CN113474454A - 可控的基因组编辑*** - Google Patents
可控的基因组编辑*** Download PDFInfo
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- CN113474454A CN113474454A CN202080012088.2A CN202080012088A CN113474454A CN 113474454 A CN113474454 A CN 113474454A CN 202080012088 A CN202080012088 A CN 202080012088A CN 113474454 A CN113474454 A CN 113474454A
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Abstract
本文提供了用于基因组编辑和修饰的组合物和方法。在一个实施方式中,所述组合物包含调控基因表达构建体,所述调控基因表达构建体包含编码RNA的核酸,所述RNA包含编码基因组编辑酶的序列和可操作地连接至所述序列的调控表达盒。在一个实施方式中,所述调控表达盒包含条件性外显子和适体结构域,所述适体结构域能够与效应分子结合以触发所述RNA的结构改变,从而调控所述条件性外显子的剪接和所述基因组编辑酶的表达。
Description
相关申请的交叉引用
本申请要求2019年01月30日提交的美国临时申请号62/798,478的优先权,其公开的内容通过引用并入本申请。
序列表
2020年1月30日创建的名称为“044903-8025WO01-SL-20200130_ST25”的文件中包含85KB(在Microsoft Windows中测量)的序列表,通过电子形式在此提交,并通过引用并入本申请。
背景技术
I.发明领域
本发明一般地涉及用于基因组编辑和修饰的组合物和方法。
II.相关现有技术说明
基因组编辑技术允许在活生物体的基因组中进行位点特异性DNA***、缺失、修饰或替换,从而彻底改变了生物医学领域。目前,基因组编辑的常用方法使用工程化的位点特异性核酸酶,在基因组中的所需位置产生双链断裂。诱导的双链断裂通过同源重组或非同源末端连接修复,导致有针对性的基因组改变。
尽管目前的基因组编辑技术为位点特异性基因组改变提供了强大的工具,但是由工程化的位点特异性核酸酶的非特异性和意外切割导致的脱靶编辑仍是一个大问题。例如,使用早期版本的CRISPR-Cas9***进行的多项研究发现,超过50%的RNA引导的核酸内切酶诱导的突变没有发生在靶点上(Fu等,(2013)Nature Biotechnology,31:822-6;Lin等,(2014)Nucleic Acid Research,42:7473-85)。担心的是,如果将基因组编辑技术用于治疗,脱靶效应可能会破坏重要的编码区,从而导致诸如癌症的基因毒性效应。
导致脱靶编辑的主要因素之一是细胞中位点特异性核酸酶的长期存在。这种位点特异性核酸酶在基因编辑后在细胞中保持活性的时间越长,脱靶编辑的机会就越大。因此,已经尝试了几种方法通过引入开和关的开关来控制细胞中位点特异性核酸酶的活性。例如,Bondy-Denomy团队使用一种天然存在的噬菌体蛋白来抑制Cas9免疫(Borges AL等,Cell(2018)174:917-25)。David Liu团队使用基于小分子激活内含肽的诱导型Cas9(DavisKM等,Nat Chem Biol.(2015)11:316-18)。博德研究所的张锋团队创造了一种能够***成雷帕霉素敏感性二聚化结构域的Cas9蛋白(Zetsche B等,Nat Biotechnol.(2015)33:139-42)。然而,这些方法将额外的可能有害的外源蛋白引入细胞。因此,持续需要开发用于基因组编辑的新的可控***。
发明内容
在一个方面,本公开内容提供了一种用于基因组编辑和修饰的组合物。在一个实施方式中,所述组合物包含调控基因表达构建体,所述调控基因表达构建体包含编码RNA的核酸,所述RNA包含编码基因组编辑酶的序列和可操作地连接至所述序列的调控表达盒。
在一个实施方式中,所述调控表达盒包含条件性外显子和适体结构域,所述适体结构域能够与效应分子结合以触发所述RNA的结构改变,从而调控所述条件性外显子的剪接和所述基因组编辑酶的表达。在某些实施方式中,在存在所述效应分子条件下的剪接过程中,跳过所述条件性外显子。
在某些实施方式中,当在所述效应分子存在的情况下将所述构建体递送至所述细胞时,所述基因组编辑酶在细胞中表达。在一个实施方式中,所述基因组编辑酶具有与SEQID NO:1至少90%(例如,90%、95%、98%、99%)同一性的序列。
在一个实施方式中,编码所述基因组编辑酶的序列被优化以包含外显子剪接增强子(ESE)。在某些实施方式中,编码所述基因组编辑酶的序列包含ESE优化的区域,所述区域具有与SEQ ID NO:10、12或14(在DNA形式中)或SEQ ID NO:11、13或15(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。
在一个实施方式中,编码所述基因组编辑酶的序列与SEQ ID NO:4、6或8(在DNA形式中)或SEQ ID NO:5、7或9(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性。
在一个实施方式中,所述适体结构域具有与SEQ ID NO:16、18或20(在DNA形式中)或SEQ ID NO:17、19或21(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。
在一个实施方式中,所述条件性外显子具有与SEQ ID NO:22(在DNA形式中)或SEQID NO:23(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。
在一个实施方式中,所述条件性外显子侧翼为上游内含子和下游内含子。在一个实施方式中,所述上游内含子具有与SEQ ID NO:24(在DNA形式中)或SEQ ID NO:25(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。在一个实施方式中,所述下游内含子具有与SEQ ID NO:26(在DNA形式中)或SEQ ID NO:27(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。
在一个实施方式中,所述调控表达盒包含与SEQ ID NO:28(在DNA形式中)或SEQID NO:29(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。在某些实施方式中,将所述调控表达盒***SEQ ID NO:10的核苷酸位置97和98之间(在DNA形式中)或者***SEQ ID NO:10的核苷酸位置498和499之间(在DNA形式中)。在某些实施方式中,所述可调控基因表达的构建体包含两个调控表达盒,其分别被***SEQ ID NO:10的核苷酸位置97和98之间和SEQ ID NO:10的核苷酸位置498和499之间。
在一个实施方式中,所述构建体包含与SEQ ID NO:30、32或34具有至少90%(例如,90%、95%、98%、99%)同一性的序列。
在一个实施方式中,所述调控表达盒包含当所述适体结构域未结合至所述效应分子时能够被miRNA识别的区域,从而导致RNA被降解。当所述适体结构域结合至所述效应分子时,RNA的结构改变阻止所述区域被miRNA所识别,从而导致所述基因组编辑酶的表达。在一个实例中,所述效应分子是四环素。
在某些实施方式中,在不存在所述效应分子的情况下,在细胞中表达所述基因组编辑酶。在某些实施方式中,在存在所述效应分子的情况下,所述调控表达盒抑制所述基因组编辑酶的表达。
在一个实施方式中,当所述适体结构域未结合至所述效应分子时,所述调控表达盒形成抗终止子茎,从而表达所述基因组编辑酶。当所述适体结合至所述效应分子时,所述调控表达盒形成终止子茎,从而抑制所述基因组编辑酶的表达。
在一个实施方式中,所述调控表达盒包含当所述适体结构域未结合至所述效应分子时被核糖体识别的核糖体结合序列,从而表达基因编辑酶。当所述适体结构域结合至所述效应分子时,所述核糖体结合序列被隔离而不能被核糖体所识别,从而抑制所述基因组编辑酶的表达。
在某些实施方式中,所述效应分子是代谢产物,例如,腺苷钴胺素、水钴胺素、cAMP、cGMP、c-di-AMP、c-di-GMP、氟化物、黄素单核苷酸、谷氨酰胺、甘氨酸、赖氨酸、镍、钴、前辫苷、嘌呤、S-腺苷甲硫氨酸、四氢叶酸、焦磷酸硫胺素、鸟嘌呤、腺嘌呤、2’-脱氧鸟苷、7-氨基甲基-7-脱氮鸟嘌呤、ZMP和ZTP。
在某些实施方式中,所述基因组编辑酶是位点特异性核酸酶或位点特异性重组酶。在一些实施方式中,所述位点特异性核酸酶选自下组:Cas9、Cas12、ZFN、TALEN和大范围核酸酶。在一些实施方式中,所述位点特异性重组酶选自下组:Cre、FLP、λ整合酶、phiC31整合酶、Bxb1整合酶、γ-δ解离酶、Tn3解离酶和Gin转化酶。
在某些实施方式中,所述构建体包含在载体中。在一个实例中,所述载体是AAV载体。
在一个实施方式中,所述基因编辑酶是Cas9,所述核酸构建体还包含编码gRNA的第二多核苷酸序列。
在另一个方面,本公开内容提供了一种在细胞中进行基因组编辑的方法。在一个实施方式中,所述方法包括将本文公开的构建体递送至细胞中。在一个实施方式中,所述方法还包括将所述效应分子递送至细胞中。
在又一个方面,本公开内容提供了一种通过将本文所述的构建体递送进入细胞中制成的修饰的细胞。
在另一个方面,本公开内容提供了一种治疗患有疾病的对象的方法。在一个实施方式中,所述方法包括将本文公开的构建体递送到所述对象的至少一个细胞中。在一个实施方式中,所述方法还包括向所述对象施用所述效应分子。
附图说明
以下附图构成本说明书的一部分并且被包括以进一步展示本公开内容的某些方面。通过参考这些附图中的一幅或多幅并结合在本文中呈现的具体实施方式的详细描述,可以更好地理解本公开内容。
图1示出了本发明核酸构建体的示例性实施方式,其中RNA转录物的结构变化调控RNA转录物的剪接。
图2示出了本发明核酸构建体的示例性实施方式,其中核酸构建体编码Cas9蛋白并被包含在AAV载体中。
图3示出了本发明核酸构建体的示例性实施方式,其中RNA转录物的结构变化调控RNA转录物的稳定性。
图4示出了本发明核酸构建体的示例性实施方式,其中RNA转录物的结构变化调控RNA转录物的翻译。
图5示出了本发明核酸构建体的示例性实施方式,其中RNA转录物的结构变化调控RNA转录物的翻译。
图6示出了在SaCas9基因中加入内含子。
图7示出了SaCas9构建体的示意图,其中SaCas9基因处于CMV启动子的控制下。可以通过ESE富集和ESS缺失并且含有一个或多个内含子、适体和条件性外显子优化SaCas9基因。
图8示出了添加内含子的SaCas9基因的EGxxFP测定的结果。
图9示出了含有适体结构域和条件性外显子的SaCas9基因的EGxxFP测定的结果。
图10示出了在不存在四环素的条件下具有双适体结构域的SaCas9基因的EGxxFP测定的结果。
图11示出了在存在四环素的条件下具有双适体结构域的SaCas9基因的EGxxFP测定的结果。
具体实施方式
在更详细地描述本公开内容之前,应当理解的是,本公开内容不限于所描述的特定实施方式,并且因此当然可以变化。还应当理解的是,本文中使用的术语仅出于描述特定实施方式的目的,而无意于限制本发明,因为本公开内容的范围将仅由所附权利要求所限定。
非另外定义,否则本申请使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本申请所述的那些类似或等同的任何方法和材料也可以用于实施或检测本公开内容,但是目前描述的是优选的方法和材料。
本说明书中引用的所有出版物和专利均通过引用并入本申请,如同每个单独的出版物或专利被具体和单独地指出通过引用并入并且通过引用并入本申请以公开和描述与所引用的出版物相关的方法和/或材料。任何出版物的引用是针对其在申请日之前公开的内容,并且不应将其解释为承认由于在先的公开使得本公开内容无权先于此类出版物。此外,所提供的公开日期可能与实际公开日期是不同的,所述实际公开日期可能需要单独确认。
本领域技术人员在阅读本公开内容之后将是显而易见的是,本申请所描述和示出的每个单独的实施方式具有离散的组件和特征,在不脱离本公开内容的范围或主旨的前提下,其可以容易地与任意其他若干实施方式的特征分离或组合在一起。任何列举的方法可以按照事件的顺序或以逻辑上可能的任何其他顺序进行。
I.定义
如在本申请中所使用的,除非上下文另外明确指出,否则单数形式“一个(a)”、“一种(an)”和“该(the)”包括复数形式。
在本公开内容中值得注意的是,诸如“包含(comprises)”、“包含(comprised)”、“包含(comprising)”、“含有(contains)”、“含有(containing)”等的术语是包容性的或开放式的,并且不排除附加的、未列举的要素或方法步骤。诸如“基本上由……组成(consisting essentially of)”和“基本上由……组成(consists essentially of)”的术语允许包含不会对要求保护的发明的基本和新颖特征产生实质性影响的附加成分或步骤。术语“由……组成(consists of)”和“由……组成(consisting of)”是封闭式的。
术语“适体”指能够特异性结合靶分子的核苷酸序列。适体通常是从大型随机序列池中选择而产生的,但也天然存在,如在核糖体开关中。
如在本申请中所使用的,“细胞”可以是原核细胞或真核细胞。原核细胞包括例如细菌。真核细胞包括例如真菌、植物细胞和动物细胞。动物细胞(例如,哺乳动物细胞或人细胞)的类型包括例如来自循环/免疫***或器官的细胞(例如,B细胞、T细胞(细胞毒性T细胞、自然杀伤T细胞、调节性T细胞、T辅助细胞)、自然杀伤细胞、粒细胞(例如,嗜碱性粒细胞、嗜酸性粒细胞、中性粒细胞和多叶核嗜中性粒细胞)、单核细胞或巨噬细胞、红细胞(例如,网织红细胞)、肥大细胞、血小板或巨核细胞和树突状细胞);来自内分泌***或器官的细胞(例如,甲状腺细胞(例如,甲状腺上皮细胞、滤泡旁细胞)、甲状旁腺细胞(例如,甲状旁腺主细胞、嗜酸性细胞),肾上腺细胞(例如,嗜铬细胞)和松果体细胞(pineal cell)(例如,松果体细胞(pinealocyte));来自神经***或器官的细胞(例如,成胶质细胞(例如,星形胶质细胞和少突胶质细胞)、小胶质细胞、巨细胞神经分泌细胞、星状细胞,伯特歇尔(boettcher)细胞和垂体细胞(例如,***、促肾上腺皮质激素、促甲状腺素、促生长激素和催乳激素细胞));来自呼吸***或器官的细胞(例如,肺细胞(I型肺细胞和II型肺细胞)、克拉拉(clara)细胞、杯状细胞、肺泡巨噬细胞);来自循环***或器官的细胞(例如,心肌细胞和周细胞);来自消化***或器官的细胞(例如,胃主细胞、壁细胞、杯状细胞、潘氏(paneth)细胞、G细胞、D细胞、ECL细胞、I细胞、K细胞、S细胞、肠内分泌细胞、肠嗜铬细胞、APUD细胞、肝脏细胞(例如,肝细胞和枯否(Kupffer)细胞));来自表皮***或器官的细胞(例如,骨细胞(例如,成骨细胞、骨细胞和破骨细胞)、牙齿细胞(例如,成牙骨质细胞和成釉细胞)、软骨细胞(例如,成软骨细胞和软骨细胞)、皮肤/毛发细胞(例如,毛细胞、角质形成细胞和黑素细胞(痣细胞))、肌肉细胞(例如,肌细胞)、脂肪细胞、成纤维细胞和肌腱细胞);来自泌尿***或器官的细胞(例如,足细胞、肾小球旁细胞、肾小球内系膜细胞、肾小球外系膜细胞、肾近端小管刷状缘细胞和致密斑细胞),以及来自生殖***或器官的细胞(例如,***、睾丸支持细胞、***细胞、卵子和***)。细胞可以是正常的、健康细胞;或者患病的或不健康的细胞(例如,癌细胞)。细胞还包括哺乳动物受精卵或干细胞,其包括胚胎干细胞、胎儿干细胞、诱导多能干细胞和成体干细胞。干细胞是能够经历细胞***周期,同时保持未分化状态并分化成特化细胞类型的细胞。干细胞可以是全能干细胞、多能干细胞(pluripotent stem cell)、多潜能干细胞(multipotent stemcell)、寡能干细胞和单能干细胞,其中任何一种都可以从体细胞诱导。干细胞还可以包括癌症干细胞。哺乳动物细胞可以是啮齿动物细胞,例如小鼠、大鼠、仓鼠细胞。哺乳动物细胞可以是兔形目细胞,例如兔细胞。哺乳动物细胞也可以是灵长类动物细胞,例如人细胞。
如在本申请中所使用的,术语“构建体”或“核酸构建体”是指其中将目标多核苷酸序列***到载体中的核酸。如在本申请中所使用的,术语“载体”指其中可以可操作地***编码蛋白的多核苷酸以引起所述蛋白表达的载体。可以将载体用于转化、转导或转染宿主细胞,以使得其携带的遗传元件在宿主细胞内表达。载体的实例包括质粒、噬菌粒、粘粒和人工染色体(如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC))、噬菌体(如λ噬菌体或M13噬菌体)和动物病毒。作为载体的动物病毒的类别包括逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒(AAV)、疱疹病毒(例如,单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒和乳多空病毒(例如,SV40)。载体可以含有用于控制表达的多种元件,包括启动子序列、转录起始序列、增强子序列、可选择元件和报告基因。此外,载体可以含有复制原点。载体还可以含有帮助其进入细胞的物质,包括但不限于病毒颗粒、脂质体或蛋白包膜。
如在本申请中所使用的,术语“双链”指沿着其长度的至少一部分杂交的一条或两条核酸链。在某些实施方式中,“双链”并不意味着核酸必须是完全双链的。相反,双链核酸可具有一个或多个单链区段和一个或多个双链区段。例如,双链核酸可以是双链DNA、双链RNA或双链DNA/RNA化合物。可以使用本领域常用的方法确定核酸的形式,如使用SYBR绿染色的分子条带和通过电泳区分。
在将核酸序列***细胞的背景下,术语“递送(deliver)”或“递送(delivered)”或“递送(delivering)”是指“转染”、或“转化”、或“转导”,并且包括涉及将核酸序列引入真核或原核细胞,其中所述核酸序列可暂时存在于细胞中或可掺入细胞的基因组(例如,染色体、质粒、质体或线粒体DNA)中,转化为自主复制子。可以使用本领域公知的任何方法将本公开内容的构建体递送进入细胞。可以使用用于转染动物细胞的各种技术,包括,例如:显微注射、逆转录病毒介导的基因转移、电穿孔、转染等(参见,例如,Keown等,Methods inEnzymology 1990,185:527-537)。在一个实施方式中,通过病毒将所述构建体递送进入细胞。
术语“外显子”指基因内的核苷酸序列,在通过RNA剪接除去内含子后,所述基因编码由该基因产生的最终成熟RNA的一部分。如在本申请中所使用的,外显子指基因内的DNA序列和在RNA转录物中的相应序列两者。
术语“基因组编辑酶”指能够改变或修饰细胞中基因序列的酶。基因组编辑酶包括单不限于位点特异性核酸酶(例如,Cas9、ZFN、TALEN和大范围核酸酶)和位点特异性重组酶(例如,Cre、FLP、λ整合酶、phiC31整合酶、Bxb1整合酶、γ-δ解离酶、Tn3解离酶和Gin转化酶)。
术语“内含子”指基因内的核苷酸序列,其在最终RNA产物的成熟过程中通过RNA剪接除去。术语“内含子”指基因内的DNA序列和在RNA转录物中的相应序列两者。
术语“修饰”或“基因修饰”指在基因组水平的破坏,其可导致细胞表达的基因的表达或活性降低或增加。示例性的修饰可以包括***、缺失、替代、移码突变、点突变、去除外显子、去除一个或多个DNAse 1-高敏位点(DHS)(例如,2、3、4或更多个DHS区)等。
在基因编辑背景下,“所需的修饰”是指目标基因修饰,这是操纵者所追求的。本公开内容的所需修饰可以是基因组区域中能够恢复、增强或改变基因的正常功能或选择的功能,或者增加或降低基因表达的修饰。“不需要的修饰”与“所需的修饰”相对,其是由不同于所需的那些随机修饰导致的不希望的修饰。在本公开内容的某些实施方式中,可以通过CRISPR相关的***产生基因组区域的一个或多个所需的修饰和/或一个或多个不需要的修饰。
术语“核酸”和“多核苷酸”可互换使用,并且指任意长度核苷酸(脱氧核糖核苷酸或核糖核苷酸,或其类似物)的聚合形式。多核苷酸可以具有任意三维结构,并且可以执行已知或未知的任何功能。多核苷酸的非限制性实例包括基因、基因片段、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核酶、cDNA、shRNA、单链的短链或长链RNA、重组多核苷酸、支链多核苷酸、质粒、载体、任何序列的分离的DNA、控制区、任何序列的分离的RNA、核酸探针和引物。核酸分子可以是线形的或环状的。
如在本申请中所使用的,“核酸酶”是能够切割核酸的核苷酸亚基之间的磷酸二酯键的酶。“位点特异性核酸酶”是指其功能取决于特定核苷酸序列的核酸酶。通常,位点特异性核酸酶识别并结合特定的核苷酸序列并切割核苷酸序列内的磷酸二酯键。在某些实施方式中,使用位点特异性核酸酶通过位点特异性切割产生双链断裂。位点特异性核酸酶的实例包括但不限于锌指核酸酶(ZFN),转录激活因子样效应物核酸酶(TALEN)、大范围核酸酶和CRISPR(成簇规则间隔短回文重复序列)相关的(Cas)核酸酶。
位点特异性核酸酶通常含有DNA结合结构域和DNA切割结构域。例如,ZFN含有通常包含3-6个独立的锌指重复序列的DNA结合结构域和由用于DNA切割的FokI限制性酶组成的核酸酶结构域。ZFN的DNA结合结构域可以识别9至18个碱基对。在含有TALE结构域和DNA切割结构域的TALEN的实例中,TALE结构域含有除了第12和第13个氨基酸之外重复的高度保守的33-34个氨基酸序列,第12和第13个氨基酸的变化显示与特异性核苷酸识别有强相关性。又例如,典型的Cas核酸酶Cas9由N末端识别结构域和C-末端的两个内切核酸酶结构域(RuvC结构域和HNH结构域)组成。
术语“可操作地连接”指元件的排布,其中这样描述的部件被配置成执行其通常的功能。当针对多核苷酸使用时,该术语指两个或多个目标多核苷酸序列以这样的方式并置(juxtaposition),其有或没有间隔区或接头,其处于允许其以预期方式起作用的关系中。例如,当编码多肽的多核苷酸与调控序列(例如,启动子、增强子、沉默子序列等)可操作地连接时,其旨在指多核苷酸序列以允许从多核苷酸调控的表达多肽的方式连接。调控序列不需要与编码序列是连续的,只要其起到指导其表达的作用即可。例如,在调控序列和编码序列之间可以存在***的未翻译但转录的序列,并且仍然可以认为调控序列与编码序列“可操作地连接”。又例如,调控序列可以包含在编码序列内(例如,内含子内),并且仍然可以认为调控序列与编码序列“可操作地连接”。
如在本申请中所使用的,“启动子”和“启动子-增强子”序列是一系列核酸控制序列,RNA聚合酶与其结合并启动转录。启动子包含靠近转录起始位点的必需的核酸序列,如在聚合酶II型启动子的情况下的TATA元件。启动子-增强子还任选地包含远端增强子或阻遏元件,其可以位于距转录起始位点多达几千个碱基对处。启动子通过指定要转录的DNA链来确定转录物的极性。真核启动子是由RNA聚合酶II使用的复杂序列排布。通用转录因子(GTFS)首先在起点附近结合特定序列,然后招募RNA聚合酶II的结合。除了这些最小的启动子元件以外,小序列元件被模块化DNA结合/反式激活蛋白(例如,AP-1、SP-1)特异性识别,这些蛋白调节给定启动子的活性。病毒启动子与细菌或真核启动子具有相同功能,并且或者提供特定的反式RNA聚合酶(噬菌体T7)、或者募集细胞因子和RNA聚合酶(SV40、RSV、CMV)。此外,启动子可以是组成型或可调控的。诱导型元件是与启动子一起起作用并且可以结合阻遏物或诱导物的DNA序列元件。在这种情况下,转录实际上被“关闭”,直至启动子被去阻遏或诱导,此时转录被“开启”。真核启动子的实例包括但不限于以下:小鼠金属硫蛋白I基因序列的启动子(Hamer等,J.Mol.Appl.Gen.(1982)1:273-288);疱疹病毒的TK启动子(McKnight,Cell(1982)31:355-365);SV40早期启动子(Benoist等,Nature(1981)290:304-310);酵母瘿基因序列启动子(Johnston等,Proc.Natl.Acad.Sci.(1982)79:6971-6975;Silver等,Proc.Natl.Acad.Sci.(1984)
81:5951-59SS)、CMV启动子、EF-1启动子、蜕皮激素响应性启动子、四环素响应性启动子等。
在通常情况下,“蛋白”是多肽(即,通过肽键彼此连接的至少两个氨基酸串)。蛋白可以包括除氨基酸以外的部分(例如,可以是糖蛋白)和/或可以以其他方式加工或修饰。本领域技术人员将意识到“蛋白”可以是由细胞产生的完整多肽链(具有或不具有信号序列),或可以是其功能部分。本领域技术人员还将意识到有时蛋白可以包含一条以上多肽链,例如其通过一个或多个二硫键连接或以其他方式缔合。
如在本申请中所使用的,术语“重组酶”或“位点特异性重组酶”指促进特定靶位点之间的DNA重排的高度特化的酶家族(Greindley等,2006;Esposito,D.和Scocca,J.J.,Nucleic Acids Research 25,3605-3614(1997);Nunes-Duby,S.E.等,Nucleic AcidsResearch 26,391-406(1998);Stark,W.M.等,Trends in Genetics 8,432-439(1992))。实际上,所有位点特异性重组酶都可以归类为两个结构和机制不同的组之一:酪氨酸(例如,Cre、Flp和λ整合酶)或丝氨酸(例如,phiC31整合酶、Bxb1整合酶、γ-δ解离酶、Tn3解离酶和Gin转化酶)重组酶。这两个重组酶家族均识别由两个反向重复结合元件组成的靶位点,这些结合元件位于发生DNA断裂和重新连接的间隔序列的侧翼。重组过程需要两个重组酶单体同时结合到每个靶位点:两个DNA结合的二聚体(四聚体),然后连接以形成突触复合物,导致交叉和链交换。
如在本申请中所使用的,术语“核糖开关”指信使RNA分子的调控区段,其与小分子结合,导致由mRNA编码的蛋白的产生发生变化。核糖开关包括但不限于钴胺素核糖开关、细胞周期蛋白AMP-GMP核糖开关、环状二AMP核糖开关、环状二GMP核糖开关、氟化物核糖开关、FMN核糖开关、glmS核糖开关、谷氨酰胺核糖开关、甘氨酸核糖开关、赖氨酸核糖开关、锰核糖开关、NiCo核糖开关、PreQ1核糖开关、嘌呤核糖开关、SAH核糖开关、SAM核糖开关、SAM-SAH核糖开关、四氢叶酸核糖开关、TPP核糖开关、ZMP/ZTP核糖开关。在某些实施方式中,小分子是代谢产物,如核糖开关代谢产物,例如,腺苷钴胺素、水钴胺素、cAMP、cGMP、c-di-AMP、c-di-GMP、氟化物、黄素单核苷酸、谷氨酰胺、甘氨酸、赖氨酸、镍、钴、前辫苷、嘌呤、S-腺苷甲硫氨酸、四氢叶酸、焦磷酸硫胺素、鸟嘌呤、腺嘌呤、2’-脱氧鸟苷、7-氨基甲基-7-脱氮鸟嘌呤、ZMP和ZTP。
如在本申请中所使用的,术语“对象”或“个体”或“动物”或“患者”指需要诊断、预后、改善、预防和/或治疗疾病或病症(如病毒感染或肿瘤)的人或非人动物,包括哺乳动物或灵长类。哺乳动物对象包括人、家畜、农场动物以及动物园、运动或宠物动物,如犬、猫、豚鼠、家兔、大鼠、小鼠、马、猪、奶牛、熊等。
在形成CRISPR复合物的背景下,“靶点”指设计为与基因组区域(即,靶序列)具有互补性的向导序列(即,gRNA),其中基因组区域和向导RNA之间的杂交促进了CRISPR复合物的形成。术语“互补性”或“互补的”用于指碱基配对规则相关的多核苷酸(即,核苷酸序列)。互补性可以是“部分的”,其中只有一些核酸碱基根据碱基配对规则匹配(例如,10个中的5、6、7、8、9、10个是50%、60%、70%、80%、90%和100%互补),或者核酸之间可能存在“完全”或“总体”互补性。核酸链之间的互补程度对其彼此杂交的效率和强度有显著影响。
“转录物”或“RNA转录物”指由用于蛋白表达的基因转录形成的RNA分子。RNA聚合酶转录初级转录物mRNA(称为前mRNA),后者被加工成成熟mRNA。因此,如在本申请中所使用的RNA转录物包括初级转录物mRNA和加工后的成熟mRNA。一种或多种转录物变体可以通过差异剪接从相同的DNA区段形成。在这样的过程中,基因的特定外显子可以包含在信使mRNA(mRNA)内或从其中排除,导致翻译的蛋白包含不同氨基酸和/或具有不同生物学功能。
如在本申请中所使用的,术语“载体”指其中可以可操作地***编码蛋白的多核苷酸以使得该蛋白能够表达的媒介(vehicle)。可以将载体用于转化、转导或转染宿主细胞,以使得其携带的遗传元件在宿主细胞内表达。载体的实例包括质粒、噬菌粒、粘粒、人工染色体(如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC))、噬菌体(如λ噬菌体或M13噬菌体)和动物病毒。作为载体的动物病毒的类别包括逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(例如,单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒和乳多空病毒(例如,SV40)。载体可以含有用于控制表达的多种元件,包括启动子序列、转录起始序列、增强子序列、可选择元件和报告基因。此外,载体可以含有复制原点。载体还可以含有帮助其进入细胞的物质,包括但不限于病毒颗粒、脂质体或蛋白包膜。
II.基因组编辑酶
在一个方面,本公开内容涉及用于基因组编辑的可控的***。在某些实施方式中,该***能够根据存在或不存在效应分子来切换基因组编辑酶的表达。
在某些实施方式中,基因组编辑酶包括但不限于位点特异性核酸酶(例如,Cas9、ZFN、TALEN和大范围核酸酶)和位点特异性重组酶(例如,Cre、FLP、λ整合酶、phiC31整合酶、Bxb1整合酶、γ-δ解离酶、Tn3解离酶和Gin转化酶)。
CRISPR(成簇规则间隔短回文重复序列)/Cas***最初是作为在原核细胞中的转录物和其他元件被发现的,其参与CRISPR相关的(“Cas”)基因表达或指导其活性,其包括编码Cas核酸酶的序列(切割核酸序列并产生双链断裂(DSB))、向导序列、反式激活CRISPR(tracr)序列、tracr-mate序列或来自CRISPR基因座的其他序列和转录物。在真核细胞中,CRISPR/Cas***包括CRISPR相关的核酸酶和小向导RNA。靶DNA序列(原型间隔区)包含“原型间隔区相邻基序”(PAM),这是一个被所使用的特定Cas蛋白识别的短DNA序列。在某些实施方式中,CRISPR***包括I型、II型和III型CRISPR/Cas***,其分别包含蛋白Cas3、Cas9和Cas10。
RNA引导的核酸内切酶Cas9是被广泛使用的II型CRISPR***的一个组分,其可在多种模型***中产生基因特异性敲除。在本公开内容的一个实施方式中,CRISPR/Cas核酸酶是“序列特异性核酸酶”。Cas9异位表达和单一向导RNA(gRNA)的引入足以导致在目标特异性基因组区域形成双链断裂(DSB),从而通过NHEJ途径导致***缺失。***缺失通常会导致移码突变,除非***/缺失的核苷酸数量是3的倍数。
与Cas核酸内切酶一起,CRISPR实验需要引入包含大约15到30个碱基序列的向导RNA,其特异性针对靶核酸(例如,DNA)。设计用于靶向目标基因组区域的gRNA(例如,编码蛋白功能性结构域的特定外显子)将在编码蛋白的每个基因中产生突变。所得到的经修饰的基因组区域可以包含一个或多个变体,其每一个在突变中是不同的。例如,突变将导致具有所需修饰的修饰的基因组区域,和/或具有不需要的修饰的修饰的基因组区域。该方法已被广泛用于在各种模型***中产生基因特异性敲除。在某些实施方式中,gRNA的长度为15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸。gRNA可以作为RNA或通过转染具有与启动子可操作连接的gRNA编码序列的载体(例如,质粒)递送到真核细胞或原核细胞中。
在某些实施方式中,Cas核酸酶和gRNA来源于相同物种。在某些实施方式中,例如,Cas核酸酶来源于金黄色葡萄球菌(Staphylococcus aureus)、表皮葡萄球菌(Staphylococcus epidermidis)、松鼠葡萄球菌(Staphylococcus sciuri)、铜绿假单胞菌(Pseudomonas aeruginosa)、屎肠球菌(Enterococcus faecium)、粪肠球菌(Enterococcusfaecalis)、大肠杆菌(Escherichia coli)、肺炎克雷伯菌(Klebsiella pneumoniae)、肺炎链球菌(Streptococcus pneumoniae)、酿脓链球菌(Streptococcus pyrogenes)、保加利亚乳杆菌(Lactobacillus bulgaricus)、嗜热链球菌(Streptococcus thermophilus)、霍乱弧菌(Vibrio cholera)、木糖氧化无色杆菌(Achromobacter xylosoxidans)、洋葱伯克霍尔德菌(Burkholderia cepacia)、异型枸橼酸杆菌(Citrobacter diversus)、弗氏柠檬酸杆菌(Citrobacter freundii)、滕黄微球菌(Micrococcus leuteus)、奇异变形杆菌(Proteus mirabilis)、普通变形杆菌(Proteus vulgaris)、路邓葡萄球菌(Staphylococcus lugdunegis)、伤寒沙门氏菌(Salmonella typhi)、A群链球菌(Streptococcus Group A)、B群链球菌(Streptococcus Group B)、粘质沙雷菌(S.marcescens)、阴沟肠杆菌(Enterobacter cloacae)、炭疽杆菌(Bacillus anthracis)、百日咳杆菌(Bordetella pertussis)、梭菌属(Clostridium sp.)、肉毒杆菌(Clostridiumbotulinum)、破伤风梭菌(Clostridium tetani)、白喉棒状杆菌(Corynebacteriumdiphtheria)、莫拉(布兰汉氏)卡他莫拉菌(Moraxalla(Brauhamella)catarrhalis)、志贺杆菌(Shigella spp.)、流感嗜血杆菌(Haemophilus influenza)、嗜麦芽窄食单胞菌(Stenotrophomonas maltophili)、假单胞菌(Pseudomonas perolens)、莓实假单胞菌(Pseuomonas fragi)、脆弱拟杆菌(Bacteroides fragilis)、梭杆菌属(Fusobacteriumsp.)、韦荣球菌属(Veillonella sp.)、鼠疫耶尔森菌(Yersinia pestis)和假结核耶尔森菌(Yersinia pseudotuberculosis)。
可以使用任何本领域公知的软件设计gRNA,如Target Finder、E-CRISPR、CasFinder和CRISPR Optimal Target Finder。
在某些实施方式中,本申请所述的组合物包含编码Cas核酸酶或gRNA的核酸,其中所述核酸包含在载体中。在一些实施方式中,所述组合物包含Cas核酸酶蛋白和编码gRNA的DNA。在一些实施方式中,所述组合物包含编码Cas核酸酶的第一核酸和编码gRNA的第二核酸,其中第一核酸和第二核酸包含在一个载体中。在一些实施方式中,第一核酸和第二核酸包含在两个单独的载体中。在一些实施方式中,至少一个载体是病毒载体。在某些实施方式中,载体是AAV载体。
锌指核酸酶(ZFN)是一种人工限制性酶,其是通过将锌指DNA结合结构域与DNA切割结构域融合而产生的。可以对锌指结构域进行工程化以靶向特定的所需DNA序列,其指导锌指核酸酶切割靶DNA序列。通常,锌指DNA结合结构域含有3至6个单独的锌指重复序列,可以识别9至18个碱基对。每个锌指重复通常包含约30个氨基酸,并且包含由锌离子稳定的ββα折叠。串联排列的相邻锌指重复序列通过接头序列连接在一起。已经开发了多种策略来设计锌指结构域以结合所需序列,包括“模块化组装”和采用噬菌体展示或细胞选择***的选择策略(Pabo CO等,“Design and Selection of Novel Cys2His2 Zinc FingerProteins”Annu.Rev.Biochem.(2001)70:313–40)。生成新的锌指DNA结合结构域的最直接方法是组合已知特异性的较小锌指重复序列。最常见的模块化组装过程包括组合三个独立的锌指重复序列,每个重复序列可以识别3个碱基对的DNA序列,以生成一个可以识别9个碱基对靶位点的3-指阵列。其他程序可以利用1-指或2-指模块来产生具有6个或更多个单独锌指重复的锌指阵列。或者,已将选择方法用于产生能够靶向所需序列的锌指DNA结合结构域。最初的选择工作利用噬菌体展示从大量部分随机化的锌指结构域中选择结合给定DNA靶点的蛋白。最近的工作已利用酵母单杂交***、细菌单杂交***和双杂交***以及哺乳动物细胞。一种选择新型锌指阵列的有前途的新方法利用细菌双杂交***将预先选择的单个锌指重复序列库组合,每个重复序列都被选择以结合给定的三联体,然后利用第二轮选择来获得能够结合所需9-bp序列的3-指重复序列(Maeder ML,等,“Rapid‘open-source’engineering of customized zinc-finger nucleases for highly efficient genemodification”.Mol.Cell(2008)31(2):294–301)。通常将来自II型限制性核酸内切酶FokI的非特异性切割结构域用作ZFN中的切割结构域。该切割结构域必须二聚化才能切割DNA,因此需要一对ZFN来靶向非回文DNA位点。标准ZFN将切割结构域融合至每个锌指结构域的C末端。为了让两个切割结构域二聚化并切割DNA,两个单独的ZFN必须结合其C端相距一定距离的相反的DNA链。锌指结构域和切割结构域之间最常用的接头序列要求每个结合位点的5’边缘间隔5到7bp。
转录激活因子样效应物核酸酶(TALEN)是一种人工限制性内切酶,其通过将转录激活因子样效应物(TALE)DNA结合结构域融合至DNA切割结构域(例如,核酸酶结构域)制备,可以对其工程化以切割特定序列。TALE是黄单胞菌属(Xanthomonas)细菌当感染植物时通过其III型分泌***分泌的蛋白。TALE DNA结合结构域包含重复的高度保守的33-34个氨基酸序列,其中第12位和第13位氨基酸存在差异,其是高度可变的并且显示出与特定核苷酸识别的强相关性。氨基酸序列与DNA识别之间的关系允许通过选择包含适当可变氨基酸的重复区段的组合来工程化特定的DNA结合结构域。可以将来自FokI核酸内切酶末端的非特异性DNA切割结构域用于构建TALEN。FokI结构域作为二聚体发挥作用,需要两个具有独特DNA结合结构域的构建体,用于靶基因组中具有适当方向和间距的位点。参见Boch,Jens“TALEs of genome targeting”Nature Biotechnology(2011)29:135–6;Boch,Jens等,“Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors”Science(2009)326:1509–12;Moscou MJ and Bogdanove AJ“A Simple Cipher GovernsDNA Recognition by TAL Effectors”Science(2009)326(5959):1501;Juillerat A等,“Optimized tuning of TALEN specificity using non-conventional RVDs”ScientificReports(2015)5:8150;Christian等,“Targeting DNA Double-Strand Breaks with TALEffector Nucleases”Genetics(2010)186(2):757–61;Li等,“TAL nucleases(TALNs):hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain”Nucleic Acids Research(2010)39:1–14。
位点特异性重组酶指介导被酶识别的特定DNA序列之间的位点特异性重组的酶家族。位点特异性重组酶的实例包括但不限于Cre重组酶、Flp重组酶、λ整合酶、γ-δ解离酶、Tn3解离酶、Sin解离酶、Gin转化酶、Hin转化酶、Tn5044解离酶、Tn3转座酶、睡美人转座酶、IS607转座酶、Bxb1整合酶、wBeta整合酶、BL3整合酶、phiR4整合酶、A118整合酶、TG1整合酶、MR11整合酶、phi370整合酶、SPBc整合酶、SV1整合酶、TP901-1整合酶、phiRV整合酶、FC1整合酶、K38整合酶、phiBT1整合酶和phiC31整合酶。
III.调控表达盒
在一个方面,本公开内容提供了一种编码RNA的调控表达的构建体,其包含控制序列(即,主编码区)表达的调控表达盒,所述主编码区通过结合至效应分子可操作地连接至调控表达盒。
本申请所述的调控表达盒是一种表达控制元件,其是要表达的RNA分子的一部分,并且在与效应分子结合时会改变状态。在一些实施方式中,调控表达盒位于主编码区的5’-非翻译区。在一些实施方式中,调控表达盒位于主编码区的3’-非翻译区。在一些实施方式中,***调控表达盒并位于主编码区内。
通常,调控表达盒包含两个独立的结构域:选择性结合效应分子的适体结构域和影响遗传控制的表达平台结构域。两个结构域之间的动态相互作用导致基因表达的控制取决于效应分子的存在。本申请公开了分离的和重组的调控表达盒,包含此类调控表达盒的重组构建体,与此类调控表达盒可操作地连接的异源序列,以及携带此类调控表达盒的转基因生物体。异源序列可以是例如编码目标蛋白或肽(包括基因组编辑酶)的序列。
所公开的调控表达盒,包括其衍生物和重组形式,通常可以来自任何来源,包括天然存在的调控表达盒和从头设计的那些。任何此类调控表达盒可以用于所公开的方法中或与所公开的方法一起使用。天然存在的调控表达盒是具有自然界中存在的调控表达盒序列(例如,核糖开关)的调控表达盒。此类天然存在的调控表达盒可以是天然存在的表达盒的分离或重组形式,因为其是在自然界中存在的。也就是说,调控表达盒具有相同的一级结构,但是已在新的遗传或核酸背景下被分离或工程化。例如,嵌合调控表达盒可以由任何或特定类别或类型的调控表达盒的调控表达盒的一部分和相同或任何不同类别或类型的调控表达盒的不同调控表达盒的一部分;任何或特定类别或类型的调控表达盒的调控表达盒的一部分和任何非调控表达盒序列或组分组成。重组调控表达盒是已在新的遗传或核酸背景下被分离或工程化的那些。
1.适体结构域
适体是能够选择性结合至特定化合物和化合物类别的核酸区段和结构。本申请所述的调控表达盒具有适体结构域,其在与效应分子结合后导致调控表达盒的状态或结构发生变化。在某些实施方式中,当效应分子与适体结构域结合时,与适体结构域连接的表达平台结构域的状态或结构发生变化。本申请所述的调控表达盒的适体结构域可以来源于任何来源,包括例如,天然存在的适体结构域,人工适体,工程化、选择的、进化的或衍生的适体或适体结构域。本申请所述的调控表达盒中的适体通常具有至少一部分,该部分可以与连接的表达平台结构域的一部分相互作用,如通过形成茎结构。该茎结构将在效应分子结合后形成或被破坏。
在现有技术中已经描述了用于产生在本申请中使用的适体结构域的适宜方法。例如,用于产生适体的一种方法是使用在例如美国专利号5,475,096和美国专利号5,270,163中所述的题目为“通过指数富集对配体进行***进化”(“SELEXTM”)的过程。SELEXTM过程是一种体外进化与靶分子具有高度特异性结合的核酸分子的方法。每个SELEXTM鉴定的核酸配体(即每个适体)都是给定目标化合物或分子的给定配体。SELEXTM过程基于独特的观点,即核酸分子有足够的能力形成各种二维和三维结构,并在其单体内具有足够的化学多功能性,以作为几乎任何化学化合物,无论是单体还是聚合物的配体(即,形成特异性结合对)。任何尺寸或组成的分子均可以作为靶点。
在通常情况下,SELEXTM方法从包含随机序列的单链寡核苷酸的大型文库或库起始。寡核苷酸可以是修饰的或未修饰的DNA、RNA或DNA/RNA杂交体。在一些实例中,该库包含100%随机或部分随机的寡核苷酸。在其他实例中,该库包含随机或部分随机的寡核苷酸,其包含至少一个引入随机序列中的固定和/或保守序列,可以将其用作例如PCR引物的杂交位点、RNA聚合酶的启动子序列、限制性位点或同聚序列,以便于目标寡核苷酸的克隆和/或测序。
通常地,初始库的寡核苷酸包含固定的5’和3’末端序列,其位于30-50个随机核苷酸的内部区域的侧翼。随机核苷酸可以通过多种方式产生,包括化学合成和从随机切割的细胞核酸中尺寸选择。还可以在选择/扩增迭代之前或期间通过诱变引入或增加在测试核酸中的序列变异。
在包含大量可能的序列和结构的初始库中,对于给定的靶点具有广泛的结合亲和性。对靶点具有较高亲和性常数的那些最有可能与靶点结合。在分配、解离和扩增之后,产生第二核酸混合物,其富含结合亲和性更高的候选物。额外轮次的选择逐渐偏向于最佳配体,直至所得到的核酸混合物主要仅由一条或几条序列组成。然后,可以将这些克隆、测序并作为纯配体或适体单独检测结合亲和性。
此前已描述了适体结构域的一些实例(参见Breaker等的美国专利号7794931,其公开的内容通过引用并入本申请)。特别地,Vogel M等已公开了一种合成的核糖开关,其可有效控制表达盒外显子响应于小分子配体四环素的选择性剪接。在存在四环素的情况下,跳过表达盒外显子,而在缺乏配体的情况下,将其包括在内(Nucleic Acid Research(2018)46:e48)。
在某些实施方式中,适体结构域具有与SEQ ID NO:16、18或20(在DNA形式中)或SEQ ID NO:17、19或21(在RNA形式中)具有至少90%(例如,90%、95%、98%、99%)同一性的序列。
2.表达平台结构域
表达平台结构域是本申请所述的调控表达盒的一部分,其影响包含调控表达盒的RNA分子的表达。在通常情况下,表达平台结构域至少有一部分可以与连接的适体结构域的一部分相互作用,如通过形成茎结构。该茎结构将在效果分子结合后形成或被破坏。该茎结构通常是或阻止形成表达调控结构。表达调控结构是允许、阻止、增强或抑制含有该结构的RNA分子表达的结构。表达平台结构域的实例包括夏因-达尔加诺(Shine-Dalgarno)序列、起始密码子、转录终止子、内含子、外显子以及稳定性和加工信号。
在某些实施方式中,表达平台结构域包含侧翼为上游内含子和下游内含子的条件性外显子。在一个实施方式中,所述条件性外显子具有与SEQ ID NO:22(在DNA形式中)或SEQ ID NO:23(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。在一个实施方式中,所述上游内含子具有与SEQ ID NO:24(在DNA形式中)或SEQ ID NO:25(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。在一个实施方式中,所述下游内含子具有与SEQ ID NO:26(在DNA形式中)或SEQ ID NO:27(在RNA形式中)至少90%(例如,90%、95%、98%、99%)同一性的序列。
3.效应分子
如本申请所使用的效应分子是能够活化调控表达盒的分子和化合物。这包括针对天然存在的调控表达盒(例如,核糖开关)的天然或正常的效应分子和能够激活调控表达盒的其他化合物。在一些合成调控表达盒的情况下,效应分子可以是针对其设计适体结构域或选择适体结构域(如在例如体外选择或在体外进化技术中)的那些分子。
在某些实施方式中,效应分子是四环素。在某些实施方式中,效应分子是代谢产物,例如,腺苷钴胺素、水钴胺素、cAMP、cGMP、c-di-AMP、c-di-GMP、氟化物、黄素单核苷酸、谷氨酰胺、甘氨酸、赖氨酸、镍、钴、前辫苷、嘌呤、S-腺苷甲硫氨酸、四氢叶酸、焦磷酸硫胺素、鸟嘌呤、腺嘌呤、2’-脱氧鸟苷、7-氨基甲基-7-脱氮鸟嘌呤、ZMP和ZTP。
4.调控表达盒的实施方式
图1示出了本发明的调控表达盒的示例性实施方式,其通过条件性外显子的选择性剪接控制基因组编辑酶的表达。参见图1,可调控的基因表达构建体包含编码基因组编辑酶的多核苷酸序列。多核苷酸序列包含基因组编辑酶的外显子1、基因组编辑酶的外显子2以及散布在外显子1和外显子2之间的条件性外显子。条件性外显子不编码基因组编辑酶的一部分,但包含终止密码子。条件性外显子前面是编码适体结构域(AD)的调控序列,其能够在与效应分子结合后改变其结构。当DNA构建体递送进入细胞后,DNA构建体转录成RNA转录物。在存在效应分子的情况下,适体结构域结合至效应分子,并且形成阻断条件性外显子的剪接受体的结构。其结果是,RNA转录物被剪接成仅包含外显子1和外显子2的成熟mRNA,并被翻译成功能性基因组编辑酶。在不存在效应分子的情况下,适体结构域形成不阻断条件性外显子的剪接受体的结构。其结果是,RNA转录物被剪接成包含外显子1、条件性外显子和外显子2的成熟mRNA。所得到的mRNA不翻译成功能性基因组编辑酶。
图2示出了本发明的调控表达盒的示例性实施方式,其通过调控RNA转录物的稳定性控制基因组编辑酶的表达。参见图2,可调控的基因表达构建体编码RNA,所述RNA包含编码基因组编辑酶(例如,Cas9)的多核苷酸序列和可操作地连接至所述多核苷酸序列的3’末端的调控表达盒。所述调控表达盒包含在结合至效应分子后能够改变结构的适体结构域。所述调控表达盒还包含能够被内源性miRNA识别的区域。当将核酸构建体递送进入细胞中时,核酸构建体被转录成RNA转录物,其包含编码基因组编辑酶的区域和随后的调控表达盒。在存在效应分子的情况下,适体结构域结合至效应分子,并且调控表达盒形成不被内源性miRNA识别的茎环结构。其结果是,RNA转录物被翻译成功能性基因组编辑酶。在不存在效应分子的情况下,适体结构域不形成茎环,并且调控表达盒被内源性miRNA识别,其导致RNA转录物的降解,例如,通过RISC途径。其结果是,不表达基因组编辑酶。
图3示出了本发明的调控表达盒的示例性实施方式,其通过调控RNA转录物的翻译控制基因组编辑酶的表达。参见图3,可调控的基因表达构建体编码RNA,所述RNA包含编码基因组编辑酶(例如,Cas9)的多核苷酸序列和可操作地连接至所述多核苷酸序列的5’末端的调控表达盒。调控表达盒包含适体结构域和表达平台结构域,当所述适体结构域不结合至效应分子时,其形成抗终止子茎,且在结合至效应分子后,其能够形成终止子。当可调控的基因表达构建体递送至细胞中时,所述构建体被转录成包含编码基因组编辑酶的区域的RNA转录物。在不存在效应分子的情况下,调控表达盒形成抗终止子茎。其结果是,RNA转录物被翻译成功能性基因组编辑酶。在存在效应分子的情况下,适体结构域结合至效应分子,并且调控表达盒形成终止子。其结果是,基因组编辑酶没有被翻译。
图4示出了本发明的调控表达盒的另一个示例性实施方式,其通过调控RNA转录物的翻译控制基因组编辑酶的表达。参见图4,可调控的基因表达构建体编码RNA,所述RNA包含编码基因组编辑酶(例如,Cas9)的多核苷酸序列和可操作地连接至所述多核苷酸序列的5’末端的调控表达盒。调控表达盒包含适体结构域,并且能够形成一种结构,当适体结构域与效应分子结合时,该结构将核糖体结合序列(RBS)隔离,使其不被核糖体识别。当构建体递送进入细胞中时,所述构建体被转录成包含编码基因组编辑酶的区域的RNA转录物。在不存在效应分子的情况下,调控表达盒形成允许RBS被核糖体识别的结构。其结果是,RNA转录物被翻译成功能性基因组编辑酶。在存在效应分子的情况下,适体结合至效应分子,并且形成使RBS不被核糖体识别的结构。其结果是,基因组编辑酶没有被翻译。
应当理解的是,在上述实施方式中所述的机制可以联合使用。例如,DNA构建体可以编码RNA,所述RNA包含编码如图1中所述的Cas9的多核苷酸序列。多核苷酸序列包含编码Cas9蛋白的5’区段的外显子1和编码Cas9蛋白的3’区段的外显子2。外显子1和外显子2穿插着包含调节性外显子的第一调控表达盒。条件性外显子前面是第一适体结构域,该结构域在与四环素结合后能够改变其结构。外显子2之后是包含第二适体结构域的第二调控表达盒,所述第二适体结构域在与四环素结合后能够形成茎环结构,该区域能够被内源性miRNA所识别。当DNA构建体递送进入细胞时,DNA构建体被转录成RNA转录物,所述RNA转录物包含外显子1、第一适体结构域、条件性外显子、外显子2和第二适体结构域。
在不存在四环素的情况下,第一适体结构域形成不阻断调节性外显子的剪接受***点的结构。其结果是,RNA转录物被剪接成成熟mRNA,其包含外显子1、条件性外显子和外显子2。所得到的mRNA没有翻译成功能性Cas9蛋白。同时,第二适体结构域不形成茎环,并且被内源性miRNA识别,其导致RNA转录物通过RISC途径降解。其结果是,不表达Cas9。
在存在四环素的情况下,第一适体结构域结合至四环素,并且形成阻断条件性外显子的剪接受体的结构。其结果是,RNA转录物被剪接成仅包含外显子1和外显子2的成熟mRNA,并被翻译成功能性Cas9蛋白。同时,第二适体结构域结合至四环素,并且形成不被内源性miRNA识别的茎环结构。其结果是,RNA转录物被翻译成功能性Cas9蛋白。IV.用于可控的基因组编辑的组合物和方法
1.组合物
可以将所公开的调控表达盒用于任何适宜的表达***。使用载体如质粒可有效地实现重组表达。载体可以包含与调控表达盒编码序列和待表达的RNA(例如,编码蛋白的RNA)可操作地连接的启动子。载体还包含转录和翻译所需的其他元件。如在本申请中所使用的,载体指包含外源性DNA的任何运载体。因此,载体是将外源性核酸转运到细胞中而不降解的试剂,并且包含在其被递送到的细胞中产生核酸表达的启动子。载体包括但不限于质粒、病毒核酸、病毒、噬菌体核酸、噬菌体、粘粒和人工染色体。可以产生适于携带可调控的基因表达构建体的多种原核和真核表达载体。此类表达载体包括例如pET、pET3d、pCR2.1、pBAD、pUC和酵母载体。例如,可以在多种体内和体外情况下使用所述载体。
病毒载体包括腺病毒、腺相关病毒、疱疹病毒、痘苗病毒、脊髓灰质炎病毒、AIDS病毒、神经嗜性病毒、辛德毕斯病毒和其他RNA病毒,包括使用HIV骨架的这些病毒。具有这些病毒性质的任何病毒家族也是有用的,这使得其适合用作载体。将在Verma(1985)中描述的逆转录病毒载体,包括小鼠马洛尼白血病病毒MMLV和表达MMLV的所需性质的逆转录病毒作为载体。通常地,病毒载体包含非结构性早期基因、结构性晚期基因、RNA聚合酶III转录物、复制和衣壳化所必需的反向末端重复序列以及控制病毒基因组转录和复制的启动子。当工程化为载体时,通常除去病毒的一个或多个早期基因,并将基因或基因/启动子表达盒***病毒基因组以代替去除的病毒DNA。
在真核宿主细胞(酵母、真菌、昆虫、植物、动物、人或有核细胞)中使用的表达载体也可以包含终止转录所必需的序列,这些序列可以影响mRNA表达。这些区域被转录为编码组织因子蛋白的mRNA的非翻译部分中的聚腺苷酸化区段。3’非翻译区还包含转录终止位点。优选地,转录单位还包含聚腺苷酸化区域。该区域的一个益处是其增加了转录单位像mRNA一样被加工和运输的可能性。表达构建体中聚腺苷酸化信号的鉴定和使用已经很好地建立起来。优选在转基因构建体中使用同源聚腺苷酸化信号。
在某些实施方式中,可调控的基因表达构建体还包含增强或促进靶基因表达的元件。在某些实施方式中,可调控基因表达构建体包含编码核定位信号(NLS)的序列,该序列融合到靶基因上,促进表达的靶蛋白进入核。在某些实施方式中,NLS是SV40 NLS或核质蛋白NLS。在某些实施方式中,编码NLS的序列是SEQ ID NO:36或38。
在某些实施方式中,可调控的基因表达构建体还包含编码与待表达的靶蛋白融合的标签的序列。在某些实施方式中,所述标签是HA标签。在某些实施方式中,编码所述标签的序列是SEQ ID NO:40。
在一些实施方式中,可调控的基因表达构建体还包含选择标志物。当这种选择标志物成功转染到宿主细胞中时,如果置于选择压力下,转化的宿主细胞可以存活。有两种广泛使用的不同类别的选择方案。第一个类别基于细胞的代谢和突变细胞系的使用,该突变细胞系缺乏不依赖于补充的培养基生长的能力。第二个类别是显性选择,其指在任何细胞类型中使用的选择方案,不需要使用突变细胞系。这些方案通常使用药物来阻止宿主细胞的生长。那些具有新基因的细胞会表达一种具有药物抗性的蛋白,并且会在选择中存活下来。这种显性选择的实例使用药物新霉素、霉酚酸或潮霉素。
基因转移可以使用遗传物质的直接转移获得,遗传物质包括但不限于质粒、病毒载体、病毒核酸、噬菌体核酸、噬菌体、粘粒和人工染色体,或者通过在细胞或运载体(如阳离子脂质体)中的遗传物质转移获得。此类方法是本领域熟知的,并且易于适用于本文所述的方法。转移载体可以是用于将基因递送到细胞中的任何核苷酸结构(例如,质粒),或作为递送基因的一般策略的一部分,例如,作为重组逆转录病毒或腺病毒的一部分(Ram等,Cancer Res.53:83-88,(1993))。例如,Wolff,J.A.,等,Science,247,1465-1468,(1990);和Wolff,J.A.Nature,352,815-818,(1991)中描述了用于转染的适当手段,包括病毒载体、化学转染子或物理机械方法,如电穿孔和DNA的直接扩散。
图5示出了一个优选的实施方式,其中可调控的基因表达构建体编码Cas9蛋白并且包含在AAV载体中。参见图5,可调控的基因表达构建体包含控制Cas9表达的AAV载体的元件,例如,AAV反向末端重复序列(ITR)、启动子和polyA区。所述构建体还可以包含编码向导RNA(sgRNA)的多核苷酸序列。核酸构建体包含编码Cas9蛋白的5’区段的外显子1和编码Cas9蛋白的3’区段的外显子2。所述构建体还包含编码调控表达盒的序列,所述调控表达盒包含适体结构域,随后是散布在第一和第二区域之间的条件性外显子。结合至四环素后,适体结构域能够改变调控表达盒的结构。当可调控的基因表达构建体递送至细胞中时,该构建体被转录成RNA转录物,其包含第一区域、适体结构域、条件性外显子和第二区域。在存在四环素的情况下,适体结构域结合至四环素并,形成阻断条件性外显子的剪接受体的结构。其结果是,RNA转录物被剪接成仅包含外显子1和外显子2的成熟mRNA,并且被翻译成功能性Cas9蛋白。在不存在四环素的情况下,适体结构域形成不阻断条件性外显子的剪接受***点的结构。其结果是,RNA转录物被剪接成成熟mRNA,其包含外显子1、条件性外显子和外显子2。所得到的mRNA不被翻译成功能性Cas9蛋白。
上文所述的可调控的基因表达构建体以及其他材料可以任何适宜的组合包装在一起作为用于进行或帮助进行所公开方法的试剂盒。如果给定试剂盒中的试剂盒组分被设计和适配以在公开的方法中一起使用,则是有用的。
2.方法
本公开内容还提供了本申请所述的可调控的基因表达构建体和组合物的用途。公开了用于调控靶基因(例如,基因组编辑酶)的表达的方法。例如,此类方法可以涉及使调控表达盒与能够激活、失活或阻断调控表达盒的效应分子接触。调控表达盒的功能是通过结合或除去效应分子来控制基因表达。例如,还可以通过从调控表达盒的存在中除去效应分子来控制靶基因的表达。因此,例如,所公开的调控基因表达的方法可以涉及从调控表达盒的存在或与之接触中除去效应分子。例如,可以通过结合不激活调控表达盒的效应分子的类似物来阻断调控表达盒。
还公开了在细胞中的基因组编辑的方法。在一个实施方式中,该方法包括将包含编码基因组编辑酶的序列的可调控的基因表达构建体递送进入细胞中。在一个实施方式中,该方法还包括将效应分子递送进入细胞中。通过在效应分子存在与否之间切换条件,调控表达盒能够开启和关闭基因组编辑酶的表达,从而控制由基因组编辑酶介导的基因编辑过程。
还公开了治疗患有疾病的对象的方法。在一个实施方式中,该方法包括将编码基因组编辑酶的可调控的基因表达构建体递送到对象的至少一个细胞中。在一个实施方式中,该方法还包括向对象施用效应分子。
可以通过本申请公开的方法治疗的疾病包括但不限于癌症、囊性纤维化、心脏疾病、糖尿病、血友病和AIDS。
V.序列相似性
应当理解的是,如本申请所讨论的,术语同源性和同一性的使用指与相似性相同的事物。因此,例如,如果在两条序列(例如,非天然存在的序列)之间使用同源性一词,则应理解的是这不一定表示这两条序列之间的进化关系,而是查看其核酸序列之间的相似性或相关性。很多用于确定两个进化相关分子之间同源性的方法通常应用于任何两个或多个核酸或蛋白,以测量序列相似性,而不管其是否在进化上相关。
在通常情况下,应当理解的是,定义本申请所公开的调控表达盒、适体结构域、表达平台结构域、基因和蛋白的任何已知变体和衍生物或者可能产生的变体和衍生物的一种方法是通过根据与特定已知序列的同源性来定义变体和衍生物。本申请公开的特定序列的这种同一性还在本申请的其他地方讨论。在通常情况下,本申请公开的调控表达盒、适体结构域、表达平台结构域、内含子、外显子、基因和蛋白的变体通常与指定序列或天然序列具有至少约70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98或99%的同源性。本领域技术人员易于理解如何确定两个蛋白或核酸(如基因)的同源性。例如,可以在对两条序列进行比对之后计算同源性,使同源性处于其最高水平。
另一种计算同源性的方法可以通过公开的算法进行。用于比较的序列的最佳比对可以通过Smith和Waterman Adv.Appl.Math.2:482(1981)的局部同源性算法,通过Needleman和Wunsch,J.Mol.Biol.48:443(1970)的同源性比对算法,通过Pearson和Lipman,Proc.Natl.Acad.Sci.U.S.A.85:2444(1988)的相似性检索方法,通过计算机化执行这些算法(Wisconsin Genetics Software Package,Genetics Computer Group,575Science Dr.,Madison,Wis.中的GAP、BESTFIT、FASTA和TFASTA)或通过目测检查进行。
针对至少与核酸比对相关的材料,通过例如在Zuker,M.Science 244:48-52,1989,Jaeger等,Proc.Natl.Acad.Sci.USA 86:7706-7710,1989,Jaeger等,MethodsEnzymol.183:281-306,1989(其通过引用并入本申请)中公开的算法,可以针对核酸获得相同类型的同源性。应当理解的是,任一方法通常都可以使用,并且在某些情况下,这些不同方法的结果可以不同,但是本领域技术人员理解,如果使用这些方法中的至少一种发现同一性,则可以说这些序列具有所述的同一性。
例如,如在本申请中所使用的,被描述为与另一序列具有特定百分比同源性的序列是指具有如通过上述任何一种或多种计算方法计算的所述同源性的序列。例如,如果使用Zuker计算方法计算出第一序列与第二序列具有80%同源性,则如本申请所定义的第一序列与第二序列具有80%同源性,即使通过任何其他计算方法计算出的第一序列与第二序列不具有80%同源性。作为另一个实例,如果使用Zuker计算方法和Pearson和Lipman计算方法计算出第一序列与第二序列具有80%同源性,则如本申请所定义的第一序列与第二序列具有80%同源性,即使通过Smith和Waterman计算方法、Needleman和Wunsch计算方法、Jaeger计算方法或任何其他计算方法计算出的第一序列与第二序列不具有80%同源性。作为又一个实例,如果使用每种计算方法计算出第一序列与第二序列具有80%同源性,则如本申请所定义的第一序列与第二序列具有80%同源性(尽管,在实践中,不同计算方法通常将导致不同的计算同源性百分比)。
VI.实施例
包括下述实施例以显示本发明的示例性实施方式。本领域技术人员应当理解的是,以下实施例中公开的技术代表发明人发现的在本发明的实践中很好地起作用的技术,并且应当仅认为是构成其实践的示例模式。根据本公开内容,本领域技术人员应当理解,在不脱离本发明的主旨和范围的前提下,可以对所公开的具体实施方式进行很多改变并仍然获得相同或相似的结果。
实施例1
本实施例示出了添加内含子的SaCas9构建体的产生。尽管Cas9基因是在细菌中鉴定的,但其没有天然的内含子和外显子。为了产生具有可正确转录和剪接的内含子的Cas9基因,本发明人优化了金黄色葡萄球菌Cas9(SaCas9)基因(SEQ ID NO:2)的三个区域(SEQID NO:10、12和14),其中富集了外显子剪接增强子(ESE)并缺失外显子剪接沉默子(ESS)。然后,发明人产生了一系列候选SaCas9基因,每个基因都有***到用ESE富集和ESS耗竭优化的区域之一的内含子(图6)。将候选SaCas9基因克隆到具有CMV启动子的载体中。
然后,在如Mashiko D等(参见Sci Rep(2013)3:3355)所述的EGxxFP测定中检测了候选SaCas9基因的活性。简言之,制备含有5’和3’EGFP片段的pCAG-EGxxFP质粒,其在普遍存在的CAG启动子下,共享482bp。将含有sgRNA靶序列的约500bp区域置于pCAG-EGxxFP质粒的EGFP片段之间。将pCAG-EGxxFP质粒与候选SaCas9构建体和sgRNA共转染到HEK293T细胞中。当候选SaCas9基因被正确转录和剪接时,EGxxFT基因中的靶序列被sgRNA向导的SaCas9蛋白消化,发生同源依赖性修复并重建EGFP表达。
如图8中所示,EGxxFP测定的结果表明,位置2、8和15是***内含子的最佳位置。
实施例2
本实施例示出了将具有由适体调控的条件性外显子的内含子***Cas9基因中。
在确认于SaCas9基因中***内含子的位置后,发明人随后测试了三个四环素适体结构域M2(SEQ ID NO:16)、M3(SEQ ID NO:18)和M4(SEQ ID NO:20),以控制条件性外显子的剪接。包含四环素适体和条件性外显子(SEQ ID NO:22)的候选SaCas9基因通过***载体制备,其侧翼有两个***位置2和8的内含子(SEQ ID NO:24和26)。然后,如实施例1所述,在EGxxFP测定中检测了候选SaCas9构建体。
如图9中所示,EGxxFP测定的结果表明,M2和M3在调控SaCas9表达方面效果良好,而M2表现最好。
实施例3
本实施例示出了具有双适体的SaCas9构建体的产生,以便在不存在四环素的情况下进一步抑制SaCas9的活性。
为产生具有两个适体结构域的候选SaCas9基因(SEQ ID NO:34),发明人将四环素适体结构域M2和条件性外显子***位置2,并将四环素适体结构域M2和条件性外显子***位置8。然后,如实施例1所述,在EGxxFP测定中检测了具有双适体的候选SaCas9基因。
EGxxFP测定的结果表明,2+8双适体基因在不存在四环素的情况下不具有高于背景的活性(图10),在存在四环素的情况下3天后,与野生型SaCas9相比,具有约40%的活性(图11)。
尽管已经参考特定实施方式(其中一些是优选的实施方式)具体地示出和描述了本公开内容,但是本领域技术人员应当理解的是,在不脱离如本文所公开的本公开内容的主旨和范围的前提下,可以在形式和细节上进行各种改变。
序列表
<110> 应用干细胞有限公司
<120> 可控的基因组编辑***
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ggctggaagg acatcaaaga atggtacgag atgctgatgg gccactgcac ctacttcccc 720
gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc cctgaacgac 780
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gccgaggacg ccctgatcat tgccaacgcc gatttcatct tcaaagagtg gaagaaactg 2160
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cccgagatcg aaaccgagca ggagtacaaa gagatcttca tcacccccca ccagatcaag 2280
cacattaagg acttcaagga ctacaagtac agccaccggg tggacaagaa gcctaataga 2340
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ctgattatgg aacagtacgg cgacgagaag aatcccctgt acaagtacta cgaggaaacc 2580
gggaactacc tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag 2640
tattacggca acaaactgaa cgcccatctg gacatcaccg acgactaccc caacagcaga 2700
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gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggcugaagcg gcggaggcgg 180
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cagcugcuga aggugcagaa ggccuaccac cagcuggacc agagcuucau cgacaccuac 600
aucgaccugc uggaaacccg gcggaccuac uaugagggac cuggcgaggg cagccccuuc 660
ggcuggaagg acaucaaaga augguacgag augcugaugg gccacugcac cuacuucccc 720
gaggaacugc ggagcgugaa guacgccuac aacgccgacc uguacaacgc ccugaacgac 780
cugaacaauc ucgugaucac cagggacgag aacgagaagc uggaauauua cgagaaguuc 840
cagaucaucg agaacguguu caagcagaag aagaagccca cccugaagca gaucgccaaa 900
gaaauccucg ugaacgaaga ggauauuaag ggcuacagag ugaccagcac cggcaagccc 960
gaguucacca accugaaggu guaccacgac aucaaggaca uuaccgcccg gaaagagauu 1020
auugagaacg ccgagcugcu ggaucagauu gccaagaucc ugaccaucua ccagagcagc 1080
gaggacaucc aggaagaacu gaccaaucug aacuccgagc ugacccagga agagaucgag 1140
cagaucucua aucugaaggg cuauaccggc acccacaacc ugagccugaa ggccaucaac 1200
cugauccugg acgagcugug gcacaccaac gacaaccaga ucgcuaucuu caaccggcug 1260
aagcuggugc ccaagaaggu ggaccugucc cagcagaaag agauccccac cacccuggug 1320
gacgacuuca uccugagccc cgucgugaag agaagcuuca uccagagcau caaagugauc 1380
aacgccauca ucaagaagua cggccugccc aacgacauca uuaucgagcu ggcccgcgag 1440
aagaacucca aggacgccca gaaaaugauc aacgagaugc agaagcggaa ccggcagacc 1500
aacgagcgga ucgaggaaau cauccggacc accggcaaag agaacgccaa guaccugauc 1560
gagaagauca agcugcacga caugcaggaa ggcaagugcc uguacagccu ggaagccauc 1620
ccucuggaag aucugcugaa caaccccuuc aacuaugagg uggaccacau cauccccaga 1680
agcguguccu ucgacaacag cuucaacaac aaggugcucg ugaagcagga agaaaacagc 1740
aagaagggca accggacccc auuccaguac cugagcagca gcgacagcaa gaucagcuac 1800
gaaaccuuca agaagcacau ccugaaucug gccaagggca agggcagaau cagcaagacc 1860
aagaaagagu aucugcugga agaacgggac aucaacaggu ucuccgugca gaaagacuuc 1920
aucaaccgga accuggugga uaccagauac gccaccagag gccugaugaa ccugcugcgg 1980
agcuacuuca gagugaacaa ccuggacgug aaagugaagu ccaucaaugg cggcuucacc 2040
agcuuucugc ggcggaagug gaaguuuaag aaagagcgga acaaggggua caagcaccac 2100
gccgaggacg cccugaucau ugccaacgcc gauuucaucu ucaaagagug gaagaaacug 2160
gacaaggcca aaaaagugau ggaaaaccag auguucgagg aaaagcaggc cgagagcaug 2220
cccgagaucg aaaccgagca ggaguacaaa gagaucuuca ucacccccca ccagaucaag 2280
cacauuaagg acuucaagga cuacaaguac agccaccggg uggacaagaa gccuaauaga 2340
gagcugauua acgacacccu guacuccacc cggaaggacg acaagggcaa cacccugauc 2400
gugaacaauc ugaacggccu guacgacaag gacaaugaca agcugaaaaa gcugaucaac 2460
aagagccccg aaaagcugcu gauguaccac cacgaccccc agaccuacca gaaacugaag 2520
cugauuaugg aacaguacgg cgacgagaag aauccccugu acaaguacua cgaggaaacc 2580
gggaacuacc ugaccaagua cuccaaaaag gacaacggcc ccgugaucaa gaagauuaag 2640
uauuacggca acaaacugaa cgcccaucug gacaucaccg acgacuaccc caacagcaga 2700
aacaaggucg ugaagcuguc ccugaagccc uacagauucg acguguaccu ggacaauggc 2760
guguacaagu ucgugaccgu gaagaaucug gaugugauca aaaaagaaaa cuacuacgaa 2820
gugaauagca agugcuauga ggaagcuaag aagcugaaga agaucagcaa ccaggccgag 2880
uuuaucgccu ccuucuacaa caacgaucug aucaagauca acggcgagcu guauagagug 2940
aucggcguga acaacgaccu gcugaaccgg aucgaaguga acaugaucga caucaccuac 3000
cgcgaguacc uggaaaacau gaacgacaag aggcccccca ggaucauuaa gacaaucgcc 3060
uccaagaccc agagcauuaa gaaguacagc acagacauuc ugggcaaccu guaugaagug 3120
aaaucuaaga agcacccuca gaucaucaaa aagggc 3156
<210> 4
<211> 3156
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 4
aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc 60
gactacgaga ctcgtgatgt tattgacgca ggcgttcgtt tgtttaaaga agctaatgtt 120
gagaataatg agggaagaag aagtaagcgt ggggctcgca ggcttaagcg aagaagaagg 180
catcggatac agcgtgtgaa gaagttgctg tttgattata atttgttgac tgatcattct 240
gagttatcag gcattaatcc ttatgaggct cgtgttaagg gtttaagtca gaagttaagt 300
gaagaagaat tttctgctgc tttgttgcat ttggctaaaa gaagaggagt tcataatgtt 360
aatgaagttg aagaggatac tggtaatgag ttaagtacta aggagcagat aagtcgtaat 420
tctaaggctt tggaagaaaa gtatgttgct gagttgcagt tggagcgttt gaagaaggat 480
ggtgaagtaa gaggaagtat taatcgtttt aagacaagtg attatgtgaa agaagcgaag 540
cagttgttga aagttcagaa ggcttatcat cagttggatc aaagttttat tgatacttat 600
attgatttgt tggagactcg tagaacttat tatgagggtc ctggtgaggg gtccccgttt 660
ggttggaagg atattaagga gtggtatgag atgttgatgg gtcattgtac ttattttcct 720
gaagaattgc ggtccgtgaa gtatgcttat aatgctgatt tgtacaacgc cctgaacgac 780
ctgaacaatc tcgtgatcac cagggacgag aacgagaagc tggaatatta cgagaagttc 840
cagatcatcg agaacgtgtt caagcagaag aagaagccca ccctgaagca gatcgccaaa 900
gaaatcctcg tgaacgaaga ggatattaag ggctacagag tgaccagcac cggcaagccc 960
gagttcacca acctgaaggt gtaccacgac atcaaggaca ttaccgcccg gaaagagatt 1020
attgagaacg ccgagctgct ggatcagatt gccaagatcc tgaccatcta ccagagcagc 1080
gaggacatcc aggaagaact gaccaatctg aactccgagc tgacccagga agagatcgag 1140
cagatctcta atctgaaggg ctataccggc acccacaacc tgagcctgaa ggccatcaac 1200
ctgatcctgg acgagctgtg gcacaccaac gacaaccaga tcgctatctt caaccggctg 1260
aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag agatccccac caccctggtg 1320
gacgacttca tcctgagccc cgtcgtgaag agaagcttca tccagagcat caaagtgatc 1380
aacgccatca tcaagaagta cggcctgccc aacgacatca ttatcgagct ggcccgcgag 1440
aagaactcca aggacgccca gaaaatgatc aacgagatgc agaagcggaa ccggcagacc 1500
aacgagcgga tcgaggaaat catccggacc accggcaaag agaacgccaa gtacctgatc 1560
gagaagatca agctgcacga catgcaggaa ggcaagtgcc tgtacagcct ggaagccatc 1620
cctctggaag atctgctgaa caaccccttc aactatgagg tggaccacat catccccaga 1680
agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg tgaagcagga agaaaacagc 1740
aagaagggca accggacccc attccagtac ctgagcagca gcgacagcaa gatcagctac 1800
gaaaccttca agaagcacat cctgaatctg gccaagggca agggcagaat cagcaagacc 1860
aagaaagagt atctgctgga agaacgggac atcaacaggt tctccgtgca gaaagacttc 1920
atcaaccgga acctggtgga taccagatac gccaccagag gcctgatgaa cctgctgcgg 1980
agctacttca gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc 2040
agctttctgc ggcggaagtg gaagtttaag aaagagcgga acaaggggta caagcaccac 2100
gccgaggacg ccctgatcat tgccaacgcc gatttcatct tcaaagagtg gaagaaactg 2160
gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg 2220
cccgagatcg aaaccgagca ggagtacaaa gagatcttca tcacccccca ccagatcaag 2280
cacattaagg acttcaagga ctacaagtac agccaccggg tggacaagaa gcctaataga 2340
gagctgatta acgacaccct gtactccacc cggaaggacg acaagggcaa caccctgatc 2400
gtgaacaatc tgaacggcct gtacgacaag gacaatgaca agctgaaaaa gctgatcaac 2460
aagagccccg aaaagctgct gatgtaccac cacgaccccc agacctacca gaaactgaag 2520
ctgattatgg aacagtacgg cgacgagaag aatcccctgt acaagtacta cgaggaaacc 2580
gggaactacc tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag 2640
tattacggca acaaactgaa cgcccatctg gacatcaccg acgactaccc caacagcaga 2700
aacaaggtcg tgaagctgtc cctgaagccc tacagattcg acgtgtacct ggacaatggc 2760
gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa ctactacgaa 2820
gtgaatagca agtgctatga ggaagctaag aagctgaaga agatcagcaa ccaggccgag 2880
tttatcgcct ccttctacaa caacgatctg atcaagatca acggcgagct gtatagagtg 2940
atcggcgtga acaacgacct gctgaaccgg atcgaagtga acatgatcga catcacctac 3000
cgcgagtacc tggaaaacat gaacgacaag aggcccccca ggatcattaa gacaatcgcc 3060
tccaagaccc agagcattaa gaagtacagc acagacattc tgggcaacct gtatgaagtg 3120
aaatctaaga agcaccctca gatcatcaaa aagggc 3156
<210> 5
<211> 3156
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 5
aagcggaacu acauccuggg ccuggacauc ggcaucacca gcgugggcua cggcaucauc 60
gacuacgaga cucgugaugu uauugacgca ggcguucguu uguuuaaaga agcuaauguu 120
gagaauaaug agggaagaag aaguaagcgu ggggcucgca ggcuuaagcg aagaagaagg 180
caucggauac agcgugugaa gaaguugcug uuugauuaua auuuguugac ugaucauucu 240
gaguuaucag gcauuaaucc uuaugaggcu cguguuaagg guuuaaguca gaaguuaagu 300
gaagaagaau uuucugcugc uuuguugcau uuggcuaaaa gaagaggagu ucauaauguu 360
aaugaaguug aagaggauac ugguaaugag uuaaguacua aggagcagau aagucguaau 420
ucuaaggcuu uggaagaaaa guauguugcu gaguugcagu uggagcguuu gaagaaggau 480
ggugaaguaa gaggaaguau uaaucguuuu aagacaagug auuaugugaa agaagcgaag 540
caguuguuga aaguucagaa ggcuuaucau caguuggauc aaaguuuuau ugauacuuau 600
auugauuugu uggagacucg uagaacuuau uaugaggguc cuggugaggg guccccguuu 660
gguuggaagg auauuaagga gugguaugag auguugaugg gucauuguac uuauuuuccu 720
gaagaauugc gguccgugaa guaugcuuau aaugcugauu uguacaacgc ccugaacgac 780
cugaacaauc ucgugaucac cagggacgag aacgagaagc uggaauauua cgagaaguuc 840
cagaucaucg agaacguguu caagcagaag aagaagccca cccugaagca gaucgccaaa 900
gaaauccucg ugaacgaaga ggauauuaag ggcuacagag ugaccagcac cggcaagccc 960
gaguucacca accugaaggu guaccacgac aucaaggaca uuaccgcccg gaaagagauu 1020
auugagaacg ccgagcugcu ggaucagauu gccaagaucc ugaccaucua ccagagcagc 1080
gaggacaucc aggaagaacu gaccaaucug aacuccgagc ugacccagga agagaucgag 1140
cagaucucua aucugaaggg cuauaccggc acccacaacc ugagccugaa ggccaucaac 1200
cugauccugg acgagcugug gcacaccaac gacaaccaga ucgcuaucuu caaccggcug 1260
aagcuggugc ccaagaaggu ggaccugucc cagcagaaag agauccccac cacccuggug 1320
gacgacuuca uccugagccc cgucgugaag agaagcuuca uccagagcau caaagugauc 1380
aacgccauca ucaagaagua cggccugccc aacgacauca uuaucgagcu ggcccgcgag 1440
aagaacucca aggacgccca gaaaaugauc aacgagaugc agaagcggaa ccggcagacc 1500
aacgagcgga ucgaggaaau cauccggacc accggcaaag agaacgccaa guaccugauc 1560
gagaagauca agcugcacga caugcaggaa ggcaagugcc uguacagccu ggaagccauc 1620
ccucuggaag aucugcugaa caaccccuuc aacuaugagg uggaccacau cauccccaga 1680
agcguguccu ucgacaacag cuucaacaac aaggugcucg ugaagcagga agaaaacagc 1740
aagaagggca accggacccc auuccaguac cugagcagca gcgacagcaa gaucagcuac 1800
gaaaccuuca agaagcacau ccugaaucug gccaagggca agggcagaau cagcaagacc 1860
aagaaagagu aucugcugga agaacgggac aucaacaggu ucuccgugca gaaagacuuc 1920
aucaaccgga accuggugga uaccagauac gccaccagag gccugaugaa ccugcugcgg 1980
agcuacuuca gagugaacaa ccuggacgug aaagugaagu ccaucaaugg cggcuucacc 2040
agcuuucugc ggcggaagug gaaguuuaag aaagagcgga acaaggggua caagcaccac 2100
gccgaggacg cccugaucau ugccaacgcc gauuucaucu ucaaagagug gaagaaacug 2160
gacaaggcca aaaaagugau ggaaaaccag auguucgagg aaaagcaggc cgagagcaug 2220
cccgagaucg aaaccgagca ggaguacaaa gagaucuuca ucacccccca ccagaucaag 2280
cacauuaagg acuucaagga cuacaaguac agccaccggg uggacaagaa gccuaauaga 2340
gagcugauua acgacacccu guacuccacc cggaaggacg acaagggcaa cacccugauc 2400
gugaacaauc ugaacggccu guacgacaag gacaaugaca agcugaaaaa gcugaucaac 2460
aagagccccg aaaagcugcu gauguaccac cacgaccccc agaccuacca gaaacugaag 2520
cugauuaugg aacaguacgg cgacgagaag aauccccugu acaaguacua cgaggaaacc 2580
gggaacuacc ugaccaagua cuccaaaaag gacaacggcc ccgugaucaa gaagauuaag 2640
uauuacggca acaaacugaa cgcccaucug gacaucaccg acgacuaccc caacagcaga 2700
aacaaggucg ugaagcuguc ccugaagccc uacagauucg acguguaccu ggacaauggc 2760
guguacaagu ucgugaccgu gaagaaucug gaugugauca aaaaagaaaa cuacuacgaa 2820
gugaauagca agugcuauga ggaagcuaag aagcugaaga agaucagcaa ccaggccgag 2880
uuuaucgccu ccuucuacaa caacgaucug aucaagauca acggcgagcu guauagagug 2940
aucggcguga acaacgaccu gcugaaccgg aucgaaguga acaugaucga caucaccuac 3000
cgcgaguacc uggaaaacau gaacgacaag aggcccccca ggaucauuaa gacaaucgcc 3060
uccaagaccc agagcauuaa gaaguacagc acagacauuc ugggcaaccu guaugaagug 3120
aaaucuaaga agcacccuca gaucaucaaa aagggc 3156
<210> 6
<211> 3156
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 6
aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc 60
gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga ggccaacgtg 120
gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg gcggaggcgg 180
catagaatcc agagagtgaa gaagctgctg ttcgactaca acctgctgac cgaccacagc 240
gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca gaagctgagc 300
gaggaagagt tctctgccgc cctgctgcac ctggccaaga gaagaggcgt gcacaacgtg 360
aacgaggtgg aagaggacac cggcaacgag ctgtccacca aagagcagat cagccggaac 420
agcaaggccc tggaagagaa atacgtggcc gaactgcagc tggaacggct gaagaaagac 480
ggcgaagtgc ggggcagcat caacagattc aagaccagcg actacgtgaa agaagccaaa 540
cagctgctga aggtgcagaa ggcctaccac cagctggacc agagcttcat cgacacctac 600
atcgacctgc tggaaacccg gcggacctac tatgagggac ctggcgaggg cagccccttc 660
ggctggaagg acatcaaaga atggtacgag atgctgatgg gccactgcac ctacttcccc 720
gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc cctgaacgac 780
ctgaacaatc tcgtgatcac cagggacgag aacgagaagc tggaatatta cgagaagttc 840
cagatcatcg agaacgtgtt caagcagaag aagaagccca ccctgaagca gatcgccaaa 900
gaaatcctcg tgaacgaaga ggatattaag ggctacagag tgaccagcac cggcaagccc 960
gagttcacca acctgaaggt gtaccacgac atcaaggaca ttaccgcccg gaaagagatt 1020
attgagaacg ccgagctgct ggatcagatt gctaagattt tgactattta tcagtcaagt 1080
gaggatattc aggaagaatt gactaatttg aattctgagt tgactcagga agaaattgag 1140
cagataagta atttgaaggg atacactggt actcataatt taagtttgaa ggctattaat 1200
ttgattttgg atgagttgtg gcatactaat gataatcaga ttgctatttt taatcgtttg 1260
aagttggttc ctaagaaagt tgatttaagt cagcagaagg agattcctac tactttggtt 1320
gatgacttta ttttaagtcc tgttgttaag cgaagtttta ttcaaagtat taaagttatt 1380
aatgctatta ttaagaagta tgggctcccg aatgatatta ttattgagtt ggctcgtgag 1440
aagaattcta aagatgctca gaagatgatt aatgagatgc agaagaggaa cagacagaca 1500
aatgaaagaa ttgaagaaat tattcggaca actggtaagg agaatgctaa gtatttgatt 1560
gagaagatta agttgcatga tatgcaggag ggtaagtgtt tgtattcttt ggaggctatt 1620
cctttggagg atttgttgaa taatcctttt aattatgaag ttgatcatat tattcctcgg 1680
tccgtaagtt ttgataattc ttttaataat aaagttttgg ttaagcagga agaaaacagc 1740
aagaagggca accggacccc attccagtac ctgagcagca gcgacagcaa gatcagctac 1800
gaaaccttca agaagcacat cctgaatctg gccaagggca agggcagaat cagcaagacc 1860
aagaaagagt atctgctgga agaacgggac atcaacaggt tctccgtgca gaaagacttc 1920
atcaaccgga acctggtgga taccagatac gccaccagag gcctgatgaa cctgctgcgg 1980
agctacttca gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc 2040
agctttctgc ggcggaagtg gaagtttaag aaagagcgga acaaggggta caagcaccac 2100
gccgaggacg ccctgatcat tgccaacgcc gatttcatct tcaaagagtg gaagaaactg 2160
gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg 2220
cccgagatcg aaaccgagca ggagtacaaa gagatcttca tcacccccca ccagatcaag 2280
cacattaagg acttcaagga ctacaagtac agccaccggg tggacaagaa gcctaataga 2340
gagctgatta acgacaccct gtactccacc cggaaggacg acaagggcaa caccctgatc 2400
gtgaacaatc tgaacggcct gtacgacaag gacaatgaca agctgaaaaa gctgatcaac 2460
aagagccccg aaaagctgct gatgtaccac cacgaccccc agacctacca gaaactgaag 2520
ctgattatgg aacagtacgg cgacgagaag aatcccctgt acaagtacta cgaggaaacc 2580
gggaactacc tgaccaagta ctccaaaaag gacaacggcc ccgtgatcaa gaagattaag 2640
tattacggca acaaactgaa cgcccatctg gacatcaccg acgactaccc caacagcaga 2700
aacaaggtcg tgaagctgtc cctgaagccc tacagattcg acgtgtacct ggacaatggc 2760
gtgtacaagt tcgtgaccgt gaagaatctg gatgtgatca aaaaagaaaa ctactacgaa 2820
gtgaatagca agtgctatga ggaagctaag aagctgaaga agatcagcaa ccaggccgag 2880
tttatcgcct ccttctacaa caacgatctg atcaagatca acggcgagct gtatagagtg 2940
atcggcgtga acaacgacct gctgaaccgg atcgaagtga acatgatcga catcacctac 3000
cgcgagtacc tggaaaacat gaacgacaag aggcccccca ggatcattaa gacaatcgcc 3060
tccaagaccc agagcattaa gaagtacagc acagacattc tgggcaacct gtatgaagtg 3120
aaatctaaga agcaccctca gatcatcaaa aagggc 3156
<210> 7
<211> 3156
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 7
aagcggaacu acauccuggg ccuggacauc ggcaucacca gcgugggcua cggcaucauc 60
gacuacgaga cacgggacgu gaucgaugcc ggcgugcggc uguucaaaga ggccaacgug 120
gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggcugaagcg gcggaggcgg 180
cauagaaucc agagagugaa gaagcugcug uucgacuaca accugcugac cgaccacagc 240
gagcugagcg gcaucaaccc cuacgaggcc agagugaagg gccugagcca gaagcugagc 300
gaggaagagu ucucugccgc ccugcugcac cuggccaaga gaagaggcgu gcacaacgug 360
aacgaggugg aagaggacac cggcaacgag cuguccacca aagagcagau cagccggaac 420
agcaaggccc uggaagagaa auacguggcc gaacugcagc uggaacggcu gaagaaagac 480
ggcgaagugc ggggcagcau caacagauuc aagaccagcg acuacgugaa agaagccaaa 540
cagcugcuga aggugcagaa ggccuaccac cagcuggacc agagcuucau cgacaccuac 600
aucgaccugc uggaaacccg gcggaccuac uaugagggac cuggcgaggg cagccccuuc 660
ggcuggaagg acaucaaaga augguacgag augcugaugg gccacugcac cuacuucccc 720
gaggaacugc ggagcgugaa guacgccuac aacgccgacc uguacaacgc ccugaacgac 780
cugaacaauc ucgugaucac cagggacgag aacgagaagc uggaauauua cgagaaguuc 840
cagaucaucg agaacguguu caagcagaag aagaagccca cccugaagca gaucgccaaa 900
gaaauccucg ugaacgaaga ggauauuaag ggcuacagag ugaccagcac cggcaagccc 960
gaguucacca accugaaggu guaccacgac aucaaggaca uuaccgcccg gaaagagauu 1020
auugagaacg ccgagcugcu ggaucagauu gcuaagauuu ugacuauuua ucagucaagu 1080
gaggauauuc aggaagaauu gacuaauuug aauucugagu ugacucagga agaaauugag 1140
cagauaagua auuugaaggg auacacuggu acucauaauu uaaguuugaa ggcuauuaau 1200
uugauuuugg augaguugug gcauacuaau gauaaucaga uugcuauuuu uaaucguuug 1260
aaguugguuc cuaagaaagu ugauuuaagu cagcagaagg agauuccuac uacuuugguu 1320
gaugacuuua uuuuaagucc uguuguuaag cgaaguuuua uucaaaguau uaaaguuauu 1380
aaugcuauua uuaagaagua ugggcucccg aaugauauua uuauugaguu ggcucgugag 1440
aagaauucua aagaugcuca gaagaugauu aaugagaugc agaagaggaa cagacagaca 1500
aaugaaagaa uugaagaaau uauucggaca acugguaagg agaaugcuaa guauuugauu 1560
gagaagauua aguugcauga uaugcaggag gguaaguguu uguauucuuu ggaggcuauu 1620
ccuuuggagg auuuguugaa uaauccuuuu aauuaugaag uugaucauau uauuccucgg 1680
uccguaaguu uugauaauuc uuuuaauaau aaaguuuugg uuaagcagga agaaaacagc 1740
aagaagggca accggacccc auuccaguac cugagcagca gcgacagcaa gaucagcuac 1800
gaaaccuuca agaagcacau ccugaaucug gccaagggca agggcagaau cagcaagacc 1860
aagaaagagu aucugcugga agaacgggac aucaacaggu ucuccgugca gaaagacuuc 1920
aucaaccgga accuggugga uaccagauac gccaccagag gccugaugaa ccugcugcgg 1980
agcuacuuca gagugaacaa ccuggacgug aaagugaagu ccaucaaugg cggcuucacc 2040
agcuuucugc ggcggaagug gaaguuuaag aaagagcgga acaaggggua caagcaccac 2100
gccgaggacg cccugaucau ugccaacgcc gauuucaucu ucaaagagug gaagaaacug 2160
gacaaggcca aaaaagugau ggaaaaccag auguucgagg aaaagcaggc cgagagcaug 2220
cccgagaucg aaaccgagca ggaguacaaa gagaucuuca ucacccccca ccagaucaag 2280
cacauuaagg acuucaagga cuacaaguac agccaccggg uggacaagaa gccuaauaga 2340
gagcugauua acgacacccu guacuccacc cggaaggacg acaagggcaa cacccugauc 2400
gugaacaauc ugaacggccu guacgacaag gacaaugaca agcugaaaaa gcugaucaac 2460
aagagccccg aaaagcugcu gauguaccac cacgaccccc agaccuacca gaaacugaag 2520
cugauuaugg aacaguacgg cgacgagaag aauccccugu acaaguacua cgaggaaacc 2580
gggaacuacc ugaccaagua cuccaaaaag gacaacggcc ccgugaucaa gaagauuaag 2640
uauuacggca acaaacugaa cgcccaucug gacaucaccg acgacuaccc caacagcaga 2700
aacaaggucg ugaagcuguc ccugaagccc uacagauucg acguguaccu ggacaauggc 2760
guguacaagu ucgugaccgu gaagaaucug gaugugauca aaaaagaaaa cuacuacgaa 2820
gugaauagca agugcuauga ggaagcuaag aagcugaaga agaucagcaa ccaggccgag 2880
uuuaucgccu ccuucuacaa caacgaucug aucaagauca acggcgagcu guauagagug 2940
aucggcguga acaacgaccu gcugaaccgg aucgaaguga acaugaucga caucaccuac 3000
cgcgaguacc uggaaaacau gaacgacaag aggcccccca ggaucauuaa gacaaucgcc 3060
uccaagaccc agagcauuaa gaaguacagc acagacauuc ugggcaaccu guaugaagug 3120
aaaucuaaga agcacccuca gaucaucaaa aagggc 3156
<210> 8
<211> 3156
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 8
aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc 60
gactacgaga cacgggacgt gatcgatgcc ggcgtgcggc tgttcaaaga ggccaacgtg 120
gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggctgaagcg gcggaggcgg 180
catagaatcc agagagtgaa gaagctgctg ttcgactaca acctgctgac cgaccacagc 240
gagctgagcg gcatcaaccc ctacgaggcc agagtgaagg gcctgagcca gaagctgagc 300
gaggaagagt tctctgccgc cctgctgcac ctggccaaga gaagaggcgt gcacaacgtg 360
aacgaggtgg aagaggacac cggcaacgag ctgtccacca aagagcagat cagccggaac 420
agcaaggccc tggaagagaa atacgtggcc gaactgcagc tggaacggct gaagaaagac 480
ggcgaagtgc ggggcagcat caacagattc aagaccagcg actacgtgaa agaagccaaa 540
cagctgctga aggtgcagaa ggcctaccac cagctggacc agagcttcat cgacacctac 600
atcgacctgc tggaaacccg gcggacctac tatgagggac ctggcgaggg cagccccttc 660
ggctggaagg acatcaaaga atggtacgag atgctgatgg gccactgcac ctacttcccc 720
gaggaactgc ggagcgtgaa gtacgcctac aacgccgacc tgtacaacgc cctgaacgac 780
ctgaacaatc tcgtgatcac cagggacgag aacgagaagc tggaatatta cgagaagttc 840
cagatcatcg agaacgtgtt caagcagaag aagaagccca ccctgaagca gatcgccaaa 900
gaaatcctcg tgaacgaaga ggatattaag ggctacagag tgaccagcac cggcaagccc 960
gagttcacca acctgaaggt gtaccacgac atcaaggaca ttaccgcccg gaaagagatt 1020
attgagaacg ccgagctgct ggatcagatt gccaagatcc tgaccatcta ccagagcagc 1080
gaggacatcc aggaagaact gaccaatctg aactccgagc tgacccagga agagatcgag 1140
cagatctcta atctgaaggg ctataccggc acccacaacc tgagcctgaa ggccatcaac 1200
ctgatcctgg acgagctgtg gcacaccaac gacaaccaga tcgctatctt caaccggctg 1260
aagctggtgc ccaagaaggt ggacctgtcc cagcagaaag agatccccac caccctggtg 1320
gacgacttca tcctgagccc cgtcgtgaag agaagcttca tccagagcat caaagtgatc 1380
aacgccatca tcaagaagta cggcctgccc aacgacatca ttatcgagct ggcccgcgag 1440
aagaactcca aggacgccca gaaaatgatc aacgagatgc agaagcggaa ccggcagacc 1500
aacgagcgga tcgaggaaat catccggacc accggcaaag agaacgccaa gtacctgatc 1560
gagaagatca agctgcacga catgcaggaa ggcaagtgcc tgtacagcct ggaagccatc 1620
cctctggaag atctgctgaa caaccccttc aactatgagg tggaccacat catccccaga 1680
agcgtgtcct tcgacaacag cttcaacaac aaggtgctcg tgaagcagga agaaaacagc 1740
aagaagggca accggacccc attccagtac ctgagcagca gcgacagcaa gatcagctac 1800
gaaaccttca agaagcacat cctgaatctg gccaagggca agggcagaat cagcaagacc 1860
aagaaagagt atctgctgga agaacgggac atcaacaggt tctccgtgca gaaagacttc 1920
atcaaccgga acctggtgga taccagatac gccaccagag gcctgatgaa cctgctgcgg 1980
agctacttca gagtgaacaa cctggacgtg aaagtgaagt ccatcaatgg cggcttcacc 2040
agctttctgc ggcggaagtg gaagtttaag aaagagcgga acaaggggta caagcaccac 2100
gccgaggacg ccctgatcat tgccaacgcc gatttcatct tcaaagagtg gaagaaactg 2160
gacaaggcca aaaaagtgat ggaaaaccag atgttcgagg aaaagcaggc cgagagcatg 2220
cccgagatcg aaaccgagca ggagtataag gagattttta taacacctca tcagattaag 2280
catattaagg attttaagga ttataagtat tctcatcgtg tggacaagaa gcctaatcgt 2340
gagttgatta atgatacttt gtattcgact cgtaaggatg acaaaggtaa caccttgatt 2400
gttaataatt tgaatggttt gtatgataag gacaatgata agttgaagaa gttgattaat 2460
aagtctcctg agaagttgtt gatgtatcat catgatccgc agacttatca gaagttgaag 2520
ttgattatgg agcagtatgg tgatgagaag aatcctttgt ataagtatta tgaagaaact 2580
ggtaattatt tgactaagta ttcgaagaag gacaatgggc ccgtgattaa gaagattaag 2640
tattatggta ataagttgaa tgctcatttg gatattactg atgactatcc taattctcgt 2700
aataaagttg ttaagttaag tttgaagcct tatcgttttg atgtttattt ggacaatggt 2760
gtttataagt ttgttactgt gaagaatttg gatgttatta agaaggagaa ttattatgaa 2820
gttaattcta agtgttatga agaagcgaag aagttgaaga agataagtaa tcaggctgag 2880
tttattgcaa gtttttataa taatgatttg attaagatta atggtgagtt gtatcgtgtt 2940
attggtgtta ataatgattt gttgaatcgt attgaagtta atatgattga tattacttat 3000
cgtgagtatt tggagaatat gaatgataag cggcccccgc gtattattaa gactattgca 3060
agtaagactc aaagtattaa gaagtattct actgatattt tgggtaattt gtatgaagtt 3120
aagtcgaaga agcatcctca gattattaag aagggt 3156
<210> 9
<211> 3156
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 9
aagcggaacu acauccuggg ccuggacauc ggcaucacca gcgugggcua cggcaucauc 60
gacuacgaga cacgggacgu gaucgaugcc ggcgugcggc uguucaaaga ggccaacgug 120
gaaaacaacg agggcaggcg gagcaagaga ggcgccagaa ggcugaagcg gcggaggcgg 180
cauagaaucc agagagugaa gaagcugcug uucgacuaca accugcugac cgaccacagc 240
gagcugagcg gcaucaaccc cuacgaggcc agagugaagg gccugagcca gaagcugagc 300
gaggaagagu ucucugccgc ccugcugcac cuggccaaga gaagaggcgu gcacaacgug 360
aacgaggugg aagaggacac cggcaacgag cuguccacca aagagcagau cagccggaac 420
agcaaggccc uggaagagaa auacguggcc gaacugcagc uggaacggcu gaagaaagac 480
ggcgaagugc ggggcagcau caacagauuc aagaccagcg acuacgugaa agaagccaaa 540
cagcugcuga aggugcagaa ggccuaccac cagcuggacc agagcuucau cgacaccuac 600
aucgaccugc uggaaacccg gcggaccuac uaugagggac cuggcgaggg cagccccuuc 660
ggcuggaagg acaucaaaga augguacgag augcugaugg gccacugcac cuacuucccc 720
gaggaacugc ggagcgugaa guacgccuac aacgccgacc uguacaacgc ccugaacgac 780
cugaacaauc ucgugaucac cagggacgag aacgagaagc uggaauauua cgagaaguuc 840
cagaucaucg agaacguguu caagcagaag aagaagccca cccugaagca gaucgccaaa 900
gaaauccucg ugaacgaaga ggauauuaag ggcuacagag ugaccagcac cggcaagccc 960
gaguucacca accugaaggu guaccacgac aucaaggaca uuaccgcccg gaaagagauu 1020
auugagaacg ccgagcugcu ggaucagauu gccaagaucc ugaccaucua ccagagcagc 1080
gaggacaucc aggaagaacu gaccaaucug aacuccgagc ugacccagga agagaucgag 1140
cagaucucua aucugaaggg cuauaccggc acccacaacc ugagccugaa ggccaucaac 1200
cugauccugg acgagcugug gcacaccaac gacaaccaga ucgcuaucuu caaccggcug 1260
aagcuggugc ccaagaaggu ggaccugucc cagcagaaag agauccccac cacccuggug 1320
gacgacuuca uccugagccc cgucgugaag agaagcuuca uccagagcau caaagugauc 1380
aacgccauca ucaagaagua cggccugccc aacgacauca uuaucgagcu ggcccgcgag 1440
aagaacucca aggacgccca gaaaaugauc aacgagaugc agaagcggaa ccggcagacc 1500
aacgagcgga ucgaggaaau cauccggacc accggcaaag agaacgccaa guaccugauc 1560
gagaagauca agcugcacga caugcaggaa ggcaagugcc uguacagccu ggaagccauc 1620
ccucuggaag aucugcugaa caaccccuuc aacuaugagg uggaccacau cauccccaga 1680
agcguguccu ucgacaacag cuucaacaac aaggugcucg ugaagcagga agaaaacagc 1740
aagaagggca accggacccc auuccaguac cugagcagca gcgacagcaa gaucagcuac 1800
gaaaccuuca agaagcacau ccugaaucug gccaagggca agggcagaau cagcaagacc 1860
aagaaagagu aucugcugga agaacgggac aucaacaggu ucuccgugca gaaagacuuc 1920
aucaaccgga accuggugga uaccagauac gccaccagag gccugaugaa ccugcugcgg 1980
agcuacuuca gagugaacaa ccuggacgug aaagugaagu ccaucaaugg cggcuucacc 2040
agcuuucugc ggcggaagug gaaguuuaag aaagagcgga acaaggggua caagcaccac 2100
gccgaggacg cccugaucau ugccaacgcc gauuucaucu ucaaagagug gaagaaacug 2160
gacaaggcca aaaaagugau ggaaaaccag auguucgagg aaaagcaggc cgagagcaug 2220
cccgagaucg aaaccgagca ggaguauaag gagauuuuua uaacaccuca ucagauuaag 2280
cauauuaagg auuuuaagga uuauaaguau ucucaucgug uggacaagaa gccuaaucgu 2340
gaguugauua augauacuuu guauucgacu cguaaggaug acaaagguaa caccuugauu 2400
guuaauaauu ugaaugguuu guaugauaag gacaaugaua aguugaagaa guugauuaau 2460
aagucuccug agaaguuguu gauguaucau caugauccgc agacuuauca gaaguugaag 2520
uugauuaugg agcaguaugg ugaugagaag aauccuuugu auaaguauua ugaagaaacu 2580
gguaauuauu ugacuaagua uucgaagaag gacaaugggc ccgugauuaa gaagauuaag 2640
uauuauggua auaaguugaa ugcucauuug gauauuacug augacuaucc uaauucucgu 2700
aauaaaguug uuaaguuaag uuugaagccu uaucguuuug auguuuauuu ggacaauggu 2760
guuuauaagu uuguuacugu gaagaauuug gauguuauua agaaggagaa uuauuaugaa 2820
guuaauucua aguguuauga agaagcgaag aaguugaaga agauaaguaa ucaggcugag 2880
uuuauugcaa guuuuuauaa uaaugauuug auuaagauua auggugaguu guaucguguu 2940
auugguguua auaaugauuu guugaaucgu auugaaguua auaugauuga uauuacuuau 3000
cgugaguauu uggagaauau gaaugauaag cggcccccgc guauuauuaa gacuauugca 3060
aguaagacuc aaaguauuaa gaaguauucu acugauauuu uggguaauuu guaugaaguu 3120
aagucgaaga agcauccuca gauuauuaag aagggu 3156
<210> 10
<211> 693
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 10
actcgtgatg ttattgacgc aggcgttcgt ttgtttaaag aagctaatgt tgagaataat 60
gagggaagaa gaagtaagcg tggggctcgc aggcttaagc gaagaagaag gcatcggata 120
cagcgtgtga agaagttgct gtttgattat aatttgttga ctgatcattc tgagttatca 180
ggcattaatc cttatgaggc tcgtgttaag ggtttaagtc agaagttaag tgaagaagaa 240
ttttctgctg ctttgttgca tttggctaaa agaagaggag ttcataatgt taatgaagtt 300
gaagaggata ctggtaatga gttaagtact aaggagcaga taagtcgtaa ttctaaggct 360
ttggaagaaa agtatgttgc tgagttgcag ttggagcgtt tgaagaagga tggtgaagta 420
agaggaagta ttaatcgttt taagacaagt gattatgtga aagaagcgaa gcagttgttg 480
aaagttcaga aggcttatca tcagttggat caaagtttta ttgatactta tattgatttg 540
ttggagactc gtagaactta ttatgagggt cctggtgagg ggtccccgtt tggttggaag 600
gatattaagg agtggtatga gatgttgatg ggtcattgta cttattttcc tgaagaattg 660
cggtccgtga agtatgctta taatgctgat ttg 693
<210> 11
<211> 693
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 11
acucgugaug uuauugacgc aggcguucgu uuguuuaaag aagcuaaugu ugagaauaau 60
gagggaagaa gaaguaagcg uggggcucgc aggcuuaagc gaagaagaag gcaucggaua 120
cagcguguga agaaguugcu guuugauuau aauuuguuga cugaucauuc ugaguuauca 180
ggcauuaauc cuuaugaggc ucguguuaag gguuuaaguc agaaguuaag ugaagaagaa 240
uuuucugcug cuuuguugca uuuggcuaaa agaagaggag uucauaaugu uaaugaaguu 300
gaagaggaua cugguaauga guuaaguacu aaggagcaga uaagucguaa uucuaaggcu 360
uuggaagaaa aguauguugc ugaguugcag uuggagcguu ugaagaagga uggugaagua 420
agaggaagua uuaaucguuu uaagacaagu gauuauguga aagaagcgaa gcaguuguug 480
aaaguucaga aggcuuauca ucaguuggau caaaguuuua uugauacuua uauugauuug 540
uuggagacuc guagaacuua uuaugagggu ccuggugagg gguccccguu ugguuggaag 600
gauauuaagg agugguauga gauguugaug ggucauugua cuuauuuucc ugaagaauug 660
cgguccguga aguaugcuua uaaugcugau uug 693
<210> 12
<211> 672
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 12
gctaagattt tgactattta tcagtcaagt gaggatattc aggaagaatt gactaatttg 60
aattctgagt tgactcagga agaaattgag cagataagta atttgaaggg atacactggt 120
actcataatt taagtttgaa ggctattaat ttgattttgg atgagttgtg gcatactaat 180
gataatcaga ttgctatttt taatcgtttg aagttggttc ctaagaaagt tgatttaagt 240
cagcagaagg agattcctac tactttggtt gatgacttta ttttaagtcc tgttgttaag 300
cgaagtttta ttcaaagtat taaagttatt aatgctatta ttaagaagta tgggctcccg 360
aatgatatta ttattgagtt ggctcgtgag aagaattcta aagatgctca gaagatgatt 420
aatgagatgc agaagaggaa cagacagaca aatgaaagaa ttgaagaaat tattcggaca 480
actggtaagg agaatgctaa gtatttgatt gagaagatta agttgcatga tatgcaggag 540
ggtaagtgtt tgtattcttt ggaggctatt cctttggagg atttgttgaa taatcctttt 600
aattatgaag ttgatcatat tattcctcgg tccgtaagtt ttgataattc ttttaataat 660
aaagttttgg tt 672
<210> 13
<211> 672
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 13
gcuaagauuu ugacuauuua ucagucaagu gaggauauuc aggaagaauu gacuaauuug 60
aauucugagu ugacucagga agaaauugag cagauaagua auuugaaggg auacacuggu 120
acucauaauu uaaguuugaa ggcuauuaau uugauuuugg augaguugug gcauacuaau 180
gauaaucaga uugcuauuuu uaaucguuug aaguugguuc cuaagaaagu ugauuuaagu 240
cagcagaagg agauuccuac uacuuugguu gaugacuuua uuuuaagucc uguuguuaag 300
cgaaguuuua uucaaaguau uaaaguuauu aaugcuauua uuaagaagua ugggcucccg 360
aaugauauua uuauugaguu ggcucgugag aagaauucua aagaugcuca gaagaugauu 420
aaugagaugc agaagaggaa cagacagaca aaugaaagaa uugaagaaau uauucggaca 480
acugguaagg agaaugcuaa guauuugauu gagaagauua aguugcauga uaugcaggag 540
gguaaguguu uguauucuuu ggaggcuauu ccuuuggagg auuuguugaa uaauccuuuu 600
aauuaugaag uugaucauau uauuccucgg uccguaaguu uugauaauuc uuuuaauaau 660
aaaguuuugg uu 672
<210> 14
<211> 912
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 14
tataaggaga tttttataac acctcatcag attaagcata ttaaggattt taaggattat 60
aagtattctc atcgtgtgga caagaagcct aatcgtgagt tgattaatga tactttgtat 120
tcgactcgta aggatgacaa aggtaacacc ttgattgtta ataatttgaa tggtttgtat 180
gataaggaca atgataagtt gaagaagttg attaataagt ctcctgagaa gttgttgatg 240
tatcatcatg atccgcagac ttatcagaag ttgaagttga ttatggagca gtatggtgat 300
gagaagaatc ctttgtataa gtattatgaa gaaactggta attatttgac taagtattcg 360
aagaaggaca atgggcccgt gattaagaag attaagtatt atggtaataa gttgaatgct 420
catttggata ttactgatga ctatcctaat tctcgtaata aagttgttaa gttaagtttg 480
aagccttatc gttttgatgt ttatttggac aatggtgttt ataagtttgt tactgtgaag 540
aatttggatg ttattaagaa ggagaattat tatgaagtta attctaagtg ttatgaagaa 600
gcgaagaagt tgaagaagat aagtaatcag gctgagttta ttgcaagttt ttataataat 660
gatttgatta agattaatgg tgagttgtat cgtgttattg gtgttaataa tgatttgttg 720
aatcgtattg aagttaatat gattgatatt acttatcgtg agtatttgga gaatatgaat 780
gataagcggc ccccgcgtat tattaagact attgcaagta agactcaaag tattaagaag 840
tattctactg atattttggg taatttgtat gaagttaagt cgaagaagca tcctcagatt 900
attaagaagg gt 912
<210> 15
<211> 912
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 15
uauaaggaga uuuuuauaac accucaucag auuaagcaua uuaaggauuu uaaggauuau 60
aaguauucuc aucgugugga caagaagccu aaucgugagu ugauuaauga uacuuuguau 120
ucgacucgua aggaugacaa agguaacacc uugauuguua auaauuugaa ugguuuguau 180
gauaaggaca augauaaguu gaagaaguug auuaauaagu cuccugagaa guuguugaug 240
uaucaucaug auccgcagac uuaucagaag uugaaguuga uuauggagca guauggugau 300
gagaagaauc cuuuguauaa guauuaugaa gaaacuggua auuauuugac uaaguauucg 360
aagaaggaca augggcccgu gauuaagaag auuaaguauu augguaauaa guugaaugcu 420
cauuuggaua uuacugauga cuauccuaau ucucguaaua aaguuguuaa guuaaguuug 480
aagccuuauc guuuugaugu uuauuuggac aaugguguuu auaaguuugu uacugugaag 540
aauuuggaug uuauuaagaa ggagaauuau uaugaaguua auucuaagug uuaugaagaa 600
gcgaagaagu ugaagaagau aaguaaucag gcugaguuua uugcaaguuu uuauaauaau 660
gauuugauua agauuaaugg ugaguuguau cguguuauug guguuaauaa ugauuuguug 720
aaucguauug aaguuaauau gauugauauu acuuaucgug aguauuugga gaauaugaau 780
gauaagcggc ccccgcguau uauuaagacu auugcaagua agacucaaag uauuaagaag 840
uauucuacug auauuuuggg uaauuuguau gaaguuaagu cgaagaagca uccucagauu 900
auuaagaagg gu 912
<210> 16
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 16
tttcaggcgc taaaacatac cagatgaaag tctggagagg tgaagaatac gaccacctag 60
cgcctgaaa 69
<210> 17
<211> 69
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 17
uuucaggcgc uaaaacauac cagaugaaag ucuggagagg ugaagaauac gaccaccuag 60
cgccugaaa 69
<210> 18
<211> 69
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 18
tttcaggcgc caaaacatac cagatgaaag tctggagagg tgaagaatac gaccacctgg 60
cgcctgaaa 69
<210> 19
<211> 69
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 19
uuucaggcgc caaaacauac cagaugaaag ucuggagagg ugaagaauac gaccaccugg 60
cgccugaaa 69
<210> 20
<211> 71
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 20
tttcaggcgc gcaaaacata ccagatgaaa gtctggagag gtgaagaata cgaccacctg 60
cgcgcctgaa a 71
<210> 21
<211> 71
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 21
uuucaggcgc gcaaaacaua ccagaugaaa gucuggagag gugaagaaua cgaccaccug 60
cgcgccugaa a 71
<210> 22
<211> 96
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 22
caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca accaaacaac 60
caaacaacca aacaaccaaa caaccaaaca acacag 96
<210> 23
<211> 96
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 23
caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca accaaacaac 60
caaacaacca aacaaccaaa caaccaaaca acacag 96
<210> 24
<211> 101
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 24
gtgagtctat gggacccttg atgttttctg catgggtagc cgctgagatg gagcctgagc 60
acacgcggcc gctgttaacg cagtgtttct ctttttttca g 101
<210> 25
<211> 101
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 25
gugagucuau gggacccuug auguuuucug cauggguagc cgcugagaug gagccugagc 60
acacgcggcc gcuguuaacg caguguuucu cuuuuuuuca g 101
<210> 26
<211> 91
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 26
gttggtgcta gctggccaag gctggattat tctgagtcca agctaggccc ttttgctaat 60
catgttcata cctcttatct tcctcccaca g 91
<210> 27
<211> 91
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 27
guuggugcua gcuggccaag gcuggauuau ucugagucca agcuaggccc uuuugcuaau 60
cauguucaua ccucuuaucu uccucccaca g 91
<210> 28
<211> 351
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 28
gtgagtctat gggacccttg atgttttttg catgggtagc cgctgagatg gagcctgagc 60
acacgcggcc gctgttaacg cagtgtttct ctttttttca ggcgctaaaa cataccagat 120
gaaagtctgg agaggtgaag aatacgacca cctagcgcct gaaacaacca aacaaccaaa 180
caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca accaaacaac 240
caaacaacca aacaacacag gttggtgcta gctggccaag gctggattat tctgagtcca 300
agctaggccc ttttgctaat catgttcata cctcttatct tcctcccaca g 351
<210> 29
<211> 351
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 29
gugagucuau gggacccuug auguuuuuug cauggguagc cgcugagaug gagccugagc 60
acacgcggcc gcuguuaacg caguguuucu cuuuuuuuca ggcgcuaaaa cauaccagau 120
gaaagucugg agaggugaag aauacgacca ccuagcgccu gaaacaacca aacaaccaaa 180
caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca accaaacaac 240
caaacaacca aacaacacag guuggugcua gcuggccaag gcuggauuau ucugagucca 300
agcuaggccc uuuugcuaau cauguucaua ccucuuaucu uccucccaca g 351
<210> 30
<211> 3507
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 30
aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc 60
gactacgaga ctcgtgatgt tattgacgca ggcgttcgtt tgtttaaaga agctaatgtt 120
gagaataatg agggaagaag aagtaagcgt ggggctcgca ggcttagtga gtctatggga 180
cccttgatgt tttttgcatg ggtagccgct gagatggagc ctgagcacac gcggccgctg 240
ttaacgcagt gtttctcttt ttttcaggcg ctaaaacata ccagatgaaa gtctggagag 300
gtgaagaata cgaccaccta gcgcctgaaa caaccaaaca accaaacaac caaacaacca 360
aacaaccaaa caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca 420
acacaggttg gtgctagctg gccaaggctg gattattctg agtccaagct aggccctttt 480
gctaatcatg ttcatacctc ttatcttcct cccacagagc gaagaagaag gcatcggata 540
cagcgtgtga agaagttgct gtttgattat aatttgttga ctgatcattc tgagttatca 600
ggcattaatc cttatgaggc tcgtgttaag ggtttaagtc agaagttaag tgaagaagaa 660
ttttctgctg ctttgttgca tttggctaaa agaagaggag ttcataatgt taatgaagtt 720
gaagaggata ctggtaatga gttaagtact aaggagcaga taagtcgtaa ttctaaggct 780
ttggaagaaa agtatgttgc tgagttgcag ttggagcgtt tgaagaagga tggtgaagta 840
agaggaagta ttaatcgttt taagacaagt gattatgtga aagaagcgaa gcagttgttg 900
aaagttcaga aggcttatca tcagttggat caaagtttta ttgatactta tattgatttg 960
ttggagactc gtagaactta ttatgagggt cctggtgagg ggtccccgtt tggttggaag 1020
gatattaagg agtggtatga gatgttgatg ggtcattgta cttattttcc tgaagaattg 1080
cggtccgtga agtatgctta taatgctgat ttgtacaacg ccctgaacga cctgaacaat 1140
ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt ccagatcatc 1200
gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa agaaatcctc 1260
gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc cgagttcacc 1320
aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat tattgagaac 1380
gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag cgaggacatc 1440
caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga gcagatctct 1500
aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa cctgatcctg 1560
gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct gaagctggtg 1620
cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt ggacgacttc 1680
atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat caacgccatc 1740
atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga gaagaactcc 1800
aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac caacgagcgg 1860
atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat cgagaagatc 1920
aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat ccctctggaa 1980
gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag aagcgtgtcc 2040
ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaaaacag caagaagggc 2100
aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta cgaaaccttc 2160
aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac caagaaagag 2220
tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt catcaaccgg 2280
aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg gagctacttc 2340
agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac cagctttctg 2400
cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca cgccgaggac 2460
gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc 2520
aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat gcccgagatc 2580
gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa gcacattaag 2640
gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag agagctgatt 2700
aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat cgtgaacaat 2760
ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa caagagcccc 2820
gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa gctgattatg 2880
gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac cgggaactac 2940
ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa gtattacggc 3000
aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag aaacaaggtc 3060
gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg cgtgtacaag 3120
ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga agtgaatagc 3180
aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga gtttatcgcc 3240
tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt gatcggcgtg 3300
aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta ccgcgagtac 3360
ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc ctccaagacc 3420
cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt gaaatctaag 3480
aagcaccctc agatcatcaa aaagggc 3507
<210> 31
<211> 3507
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 31
aagcggaacu acauccuggg ccuggacauc ggcaucacca gcgugggcua cggcaucauc 60
gacuacgaga cucgugaugu uauugacgca ggcguucguu uguuuaaaga agcuaauguu 120
gagaauaaug agggaagaag aaguaagcgu ggggcucgca ggcuuaguga gucuauggga 180
cccuugaugu uuuuugcaug gguagccgcu gagauggagc cugagcacac gcggccgcug 240
uuaacgcagu guuucucuuu uuuucaggcg cuaaaacaua ccagaugaaa gucuggagag 300
gugaagaaua cgaccaccua gcgccugaaa caaccaaaca accaaacaac caaacaacca 360
aacaaccaaa caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca 420
acacagguug gugcuagcug gccaaggcug gauuauucug aguccaagcu aggcccuuuu 480
gcuaaucaug uucauaccuc uuaucuuccu cccacagagc gaagaagaag gcaucggaua 540
cagcguguga agaaguugcu guuugauuau aauuuguuga cugaucauuc ugaguuauca 600
ggcauuaauc cuuaugaggc ucguguuaag gguuuaaguc agaaguuaag ugaagaagaa 660
uuuucugcug cuuuguugca uuuggcuaaa agaagaggag uucauaaugu uaaugaaguu 720
gaagaggaua cugguaauga guuaaguacu aaggagcaga uaagucguaa uucuaaggcu 780
uuggaagaaa aguauguugc ugaguugcag uuggagcguu ugaagaagga uggugaagua 840
agaggaagua uuaaucguuu uaagacaagu gauuauguga aagaagcgaa gcaguuguug 900
aaaguucaga aggcuuauca ucaguuggau caaaguuuua uugauacuua uauugauuug 960
uuggagacuc guagaacuua uuaugagggu ccuggugagg gguccccguu ugguuggaag 1020
gauauuaagg agugguauga gauguugaug ggucauugua cuuauuuucc ugaagaauug 1080
cgguccguga aguaugcuua uaaugcugau uuguacaacg cccugaacga ccugaacaau 1140
cucgugauca ccagggacga gaacgagaag cuggaauauu acgagaaguu ccagaucauc 1200
gagaacgugu ucaagcagaa gaagaagccc acccugaagc agaucgccaa agaaauccuc 1260
gugaacgaag aggauauuaa gggcuacaga gugaccagca ccggcaagcc cgaguucacc 1320
aaccugaagg uguaccacga caucaaggac auuaccgccc ggaaagagau uauugagaac 1380
gccgagcugc uggaucagau ugccaagauc cugaccaucu accagagcag cgaggacauc 1440
caggaagaac ugaccaaucu gaacuccgag cugacccagg aagagaucga gcagaucucu 1500
aaucugaagg gcuauaccgg cacccacaac cugagccuga aggccaucaa ccugauccug 1560
gacgagcugu ggcacaccaa cgacaaccag aucgcuaucu ucaaccggcu gaagcuggug 1620
cccaagaagg uggaccuguc ccagcagaaa gagaucccca ccacccuggu ggacgacuuc 1680
auccugagcc ccgucgugaa gagaagcuuc auccagagca ucaaagugau caacgccauc 1740
aucaagaagu acggccugcc caacgacauc auuaucgagc uggcccgcga gaagaacucc 1800
aaggacgccc agaaaaugau caacgagaug cagaagcgga accggcagac caacgagcgg 1860
aucgaggaaa ucauccggac caccggcaaa gagaacgcca aguaccugau cgagaagauc 1920
aagcugcacg acaugcagga aggcaagugc cuguacagcc uggaagccau cccucuggaa 1980
gaucugcuga acaaccccuu caacuaugag guggaccaca ucauccccag aagcgugucc 2040
uucgacaaca gcuucaacaa caaggugcuc gugaagcagg aagaaaacag caagaagggc 2100
aaccggaccc cauuccagua ccugagcagc agcgacagca agaucagcua cgaaaccuuc 2160
aagaagcaca uccugaaucu ggccaagggc aagggcagaa ucagcaagac caagaaagag 2220
uaucugcugg aagaacggga caucaacagg uucuccgugc agaaagacuu caucaaccgg 2280
aaccuggugg auaccagaua cgccaccaga ggccugauga accugcugcg gagcuacuuc 2340
agagugaaca accuggacgu gaaagugaag uccaucaaug gcggcuucac cagcuuucug 2400
cggcggaagu ggaaguuuaa gaaagagcgg aacaaggggu acaagcacca cgccgaggac 2460
gcccugauca uugccaacgc cgauuucauc uucaaagagu ggaagaaacu ggacaaggcc 2520
aaaaaaguga uggaaaacca gauguucgag gaaaagcagg ccgagagcau gcccgagauc 2580
gaaaccgagc aggaguacaa agagaucuuc aucacccccc accagaucaa gcacauuaag 2640
gacuucaagg acuacaagua cagccaccgg guggacaaga agccuaauag agagcugauu 2700
aacgacaccc uguacuccac ccggaaggac gacaagggca acacccugau cgugaacaau 2760
cugaacggcc uguacgacaa ggacaaugac aagcugaaaa agcugaucaa caagagcccc 2820
gaaaagcugc ugauguacca ccacgacccc cagaccuacc agaaacugaa gcugauuaug 2880
gaacaguacg gcgacgagaa gaauccccug uacaaguacu acgaggaaac cgggaacuac 2940
cugaccaagu acuccaaaaa ggacaacggc cccgugauca agaagauuaa guauuacggc 3000
aacaaacuga acgcccaucu ggacaucacc gacgacuacc ccaacagcag aaacaagguc 3060
gugaagcugu cccugaagcc cuacagauuc gacguguacc uggacaaugg cguguacaag 3120
uucgugaccg ugaagaaucu ggaugugauc aaaaaagaaa acuacuacga agugaauagc 3180
aagugcuaug aggaagcuaa gaagcugaag aagaucagca accaggccga guuuaucgcc 3240
uccuucuaca acaacgaucu gaucaagauc aacggcgagc uguauagagu gaucggcgug 3300
aacaacgacc ugcugaaccg gaucgaagug aacaugaucg acaucaccua ccgcgaguac 3360
cuggaaaaca ugaacgacaa gaggcccccc aggaucauua agacaaucgc cuccaagacc 3420
cagagcauua agaaguacag cacagacauu cugggcaacc uguaugaagu gaaaucuaag 3480
aagcacccuc agaucaucaa aaagggc 3507
<210> 32
<211> 3507
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 32
aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc 60
gactacgaga ctcgtgatgt tattgacgca ggcgttcgtt tgtttaaaga agctaatgtt 120
gagaataatg agggaagaag aagtaagcgt ggggctcgca ggcttaagcg aagaagaagg 180
catcggatac agcgtgtgaa gaagttgctg tttgattata atttgttgac tgatcattct 240
gagttatcag gcattaatcc ttatgaggct cgtgttaagg gtttaagtca gaagttaagt 300
gaagaagaat tttctgctgc tttgttgcat ttggctaaaa gaagaggagt tcataatgtt 360
aatgaagttg aagaggatac tggtaatgag ttaagtacta aggagcagat aagtcgtaat 420
tctaaggctt tggaagaaaa gtatgttgct gagttgcagt tggagcgttt gaagaaggat 480
ggtgaagtaa gaggaagtat taatcgtttt aagacaagtg attatgtgaa agaagcgaag 540
cagttgttga aagttcagaa ggcttatgtg agtctatggg acccttgatg ttttctgcat 600
gggtagccgc tgagatggag cctgagcaca cgcggccgct gttaacgcag tgtttctctt 660
tttttcaggc gctaaaacat accagatgaa agtctggaga ggtgaagaat acgaccacct 720
agcgcctgaa acaaccaaac aaccaaacaa ccaaacaacc aaacaaccaa acaaccaaac 780
aaccaaacaa ccaaacaacc aaacaaccaa acaaccaaac aacacaggtt ggtgctagct 840
ggccaaggct ggattattct gagtccaagc taggcccttt tgctaatcat gttcatacct 900
cttatcttcc tcccacagca tcagttggat caaagtttta ttgatactta tattgatttg 960
ttggagactc gtagaactta ttatgagggt cctggtgagg ggtccccgtt tggttggaag 1020
gatattaagg agtggtatga gatgttgatg ggtcattgta cttattttcc tgaagaattg 1080
cggtccgtga agtatgctta taatgctgat ttgtacaacg ccctgaacga cctgaacaat 1140
ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt ccagatcatc 1200
gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa agaaatcctc 1260
gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc cgagttcacc 1320
aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat tattgagaac 1380
gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag cgaggacatc 1440
caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga gcagatctct 1500
aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa cctgatcctg 1560
gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct gaagctggtg 1620
cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt ggacgacttc 1680
atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat caacgccatc 1740
atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga gaagaactcc 1800
aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac caacgagcgg 1860
atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat cgagaagatc 1920
aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat ccctctggaa 1980
gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag aagcgtgtcc 2040
ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaaaacag caagaagggc 2100
aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta cgaaaccttc 2160
aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac caagaaagag 2220
tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt catcaaccgg 2280
aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg gagctacttc 2340
agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac cagctttctg 2400
cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca cgccgaggac 2460
gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc 2520
aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat gcccgagatc 2580
gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa gcacattaag 2640
gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag agagctgatt 2700
aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat cgtgaacaat 2760
ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa caagagcccc 2820
gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa gctgattatg 2880
gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac cgggaactac 2940
ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa gtattacggc 3000
aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag aaacaaggtc 3060
gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg cgtgtacaag 3120
ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga agtgaatagc 3180
aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga gtttatcgcc 3240
tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt gatcggcgtg 3300
aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta ccgcgagtac 3360
ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc ctccaagacc 3420
cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt gaaatctaag 3480
aagcaccctc agatcatcaa aaagggc 3507
<210> 33
<211> 3507
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 33
aagcggaacu acauccuggg ccuggacauc ggcaucacca gcgugggcua cggcaucauc 60
gacuacgaga cucgugaugu uauugacgca ggcguucguu uguuuaaaga agcuaauguu 120
gagaauaaug agggaagaag aaguaagcgu ggggcucgca ggcuuaagcg aagaagaagg 180
caucggauac agcgugugaa gaaguugcug uuugauuaua auuuguugac ugaucauucu 240
gaguuaucag gcauuaaucc uuaugaggcu cguguuaagg guuuaaguca gaaguuaagu 300
gaagaagaau uuucugcugc uuuguugcau uuggcuaaaa gaagaggagu ucauaauguu 360
aaugaaguug aagaggauac ugguaaugag uuaaguacua aggagcagau aagucguaau 420
ucuaaggcuu uggaagaaaa guauguugcu gaguugcagu uggagcguuu gaagaaggau 480
ggugaaguaa gaggaaguau uaaucguuuu aagacaagug auuaugugaa agaagcgaag 540
caguuguuga aaguucagaa ggcuuaugug agucuauggg acccuugaug uuuucugcau 600
ggguagccgc ugagauggag ccugagcaca cgcggccgcu guuaacgcag uguuucucuu 660
uuuuucaggc gcuaaaacau accagaugaa agucuggaga ggugaagaau acgaccaccu 720
agcgccugaa acaaccaaac aaccaaacaa ccaaacaacc aaacaaccaa acaaccaaac 780
aaccaaacaa ccaaacaacc aaacaaccaa acaaccaaac aacacagguu ggugcuagcu 840
ggccaaggcu ggauuauucu gaguccaagc uaggcccuuu ugcuaaucau guucauaccu 900
cuuaucuucc ucccacagca ucaguuggau caaaguuuua uugauacuua uauugauuug 960
uuggagacuc guagaacuua uuaugagggu ccuggugagg gguccccguu ugguuggaag 1020
gauauuaagg agugguauga gauguugaug ggucauugua cuuauuuucc ugaagaauug 1080
cgguccguga aguaugcuua uaaugcugau uuguacaacg cccugaacga ccugaacaau 1140
cucgugauca ccagggacga gaacgagaag cuggaauauu acgagaaguu ccagaucauc 1200
gagaacgugu ucaagcagaa gaagaagccc acccugaagc agaucgccaa agaaauccuc 1260
gugaacgaag aggauauuaa gggcuacaga gugaccagca ccggcaagcc cgaguucacc 1320
aaccugaagg uguaccacga caucaaggac auuaccgccc ggaaagagau uauugagaac 1380
gccgagcugc uggaucagau ugccaagauc cugaccaucu accagagcag cgaggacauc 1440
caggaagaac ugaccaaucu gaacuccgag cugacccagg aagagaucga gcagaucucu 1500
aaucugaagg gcuauaccgg cacccacaac cugagccuga aggccaucaa ccugauccug 1560
gacgagcugu ggcacaccaa cgacaaccag aucgcuaucu ucaaccggcu gaagcuggug 1620
cccaagaagg uggaccuguc ccagcagaaa gagaucccca ccacccuggu ggacgacuuc 1680
auccugagcc ccgucgugaa gagaagcuuc auccagagca ucaaagugau caacgccauc 1740
aucaagaagu acggccugcc caacgacauc auuaucgagc uggcccgcga gaagaacucc 1800
aaggacgccc agaaaaugau caacgagaug cagaagcgga accggcagac caacgagcgg 1860
aucgaggaaa ucauccggac caccggcaaa gagaacgcca aguaccugau cgagaagauc 1920
aagcugcacg acaugcagga aggcaagugc cuguacagcc uggaagccau cccucuggaa 1980
gaucugcuga acaaccccuu caacuaugag guggaccaca ucauccccag aagcgugucc 2040
uucgacaaca gcuucaacaa caaggugcuc gugaagcagg aagaaaacag caagaagggc 2100
aaccggaccc cauuccagua ccugagcagc agcgacagca agaucagcua cgaaaccuuc 2160
aagaagcaca uccugaaucu ggccaagggc aagggcagaa ucagcaagac caagaaagag 2220
uaucugcugg aagaacggga caucaacagg uucuccgugc agaaagacuu caucaaccgg 2280
aaccuggugg auaccagaua cgccaccaga ggccugauga accugcugcg gagcuacuuc 2340
agagugaaca accuggacgu gaaagugaag uccaucaaug gcggcuucac cagcuuucug 2400
cggcggaagu ggaaguuuaa gaaagagcgg aacaaggggu acaagcacca cgccgaggac 2460
gcccugauca uugccaacgc cgauuucauc uucaaagagu ggaagaaacu ggacaaggcc 2520
aaaaaaguga uggaaaacca gauguucgag gaaaagcagg ccgagagcau gcccgagauc 2580
gaaaccgagc aggaguacaa agagaucuuc aucacccccc accagaucaa gcacauuaag 2640
gacuucaagg acuacaagua cagccaccgg guggacaaga agccuaauag agagcugauu 2700
aacgacaccc uguacuccac ccggaaggac gacaagggca acacccugau cgugaacaau 2760
cugaacggcc uguacgacaa ggacaaugac aagcugaaaa agcugaucaa caagagcccc 2820
gaaaagcugc ugauguacca ccacgacccc cagaccuacc agaaacugaa gcugauuaug 2880
gaacaguacg gcgacgagaa gaauccccug uacaaguacu acgaggaaac cgggaacuac 2940
cugaccaagu acuccaaaaa ggacaacggc cccgugauca agaagauuaa guauuacggc 3000
aacaaacuga acgcccaucu ggacaucacc gacgacuacc ccaacagcag aaacaagguc 3060
gugaagcugu cccugaagcc cuacagauuc gacguguacc uggacaaugg cguguacaag 3120
uucgugaccg ugaagaaucu ggaugugauc aaaaaagaaa acuacuacga agugaauagc 3180
aagugcuaug aggaagcuaa gaagcugaag aagaucagca accaggccga guuuaucgcc 3240
uccuucuaca acaacgaucu gaucaagauc aacggcgagc uguauagagu gaucggcgug 3300
aacaacgacc ugcugaaccg gaucgaagug aacaugaucg acaucaccua ccgcgaguac 3360
cuggaaaaca ugaacgacaa gaggcccccc aggaucauua agacaaucgc cuccaagacc 3420
cagagcauua agaaguacag cacagacauu cugggcaacc uguaugaagu gaaaucuaag 3480
aagcacccuc agaucaucaa aaagggc 3507
<210> 34
<211> 3858
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 34
aagcggaact acatcctggg cctggacatc ggcatcacca gcgtgggcta cggcatcatc 60
gactacgaga ctcgtgatgt tattgacgca ggcgttcgtt tgtttaaaga agctaatgtt 120
gagaataatg agggaagaag aagtaagcgt ggggctcgca ggcttagtga gtctatggga 180
cccttgatgt tttttgcatg ggtagccgct gagatggagc ctgagcacac gcggccgctg 240
ttaacgcagt gtttctcttt ttttcaggcg ctaaaacata ccagatgaaa gtctggagag 300
gtgaagaata cgaccaccta gcgcctgaaa caaccaaaca accaaacaac caaacaacca 360
aacaaccaaa caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca 420
acacaggttg gtgctagctg gccaaggctg gattattctg agtccaagct aggccctttt 480
gctaatcatg ttcatacctc ttatcttcct cccacagagc gaagaagaag gcatcggata 540
cagcgtgtga agaagttgct gtttgattat aatttgttga ctgatcattc tgagttatca 600
ggcattaatc cttatgaggc tcgtgttaag ggtttaagtc agaagttaag tgaagaagaa 660
ttttctgctg ctttgttgca tttggctaaa agaagaggag ttcataatgt taatgaagtt 720
gaagaggata ctggtaatga gttaagtact aaggagcaga taagtcgtaa ttctaaggct 780
ttggaagaaa agtatgttgc tgagttgcag ttggagcgtt tgaagaagga tggtgaagta 840
agaggaagta ttaatcgttt taagacaagt gattatgtga aagaagcgaa gcagttgttg 900
aaagttcaga aggcttatgt gagtctatgg gacccttgat gttttctgca tgggtagccg 960
ctgagatgga gcctgagcac acgcggccgc tgttaacgca gtgtttctct ttttttcagg 1020
cgctaaaaca taccagatga aagtctggag aggtgaagaa tacgaccacc tagcgcctga 1080
aacaaccaaa caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca 1140
accaaacaac caaacaacca aacaaccaaa caacacaggt tggtgctagc tggccaaggc 1200
tggattattc tgagtccaag ctaggccctt ttgctaatca tgttcatacc tcttatcttc 1260
ctcccacagc atcagttgga tcaaagtttt attgatactt atattgattt gttggagact 1320
cgtagaactt attatgaggg tcctggtgag gggtccccgt ttggttggaa ggatattaag 1380
gagtggtatg agatgttgat gggtcattgt acttattttc ctgaagaatt gcggtccgtg 1440
aagtatgctt ataatgctga tttgtacaac gccctgaacg acctgaacaa tctcgtgatc 1500
accagggacg agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg 1560
ttcaagcaga agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa 1620
gaggatatta agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag 1680
gtgtaccacg acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg 1740
ctggatcaga ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa 1800
ctgaccaatc tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag 1860
ggctataccg gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg 1920
tggcacacca acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag 1980
gtggacctgt cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc 2040
cccgtcgtga agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag 2100
tacggcctgc ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc 2160
cagaaaatga tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa 2220
atcatccgga ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac 2280
gacatgcagg aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg 2340
aacaacccct tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac 2400
agcttcaaca acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc 2460
ccattccagt acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac 2520
atcctgaatc tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg 2580
gaagaacggg acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg 2640
gataccagat acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac 2700
aacctggacg tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag 2760
tggaagttta agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc 2820
attgccaacg ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg 2880
atggaaaacc agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag 2940
caggagtaca aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag 3000
gactacaagt acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc 3060
ctgtactcca cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc 3120
ctgtacgaca aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg 3180
ctgatgtacc accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac 3240
ggcgacgaga agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag 3300
tactccaaaa aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg 3360
aacgcccatc tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg 3420
tccctgaagc cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc 3480
gtgaagaatc tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat 3540
gaggaagcta agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac 3600
aacaacgatc tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac 3660
ctgctgaacc ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac 3720
atgaacgaca agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt 3780
aagaagtaca gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct 3840
cagatcatca aaaagggc 3858
<210> 35
<211> 3858
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 35
aagcggaacu acauccuggg ccuggacauc ggcaucacca gcgugggcua cggcaucauc 60
gacuacgaga cucgugaugu uauugacgca ggcguucguu uguuuaaaga agcuaauguu 120
gagaauaaug agggaagaag aaguaagcgu ggggcucgca ggcuuaguga gucuauggga 180
cccuugaugu uuuuugcaug gguagccgcu gagauggagc cugagcacac gcggccgcug 240
uuaacgcagu guuucucuuu uuuucaggcg cuaaaacaua ccagaugaaa gucuggagag 300
gugaagaaua cgaccaccua gcgccugaaa caaccaaaca accaaacaac caaacaacca 360
aacaaccaaa caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca 420
acacagguug gugcuagcug gccaaggcug gauuauucug aguccaagcu aggcccuuuu 480
gcuaaucaug uucauaccuc uuaucuuccu cccacagagc gaagaagaag gcaucggaua 540
cagcguguga agaaguugcu guuugauuau aauuuguuga cugaucauuc ugaguuauca 600
ggcauuaauc cuuaugaggc ucguguuaag gguuuaaguc agaaguuaag ugaagaagaa 660
uuuucugcug cuuuguugca uuuggcuaaa agaagaggag uucauaaugu uaaugaaguu 720
gaagaggaua cugguaauga guuaaguacu aaggagcaga uaagucguaa uucuaaggcu 780
uuggaagaaa aguauguugc ugaguugcag uuggagcguu ugaagaagga uggugaagua 840
agaggaagua uuaaucguuu uaagacaagu gauuauguga aagaagcgaa gcaguuguug 900
aaaguucaga aggcuuaugu gagucuaugg gacccuugau guuuucugca uggguagccg 960
cugagaugga gccugagcac acgcggccgc uguuaacgca guguuucucu uuuuuucagg 1020
cgcuaaaaca uaccagauga aagucuggag aggugaagaa uacgaccacc uagcgccuga 1080
aacaaccaaa caaccaaaca accaaacaac caaacaacca aacaaccaaa caaccaaaca 1140
accaaacaac caaacaacca aacaaccaaa caacacaggu uggugcuagc uggccaaggc 1200
uggauuauuc ugaguccaag cuaggcccuu uugcuaauca uguucauacc ucuuaucuuc 1260
cucccacagc aucaguugga ucaaaguuuu auugauacuu auauugauuu guuggagacu 1320
cguagaacuu auuaugaggg uccuggugag ggguccccgu uugguuggaa ggauauuaag 1380
gagugguaug agauguugau gggucauugu acuuauuuuc cugaagaauu gcgguccgug 1440
aaguaugcuu auaaugcuga uuuguacaac gcccugaacg accugaacaa ucucgugauc 1500
accagggacg agaacgagaa gcuggaauau uacgagaagu uccagaucau cgagaacgug 1560
uucaagcaga agaagaagcc cacccugaag cagaucgcca aagaaauccu cgugaacgaa 1620
gaggauauua agggcuacag agugaccagc accggcaagc ccgaguucac caaccugaag 1680
guguaccacg acaucaagga cauuaccgcc cggaaagaga uuauugagaa cgccgagcug 1740
cuggaucaga uugccaagau ccugaccauc uaccagagca gcgaggacau ccaggaagaa 1800
cugaccaauc ugaacuccga gcugacccag gaagagaucg agcagaucuc uaaucugaag 1860
ggcuauaccg gcacccacaa ccugagccug aaggccauca accugauccu ggacgagcug 1920
uggcacacca acgacaacca gaucgcuauc uucaaccggc ugaagcuggu gcccaagaag 1980
guggaccugu cccagcagaa agagaucccc accacccugg uggacgacuu cauccugagc 2040
cccgucguga agagaagcuu cauccagagc aucaaaguga ucaacgccau caucaagaag 2100
uacggccugc ccaacgacau cauuaucgag cuggcccgcg agaagaacuc caaggacgcc 2160
cagaaaauga ucaacgagau gcagaagcgg aaccggcaga ccaacgagcg gaucgaggaa 2220
aucauccgga ccaccggcaa agagaacgcc aaguaccuga ucgagaagau caagcugcac 2280
gacaugcagg aaggcaagug ccuguacagc cuggaagcca ucccucugga agaucugcug 2340
aacaaccccu ucaacuauga gguggaccac aucaucccca gaagcguguc cuucgacaac 2400
agcuucaaca acaaggugcu cgugaagcag gaagaaaaca gcaagaaggg caaccggacc 2460
ccauuccagu accugagcag cagcgacagc aagaucagcu acgaaaccuu caagaagcac 2520
auccugaauc uggccaaggg caagggcaga aucagcaaga ccaagaaaga guaucugcug 2580
gaagaacggg acaucaacag guucuccgug cagaaagacu ucaucaaccg gaaccuggug 2640
gauaccagau acgccaccag aggccugaug aaccugcugc ggagcuacuu cagagugaac 2700
aaccuggacg ugaaagugaa guccaucaau ggcggcuuca ccagcuuucu gcggcggaag 2760
uggaaguuua agaaagagcg gaacaagggg uacaagcacc acgccgagga cgcccugauc 2820
auugccaacg ccgauuucau cuucaaagag uggaagaaac uggacaaggc caaaaaagug 2880
auggaaaacc agauguucga ggaaaagcag gccgagagca ugcccgagau cgaaaccgag 2940
caggaguaca aagagaucuu caucaccccc caccagauca agcacauuaa ggacuucaag 3000
gacuacaagu acagccaccg gguggacaag aagccuaaua gagagcugau uaacgacacc 3060
cuguacucca cccggaagga cgacaagggc aacacccuga ucgugaacaa ucugaacggc 3120
cuguacgaca aggacaauga caagcugaaa aagcugauca acaagagccc cgaaaagcug 3180
cugauguacc accacgaccc ccagaccuac cagaaacuga agcugauuau ggaacaguac 3240
ggcgacgaga agaauccccu guacaaguac uacgaggaaa ccgggaacua ccugaccaag 3300
uacuccaaaa aggacaacgg ccccgugauc aagaagauua aguauuacgg caacaaacug 3360
aacgcccauc uggacaucac cgacgacuac cccaacagca gaaacaaggu cgugaagcug 3420
ucccugaagc ccuacagauu cgacguguac cuggacaaug gcguguacaa guucgugacc 3480
gugaagaauc uggaugugau caaaaaagaa aacuacuacg aagugaauag caagugcuau 3540
gaggaagcua agaagcugaa gaagaucagc aaccaggccg aguuuaucgc cuccuucuac 3600
aacaacgauc ugaucaagau caacggcgag cuguauagag ugaucggcgu gaacaacgac 3660
cugcugaacc ggaucgaagu gaacaugauc gacaucaccu accgcgagua ccuggaaaac 3720
augaacgaca agaggccccc caggaucauu aagacaaucg ccuccaagac ccagagcauu 3780
aagaaguaca gcacagacau ucugggcaac cuguaugaag ugaaaucuaa gaagcacccu 3840
cagaucauca aaaagggc 3858
<210> 36
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 36
ccaaagaaga agcggaaggt c 21
<210> 37
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 37
ccaaagaaga agcggaaggu c 21
<210> 38
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 38
aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atcc 54
<210> 39
<211> 54
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 39
aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg aucc 54
<210> 40
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成的
<400> 40
tacccatacg atgttccaga ttacgct 27
<210> 41
<211> 27
<212> RNA
<213> 人工序列
<220>
<223> 合成的
<400> 41
uacccauacg auguuccaga uuacgcu 27
Claims (26)
1.一种可调控基因表达的构建体,其包含编码RNA的核酸,所述RNA包含
(1)编码基因组编辑酶的序列;和
(2)可操作地连接至所述序列的调控表达盒,所述调控表达盒包含
(i)侧翼为上游内含子和下游内含子的条件性外显子,和
(ii)可操作地连接至所述条件性外显子的适体结构域,其中所述适体结构域能够与效应分子结合,以触发所述RNA的结构改变,从而调控所述条件性外显子的剪接和所述基因组编辑酶的表达。
2.根据权利要求1所述的构建体,其中所述基因组编辑酶在存在所述效应分子的条件下表达。
3.根据权利要求1所述的构建体,其中在存在所述效应分子条件下的剪接过程中,跳过所述条件性外显子。
4.根据前述任一项权利要求所述的构建体,其中所述效应分子是四环素。
5.根据前述任一项权利要求所述的构建体,其中优化所述序列以包含外显子剪接增强子。
6.根据前述任一项权利要求所述的构建体,其中所述基因组编辑酶是位点特异性核酸酶或位点特异性重组酶。
7.根据权利要求6所述的构建体,其中所述位点特异性核酸酶选自下组:Cas9、Cas12、ZFN、TALEN和大范围核酸酶。
8.根据权利要求6所述的构建体,其中所述位点特异性重组酶选自下组:Cre、FLP、λ整合酶、phiC31整合酶、Bxb1整合酶、γ-δ解离酶、Tn3解离酶和Gin转化酶。
9.根据前述任一项权利要求所述的构建体,其中所述基因组编辑酶具有与SEQ ID NO:1至少90%同一性的序列。
10.根据前述任一项权利要求所述的构建体,其中所述序列与SEQ ID NO:5、7或9具有至少90%同一性。
11.根据前述任一项权利要求所述的构建体,其中所述序列包含外显子剪接增强子(ESE)优化的区域,所述区域与SEQ ID NO:11、13或15具有至少90%同一性。
12.根据前述任一项权利要求所述的构建体,其中所述适体结构域具有与SEQ ID NO:17、19或21至少90%同一性的序列。
13.根据前述任一项权利要求所述的构建体,其中所述条件性外显子具有与SEQ IDNO:23至少90%同一性的序列。
14.根据前述任一项权利要求所述的构建体,其中所述上游内含子具有与SEQ ID NO:25至少90%同一性的序列。
15.根据前述任一项权利要求所述的构建体,其中所述下游内含子具有与SEQ ID NO:27至少90%同一性的序列。
16.根据前述任一项权利要求所述的构建体,其中所述调控表达盒包含与SEQ ID NO:29至少90%同一性的序列。
17.根据前述任一项权利要求所述的构建体,其中将所述调控表达盒插在以下之间(1)SEQ ID NO:11的核苷酸位置97和98;或者
(2)SEQ ID NO:11的核苷酸位置498和499。
18.根据前述任一项权利要求所述的构建体,其包含SEQ ID NO:30、32或34。
19.根据前述任一项权利要求所述的构建体,其被包含在载体中。
20.根据权利要求19所述的构建体,其中所述载体是AAV载体。
21.根据权利要求1所述的构建体,其中所述基因编辑酶是Cas9,且其中所述构建体包含编码gRNA的第二多核苷酸序列。
22.一种在细胞中进行基因组编辑的方法,所述方法包括将权利要求1-21中任一项所述的构建体递送至所述细胞中。
23.根据权利要求22所述的方法,其还包括将所述效应分子递送至所述细胞。
24.一种修饰的细胞,其通过将权利要求1-21中任一项所述的构建体递送至所述细胞中制备。
25.一种治疗患有疾病的对象的方法,所述方法包括将根据权利要求1-21中任一项所述的构建体递送至所述对象的至少一个细胞中。
26.根据权利要求25所述的方法,其还包括向所述对象施用所述效应细胞。
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