacauu uuucacagcg ugucgcgcgc ccgaccccgc ucauuacucu ucggccuacu       9420
ccuacuuuuc guagggguag gccucuuccu acuccccgcu cgguagagcg gcacacacua       9480
gguacacucc auagcuaacu guuccuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu       9540
uuuuuuuucu uuuuuuuuuu uuucccucuu ucuucccuuc ucaucuuauu cuacuuucuu       9600
ucuugguggc uccaucuuag cccuagucac ggcuagcugu gaaagguccg ugagccgcau       9660
gacugcagag agugccguaa cuggucucuc ugcagaucau gucuaga                     9707
<210>16
<211>17
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>16
cgggagagcc atagtgg                                                      17
<210>17
<211>19
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>17
agtaccacaa ggcctttcg                                                    19
<210>18
<211>21
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>18
ctgcggaacc ggtgagtaca c                                                 21
<210>19
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>19
aacaagatgg attgcacgca                                                   20
<210>20
<211>20
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：引物
<400>20
cgtcaagaag gcgatagaag                                                   20
<210>21
<211>11969
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：来源于表达载体rFGR-JFH1/Luc的复制子RNA
<400>21
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gaagacgcca aaaacauaaa       420
gaaaggcccg gcgccauucu auccucugga ggauggaacc gcuggagagc aacugcauaa       480
ggcuaugaag agauacgccc ugguuccugg aacaauugcu uuuacagaug cacauaucga       540
ggugaacauc acguacgcgg aauacuucga aauguccguu cgguuggcag aagcuaugaa       600
acgauauggg cugaauacaa aucacagaau cgucguaugc agugaaaacu cucuucaauu       660
cuuuaugccg guguugggcg cguuauuuau cggaguugca guugcgcccg cgaacgacau       720
uuauaaugaa cgugaauugc ucaacaguau gaacauuucg cagccuaccg uaguguuugu       780
uuccaaaaag ggguugcaaa aaauuuugaa cgugcaaaaa aaauuaccaa uaauccagaa       840
aauuauuauc auggauucua aaacggauua ccagggauuu cagucgaugu acacguucgu       900
cacaucucau cuaccucccg guuuuaauga auacgauuuu guaccagagu ccuuugaucg       960
ugacaaaaca auugcacuga uaaugaacuc cucuggaucu acuggguuac cuaagggugu       1020
ggcccuuccg cauagaacug ccugcgucag auucucgcau gccagagauc cuauuuuugg       1080
caaucaaauc auuccggaua cugcgauuuu aaguguuguu ccauuccauc acgguuuugg       1140
aauguuuacu acacucggau auuugauaug uggauuucga gucgucuuaa uguauagauu       1200
ugaagaagag cuguuuuuac gaucccuuca ggauuacaaa auucaaagug cguugcuagu       1260
accaacccua uuuucauucu ucgccaaaag cacucugauu gacaaauacg auuuaucuaa       1320
uuuacacgaa auugcuucug ggggcgcacc ucuuucgaaa gaagucgggg aagcgguugc       1380
aaaacgcuuc caucuuccag ggauacgaca aggauauggg cucacugaga cuacaucagc       1440
uauucugauu acacccgagg gggaugauaa accgggcgcg gucgguaaag uuguuccauu       1500
uuuugaagcg aagguugugg aucuggauac cgggaaaacg cugggcguua aucagagagg       1560
cgaauuaugu gucagaggac cuaugauuau guccgguuau guaaacaauc cggaagcgac       1620
caacgccuug auugacaagg auggauggcu acauucugga gacauagcuu acugggacga       1680
agacgaacac uucuucauag uugaccgcuu gaagucuuua auuaaauaca aaggauauca       1740
gguggccccc gcugaauugg aaucgauauu guuacaacac cccaacaucu ucgacgcggg       1800
cguggcaggu cuucccgacg augacgccgg ugaacuuccc gccgccguug uuguuuugga       1860
gcacggaaag acgaugacgg aaaaagagau cguggauuac gucgccaguc aaguaacaac       1920
cgcgaaaaag uugcgcggag gaguuguguu uguggacgaa guaccgaaag gucuuaccgg       1980
aaaacucgac gcaagaaaaa ucagagagau ccucauaaag gccaagaagg gcggaaaguc       2040
caaauuguaa guuuaaaccc ucucccuccc cccccccuaa cguuacuggc cgaagccgcu       2100
uggaauaagg ccggugugcg uuugucuaua uguuauuuuc caccauauug ccgucuuuug       2160
gcaaugugag ggcccggaaa ccuggcccug ucuucuugac gagcauuccu aggggucuuu       2220
ccccucucgc caaaggaaug caaggucugu ugaaugucgu gaaggaagca guuccucugg       2280
aagcuucuug aagacaaaca acgucuguag cgacccuuug caggcagcgg aaccccccac       2340
cuggcgacag gugccucugc ggccaaaagc cacguguaua agauacaccu gcaaaggcgg       2400
cacaacccca gugccacguu gugaguugga uaguugugga aagagucaaa uggcucuccu       2460
caagcguauu caacaagggg cugaaggaug cccagaaggu accccauugu augggaucug       2520
aucuggggcc ucggugcaca ugcuuuacau guguuuaguc gagguuaaaa aaacgucuag       2580
gccccccgaa ccacggggac gugguuuucc uuugaaaaac acgaugauac caugagcaca       2640
aauccuaaac cucaaagaaa aaccaaaaga aacaccaacc gucgcccaga agacguuaag       2700
uucccgggcg gcggccagau cguuggcgga guauacuugu ugccgcgcag gggccccagg       2760
uugggugugc gcacgacaag gaaaacuucg gagcgguccc agccacgugg gagacgccag       2820
cccaucccca aagaucggcg cuccacuggc aaggccuggg gaaaaccagg ucgccccugg       2880
ccccuauaug ggaaugaggg acucggcugg gcaggauggc uccugucccc ccgaggcucu       2940
cgccccuccu ggggccccac ugacccccgg cauaggucgc gcaacguggg uaaagucauc       3000
gacacccuaa cguguggcuu ugccgaccuc augggguaca uccccgucgu aggcgccccg       3060
cuuaguggcg ccgccagagc ugucgcgcac ggcgugagag uccuggagga cgggguuaau       3120
uaugcaacag ggaaccuacc cgguuucccc uuuucuaucu ucuugcuggc ccuguugucc       3180
ugcaucaccg uuccggucuc ugcugcccag gugaagaaua ccaguagcag cuacauggug       3240
accaaugacu gcuccaauga cagcaucacu uggcagcucg aggcugcggu ucuccacguc       3300
cccgggugcg ucccgugcga gagagugggg aauacgucac gguguugggu gccagucucg       3360
ccaaacaugg cugugcggca gcccggugcc cucacgcagg gucugcggac gcacaucgau       3420
augguuguga uguccgccac cuucugcucu gcucucuacg ugggggaccu cuguggcggg       3480
gugaugcucg cggcccaggu guucaucguc ucgccgcagu accacugguu ugugcaagaa       3540
ugcaauugcu ccaucuaccc uggcaccauc acuggacacc gcauggcaug ggacaugaug       3600
augaacuggu cgcccacggc caccaugauc cuggcguacg ugaugcgcgu ccccgagguc       3660
aucauagaca ucguuagcgg ggcucacugg ggcgucaugu ucggcuuggc cuacuucucu       3720
augcagggag cgugggcgaa ggucauuguc auccuucugc uggccgcugg gguggacgcg       3780
ggcaccacca ccguuggagg cgcuguugca cguuccacca acgugauugc cggcguguuc       3840
agccauggcc cucagcagaa cauucagcuc auuaacacca acggcaguug gcacaucaac       3900
cguacugccu ugaauugcaa ugacuccuug aacaccggcu uucucgcggc cuuguucuac       3960
accaaccgcu uuaacucguc agggugucca gggcgccugu ccgccugccg caacaucgag       4020
gcuuuccgga uagggugggg cacccuacag uacgaggaua augucaccaa uccagaggau       4080
augaggccgu acugcuggca cuacccccca aagccgugug gcguaguccc cgcgaggucu       4140
guguguggcc caguguacug uuucaccccc agcccgguag uagugggcac gaccgacaga       4200
cguggagugc ccaccuacac auggggagag aaugagacag augucuuccu acugaacagc       4260
acccgaccgc cgcagggcuc augguucggc ugcacgugga ugaacuccac ugguuucacc       4320
aagacuugug gcgcgccacc uugccgcacc agagcugacu ucaacgccag cacggacuug       4380
uugugcccua cggauuguuu uaggaagcau ccugaugcca cuuauauuaa gugugguucu       4440
gggcccuggc ucacaccaaa gugccugguc cacuacccuu acagacucug gcauuacccc       4500
ugcacaguca auuuuaccau cuucaagaua agaauguaug uagggggggu ugagcacagg       4560
cucacggccg caugcaacuu cacucguggg gaucgcugcg acuuggagga cagggacagg       4620
agucagcugu cuccucuguu gcacucuacc acggaauggg ccauccugcc cugcaccuac       4680
ucagacuuac ccgcuuuguc aacuggucuu cuccaccuuc accagaacau cguggacgua       4740
caauacaugu auggccucuc accugcuauc acaaaauacg ucguucgaug ggagugggug       4800
guacucuuau uccugcucuu agcggacgcc agagucugcg ccugcuugug gaugcucauc       4860
uuguugggcc aggccgaagc agcauuggag aaguuggucg ucuugcacgc ugcgagugcg       4920
gcuaacugcc auggccuccu auauuuugcc aucuucuucg uggcagcuug gcacaucagg       4980
ggucgggugg uccccuugac caccuauugc cucacuggcc uauggcccuu cugccuacug       5040
cucauggcac ugccccggca ggcuuaugcc uaugacgcac cugugcacgg acagauaggc       5100
guggguuugu ugauauugau cacccucuuc acacucaccc cgggguauaa gacccuccuc       5160
ggccaguguc uguggugguu gugcuaucuc cugacccugg gggaagccau gauucaggag       5220
uggguaccac ccaugcaggu gcgcggcggc cgcgauggca ucgcgugggc cgucacuaua       5280
uucugcccgg gugugguguu ugacauuacc aaauggcuuu uggcguugcu ugggccugcu       5340
uaccucuuaa gggccgcuuu gacacaugug ccguacuucg ucagagcuca cgcucugaua       5400
aggguaugcg cuuuggugaa gcagcucgcg ggggguaggu auguucaggu ggcgcuauug       5460
gcccuuggca gguggacugg caccuacauc uaugaccacc ucacaccuau gucggacugg       5520
gccgcuagcg gccugcgcga cuuagcgguc gccguggaac ccaucaucuu caguccgaug       5580
gagaagaagg ucaucgucug gggagcggag acggcugcau guggggacau ucuacaugga       5640
cuucccgugu ccgcccgacu cggccaggag auccuccucg gcccagcuga uggcuacacc       5700
uccaaggggu ggaagcuccu ugcucccauc acugcuuaug cccagcaaac acgaggccuc       5760
cugggcgcca uaguggugag uaugacgggg cgugacagga cagaacaggc cggggaaguc       5820
caaauccugu ccacagucuc ucaguccuuc cucggaacaa ccaucucggg gguuuugugg       5880
acuguuuacc acggagcugg caacaagacu cuagccggcu uacggggucc ggucacgcag       5940
auguacucga gugcugaggg ggacuuggua ggcuggccca gccccccugg gaccaagucu       6000
uuggagccgu gcaagugugg agccgucgac cuauaucugg ucacgcggaa cgcugauguc       6060
aucccggcuc ggagacgcgg ggacaagcgg ggagcauugc ucuccccgag acccauuucg       6120
accuugaagg gguccucggg ggggccggug cucugcccua ggggccacgu cguugggcuc       6180
uuccgagcag cugugugcuc ucggggcgug gccaaaucca ucgauuucau ccccguugag       6240
acacucgacg uuguuacaag gucucccacu uucagugaca acagcacgcc accggcugug       6300
ccccagaccu aucaggucgg guacuugcau gcuccaacug gcaguggaaa gagcaccaag       6360
gucccugucg cguaugccgc ccagggguac aaaguacuag ugcuuaaccc cucgguagcu       6420
gccacccugg gguuuggggc guaccuaucc aaggcacaug gcaucaaucc caacauuagg       6480
acuggaguca ggaccgugau gaccggggag gccaucacgu acuccacaua uggcaaauuu       6540
cucgccgaug ggggcugcgc uagcggcgcc uaugacauca ucauaugcga ugaaugccac       6600
gcuguggaug cuaccuccau ucucggcauc ggaacggucc uugaucaagc agagacagcc       6660
ggggucagac uaacugugcu ggcuacggcc acaccccccg ggucagugac aaccccccau       6720
cccgauauag aagagguagg ccucgggcgg gagggugaga uccccuucua ugggagggcg       6780
auuccccuau ccugcaucaa gggagggaga caccugauuu ucugccacuc aaagaaaaag       6840
ugugacgagc ucgcggcggc ccuucggggc augggcuuga augccguggc auacuauaga       6900
ggguuggacg ucuccauaau accagcucag ggagaugugg uggucgucgc caccgacgcc       6960
cucaugacgg gguacacugg agacuuugac uccgugaucg acugcaaugu agcggucacc       7020
caagcugucg acuucagccu ggaccccacc uucacuauaa ccacacagac ugucccacaa       7080
gacgcugucu cacgcaguca gcgccgcggg cgcacaggua gaggaagaca gggcacuuau       7140
agguauguuu ccacugguga acgagccuca ggaauguuug acaguguagu gcuuugugag       7200
ugcuacgacg caggggcugc gugguacgau cucacaccag cggagaccac cgucaggcuu       7260
agagcguauu ucaacacgcc cggccuaccc gugugucaag accaucuuga auuuugggag       7320
gcaguuuuca ccggccucac acacauagac gcccacuucc ucucccaaac aaagcaagcg       7380
ggggagaacu ucgcguaccu aguagccuac caagcuacgg ugugcgccag agccaaggcc       7440
ccucccccgu ccugggacgc cauguggaag ugccuggccc gacucaagcc uacgcuugcg       7500
ggccccacac cucuccugua ccguuugggc ccuauuacca augaggucac ccucacacac       7560
ccugggacga aguacaucgc cacaugcaug caagcugacc uugaggucau gaccagcacg       7620
uggguccuag cuggaggagu ccuggcagcc gucgccgcau auugccuggc gacuggaugc       7680
guuuccauca ucggccgcuu gcacgucaac cagcgagucg ucguugcgcc ggauaaggag       7740
guccuguaug aggcuuuuga ugagauggag gaaugcgccu cuagggcggc ucucaucgaa       7800
gaggggcagc ggauagccga gauguugaag uccaagaucc aaggcuugcu gcagcaggcc       7860
ucuaagcagg cccaggacau acaacccgcu augcaggcuu cauggcccaa aguggaacaa       7920
uuuugggcca gacacaugug gaacuucauu agcggcaucc aauaccucgc aggauuguca       7980
acacugccag ggaaccccgc gguggcuucc augauggcau ucagugccgc ccucaccagu       8040
ccguugucga ccaguaccac cauccuucuc aacaucaugg gaggcugguu agcgucccag       8100
aucgcaccac ccgcgggggc caccggcuuu gucgucagug gccugguggg ggcugccgug       8160
ggcagcauag gccuggguaa ggugcuggug gacauccugg caggauaugg ugcgggcauu       8220
ucgggggccc ucgucgcauu caagaucaug ucuggcgaga agcccucuau ggaagauguc       8280
aucaaucuac ugccugggau ccugucuccg ggagcccugg uggugggggu caucugcgcg       8340
gccauucugc gccgccacgu gggaccgggg gagggcgcgg uccaauggau gaacaggcuu       8400
auugccuuug cuuccagagg aaaccacguc gccccuacuc acuacgugac ggagucggau       8460
gcgucgcagc gugugaccca acuacuuggc ucucuuacua uaaccagccu acucagaaga       8520
cuccacaauu ggauaacuga ggacugcccc aucccaugcu ccggauccug gcuccgcgac       8580
gugugggacu ggguuugcac caucuugaca gacuucaaaa auuggcugac cucuaaauug       8640
uuccccaagc ugcccggccu ccccuucauc ucuugucaaa agggguacaa gggugugugg       8700
gccggcacug gcaucaugac cacgcgcugc ccuugcggcg ccaacaucuc uggcaauguc       8760
cgccugggcu cuaugaggau cacagggccu aaaaccugca ugaacaccug gcaggggacc       8820
uuuccuauca auugcuacac ggagggccag ugcgcgccga aaccccccac gaacuacaag       8880
accgccaucu ggaggguggc ggccucggag uacgcggagg ugacgcagca ugggucguac       8940
uccuauguaa caggacugac cacugacaau cugaaaauuc cuugccaacu accuucucca       9000
gaguuuuucu ccugggugga cggugugcag auccauaggu uugcacccac accaaagccg       9060
uuuuuccggg augaggucuc guucugcguu gggcuuaauu ccuaugcugu cgggucccag       9120
cuucccugug aaccugagcc cgacgcagac guauugaggu ccaugcuaac agauccgccc       9180
cacaucacgg cggagacugc ggcgcggcgc uuggcacggg gaucaccucc aucugaggcg       9240
agcuccucag ugagccagcu aucagcaccg ucgcugcggg ccaccugcac cacccacagc       9300
aacaccuaug acguggacau ggucgaugcc aaccugcuca uggagggcgg uguggcucag       9360
acagagccug aguccagggu gcccguucug gacuuucucg agccaauggc cgaggaagag       9420
agcgaccuug agcccucaau accaucggag ugcaugcucc ccaggagcgg guuuccacgg       9480
gccuuaccgg cuugggcacg gccugacuac aacccgccgc ucguggaauc guggaggagg       9540
ccagauuacc aaccgcccac cguugcuggu ugugcucucc ccccccccaa gaaggccccg       9600
acgccucccc caaggagacg ccggacagug ggucugagcg agagcaccau aucagaagcc       9660
cuccagcaac uggccaucaa gaccuuuggc cagccccccu cgagcgguga ugcaggcucg       9720
uccacggggg cgggcgccgc cgaauccggc gguccgacgu ccccugguga gccggccccc       9780
ucagagacag guuccgccuc cucuaugccc ccccucgagg gggagccugg agauccggac       9840
cuggagucug aucagguaga gcuucaaccu cccccccagg gggggggggu agcucccggu       9900
ucgggcucgg ggucuugguc uacuugcucc gaggaggacg auaccaccgu gugcugcucc       9960
augucauacu ccuggaccgg ggcucuaaua acucccugua gccccgaaga ggaaaaguug       10020
ccaaucaacc cuuugaguaa cucgcuguug cgauaccaua acaaggugua cuguacaaca       10080
ucaaagagcg ccucacagag ggcuaaaaag guaacuuuug acaggacgca agugcucgac       10140
gcccauuaug acucagucuu aaaggacauc aagcuagcgg cuuccaaggu cagcgcaagg       10200
cuccucaccu uggaggaggc gugccaguug acuccacccc auucugcaag auccaaguau       10260
ggauucgggg ccaaggaggu ccgcagcuug uccgggaggg ccguuaacca caucaagucc       10320
guguggaagg accuccugga agacccacaa acaccaauuc ccacaaccau cauggccaaa       10380
aaugaggugu ucugcgugga ccccgccaag ggggguaaga aaccagcucg ccucaucguu       10440
uacccugacc ucggcguccg ggucugcgag aaaauggccc ucuaugacau uacacaaaag       10500
cuuccucagg cgguaauggg agcuuccuau ggcuuccagu acuccccugc ccaacgggug       10560
gaguaucucu ugaaagcaug ggcggaaaag aaggacccca uggguuuuuc guaugauacc       10620
cgaugcuucg acucaaccgu cacugagaga gacaucagga ccgaggaguc cauauaccag       10680
gccugcuccc ugcccgagga ggcccgcacu gccauacacu cgcugacuga gagacuuuac       10740
guaggagggc ccauguucaa cagcaagggu caaaccugcg guuacagacg uugccgcgcc       10800
agcggggugc uaaccacuag cauggguaac accaucacau gcuaugugaa agcccuagcg       10860
gccugcaagg cugcggggau aguugcgccc acaaugcugg uaugcggcga ugaccuagua       10920
gucaucucag aaagccaggg gacugaggag gacgagcgga accugagagc cuucacggag       10980
gccaugacca gguacucugc cccuccuggu gaucccccca gaccggaaua ugaccuggag       11040
cuaauaacau ccuguuccuc aaaugugucu guggcguugg gcccgcgggg ccgccgcaga       11100
uacuaccuga ccagagaccc aaccacucca cucgcccggg cugccuggga aacaguuaga       11160
cacuccccua ucaauucaug gcugggaaac aucauccagu augcuccaac cauauggguu       11220
cgcauggucc uaaugacaca cuucuucucc auucucaugg uccaagacac ccuggaccag       11280
aaccucaacu uugagaugua uggaucagua uacuccguga auccuuugga ccuuccagcc       11340
auaauugaga gguuacacgg gcuugacgcc uuuucuaugc acacauacuc ucaccacgaa       11400
cugacgcggg uggcuucagc ccucagaaaa cuuggggcgc caccccucag gguguggaag       11460
agucgggcuc gcgcagucag ggcgucccuc aucucccgug gagggaaagc ggccguuugc       11520
ggccgauauc ucuucaauug ggcggugaag accaagcuca aacucacucc auugccggag       11580
gcgcgccuac uggacuuauc caguugguuc accgucggcg ccggcggggg cgacauuuuu       11640
cacagcgugu cgcgcgcccg accccgcuca uuacucuucg gccuacuccu acuuuucgua       11700
gggguaggcc ucuuccuacu ccccgcucgg uagagcggca cacacuaggu acacuccaua       11760
gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuucuuuu       11820
uuuuuuuuuu cccucuuucu ucccuucuca ucuuauucua cuuucuuucu ugguggcucc       11880
aucuuagccc uagucacggc uagcugugaa agguccguga gccgcaugac ugcagagagu       11940
gccguaacug gucucucugc agaucaugu                                         11969
<210>22
<211>11969
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：来源于表达载体rFGR-JFH1/Luc/GND的复制子RNA
<400>22
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gaagacgcca aaaacauaaa       420
gaaaggcccg gcgccauucu auccucugga ggauggaacc gcuggagagc aacugcauaa       480
ggcuaugaag agauacgccc ugguuccugg aacaauugcu uuuacagaug cacauaucga       540
ggugaacauc acguacgcgg aauacuucga aauguccguu cgguuggcag aagcuaugaa       600
acgauauggg cugaauacaa aucacagaau cgucguaugc agugaaaacu cucuucaauu       660
cuuuaugccg guguugggcg cguuauuuau cggaguugca guugcgcccg cgaacgacau       720
uuauaaugaa cgugaauugc ucaacaguau gaacauuucg cagccuaccg uaguguuugu       780
uuccaaaaag ggguugcaaa aaauuuugaa cgugcaaaaa aaauuaccaa uaauccagaa       840
aauuauuauc auggauucua aaacggauua ccagggauuu cagucgaugu acacguucgu       900
cacaucucau cuaccucccg guuuuaauga auacgauuuu guaccagagu ccuuugaucg       960
ugacaaaaca auugcacuga uaaugaacuc cucuggaucu acuggguuac cuaagggugu       1020
ggcccuuccg cauagaacug ccugcgucag auucucgcau gccagagauc cuauuuuugg       1080
caaucaaauc auuccggaua cugcgauuuu aaguguuguu ccauuccauc acgguuuugg       1140
aauguuuacu acacucggau auuugauaug uggauuucga gucgucuuaa uguauagauu       1200
ugaagaagag cuguuuuuac gaucccuuca ggauuacaaa auucaaagug cguugcuagu       1260
accaacccua uuuucauucu ucgccaaaag cacucugauu gacaaauacg auuuaucuaa       1320
uuuacacgaa auugcuucug ggggcgcacc ucuuucgaaa gaagucgggg aagcgguugc       1380
aaaacgcuuc caucuuccag ggauacgaca aggauauggg cucacugaga cuacaucagc       1440
uauucugauu acacccgagg gggaugauaa accgggcgcg gucgguaaag uuguuccauu       1500
uuuugaagcg aagguugugg aucuggauac cgggaaaacg cugggcguua aucagagagg       1560
cgaauuaugu gucagaggac cuaugauuau guccgguuau guaaacaauc cggaagcgac       1620
caacgccuug auugacaagg auggauggcu acauucugga gacauagcuu acugggacga       1680
agacgaacac uucuucauag uugaccgcuu gaagucuuua auuaaauaca aaggauauca       1740
gguggccccc gcugaauugg aaucgauauu guuacaacac cccaacaucu ucgacgcggg       1800
cguggcaggu cuucccgacg augacgccgg ugaacuuccc gccgccguug uuguuuugga       1860
gcacggaaag acgaugacgg aaaaagagau cguggauuac gucgccaguc aaguaacaac       1920
cgcgaaaaag uugcgcggag gaguuguguu uguggacgaa guaccgaaag gucuuaccgg       1980
aaaacucgac gcaagaaaaa ucagagagau ccucauaaag gccaagaagg gcggaaaguc       2040
caaauuguaa guuuaaaccc ucucccuccc cccccccuaa cguuacuggc cgaagccgcu       2100
uggaauaagg ccggugugcg uuugucuaua uguuauuuuc caccauauug ccgucuuuug       2160
gcaaugugag ggcccggaaa ccuggcccug ucuucuugac gagcauuccu aggggucuuu       2220
ccccucucgc caaaggaaug caaggucugu ugaaugucgu gaaggaagca guuccucugg       2280
aagcuucuug aagacaaaca acgucuguag cgacccuuug caggcagcgg aaccccccac       2340
cuggcgacag gugccucugc ggccaaaagc cacguguaua agauacaccu gcaaaggcgg       2400
cacaacccca gugccacguu gugaguugga uaguugugga aagagucaaa uggcucuccu       2460
caagcguauu caacaagggg cugaaggaug cccagaaggu accccauugu augggaucug       2520
aucuggggcc ucggugcaca ugcuuuacau guguuuaguc gagguuaaaa aaacgucuag       2580
gccccccgaa ccacggggac gugguuuucc uuugaaaaac acgaugauac caugagcaca       2640
aauccuaaac cucaaagaaa aaccaaaaga aacaccaacc gucgcccaga agacguuaag       2700
uucccgggcg gcggccagau cguuggcgga guauacuugu ugccgcgcag gggccccagg       2760
uugggugugc gcacgacaag gaaaacuucg gagcgguccc agccacgugg gagacgccag       2820
cccaucccca aagaucggcg cuccacuggc aaggccuggg gaaaaccagg ucgccccugg       2880
ccccuauaug ggaaugaggg acucggcugg gcaggauggc uccugucccc ccgaggcucu       2940
cgccccuccu ggggccccac ugacccccgg cauaggucgc gcaacguggg uaaagucauc       3000
gacacccuaa cguguggcuu ugccgaccuc augggguaca uccccgucgu aggcgccccg       3060
cuuaguggcg ccgccagagc ugucgcgcac ggcgugagag uccuggagga cgggguuaau       3120
uaugcaacag ggaaccuacc cgguuucccc uuuucuaucu ucuugcuggc ccuguugucc       3180
ugcaucaccg uuccggucuc ugcugcccag gugaagaaua ccaguagcag cuacauggug       3240
accaaugacu gcuccaauga cagcaucacu uggcagcucg aggcugcggu ucuccacguc       3300
cccgggugcg ucccgugcga gagagugggg aauacgucac gguguugggu gccagucucg       3360
ccaaacaugg cugugcggca gcccggugcc cucacgcagg gucugcggac gcacaucgau       3420
augguuguga uguccgccac cuucugcucu gcucucuacg ugggggaccu cuguggcggg       3480
gugaugcucg cggcccaggu guucaucguc ucgccgcagu accacugguu ugugcaagaa       3540
ugcaauugcu ccaucuaccc uggcaccauc acuggacacc gcauggcaug ggacaugaug       3600
augaacuggu cgcccacggc caccaugauc cuggcguacg ugaugcgcgu ccccgagguc       3660
aucauagaca ucguuagcgg ggcucacugg ggcgucaugu ucggcuuggc cuacuucucu       3720
augcagggag cgugggcgaa ggucauuguc auccuucugc uggccgcugg gguggacgcg       3780
ggcaccacca ccguuggagg cgcuguugca cguuccacca acgugauugc cggcguguuc       3840
agccauggcc cucagcagaa cauucagcuc auuaacacca acggcaguug gcacaucaac       3900
cguacugccu ugaauugcaa ugacuccuug aacaccggcu uucucgcggc cuuguucuac       3960
accaaccgcu uuaacucguc agggugucca gggcgccugu ccgccugccg caacaucgag       4020
gcuuuccgga uagggugggg cacccuacag uacgaggaua augucaccaa uccagaggau       4080
augaggccgu acugcuggca cuacccccca aagccgugug gcguaguccc cgcgaggucu       4140
guguguggcc caguguacug uuucaccccc agcccgguag uagugggcac gaccgacaga       4200
cguggagugc ccaccuacac auggggagag aaugagacag augucuuccu acugaacagc       4260
acccgaccgc cgcagggcuc augguucggc ugcacgugga ugaacuccac ugguuucacc       4320
aagacuugug gcgcgccacc uugccgcacc agagcugacu ucaacgccag cacggacuug       4380
uugugcccua cggauuguuu uaggaagcau ccugaugcca cuuauauuaa gugugguucu       4440
gggcccuggc ucacaccaaa gugccugguc cacuacccuu acagacucug gcauuacccc       4500
ugcacaguca auuuuaccau cuucaagaua agaauguaug uagggggggu ugagcacagg       4560
cucacggccg caugcaacuu cacucguggg gaucgcugcg acuuggagga cagggacagg       4620
agucagcugu cuccucuguu gcacucuacc acggaauggg ccauccugcc cugcaccuac       4680
ucagacuuac ccgcuuuguc aacuggucuu cuccaccuuc accagaacau cguggacgua       4740
caauacaugu auggccucuc accugcuauc acaaaauacg ucguucgaug ggagugggug       4800
guacucuuau uccugcucuu agcggacgcc agagucugcg ccugcuugug gaugcucauc       4860
uuguugggcc aggccgaagc agcauuggag aaguuggucg ucuugcacgc ugcgagugcg       4920
gcuaacugcc auggccuccu auauuuugcc aucuucuucg uggcagcuug gcacaucagg       4980
ggucgggugg uccccuugac caccuauugc cucacuggcc uauggcccuu cugccuacug       5040
cucauggcac ugccccggca ggcuuaugcc uaugacgcac cugugcacgg acagauaggc       5100
guggguuugu ugauauugau cacccucuuc acacucaccc cgggguauaa gacccuccuc       5160
ggccaguguc uguggugguu gugcuaucuc cugacccugg gggaagccau gauucaggag       5220
uggguaccac ccaugcaggu gcgcggcggc cgcgauggca ucgcgugggc cgucacuaua       5280
uucugcccgg gugugguguu ugacauuacc aaauggcuuu uggcguugcu ugggccugcu       5340
uaccucuuaa gggccgcuuu gacacaugug ccguacuucg ucagagcuca cgcucugaua       5400
aggguaugcg cuuuggugaa gcagcucgcg ggggguaggu auguucaggu ggcgcuauug       5460
gcccuuggca gguggacugg caccuacauc uaugaccacc ucacaccuau gucggacugg       5520
gccgcuagcg gccugcgcga cuuagcgguc gccguggaac ccaucaucuu caguccgaug       5580
gagaagaagg ucaucgucug gggagcggag acggcugcau guggggacau ucuacaugga       5640
cuucccgugu ccgcccgacu cggccaggag auccuccucg gcccagcuga uggcuacacc       5700
uccaaggggu ggaagcuccu ugcucccauc acugcuuaug cccagcaaac acgaggccuc       5760
cugggcgcca uaguggugag uaugacgggg cgugacagga cagaacaggc cggggaaguc       5820
caaauccugu ccacagucuc ucaguccuuc cucggaacaa ccaucucggg gguuuugugg       5880
acuguuuacc acggagcugg caacaagacu cuagccggcu uacggggucc ggucacgcag       5940
auguacucga gugcugaggg ggacuuggua ggcuggccca gccccccugg gaccaagucu       6000
uuggagccgu gcaagugugg agccgucgac cuauaucugg ucacgcggaa cgcugauguc       6060
aucccggcuc ggagacgcgg ggacaagcgg ggagcauugc ucuccccgag acccauuucg       6120
accuugaagg gguccucggg ggggccggug cucugcccua ggggccacgu cguugggcuc       6180
uuccgagcag cugugugcuc ucggggcgug gccaaaucca ucgauuucau ccccguugag       6240
acacucgacg uuguuacaag gucucccacu uucagugaca acagcacgcc accggcugug       6300
ccccagaccu aucaggucgg guacuugcau gcuccaacug gcaguggaaa gagcaccaag       6360
gucccugucg cguaugccgc ccagggguac aaaguacuag ugcuuaaccc cucgguagcu       6420
gccacccugg gguuuggggc guaccuaucc aaggcacaug gcaucaaucc caacauuagg       6480
acuggaguca ggaccgugau gaccggggag gccaucacgu acuccacaua uggcaaauuu       6540
cucgccgaug ggggcugcgc uagcggcgcc uaugacauca ucauaugcga ugaaugccac       6600
gcuguggaug cuaccuccau ucucggcauc ggaacggucc uugaucaagc agagacagcc       6660
ggggucagac uaacugugcu ggcuacggcc acaccccccg ggucagugac aaccccccau       6720
cccgauauag aagagguagg ccucgggcgg gagggugaga uccccuucua ugggagggcg       6780
auuccccuau ccugcaucaa gggagggaga caccugauuu ucugccacuc aaagaaaaag       6840
ugugacgagc ucgcggcggc ccuucggggc augggcuuga augccguggc auacuauaga       6900
ggguuggacg ucuccauaau accagcucag ggagaugugg uggucgucgc caccgacgcc       6960
cucaugacgg gguacacugg agacuuugac uccgugaucg acugcaaugu agcggucacc       7020
caagcugucg acuucagccu ggaccccacc uucacuauaa ccacacagac ugucccacaa       7080
gacgcugucu cacgcaguca gcgccgcggg cgcacaggua gaggaagaca gggcacuuau       7140
agguauguuu ccacugguga acgagccuca ggaauguuug acaguguagu gcuuugugag       7200
ugcuacgacg caggggcugc gugguacgau cucacaccag cggagaccac cgucaggcuu       7260
agagcguauu ucaacacgcc cggccuaccc gugugucaag accaucuuga auuuugggag       7320
gcaguuuuca ccggccucac acacauagac gcccacuucc ucucccaaac aaagcaagcg       7380
ggggagaacu ucgcguaccu aguagccuac caagcuacgg ugugcgccag agccaaggcc       7440
ccucccccgu ccugggacgc cauguggaag ugccuggccc gacucaagcc uacgcuugcg       7500
ggccccacac cucuccugua ccguuugggc ccuauuacca augaggucac ccucacacac       7560
ccugggacga aguacaucgc cacaugcaug caagcugacc uugaggucau gaccagcacg       7620
uggguccuag cuggaggagu ccuggcagcc gucgccgcau auugccuggc gacuggaugc       7680
guuuccauca ucggccgcuu gcacgucaac cagcgagucg ucguugcgcc ggauaaggag       7740
guccuguaug aggcuuuuga ugagauggag gaaugcgccu cuagggcggc ucucaucgaa       7800
gaggggcagc ggauagccga gauguugaag uccaagaucc aaggcuugcu gcagcaggcc       7860
ucuaagcagg cccaggacau acaacccgcu augcaggcuu cauggcccaa aguggaacaa       7920
uuuugggcca gacacaugug gaacuucauu agcggcaucc aauaccucgc aggauuguca       7980
acacugccag ggaaccccgc gguggcuucc augauggcau ucagugccgc ccucaccagu       8040
ccguugucga ccaguaccac cauccuucuc aacaucaugg gaggcugguu agcgucccag       8100
aucgcaccac ccgcgggggc caccggcuuu gucgucagug gccugguggg ggcugccgug       8160
ggcagcauag gccuggguaa ggugcuggug gacauccugg caggauaugg ugcgggcauu       8220
ucgggggccc ucgucgcauu caagaucaug ucuggcgaga agcccucuau ggaagauguc       8280
aucaaucuac ugccugggau ccugucuccg ggagcccugg uggugggggu caucugcgcg       8340
gccauucugc gccgccacgu gggaccgggg gagggcgcgg uccaauggau gaacaggcuu       8400
auugccuuug cuuccagagg aaaccacguc gccccuacuc acuacgugac ggagucggau       8460
gcgucgcagc gugugaccca acuacuuggc ucucuuacua uaaccagccu acucagaaga       8520
cuccacaauu ggauaacuga ggacugcccc aucccaugcu ccggauccug gcuccgcgac       8580
gugugggacu ggguuugcac caucuugaca gacuucaaaa auuggcugac cucuaaauug       8640
uuccccaagc ugcccggccu ccccuucauc ucuugucaaa agggguacaa gggugugugg       8700
gccggcacug gcaucaugac cacgcgcugc ccuugcggcg ccaacaucuc uggcaauguc       8760
cgccugggcu cuaugaggau cacagggccu aaaaccugca ugaacaccug gcaggggacc       8820
uuuccuauca auugcuacac ggagggccag ugcgcgccga aaccccccac gaacuacaag       8880
accgccaucu ggaggguggc ggccucggag uacgcggagg ugacgcagca ugggucguac       8940
uccuauguaa caggacugac cacugacaau cugaaaauuc cuugccaacu accuucucca       9000
gaguuuuucu ccugggugga cggugugcag auccauaggu uugcacccac accaaagccg       9060
uuuuuccggg augaggucuc guucugcguu gggcuuaauu ccuaugcugu cgggucccag       9120
cuucccugug aaccugagcc cgacgcagac guauugaggu ccaugcuaac agauccgccc       9180
cacaucacgg cggagacugc ggcgcggcgc uuggcacggg gaucaccucc aucugaggcg       9240
agcuccucag ugagccagcu aucagcaccg ucgcugcggg ccaccugcac cacccacagc       9300
aacaccuaug acguggacau ggucgaugcc aaccugcuca uggagggcgg uguggcucag       9360
acagagccug aguccagggu gcccguucug gacuuucucg agccaauggc cgaggaagag       9420
agcgaccuug agcccucaau accaucggag ugcaugcucc ccaggagcgg guuuccacgg       9480
gccuuaccgg cuugggcacg gccugacuac aacccgccgc ucguggaauc guggaggagg       9540
ccagauuacc aaccgcccac cguugcuggu ugugcucucc ccccccccaa gaaggccccg       9600
acgccucccc caaggagacg ccggacagug ggucugagcg agagcaccau aucagaagcc       9660
cuccagcaac uggccaucaa gaccuuuggc cagccccccu cgagcgguga ugcaggcucg       9720
uccacggggg cgggcgccgc cgaauccggc gguccgacgu ccccugguga gccggccccc       9780
ucagagacag guuccgccuc cucuaugccc ccccucgagg gggagccugg agauccggac       9840
cuggagucug aucagguaga gcuucaaccu cccccccagg gggggggggu agcucccggu       9900
ucgggcucgg ggucuugguc uacuugcucc gaggaggacg auaccaccgu gugcugcucc       9960
augucauacu ccuggaccgg ggcucuaaua acucccugua gccccgaaga ggaaaaguug       10020
ccaaucaacc cuuugaguaa cucgcuguug cgauaccaua acaaggugua cuguacaaca       10080
ucaaagagcg ccucacagag ggcuaaaaag guaacuuuug acaggacgca agugcucgac       10140
gcccauuaug acucagucuu aaaggacauc aagcuagcgg cuuccaaggu cagcgcaagg       10200
cuccucaccu uggaggaggc gugccaguug acuccacccc auucugcaag auccaaguau       10260
ggauucgggg ccaaggaggu ccgcagcuug uccgggaggg ccguuaacca caucaagucc       10320
guguggaagg accuccugga agacccacaa acaccaauuc ccacaaccau cauggccaaa       10380
aaugaggugu ucugcgugga ccccgccaag ggggguaaga aaccagcucg ccucaucguu       10440
uacccugacc ucggcguccg ggucugcgag aaaauggccc ucuaugacau uacacaaaag       10500
cuuccucagg cgguaauggg agcuuccuau ggcuuccagu acuccccugc ccaacgggug       10560
gaguaucucu ugaaagcaug ggcggaaaag aaggacccca uggguuuuuc guaugauacc       10620
cgaugcuucg acucaaccgu cacugagaga gacaucagga ccgaggaguc cauauaccag       10680
gccugcuccc ugcccgagga ggcccgcacu gccauacacu cgcugacuga gagacuuuac       10740
guaggagggc ccauguucaa cagcaagggu caaaccugcg guuacagacg uugccgcgcc       10800
agcggggugc uaaccacuag cauggguaac accaucacau gcuaugugaa agcccuagcg       10860
gccugcaagg cugcggggau aguugcgccc acaaugcugg uaugcggcaa ugaccuagua       10920
gucaucucag aaagccaggg gacugaggag gacgagcgga accugagagc cuucacggag       10980
gccaugacca gguacucugc cccuccuggu gaucccccca gaccggaaua ugaccuggag       11040
cuaauaacau ccuguuccuc aaaugugucu guggcguugg gcccgcgggg ccgccgcaga       11100
uacuaccuga ccagagaccc aaccacucca cucgcccggg cugccuggga aacaguuaga       11160
cacuccccua ucaauucaug gcugggaaac aucauccagu augcuccaac cauauggguu       11220
cgcauggucc uaaugacaca cuucuucucc auucucaugg uccaagacac ccuggaccag       11280
aaccucaacu uugagaugua uggaucagua uacuccguga auccuuugga ccuuccagcc       11340
auaauugaga gguuacacgg gcuugacgcc uuuucuaugc acacauacuc ucaccacgaa       11400
cugacgcggg uggcuucagc ccucagaaaa cuuggggcgc caccccucag gguguggaag       11460
agucgggcuc gcgcagucag ggcgucccuc aucucccgug gagggaaagc ggccguuugc       11520
ggccgauauc ucuucaauug ggcggugaag accaagcuca aacucacucc auugccggag       11580
gcgcgccuac uggacuuauc caguugguuc accgucggcg ccggcggggg cgacauuuuu       11640
cacagcgugu cgcgcgcccg accccgcuca uuacucuucg gccuacuccu acuuuucgua       11700
gggguaggcc ucuuccuacu ccccgcucgg uagagcggca cacacuaggu acacuccaua       11760
gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuucuuuu       11820
uuuuuuuuuu cccucuuucu ucccuucuca ucuuauucua cuuucuuucu ugguggcucc       11880
aucuuagccc uagucacggc uagcugugaa agguccguga gccgcaugac ugcagagagu       11940
gccguaacug gucucucugc agaucaugu                                         11969
<210>23
<211>11036
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：来源于表达载体rFGR-JFH1/EGFP的复制子RNA
<400>23
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gugagcaagg gcgaggagcu       420
guucaccggg guggugccca uccuggucga gcuggacggc gacguaaacg gccacaaguu       480
cagcgugucc ggcgagggcg agggcgaugc caccuacggc aagcugaccc ugaaguucau       540
cugcaccacc ggcaagcugc ccgugcccug gcccacccuc gugaccaccc ugaccuacgg       600
cgugcagugc uucagccgcu accccgacca caugaagcag cacgacuucu ucaaguccgc       660
caugcccgaa ggcuacgucc aggagcgcac caucuucuuc aaggacgacg gcaacuacaa       720
gacccgcgcc gaggugaagu ucgagggcga cacccuggug aaccgcaucg agcugaaggg       780
caucgacuuc aaggaggacg gcaacauccu ggggcacaag cuggaguaca acuacaacag       840
ccacaacguc uauaucaugg ccgacaagca gaagaacggc aucaagguga acuucaagau       900
ccgccacaac aucgaggacg gcagcgugca gcucgccgac cacuaccagc agaacacccc       960
caucggcgac ggccccgugc ugcugcccga caaccacuac cugagcaccc aguccgcccu       1020
gagcaaagac cccaacgaga agcgcgauca caugguccug cuggaguucg ugaccgccgc       1080
cgggaucacu cucggcaugg acgagcugua caaguaaguu uaaacccucu cccucccccc       1140
ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu       1200
uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu       1260
ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga       1320
augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga       1380
cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac       1440
guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag       1500
uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc       1560
agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug       1620
uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu       1680
gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac       1740
accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua       1800
uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag       1860
cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag       1920
gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca       1980
ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau       2040
aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug       2100
ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc       2160
gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu       2220
ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug       2280
aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg       2340
cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau       2400
acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc       2460
acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu       2520
cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg       2580
ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu       2640
ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug       2700
gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc       2760
gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc       2820
cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu       2880
uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu       2940
aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac       3000
accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg       3060
cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac       3120
gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag       3180
ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc       3240
ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau       3300
gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc       3360
acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga       3420
gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu       3480
gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac       3540
uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga       3600
auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau       3660
cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg       3720
gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc       3780
caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca       3840
aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga       3900
gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag       3960
uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc       4020
uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc       4080
acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau       4140
gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca       4200
cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug       4260
acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc       4320
gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa       4380
uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg       4440
uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg       4500
gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau       4560
gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc       4620
guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg       4680
gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc       4740
cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu       4800
gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu       4860
gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc       4920
ggaacaacca ucucgggggu uuuguggacu guuuaccacg gagcuggcaa caagacucua       4980
gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc       5040
uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua       5100
uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga       5160
gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc       5220
ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc       5280
aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc       5340
agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu       5400
ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa       5460
guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag       5520
gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc       5580
aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau       5640
gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga       5700
acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca       5760
ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag       5820
ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac       5880
cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug       5940
ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga       6000
gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc       6060
gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc       6120
acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc       6180
acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga       6240
auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc       6300
acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug       6360
ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc       6420
cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa       6480
gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc       6540
cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu       6600
auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa       6660
gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc       6720
gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag       6780
cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa       6840
ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc       6900
aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug       6960
caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc       7020
ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug       7080
auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac       7140
aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc       7200
gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac       7260
auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu       7320
ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga       7380
gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag       7440
ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc       7500
ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu       7560
cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc       7620
ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac       7680
uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu       7740
ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu       7800
ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa       7860
accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc       7920
gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac       7980
gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug       8040
aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc       8100
cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg       8160
cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua       8220
uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug       8280
gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg       8340
cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac       8400
cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac       8460
uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc       8520
augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac       8580
ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu       8640
gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu       8700
cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag       8760
ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu       8820
ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc       8880
cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc       8940
ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag       9000
gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu       9060
cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga       9120
uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua       9180
acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag       9240
cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu       9300
ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc       9360
gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca       9420
ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg       9480
gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa       9540
auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc       9600
uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag       9660
gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac       9720
aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc       9780
auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa       9840
accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc       9900
aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca       9960
augcugguau gcggcgauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac       10020
gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau       10080
ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug       10140
gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc       10200
gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc       10260
auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu       10320
cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac       10380
uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu       10440
ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu       10500
ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc       10560
ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc       10620
aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc       10680
gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua       10740
cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag       10800
agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu       10860
uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu       10920
uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg       10980
uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcaga ucaugu           11036
<210>24
<211>11036
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：来源于表达载体rFGR-JFH1/EGFP/GND的复制子RNA
<400>24
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug gugagcaagg gcgaggagcu       420
guucaccggg guggugccca uccuggucga gcuggacggc gacguaaacg gccacaaguu       480
cagcgugucc ggcgagggcg agggcgaugc caccuacggc aagcugaccc ugaaguucau       540
cugcaccacc ggcaagcugc ccgugcccug gcccacccuc gugaccaccc ugaccuacgg       600
cgugcagugc uucagccgcu accccgacca caugaagcag cacgacuucu ucaaguccgc       660
caugcccgaa ggcuacgucc aggagcgcac caucuucuuc aaggacgacg gcaacuacaa       720
gacccgcgcc gaggugaagu ucgagggcga cacccuggug aaccgcaucg agcugaaggg       780
caucgacuuc aaggaggacg gcaacauccu ggggcacaag cuggaguaca acuacaacag       840
ccacaacguc uauaucaugg ccgacaagca gaagaacggc aucaagguga acuucaagau       900
ccgccacaac aucgaggacg gcagcgugca gcucgccgac cacuaccagc agaacacccc       960
caucggcgac ggccccgugc ugcugcccga caaccacuac cugagcaccc aguccgcccu       1020
gagcaaagac cccaacgaga agcgcgauca caugguccug cuggaguucg ugaccgccgc       1080
cgggaucacu cucggcaugg acgagcugua caaguaaguu uaaacccucu cccucccccc       1140
ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu       1200
uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu       1260
ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga       1320
augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga       1380
cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac       1440
guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag       1500
uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc       1560
agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug       1620
uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu       1680
gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac       1740
accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua       1800
uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag       1860
cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag       1920
gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca       1980
ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau       2040
aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug       2100
ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc       2160
gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu       2220
ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug       2280
aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg       2340
cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau       2400
acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc       2460
acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu       2520
cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg       2580
ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu       2640
ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug       2700
gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc       2760
gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc       2820
cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu       2880
uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu       2940
aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac       3000
accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg       3060
cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac       3120
gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag       3180
ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc       3240
ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau       3300
gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc       3360
acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga       3420
gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu       3480
gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac       3540
uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga       3600
auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau       3660
cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg       3720
gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc       3780
caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca       3840
aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga       3900
gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag       3960
uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc       4020
uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc       4080
acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau       4140
gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca       4200
cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug       4260
acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc       4320
gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa       4380
uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg       4440
uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg       4500
gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau       4560
gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc       4620
guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg       4680
gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc       4740
cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu       4800
gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu       4860
gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc       4920
ggaacaaccaucucgggggu  uuuguggacu guuuaccacg gagcuggcaa caagacucua       4980
gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc       5040
uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua       5100
uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga       5160
gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc       5220
ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc       5280
aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc       5340
agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu       5400
ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa       5460
guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag       5520
gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc       5580
aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau       5640
gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga       5700
acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca       5760
ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag       5820
ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac       5880
cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug       5940
ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga       6000
gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc       6060
gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc       6120
acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc       6180
acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga       6240
auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc       6300
acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug       6360
ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc       6420
cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa       6480
gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc       6540
cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu       6600
auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa       6660
gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc       6720
gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag       6780
cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa       6840
ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc       6900
aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug       6960
caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc       7020
ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug       7080
auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac       7140
aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc       7200
gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac       7260
auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu       7320
ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga       7380
gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag       7440
ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc       7500
ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu       7560
cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc       7620
ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac       7680
uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu       7740
ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu       7800
ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa       7860
accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc       7920
gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac       7980
gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug       8040
aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc       8100
cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg       8160
cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua       8220
uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug       8280
gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg       8340
cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac       8400
cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac       8460
uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc       8520
augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac       8580
ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu       8640
gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu       8700
cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag       8760
ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu       8820
ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc       8880
cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc       8940
ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag       9000
gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu       9060
cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga       9120
uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua       9180
acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag       9240
cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu       9300
ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc       9360
gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca       9420
ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg       9480
gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa       9540
auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc       9600
uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag       9660
gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac       9720
aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc       9780
auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa       9840
accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc       9900
aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca       9960
augcugguau gcggcaauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac       10020
gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau       10080
ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug       10140
gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc       10200
gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc       10260
auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu       10320
cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac       10380
uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu       10440
ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu       10500
ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc       10560
ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc       10620
aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc       10680
gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua       10740
cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag       10800
agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu       10860
uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu       10920
uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg       10980
uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcagaucaugu            11036
<210>25
<211>11876
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：来源于表达载体rFGR-JFH1/SEAP的复制子RNA
<400>25
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug cugcugcugc ugcugcugcu       420
gggccugagg cuacagcucu cccugggcau caucccaguu gaggaggaga acccggacuu       480
cuggaaccgc gaggcagccg aggcccuggg ugccgccaag aagcugcagc cugcacagac       540
agccgccaag aaccucauca ucuuccuggg cgaugggaug ggggugucua cggugacagc       600
ugccaggauc cuaaaagggc agaagaagga caaacugggg ccugagauac cccuggccau       660
ggaccgcuuc ccauaugugg cucuguccaa gacauacaau guagacaaac augugccaga       720
caguggagcc acagccacgg ccuaccugug cggggucaag ggcaacuucc agaccauugg       780
cuugagugca gccgcccgcu uuaaccagug caacacgaca cgcggcaacg aggucaucuc       840
cgugaugaau cgggccaaga aagcagggaa gucaguggga gugguaacca ccacacgagu       900
gcagcacgcc ucgccagccg gcaccuacgc ccacacggug aaccgcaacu gguacucgga       960
cgccgacgug ccugccucgg cccgccagga ggggugccag gacaucgcua cgcagcucau       1020
cuccaacaug gacauugacg ugauccuagg uggaggccga aaguacaugu uucgcauggg       1080
aaccccagac ccugaguacc cagaugacua cagccaaggu gggaccaggc uggacgggaa       1140
gaaucuggu gcaggaauggc uggcgaagcg ccagggugcc cgguaugugu ggaaccgcac       1200
ugagcucaug caggcuuccc uggacccguc ugugacccau cucauggguc ucuuugagcc       1260
uggagacaug aaauacgaga uccaccgaga cuccacacug gaccccuccc ugauggagau       1320
gacagaggcu gcccugcgcc ugcugagcag gaacccccgc ggcuucuucc ucuucgugga       1380
ggguggucgc aucgaccaug gucaucauga aagcagggcu uaccgggcac ugacugagac       1440
gaucauguuc gacgacgcca uugagagggc gggccagcuc accagcgagg aggacacgcu       1500
gagccucguc acugccgacc acucccacgu cuucuccuuc ggaggcuacc cccugcgagg       1560
gagcuccauc uucgggcugg ccccuggcaa ggcccgggac aggaaggccu acacgguccu       1620
ccuauacgga aacgguccag gcuaugugcu caaggacggc gcccggccgg auguuaccga       1680
gagcgagagc gggagccccg aguaucggca gcagucagca gugccccugg acgaagagac       1740
ccacgcaggc gaggacgugg cgguguucgc gcgcggcccg caggcgcacc ugguucacgg       1800
cgugcaggag cagaccuuca uagcgcacgu cauggccuuc gccgccugcc uggagcccua       1860
caccgccugc gaccuggcgc cccccgccgg caccaccgac gccgcgcacc cggguuacuc       1920
uagagucggg gcggccggcc gcuucgagca gacaugaguu uaaacccucu cccucccccc       1980
ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu       2040
uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu       2100
ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga       2160
augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga       2220
cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac       2280
guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag       2340
uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc       2400
agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug       2460
uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu       2520
gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac       2580
accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua       2640
uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag       2700
cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag       2760
gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca       2820
ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau       2880
aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug       2940
ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc       3000
gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu       3060
ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug       3120
aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg       3180
cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau       3240
acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc       3300
acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu       3360
cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg       3420
ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu       3480
ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug       3540
gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc       3600
gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc       3660
cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu       3720
uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu       3780
aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac       3840
accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg       3900
cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac       3960
gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag       4020
ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc       4080
ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau       4140
gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc       4200
acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga       4260
gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu       4320
gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac       4380
uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga       4440
auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau       4500
cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg       4560
gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc       4620
caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca       4680
aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga       4740
gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag       4800
uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc       4860
uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc       4920
acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau       4980
gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca       5040
cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug       5100
acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc       5160
gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa       5220
uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg       5280
uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg       5340
gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau       5400
gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc       5460
guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg       5520
gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc       5580
cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu       5640
gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu       5700
gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc       5760
ggaacaacca ucucgggggu uuuguggacu guuuaccacg gagcuggcaa caagacucua       5820
gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc       5880
uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua       5940
uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga       6000
gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc       6060
ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc       6120
aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc       6180
agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu       6240
ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa       6300
guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag       6360
gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc       6420
aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau       6480
gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga       6540
acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca       6600
ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag       6660
ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac       6720
cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug       6780
ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga       6840
gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc       6900
gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc       6960
acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc       7020
acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga       7080
auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc       7140
acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug       7200
ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc       7260
cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa       7320
gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc       7380
cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu       7440
auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa       7500
gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc       7560
gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag       7620
cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa       7680
ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc       7740
aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug       7800
caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc       7860
ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug       7920
auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac       7980
aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc       8040
gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac       8100
auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu       8160
ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga       8220
gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag       8280
ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc       8340
ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu       8400
cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc       8460
ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac       8520
uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu       8580
ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu       8640
ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa       8700
accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc       8760
gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac       8820
gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug       8880
aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc       8940
cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg       9000
cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua       9060
uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug       9120
gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg       9180
cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac       9240
cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac       9300
uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc       9360
augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac       9420
ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu       9480
gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu       9540
cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag       9600
ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu       9660
ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc       9720
cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc       9780
ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag       9840
gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu       9900
cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga       9960
uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua       10020
acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag       10080
cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu       10140
ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc       10200
gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca       10260
ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg       10320
gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa       10380
auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc       10440
uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag       10500
gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac       10560
aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc       10620
auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa       10680
accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc       10740
aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca       10800
augcugguau gcggcgauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac       10860
gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau       10920
ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug       10980
gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc       11040
gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc       11100
auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu       11160
cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac       11220
uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu       11280
ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu       11340
ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc       11400
ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc       11460
aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc       11520
gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua       11580
cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag       11640
agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu       11700
uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu       11760
uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg       11820
uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcaga ucaugu           11876
<210>26
<211>11876
<212>RNA
<213>人工序列
<220>
<223>人工序列的描述：来源于表达载体rFGR-JFH1/sEAP/GND的复制子RNA
<400>26
accugccccu aauaggggcg acacuccgcc augaaucacu ccccugugag gaacuacugu       60
cuucacgcag aaagcgccua gccauggcgu uaguaugagu gucguacagc cuccaggccc       120
cccccucccg ggagagccau aguggucugc ggaaccggug aguacaccgg aauugccggg       180
aagacugggu ccuuucuugg auaaacccac ucuaugcccg gccauuuggg cgugcccccg       240
caagacugcu agccgaguag cguuggguug cgaaaggccu ugugguacug ccugauaggg       300
cgcuugcgag ugccccggga ggucucguag accgugcacc augagcacaa auccuaaacc       360
ucaaagaaaa accaaaagaa acaccaaccg acgcguaaug cugcugcugc ugcugcugcu       420
gggccugagg cuacagcucu cccugggcau caucccaguu gaggaggaga acccggacuu       480
cuggaaccgc gaggcagccg aggcccuggg ugccgccaag aagcugcagc cugcacagac       540
agccgccaag aaccucauca ucuuccuggg cgaugggaug ggggugucua cggugacagc       600
ugccaggauc cuaaaagggc agaagaagga caaacugggg ccugagauac cccuggccau       660
ggaccgcuuc ccauaugugg cucuguccaa gacauacaau guagacaaac augugccaga       720
caguggagcc acagccacgg ccuaccugug cggggucaag ggcaacuucc agaccauugg       780
cuugagugca gccgcccgcu uuaaccagug caacacgaca cgcggcaacg aggucaucuc       840
cgugaugaau cgggccaaga aagcagggaa gucaguggga gugguaacca ccacacgagu       900
gcagcacgcc ucgccagccg gcaccuacgc ccacacggug aaccgcaacu gguacucgga       960
cgccgacgug ccugccucgg cccgccagga ggggugccag gacaucgcua cgcagcucau       1020
cuccaacaug gacauugacg ugauccuagg uggaggccga aaguacaugu uucgcauggg       1080
aaccccagac ccugaguacc cagaugacua cagccaaggu gggaccaggc uggacgggaa       1140
gaaucuggug caggaauggc uggcgaagcg ccagggugcc cgguaugugu ggaaccgcac       1200
ugagcucaug caggcuuccc uggacccguc ugugacccau cucauggguc ucuuugagcc       1260
uggagacaug aaauacgaga uccaccgaga cuccacacug gaccccuccc ugauggagau       1320
gacagaggcu gcccugcgcc ugcugagcag gaacccccgc ggcuucuucc ucuucgugga       1380
ggguggucgc aucgaccaug gucaucauga aagcagggcu uaccgggcac ugacugagac       1440
gaucauguuc gacgacgcca uugagagggc gggccagcuc accagcgagg aggacacgcu       1500
gagccucguc acugccgacc acucccacgu cuucuccuuc ggaggcuacc cccugcgagg       1560
gagcuccauc uucgggcugg ccccuggcaa ggcccgggac aggaaggccu acacgguccu       1620
ccuauacgga aacgguccag gcuaugugcu caaggacggc gcccggccgg auguuaccga       1680
gagcgagagc gggagccccg aguaucggca gcagucagca gugccccugg acgaagagac       1740
ccacgcaggc gaggacgugg cgguguucgc gcgcggcccg caggcgcacc ugguucacgg       1800
cgugcaggag cagaccuuca uagcgcacgu cauggccuuc gccgccugcc uggagcccua       1860
caccgccugc gaccuggcgc cccccgccgg caccaccgac gccgcgcacc cggguuacuc       1920
uagagucggg gcggccggcc gcuucgagca gacaugaguu uaaacccucu cccucccccc       1980
ccccuaacgu uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu       2040
uauuuuccac cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu       2100
ucuugacgag cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga       2160
augucgugaa ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga       2220
cccuuugcag gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac       2280
guguauaaga uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag       2340
uuguggaaag agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc       2400
agaagguacc ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug       2460
uuuagucgag guuaaaaaaa cgucuaggcc ccccgaacca cggggacgug guuuuccuuu       2520
gaaaaacacg augauaccau gagcacaaau ccuaaaccuc aaagaaaaac caaaagaaac       2580
accaaccguc gcccagaaga cguuaaguuc ccgggcggcg gccagaucgu uggcggagua       2640
uacuuguugc cgcgcagggg ccccagguug ggugugcgca cgacaaggaa aacuucggag       2700
cggucccagc cacgugggag acgccagccc auccccaaag aucggcgcuc cacuggcaag       2760
gccuggggaa aaccaggucg ccccuggccc cuauauggga augagggacu cggcugggca       2820
ggauggcucc uguccccccg aggcucucgc cccuccuggg gccccacuga cccccggcau       2880
aggucgcgca acguggguaa agucaucgac acccuaacgu guggcuuugc cgaccucaug       2940
ggguacaucc ccgucguagg cgccccgcuu aguggcgccg ccagagcugu cgcgcacggc       3000
gugagagucc uggaggacgg gguuaauuau gcaacaggga accuacccgg uuuccccuuu       3060
ucuaucuucu ugcuggcccu guuguccugc aucaccguuc cggucucugc ugcccaggug       3120
aagaauacca guagcagcua cauggugacc aaugacugcu ccaaugacag caucacuugg       3180
cagcucgagg cugcgguucu ccacgucccc gggugcgucc cgugcgagag aguggggaau       3240
acgucacggu guugggugcc agucucgcca aacauggcug ugcggcagcc cggugcccuc       3300
acgcaggguc ugcggacgca caucgauaug guugugaugu ccgccaccuu cugcucugcu       3360
cucuacgugg gggaccucug uggcggggug augcucgcgg cccagguguu caucgucucg       3420
ccgcaguacc acugguuugu gcaagaaugc aauugcucca ucuacccugg caccaucacu       3480
ggacaccgca uggcauggga caugaugaug aacuggucgc ccacggccac caugauccug       3540
gcguacguga ugcgcguccc cgaggucauc auagacaucg uuagcggggc ucacuggggc       3600
gucauguucg gcuuggccua cuucucuaug cagggagcgu gggcgaaggu cauugucauc       3660
cuucugcugg ccgcuggggu ggacgcgggc accaccaccg uuggaggcgc uguugcacgu       3720
uccaccaacg ugauugccgg cguguucagc cauggcccuc agcagaacau ucagcucauu       3780
aacaccaacg gcaguuggca caucaaccgu acugccuuga auugcaauga cuccuugaac       3840
accggcuuuc ucgcggccuu guucuacacc aaccgcuuua acucgucagg guguccaggg       3900
cgccuguccg ccugccgcaa caucgaggcu uuccggauag gguggggcac ccuacaguac       3960
gaggauaaug ucaccaaucc agaggauaug aggccguacu gcuggcacua ccccccaaag       4020
ccguguggcg uaguccccgc gaggucugug uguggcccag uguacuguuu cacccccagc       4080
ccgguaguag ugggcacgac cgacagacgu ggagugccca ccuacacaug gggagagaau       4140
gagacagaug ucuuccuacu gaacagcacc cgaccgccgc agggcucaug guucggcugc       4200
acguggauga acuccacugg uuucaccaag acuuguggcg cgccaccuug ccgcaccaga       4260
gcugacuuca acgccagcac ggacuuguug ugcccuacgg auuguuuuag gaagcauccu       4320
gaugccacuu auauuaagug ugguucuggg cccuggcuca caccaaagug ccugguccac       4380
uacccuuaca gacucuggca uuaccccugc acagucaauu uuaccaucuu caagauaaga       4440
auguauguag gggggguuga gcacaggcuc acggccgcau gcaacuucac ucguggggau       4500
cgcugcgacu uggaggacag ggacaggagu cagcugucuc cucuguugca cucuaccacg       4560
gaaugggcca uccugcccug caccuacuca gacuuacccg cuuugucaac uggucuucuc       4620
caccuucacc agaacaucgu ggacguacaa uacauguaug gccucucacc ugcuaucaca       4680
aaauacgucg uucgauggga guggguggua cucuuauucc ugcucuuagc ggacgccaga       4740
gucugcgccu gcuuguggau gcucaucuug uugggccagg ccgaagcagc auuggagaag       4800
uuggucgucu ugcacgcugc gagugcggcu aacugccaug gccuccuaua uuuugccauc       4860
uucuucgugg cagcuuggca caucaggggu cggguggucc ccuugaccac cuauugccuc       4920
acuggccuau ggcccuucug ccuacugcuc auggcacugc cccggcaggc uuaugccuau       4980
gacgcaccug ugcacggaca gauaggcgug gguuuguuga uauugaucac ccucuucaca       5040
cucaccccgg gguauaagac ccuccucggc cagugucugu ggugguugug cuaucuccug       5100
acccuggggg aagccaugau ucaggagugg guaccaccca ugcaggugcg cggcggccgc       5160
gauggcaucg cgugggccgu cacuauauuc ugcccgggug ugguguuuga cauuaccaaa       5220
uggcuuuugg cguugcuugg gccugcuuac cucuuaaggg ccgcuuugac acaugugccg       5280
uacuucguca gagcucacgc ucugauaagg guaugcgcuu uggugaagca gcucgcgggg       5340
gguagguaug uucagguggc gcuauuggcc cuuggcaggu ggacuggcac cuacaucuau       5400
gaccaccuca caccuauguc ggacugggcc gcuagcggcc ugcgcgacuu agcggucgcc       5460
guggaaccca ucaucuucag uccgauggag aagaagguca ucgucugggg agcggagacg       5520
gcugcaugug gggacauucu acauggacuu cccguguccg cccgacucgg ccaggagauc       5580
cuccucggcc cagcugaugg cuacaccucc aaggggugga agcuccuugc ucccaucacu       5640
gcuuaugccc agcaaacacg aggccuccug ggcgccauag uggugaguau gacggggcgu       5700
gacaggacag aacaggccgg ggaaguccaa auccugucca cagucucuca guccuuccuc       5760
ggaacaacca ucucgggggu uuuguggacu guuuaccacg gagcuggcaa caagacucua       5820
gccggcuuac gggguccggu cacgcagaug uacucgagug cugaggggga cuugguaggc       5880
uggcccagcc ccccugggac caagucuuug gagccgugca aguguggagc cgucgaccua       5940
uaucugguca cgcggaacgc ugaugucauc ccggcucgga gacgcgggga caagcgggga       6000
gcauugcucu ccccgagacc cauuucgacc uugaaggggu ccucgggggg gccggugcuc       6060
ugcccuaggg gccacgucgu ugggcucuuc cgagcagcug ugugcucucg gggcguggcc       6120
aaauccaucg auuucauccc cguugagaca cucgacguug uuacaagguc ucccacuuuc       6180
agugacaaca gcacgccacc ggcugugccc cagaccuauc aggucgggua cuugcaugcu       6240
ccaacuggca guggaaagag caccaagguc ccugucgcgu augccgccca gggguacaaa       6300
guacuagugc uuaaccccuc gguagcugcc acccuggggu uuggggcgua ccuauccaag       6360
gcacauggca ucaaucccaa cauuaggacu ggagucagga ccgugaugac cggggaggcc       6420
aucacguacu ccacauaugg caaauuucuc gccgaugggg gcugcgcuag cggcgccuau       6480
gacaucauca uaugcgauga augccacgcu guggaugcua ccuccauucu cggcaucgga       6540
acgguccuug aucaagcaga gacagccggg gucagacuaa cugugcuggc uacggccaca       6600
ccccccgggu cagugacaac cccccauccc gauauagaag agguaggccu cgggcgggag       6660
ggugagaucc ccuucuaugg gagggcgauu ccccuauccu gcaucaaggg agggagacac       6720
cugauuuucu gccacucaaa gaaaaagugu gacgagcucg cggcggcccu ucggggcaug       6780
ggcuugaaug ccguggcaua cuauagaggg uuggacgucu ccauaauacc agcucaggga       6840
gauguggugg ucgucgccac cgacgcccuc augacggggu acacuggaga cuuugacucc       6900
gugaucgacu gcaauguagc ggucacccaa gcugucgacu ucagccugga ccccaccuuc       6960
acuauaacca cacagacugu cccacaagac gcugucucac gcagucagcg ccgcgggcgc       7020
acagguagag gaagacaggg cacuuauagg uauguuucca cuggugaacg agccucagga       7080
auguuugaca guguagugcu uugugagugc uacgacgcag gggcugcgug guacgaucuc       7140
acaccagcgg agaccaccgu caggcuuaga gcguauuuca acacgcccgg ccuacccgug       7200
ugucaagacc aucuugaauu uugggaggca guuuucaccg gccucacaca cauagacgcc       7260
cacuuccucu cccaaacaaa gcaagcgggg gagaacuucg cguaccuagu agccuaccaa       7320
gcuacggugu gcgccagagc caaggccccu cccccguccu gggacgccau guggaagugc       7380
cuggcccgac ucaagccuac gcuugcgggc cccacaccuc uccuguaccg uuugggcccu       7440
auuaccaaug aggucacccu cacacacccu gggacgaagu acaucgccac augcaugcaa       7500
gcugaccuug aggucaugac cagcacgugg guccuagcug gaggaguccu ggcagccguc       7560
gccgcauauu gccuggcgac uggaugcguu uccaucaucg gccgcuugca cgucaaccag       7620
cgagucgucg uugcgccgga uaaggagguc cuguaugagg cuuuugauga gauggaggaa       7680
ugcgccucua gggcggcucu caucgaagag gggcagcgga uagccgagau guugaagucc       7740
aagauccaag gcuugcugca gcaggccucu aagcaggccc aggacauaca acccgcuaug       7800
caggcuucau ggcccaaagu ggaacaauuu ugggccagac acauguggaa cuucauuagc       7860
ggcauccaau accucgcagg auugucaaca cugccaggga accccgcggu ggcuuccaug       7920
auggcauuca gugccgcccu caccaguccg uugucgacca guaccaccau ccuucucaac       7980
aucaugggag gcugguuagc gucccagauc gcaccacccg cgggggccac cggcuuuguc       8040
gucaguggcc uggugggggc ugccgugggc agcauaggcc uggguaaggu gcugguggac       8100
auccuggcag gauauggugc gggcauuucg ggggcccucg ucgcauucaa gaucaugucu       8160
ggcgagaagc ccucuaugga agaugucauc aaucuacugc cugggauccu gucuccggga       8220
gcccuggugg ugggggucau cugcgcggcc auucugcgcc gccacguggg accgggggag       8280
ggcgcggucc aauggaugaa caggcuuauu gccuuugcuu ccagaggaaa ccacgucgcc       8340
ccuacucacu acgugacgga gucggaugcg ucgcagcgug ugacccaacu acuuggcucu       8400
cuuacuauaa ccagccuacu cagaagacuc cacaauugga uaacugagga cugccccauc       8460
ccaugcuccg gauccuggcu ccgcgacgug ugggacuggg uuugcaccau cuugacagac       8520
uucaaaaauu ggcugaccuc uaaauuguuc cccaagcugc ccggccuccc cuucaucucu       8580
ugucaaaagg gguacaaggg ugugugggcc ggcacuggca ucaugaccac gcgcugcccu       8640
ugcggcgcca acaucucugg caauguccgc cugggcucua ugaggaucac agggccuaaa       8700
accugcauga acaccuggca ggggaccuuu ccuaucaauu gcuacacgga gggccagugc       8760
gcgccgaaac cccccacgaa cuacaagacc gccaucugga ggguggcggc cucggaguac       8820
gcggagguga cgcagcaugg gucguacucc uauguaacag gacugaccac ugacaaucug       8880
aaaauuccuu gccaacuacc uucuccagag uuuuucuccu ggguggacgg ugugcagauc       8940
cauagguuug cacccacacc aaagccguuu uuccgggaug aggucucguu cugcguuggg       9000
cuuaauuccu augcugucgg gucccagcuu cccugugaac cugagcccga cgcagacgua       9060
uugaggucca ugcuaacaga uccgccccac aucacggcgg agacugcggc gcggcgcuug       9120
gcacggggau caccuccauc ugaggcgagc uccucaguga gccagcuauc agcaccgucg       9180
cugcgggcca ccugcaccac ccacagcaac accuaugacg uggacauggu cgaugccaac       9240
cugcucaugg agggcggugu ggcucagaca gagccugagu ccagggugcc cguucuggac       9300
uuucucgagc caauggccga ggaagagagc gaccuugagc ccucaauacc aucggagugc       9360
augcucccca ggagcggguu uccacgggcc uuaccggcuu gggcacggcc ugacuacaac       9420
ccgccgcucg uggaaucgug gaggaggcca gauuaccaac cgcccaccgu ugcugguugu       9480
gcucuccccc cccccaagaa ggccccgacg ccucccccaa ggagacgccg gacagugggu       9540
cugagcgaga gcaccauauc agaagcccuc cagcaacugg ccaucaagac cuuuggccag       9600
ccccccucga gcggugaugc aggcucgucc acgggggcgg gcgccgccga auccggcggu       9660
ccgacguccc cuggugagcc ggcccccuca gagacagguu ccgccuccuc uaugcccccc       9720
cucgaggggg agccuggaga uccggaccug gagucugauc agguagagcu ucaaccuccc       9780
ccccaggggg gggggguagc ucccgguucg ggcucggggu cuuggucuac uugcuccgag       9840
gaggacgaua ccaccgugug cugcuccaug ucauacuccu ggaccggggc ucuaauaacu       9900
cccuguagcc ccgaagagga aaaguugcca aucaacccuu ugaguaacuc gcuguugcga       9960
uaccauaaca agguguacug uacaacauca aagagcgccu cacagagggc uaaaaaggua       10020
acuuuugaca ggacgcaagu gcucgacgcc cauuaugacu cagucuuaaa ggacaucaag       10080
cuagcggcuu ccaaggucag cgcaaggcuc cucaccuugg aggaggcgug ccaguugacu       10140
ccaccccauu cugcaagauc caaguaugga uucggggcca aggagguccg cagcuugucc       10200
gggagggccg uuaaccacau caaguccgug uggaaggacc uccuggaaga cccacaaaca       10260
ccaauuccca caaccaucau ggccaaaaau gagguguucu gcguggaccc cgccaagggg       10320
gguaagaaac cagcucgccu caucguuuac ccugaccucg gcguccgggu cugcgagaaa       10380
auggcccucu augacauuac acaaaagcuu ccucaggcgg uaaugggagc uuccuauggc       10440
uuccaguacu ccccugccca acggguggag uaucucuuga aagcaugggc ggaaaagaag       10500
gaccccaugg guuuuucgua ugauacccga ugcuucgacu caaccgucac ugagagagac       10560
aucaggaccg aggaguccau auaccaggcc ugcucccugc ccgaggaggc ccgcacugcc       10620
auacacucgc ugacugagag acuuuacgua ggagggccca uguucaacag caagggucaa       10680
accugcgguu acagacguug ccgcgccagc ggggugcuaa ccacuagcau ggguaacacc       10740
aucacaugcu augugaaagc ccuagcggcc ugcaaggcug cggggauagu ugcgcccaca       10800
augcugguau gcggcaauga ccuaguaguc aucucagaaa gccaggggac ugaggaggac       10860
gagcggaacc ugagagccuu cacggaggcc augaccaggu acucugcccc uccuggugau       10920
ccccccagac cggaauauga ccuggagcua auaacauccu guuccucaaa ugugucugug       10980
gcguugggcc cgcggggccg ccgcagauac uaccugacca gagacccaac cacuccacuc       11040
gcccgggcug ccugggaaac aguuagacac uccccuauca auucauggcu gggaaacauc       11100
auccaguaug cuccaaccau auggguucgc augguccuaa ugacacacuu cuucuccauu       11160
cucauggucc aagacacccu ggaccagaac cucaacuuug agauguaugg aucaguauac       11220
uccgugaauc cuuuggaccu uccagccaua auugagaggu uacacgggcu ugacgccuuu       11280
ucuaugcaca cauacucuca ccacgaacug acgcgggugg cuucagcccu cagaaaacuu       11340
ggggcgccac cccucagggu guggaagagu cgggcucgcg cagucagggc gucccucauc       11400
ucccguggag ggaaagcggc cguuugcggc cgauaucucu ucaauugggc ggugaagacc       11460
aagcucaaac ucacuccauu gccggaggcg cgccuacugg acuuauccag uugguucacc       11520
gucggcgccg gcgggggcga cauuuuucac agcgugucgc gcgcccgacc ccgcucauua       11580
cucuucggcc uacuccuacu uuucguaggg guaggccucu uccuacuccc cgcucgguag       11640
agcggcacac acuagguaca cuccauagcu aacuguuccu uuuuuuuuuu uuuuuuuuuu       11700
uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuccc ucuuucuucc cuucucaucu       11760
uauucuacuu ucuuucuugg uggcuccauc uuagcccuag ucacggcuag cugugaaagg       11820
uccgugagcc gcaugacugc agagagugcc guaacugguc ucucugcaga ucaugu           11876

Claims (26)

1.一种复制子RNA，所述RNA含有的核苷酸序列包含基因型2a丙型肝炎病毒基因组RNA的5′非翻译区、核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列、NS5B蛋白编码序列和3′非翻译区，至少一种选择标记基因和/或至少一种报告基因以及至少一种IRES序列。
2.权利要求1的复制子RNA，其中所述核苷酸序列以5′-3′方向按以下顺序包含5′非翻译区、至少一种选择标记基因和/或至少一种报告基因、至少一种IRES序列，以及核心蛋白编码序列、E1蛋白编码序列、E2蛋白编码序列、NS2蛋白编码序列、NS3蛋白编码序列、NS4A蛋白编码序列、NS4B蛋白编码序列、NS5A蛋白编码序列、NS5B蛋白编码序列和3′非翻译区。
3.权利要求1或2的复制子RNA，其中基因型2a丙型肝炎病毒基因组RNA是包含SEQ ID NO：12所示核苷酸序列的RNA。
4.权利要求1-3中任一项的复制子RNA，其中5′非翻译区含SEQID NO：1所示核苷酸序列，核心蛋白编码序列含SEQ ID NO：2所示核苷酸序列，E1蛋白编码序列含SEQ ID NO：3所示核苷酸序列，E2蛋白编码序列含SEQ ID NO：4所示核苷酸序列，NS2蛋白编码序列含SEQ ID NO：5所示核苷酸序列，NS3蛋白编码序列含SEQ ID NO：6所示核苷酸序列，NS4A蛋白编码序列含SEQ ID NO：7所示核苷酸序列，NS4B蛋白编码序列含SEQ ID NO：8所示核苷酸序列，NS5A蛋白编码序列含SEQ ID NO：9所示核苷酸序列，NS5B蛋白编码序列含SEQ ID NO：10所示核苷酸序列，而3′非翻译区含SEQ ID NO：11所示核苷酸序列。
5.一种复制子RNA，所述RNA包含以下(a)或(b)的RNA：
(a)含SEQ ID NO：13所示核苷酸序列的RNA；或
(b)含通过缺失、置换或添加1-100个核苷酸而衍生自SEQ ID NO：13所示核苷酸序列的核苷酸序列、并具有自主复制能力和病毒颗粒生产能力的RNA。
6.一种生产细胞的方法，其中所述细胞复制复制子RNA并生产病毒颗粒，所述方法包括将权利要求1-5中任一项的复制子RNA导入到细胞中。
7.权利要求6的方法，其中所述细胞为增殖性细胞。
8.权利要求6或7的方法，其中所述细胞为真核细胞。
9.权利要求8的方法，其中所述真核细胞为人肝源细胞、人子宫颈源细胞或人胎肾源细胞。
10.权利要求8的方法，其中所述真核细胞为Huh7细胞、HepG2细胞、IMY-N9细胞、HeLa细胞或293细胞。
11.权利要求6-10中任一项的方法可获得的细胞，所述细胞复制复制子RNA并生产病毒颗粒。
12.一种生产丙型肝炎病毒颗粒的方法，所述方法包括培养权利要求11的细胞，以使该细胞生产病毒颗粒。
13.一种丙型肝炎病毒颗粒，所述颗粒可利用权利要求12的方法获得。
14.一种生产丙型肝炎病毒感染细胞的方法，所述方法包括培养权利要求11的细胞，并周培养物中的病毒颗粒感染其它细胞。
15.一种丙型肝炎病毒感染细胞，所述细胞可利用权利要求14的方法获得。
16.一种筛选抗丙型肝炎病毒的物质的方法，所述方法包括在测试物存在下培养选自以下(a)、(b)和(c)中的至少一种：
(a)权利要求11的细胞，
(b)权利要求15的丙型肝炎病毒感染细胞，
(c)权利要求13的丙型肝炎病毒颗粒和丙型肝炎病毒允许细胞；
并检测产生的培养物中的复制子RNA或病毒颗粒。
17.一种丙型肝炎疫苗，所述疫苗包含权利要求13的丙型肝炎病毒颗粒或其部分。
18.一种生产丙型肝炎疫苗的方法，所述方法使用权利要求13的丙型肝炎病毒颗粒或其部分作为抗原。
19.一种生产用于基因治疗的嗜肝病毒载体的方法，所述方法使用权利要求1-5中任一项的复制子RNA。
20.一种嗜肝病毒载体，所述载体可利用权利要求18的方法获得。
21.一种在细胞中复制和/或表达外源基因的方法，所述方法包括将外源基因编码RNA插入到权利要求1-5中任一项的复制子RNA中，并将其导入到所述细胞中。
22.一种生产细胞的方法，其中所述细胞复制RNA并生产病毒颗粒，所述方法包括将含SEQ ID NO：12所示核苷酸序列的RNA导入到细胞中。
23.一种生产丙型肝炎病毒颗粒的方法，所述方法包括将含SEQID NO：12所示核苷酸序列的RNA导入到细胞中，并培养细胞，以使该细胞生产病毒颗粒。
24.权利要求21或22的方法，其中所述细胞是增殖性细胞。
25.一种生产含外源基因的病毒载体的方法，所述方法包括将外源基因编码RNA插入到含SEQ ID NO：12所示核苷酸序列的RNA中，将其导入到细胞中，并培养细胞，以使其生产病毒颗粒。
26.一种抗体，所述抗体抗权利要求13的丙型肝炎病毒颗粒。
CN2005800115150A 2004-02-20 2005-02-21 含人丙型肝炎病毒全长基因组的核酸构建物、核酸构建物转入其中的重组全长病毒基因组复制型细胞和生产丙型肝炎病毒颗粒的方法 Expired - Fee Related CN1942585B (zh)

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