CN114828875A - 新颖的酶 - Google Patents

新颖的酶 Download PDF

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CN114828875A
CN114828875A CN202080086562.6A CN202080086562A CN114828875A CN 114828875 A CN114828875 A CN 114828875A CN 202080086562 A CN202080086562 A CN 202080086562A CN 114828875 A CN114828875 A CN 114828875A
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sequence
nucleic acid
editing
dna
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金主汉
本杰明·米杰茨
阿米尔·米尔
安德鲁·加斯特
凯尔·西蒙
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Inscripta Inc
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Abstract

本公开内容提供了用于对活细胞基因组进行合理和直接编辑的新颖的RNA引导的酶。本公开内容提供了具有在来自不同生物体的细胞中的不同活性的新颖的MAD系列核酸酶。因此，提供了具有密码子优化的核酸序列的新颖的MAD系列核酸酶，所述密码子优化的核酸序列包含与SEQ ID NO:3‑7、11、13、15‑22和24中任一项的至少65％同源性。

Description

新颖的酶
相关申请的交叉引用
本申请是2019年12月13日提交的美国专利申请号16/714,320的国际PCT申请，并通过引用并入本文。
发明领域
本发明涉及用于对活细胞基因组进行合理和直接编辑的新颖的酶。
发明背景
在以下讨论中，将出于背景和介绍的目的描述某些文章和方法。本文包含的任何内容不被解释为对现有技术的“承认”。本申请人明确地保留根据情况证明根据可适用的法定条文由本文引用的方法不构成现有技术的权利。
对活细胞的基因组进行精确的、靶向的改变的能力一直是生物医学研究和开发的长期目标。最近，已经鉴定出允许操纵基因序列从而操纵基因功能的多种核酸酶。这些核酸酶包括核酸引导的核酸酶。然而，核酸引导的核酸酶可以识别的靶序列的范围受需要特定PAM定位于期望的靶序列附近的限制。PAM是由gRNA/核酸酶复合体识别的短核苷酸序列，在该处gRNA/核酸酶复合体指导靶序列的编辑。用于不同的核酸引导的核酸酶的精确的PAM序列和PAM长度要求不同；然而，PAM通常是与靶序列邻近或接近的2-7个碱基对序列，并且取决于核酸酶，可以在靶序列的5’或3’。跨物种筛选存在的核酸引导的核酸酶的天然多样性可以允许发现在特定生物体中使用时具有增强的核酸酶活性或增加的裂解保真度的酶；这两种变化都可以增加核酸引导的核酸酶对某些编辑任务的多功能性。
因此，在核酸引导的核酸酶基因编辑领域中存在对具有在来自不同生物体的细胞中的不同活性和/或改变的酶保真度的改进的核酸酶的需求。本文描述的新颖的MAD系列核酸酶满足了这一需求。
发明概述
提供该概述是为了以简化的形式介绍概念的选择，所述概念在下文的详述中进一步描述。该概述既不意图确定所要求保护的主题的关键或基本特征，也不意图用于限制所要求保护的主题的范围。所要求保护的主题的其他特征、细节、效用和优势将是从下面撰写的详述，包括附图中阐明的和所附的权利要求中限定的那些方面明显的。
本公开内容提供了具有在来自不同生物体的细胞中的不同活性的新颖的MAD系列核酸酶。
因此，提供了新颖的MAD系列核酸酶，其具有密码子优化的核酸序列，该密码子优化的核酸序列包含与SEQ ID No.3-7、11、13、15-22和24中任一项的至少65％同源性。在一些方面，具有密码子优化的核酸序列的新颖的MAD系列核酸酶包含与SEQ ID No.3-7、11、13、15-22和24中任一项的至少66％、67％、68％、69％、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％、99％或100％同源性。
在一些方面，新颖的MAD系列核酸酶在包含gRNA的系统中，该gRNA具有包含UGUU、UCUU或UAUU的最佳crRNA可变环。
还提供了用于在细菌中编辑的新颖的MAD系列核酸酶，其包含与SEQ ID No.4、11、15、16、17、19、21、22或24中任一项的至少80％同源性；和用于在酵母中编辑的新颖的MAD系列核酸酶，其包含与SEQ ID No.3-6、13、15-22或24中任一项的至少80％同源性。
下面更详细地描述本发明的这些方面和其他特征和优势。
附图简述
图1描绘了crRNA序列的最小结构，勾画了支架(可变环序列)、核酸酶靶向引导序列的位置和延伸的手柄结构。
图2是用于鉴定、产生和筛选新颖的MAD系列酶的靶向核酸酶活性的示例性工作流程。
图3显示了具有所示可变环序列的所示的MAD系列核酸酶在大肠杆菌(E.coli)中的编辑结果，如通过比色筛选galK基因座中的精确编辑评估的。
图4显示了使用具有所示可变环序列的所示的MAD系列核酸酶在酿酒酵母(S.cerevisiae)中的编辑结果，如通过Can1基因座中的精确编辑诱导的在含刀豆氨酸培养基中的生长评估的。
图5显示了使用另外的可变环支架，由MAD7和MAD41在酿酒酵母中编辑的结果。
图6显示了由具有所示可变环的所示的核酸酶诱导的HEK293T人类细胞的位点特异性核酸酶裂解诱导的插入缺失的比率。
详细描述
下文结合所附的附图阐述的描述意图描述所公开主题的多种说明性实施方案。结合每种说明性实施方案描述了特定特征和功能；然而，对本领域技术人员将明显的是，所公开的实施方案可以在没有这些特定特征和功能的每一种的情况下实践。此外，除非明确声明或者特征或功能与另外的实施方案不兼容，否则结合一种实施方案描述的所有功能意图适用于本文描述的另外的实施方案。例如，除非特征或功能与替代实施方案不兼容，否则在结合一种实施方案明确描述给定特征或功能但没有结合替代实施方案明确提及的情况下，应当理解，该特征或功能可以结合替代实施方案来部署、利用或实现。
除非另外指出，否则本文描述的技术的实践可以采用有机化学、聚合物技术、分子生物学(包括重组技术)、细胞生物学、生物化学、生物乳液产生和测序技术的常规技术和描述，这些都在本领域从业的人员的技术内。这样的常规的技术包括聚合物阵列合成、多核苷酸的杂交和连接以及使用标记物的杂交检测。合适的技术的具体的说明可以通过参考本文的实例获得。然而，当然，也可以使用其他等同的常规程序。这样的常规技术和描述可以见于标准实验室手册，诸如Green等人编著(1999),Genome Analysis:A Laboratory ManualSeries(卷I-IV)；Weiner,Gabriel,Stephens,编著(2007),Genetic Variation:ALaboratory Manual；Dieffenbach,Dveksler,编著(2003),PCR Primer:A LaboratoryManual；Bowtell和Sambrook(2003),DNA Microarrays:A Molecular Cloning Manual；Mount(2004),Bioinformatics:Sequence and Genome Analysis；Sambrook和Russell(2006),Condensed Protocols from Molecular Cloning:A Laboratory Manual；以及Sambrook和Russell(2002),Molecular Cloning:A Laboratory Manual(全部来自ColdSpring Harbor Laboratory Press)；Stryer,L.(1995)Biochemistry(第4版)W.H.Freeman,New York N.Y.；Gait,“Oligonucleotide Synthesis:A PracticalApproach”1984,IRL Press,London；Nelson和Cox(2000),Lehninger,Principles ofBiochemistry第3版,W.H.Freeman Pub.,New York,N.Y.；Berg等人(2002)Biochemistry,第5版,W.H.Freeman Pub.,New York,N.Y.；Cell and Tissue Culture:LaboratoryProcedures in Biotechnology(Doyle&Griffiths,编著,John Wiley&Sons 1998)；Mammalian Chromosome Engineering–Methods and Protocols(G.Hadlaczky,编著,Humana Press2011)；Essential Stem Cell Methods,(Lanza和Klimanskaya,编著,Academic Press 2011)，所有文献出于所有目的通过引用以其整体并入本文。核酸酶特异性技术可以见于，例如，Genome Editing and Engineering From TALENs and CRISPRs toMolecular Surgery,Appasani和Church,2018；以及CRISPR:Methods and Protocols,Lindgren和Charpentier,2015；这两篇文献出于所有目的通过引用以其整体并入本文。酶工程化的基本方法可以见于，Enzyme Engineering Methods and Protocols,Samuelson,编著,2013；Protein Engineering,Kaumaya,编著,(2012)；以及Kaur和Sharma,“DirectedEvolution:An Approach to Engineer Enzymes”,Crit.Rev.Biotechnology,26:165-69(2006)。
注意，除非上下文另外清楚指示，如本文和所附的权利要求书中使用的，单数形式“一(a)”、“一(an)”和“该(the)”包括复数指代物。因此，例如，提及“寡核苷酸”是指一种或更多种寡核苷酸，并且提及“自动化系统”包括提及本领域技术人员已知的用于该系统的等同步骤和方法，等等。此外，应当理解，本文可以使用的术语诸如“左”、“右”、“顶”、“底”、“前”、“后”、“侧”、“高度”、“长度”、“宽度”、“上”、“下”、“内部(interior)”、“外部(exterior)”、“内(inner)”、“外(outer)”仅描述参考点，并且不必然将本公开内容的实施方案限制为任何特定的方向或配置。此外，术语诸如“第一”、“第二”、“第三”等，仅标识如本文公开的许多部分、组件、步骤、操作、功能和/或参考点中的一个，并且同样不必然将本公开内容的实施方案限制为任何特定的配置或方向。
除非另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域内的普通技术人员通常理解的相同含义。本文提及的所有的出版物为了描述和公开可以与本文描述的发明结合使用的设备、方法和细胞群体的目的，以引用方式并入。
在提供值的范围情况下，应理解，在该范围的上限值和下限值之间的每一个中间值和该规定的范围内的任何其他规定的值或中间值被涵盖在本发明内。这些较小的范围的上限值和下限值可以独立地被包括在较小的范围内，并且也被涵盖在本发明内，受限于规定的范围内的任何特定地排除的限值。在规定的范围包括限值中的一个或两个的情况下，将那些所包括的限值中的任一个或两个排除的范围也被包括在本发明中。
在以下的描述中，阐述了许多具体细节，以提供对本发明的更充分理解。然而，对本领域普通技术人员将明显的是，可以在没有一个或更多个这些具体细节的情况下，实践本发明。在其他情况下，为了避免使本发明含混不清，尚未描述本领域技术人员熟知的特征和熟知的程序。
如本文使用的术语“互补”是指核苷酸之间的Watson-Crick碱基配对，并且特别地是指彼此形成氢键的核苷酸，其中胸腺嘧啶或尿嘧啶残基通过两个氢键与腺嘌呤残基连接，并且胞嘧啶和鸟嘌呤残基通过三个氢键连接。通常，核酸包含被描述为与指定的第二核苷酸序列具有“互补性百分比”或“同源性百分比”的核苷酸序列。例如，核苷酸序列可以与指定的第二核苷酸序列具有80％、90％或100％的互补性，指示序列的10个核苷酸中的8个、10个核苷酸中的9个或10个核苷酸中的10个与指定的第二核苷酸序列互补。例如，核苷酸序列3’-TCGA-5’与核苷酸序列5’-AGCT-3’是100％互补的；并且核苷酸序列3’-TCGA-5’与核苷酸序列5’-TAGCTG-3’的区域是100％互补的。
术语DNA“控制序列”统指启动子序列、多腺苷酸化信号、转录终止序列、上游调控结构域、复制起点、内部核糖体进入位点、核定位序列、增强子等，它们共同地提供编码序列在接受者细胞中的复制、转录和翻译。只要选择的编码序列能够在适当的宿主细胞中被复制、转录和(对于一些组分)翻译，则并非所有这些类型的控制序列都需要存在。
如本文使用的，术语“供体DNA”或“供体核酸”是指被设计成通过使用核酸引导的核酸酶的同源重组将DNA序列修饰(插入、缺失、取代)引入基因座的核酸。对于同源指导的修复，供体DNA必须与基因组靶序列中的“切割位点”或待编辑位点侧翼的区具有足够的同源性。一条或更多条同源臂的长度将取决于，例如，所做修饰的类型和大小。在许多情况下，并且优选地，供体DNA将与基因组靶基因座具有两个序列同源性区(例如，两个同源臂)。优选地，“插入物(insert)”区或“DNA序列修饰”区(期望引入细胞中的基因组靶基因座的核酸修饰)将位于两个同源区之间。DNA序列修饰可以改变一个特定位点或多于一个特定位点处的靶基因组DNA序列的一个或更多个碱基。改变可以包括改变靶序列的1个、2个、3个、4个、5个、10个、15个、20个、25个、30个、35个、40个、50个、75个、100个、150个、200个、300个、400个或500个或更多个碱基对。缺失或插入可以是靶序列的1个、2个、3个、4个、5个、10个、15个、20个、25个、30个、40个、50个、75个、100个、150个、200个、300个、400个或500个或更多个碱基对的缺失或插入。
术语“引导核酸”或“引导RNA”或“gRNA”或“crRNA”是指包含以下的多核苷酸：1)能够与基因组靶基因座杂交的引导序列和2)能够与核酸引导的核酸酶相互作用或复合的支架序列(参见例如，图1)。
“同源性”或“同一性”或“相似性”是指两个肽之间的序列相似性，或者在本公开内容的上下文中，更常见地是指两个核酸分子之间的序列相似性。术语“同源区”或“同源臂”是指供体DNA上与靶基因组DNA序列具有一定程度同源性的区域。同源性可以通过比较每个序列中的位置来确定，所述每个序列可以出于比较的目的而被对齐。当比较的序列中的一个位置被相同的碱基或氨基酸占据时，那么分子在该位置处是同源的。序列之间的同源性程度随着序列共有的匹配或同源位置数而变化。
“可操作地连接”指其中如此描述的组分被配置以执行它们的通常功能的元件布置。因此，可操作地连接至编码序列的控制序列能够影响编码序列的转录，并且在一些情况下，能够影响编码序列的翻译。只要控制序列起作用以指导编码序列的表达，控制序列不必与编码序列邻接。因此，例如，不翻译但转录的间插序列可以存在于启动子序列和编码序列之间，且启动子序列仍可以被认为是“可操作地连接”至编码序列。事实上，这样的序列不必驻留于同一连续DNA分子(即染色体)上，并且仍可以具有引起调控改变的相互作用。
“启动子”或“启动子序列”是能够与RNA聚合酶结合并启动多核苷酸或多肽编码序列(诸如信使RNA、核糖体RNA、小核RNA(small nuclear RNA)或核仁小RNA(smallnucleolar RNA)、引导RNA或由任何类别的任何RNA聚合酶I、II或III转录的任何种类的RNA)的转录的DNA调控区。启动子可以是组成型或诱导型，并且在一些实施方案中，特别地在许多采用选择的实施方案中，核酸引导的核酸酶编辑系统的至少一种组分的转录处于诱导型启动子的控制下。
如本文使用的术语“选择标记(selectable marker)”是指引入细胞中的、赋予适于人工选择的性状的基因。一般使用的选择标记是本领域普通技术人员熟知的。可以采用药物选择标记，诸如氨苄青霉素/羧苄青霉素、卡那霉素、氯霉素、红霉素、四环素、庆大霉素、博莱霉素、链霉素、鼠李糖、嘌呤霉素、潮霉素、杀稻瘟素和G418。在其他实施方案中，选择标记包括但不限于人类神经生长因子受体(用MAb检测，诸如美国专利第6,365,373号中描述的)；截短的人类生长因子受体(用MAb检测)；突变体人类二氢叶酸还原酶(DHFR；可用荧光MTX底物)；分泌型碱性磷酸酶(SEAP；可用荧光底物)；人类胸苷酸合酶(TS；赋予对抗癌剂氟脱氧尿苷的抗性)；人类谷胱甘肽S-转移酶α(GSTA1；将谷胱甘肽与干细胞选择性烷化剂白消安缀合；CD34+细胞中的化学保护性选择标记)；造血干细胞中的CD24细胞表面抗原；赋予对N-膦酰乙酰基-L-天冬氨酸(PALA)的抗性的人类CAD基因；人类多耐药性-1(MDR-1；可通过增加的耐药性选择或通过FACS富集P-糖蛋白表面蛋白)；人类CD25(IL-2α；可通过Mab-FITC检测)；甲基鸟嘌呤-DNA甲基转移酶(MGMT；可通过卡莫司汀(carmustine)选择)；和胞苷脱氨酶(CD；可通过Ara-C选择)。如本文使用的“选择性培养基”是指向其中添加了选择选择标记或针对选择标记进行选择的化学化合物或生物部分的细胞生长培养基。
术语“靶基因组DNA序列”、“靶序列”或“基因组靶基因座”是指体外或体内，或者细胞或细胞群体的核酸(例如基因组)中的期望使用核酸引导的核酸酶编辑系统对至少一个核苷酸进行改变的任何基因座。靶序列可以是基因组基因座或染色体外基因座。
“载体”是包含待递送至细胞和/或在细胞中表达的期望的一种序列或更多种序列的多种核酸中的任一种。载体通常由DNA构成，但是RNA载体也是可用的。载体包括但不限于质粒、F粘粒(fosmid)、噬菌粒、病毒基因组、合成染色体等。如本文使用的，短语“引擎载体”包含用于本公开内容的核酸引导的核酸酶系统和方法中的核酸酶的编码序列。在细菌系统中，引擎载体还可以包含λRed重组工程系统或其等同物。引擎载体通常还包含选择标记。如本文使用的，短语“编辑载体”包含供体核酸和gRNA编码序列，所述供体核酸任选地包括对靶序列的改变，所述改变在编辑发生后阻止核酸酶在靶序列中的PAM或间隔区(spacer)处结合。编辑载体还可以包括选择标记和/或条形码。在一些实施方案中，可以将引擎载体和编辑载体组合；即，引擎载体的内容物可以在编辑载体上找到。此外，引擎载体和编辑载体包含可操作地连接至例如核酸酶编码序列、重组工程系统编码序列(如果存在)、供体核酸、引导核酸和一种或更多种选择标记的控制序列。
核酸引导的核酸酶在基因组系统中的编辑
核酸引导的核酸酶已被用于将各种生物体的基因组工程化；然而，不同生物体内的固有DNA切割活性、蛋白质表达水平、细胞毒性和活性的差异仍然是巨大的挑战，这要求在每个生物体内筛选许多进行编辑的候选酶。因此，在体外和/或体内在细菌、真菌或哺乳动物中证明具有活性的核酸引导的核酸酶是非常有用的。本公开内容提供了具有在不同类型细胞中的不同PAM偏好、改变的RNA引导的酶保真度和/或改变的细胞毒性或活性的新颖的基因编辑MAD系列核酸酶。也就是说，新颖的MAD系列核酸酶可以用于编辑不同的细胞类型，包括古细菌(archaeal)、原核细胞和真核(例如，酵母、真菌、植物和动物)细胞。
本文描述的新颖的MAD系列核酸酶改进了RNA引导的酶编辑系统，其中使用核酸引导的核酸酶(例如，RNA引导的核酸酶)来编辑生物体的基因组中的特定靶区域。与适当的合成的引导核酸在细胞中复合的核酸引导的核酸酶可以在期望的位置处切割细胞的基因组。引导核酸有助于核酸引导的核酸酶识别和切割特定靶序列处的DNA。通过操纵引导核酸的核苷酸序列，核酸引导的核酸酶可以被编程为靶向任何DNA序列用于裂解，只要适当的前间区邻近基序(protospacer adjacent motif,PAM)在附近。
可以将新颖的MAD系列核酸酶作为多肽递送到待编辑的细胞；可选地，将编码新颖的MAD系列核酸酶的多核苷酸序列转化或转染到待编辑的细胞中。编码新颖的MAD系列核酸酶的多核苷酸序列可以针对特定细胞诸如古细菌、原核细胞或真核细胞中的表达被密码子优化。真核细胞可以是酵母、真菌、藻类、植物、动物或人类的细胞。真核细胞可以是特定生物体的细胞或来源于特定生物体的细胞，所述特定生物体诸如哺乳动物，包括但不限于人类、小鼠、大鼠、兔、犬或非人类哺乳动物，包括非人类灵长类动物。待采用的新颖的MAD系列核酸酶的选择取决于许多因素，诸如在靶序列中待进行何种类型的编辑，以及适当的PAM是否位于期望的靶序列附近。新颖的MAD系列核酸酶可以由载体(例如，引擎载体)上的DNA序列编码，并且处于组成型或诱导型启动子的控制下。在一些实施方案中，编码核酸酶的序列处于诱导型启动子的控制下，并且该诱导型启动子可以与控制引导核酸转录的诱导型启动子分开但相同；即，分开的诱导型启动子可驱动核酸酶和引导核酸序列的转录，但是这两个诱导型启动子可以是相同类型的诱导型启动子(例如，二者都是pL启动子)。可选地，控制核酸酶表达的诱导型启动子可以与控制引导核酸转录的诱导型启动子不同；即，例如，核酸酶可以处于pBAD诱导型启动子的控制下，并且引导核酸可以处于pL诱导型启动子的控制下。
通常，引导核酸(例如，gRNA)，也称为CRISPR RNA(例如，crRNA)，可以与相容的核酸引导的核酸酶复合，并且然后可以与靶序列杂交，从而将核酸酶指导至靶序列。gRNA可以由多核苷酸分子诸如质粒、线性构建体上的DNA序列编码，或者编码序列可以驻留于编辑盒内，并且处于组成型启动子的控制下，或者在一些实施方案中，在如下文描述的诱导型启动子的控制下。图1描绘了crRNA序列的最小结构，勾画了支架(可变环序列)以及核酸酶靶向引导序列的位置、假结(pseudoknot)和延伸的手柄结构。
引导核酸包含引导序列，其中引导序列是具有与靶序列足够互补性以与靶序列杂交并且指导复合的核酸引导的核酸酶与靶序列的序列特异性结合的多核苷酸序列。引导序列和对应的靶序列之间的互补性程度在使用合适的比对算法进行最佳比对时是约或多于约50％、60％、75％、80％、85％、90％、95％、97.5％、99％或更多。最佳比对可以通过使用用于比对序列的任何合适的算法来确定。在一些实施方案中，引导序列的长度是约或多于约10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、35个、40个、45个、50个、75个或更多个核苷酸。在一些实施方案中，引导序列的长度为少于约75个、50个、45个、40个、35个、30个、25个、20个核苷酸。优选地，引导序列是10-30个或15-20个核苷酸长，或者长度是15个、16个、17个、18个、19个或20个核苷酸。
在本发明的方法和组合物中，引导核酸作为待由质粒或载体表达的序列提供，并且包含引导序列和支架序列二者作为启动子控制下的，并且在一些实施方案中，诱导型启动子控制下的单一转录物。通过改变引导序列，使得引导序列与期望的靶序列互补，从而允许引导序列和靶序列之间的杂交，可以将引导核酸工程化成靶向期望的靶序列。通常，为了在靶序列中产生编辑，gRNA/核酸酶复合体与如由引导RNA确定的靶序列结合，并且核酸酶识别与靶序列邻近的前间区邻近基序(PAM)序列。靶序列可以是对原核细胞或真核细胞而言为内源或外源的任何多核苷酸，或体外的任何多核苷酸。例如，靶序列可以是驻留于真核细胞的细胞核中的多核苷酸。靶序列可以是编码基因产物(例如，蛋白)的序列或非编码序列(例如，调控多核苷酸、内含子、PAM或“垃圾”DNA(“junk”DNA))。
引导核酸可以是编码供体核酸的编辑盒的一部分。可选地，引导核酸可以不是编辑盒的一部分，而是可以被编码在引擎或编辑载体骨架上。例如，可以首先将编码引导核酸的序列组装或插入载体骨架中，随后将供体核酸插入例如编辑盒中。在其他情况下，可以首先将例如编辑盒中的供体核酸插入或组装到载体骨架中，随后插入编码引导核酸的序列。在又其他情况下，编码引导核酸和供体核酸(例如，插入编辑盒中)的序列同时但分开插入或组装到载体中。在又其他实施方案中，编码引导核酸的序列和编码供体核酸的序列二者均包含在编辑盒中。
靶序列与PAM相关，PAM是由gRNA/核酸酶复合体识别的短核苷酸序列。用于不同的核酸引导的核酸酶的精确的PAM序列和长度要求不同；然而，PAM通常是与靶序列邻近或接近的2-7个碱基对序列，并且取决于核酸酶，可以在靶序列的5’或3’。核酸引导的核酸酶的PAM相互作用结构域的工程化可以允许改变PAM特异性、改进保真度或降低保真度。在某些实施方案中，靶序列的基因组编辑既将期望的DNA改变引入靶序列，例如细胞的基因组DNA，又去除靶序列中的前间区突变(PAM)区，使靶序列中的前间区突变(PAM)区突变或失活。使靶序列处的PAM失活排除了对该靶序列处细胞基因组的另外的编辑，例如，在后续的编辑轮中随后暴露于与合成的引导核酸复合的核酸引导的核酸酶时。因此，具有期望的靶序列编辑和改变的PAM的细胞可以使用与合成的引导核酸复合的核酸引导的核酸酶来选择，所述合成的引导核酸与靶序列互补。没有经历第一编辑事件的细胞会被切割，引起双链DNA断裂，并且因此会无法继续存活。包含期望的靶序列编辑和PAM改变的细胞不会被切割，因为这些编辑的细胞不再包含必需的PAM位点，并且会继续生长和繁殖。
核酸引导的核酸酶系统的另一组分是供体核酸。在一些实施方案中，供体核酸与引导核酸在同一多核苷酸(例如，编辑载体或编辑盒)上，并且可以(但不必然)处于与引导核酸相同的启动子的控制下(例如，驱动引导核酸和供体核酸二者转录的单一启动子)。供体核酸被设计成用作与靶序列同源重组的模板，该靶序列被作为gRNA/核酸酶复合体的一部分的核酸引导的核酸酶切口或裂解。供体核酸多核苷酸可以具有任何合适的长度，诸如约或多于约20个、25个、50个、75个、100个、150个、200个、500个或1000个核苷酸的长度。在某些优选的方面，供体核酸可以以20-300个核苷酸之间，更优选地50-250个核苷酸之间的寡核苷酸提供。供体核酸包含与靶序列的一部分互补的区域(例如同源臂)。当最佳比对时，供体核酸与靶序列重叠(互补)例如约20个、25个、30个、35个、40个、50个、60个、70个、80个、90个或更多个核苷酸。在许多实施方案中，供体核酸包含位于供体核酸和靶模板之间的突变或差异的侧翼的两个同源臂(与靶序列互补的区域)。供体核酸包含与靶序列相比的至少一个突变或改变，诸如与靶序列相比的插入、缺失、修饰或其任何组合。
通常，供体核酸以编辑盒提供，其被插入到载体骨架中，其中载体骨架可以包含驱动gRNA转录的启动子和gRNA编码序列，或者载体骨架可以包含驱动gRNA转录的启动子，但不包含gRNA本身。此外，可以存在多于一个，例如两个、三个、四个或更多个引导核酸/供体核酸盒插入到引擎载体中，其中每个引导核酸处于分开的不同启动子、分开的相似启动子的控制下，或者其中所有引导核酸/供体核酸对处于单个启动子的控制下。在一些实施方案中——诸如采用细胞选择的实施方案中——驱动gRNA和供体核酸(或驱动多于一个gRNA/供体核酸对)转录的启动子是诱导型启动子。诱导型编辑的优点在于，在启动编辑前，单个化的细胞可以生长几倍至许多倍的细胞倍增，这增加具有编辑的细胞将存活的可能性，因为由有效编辑(active editing)引起的双链切割对细胞有很大毒性。这种毒性既导致编辑的集落中的细胞死亡，也导致确实存活但必须在编辑后修复和恢复(recover)的编辑的细胞生长延滞。然而，在编辑的细胞具有恢复的机会后，编辑的细胞集落的尺寸最终会赶上未编辑的细胞集落的尺寸。参见例如，2019年4月30日提交的USSN 16/399,988；2019年6月26日提交的USSN16/454,865；和2019年8月14日提交的USSN 16/540,606。此外，引导核酸可以有效指导编辑盒中多于一个供体核酸的编辑；例如，如果期望的编辑在靶序列中彼此接近。
除了供体核酸之外，编辑盒可以包含一个或更多个引物位点。引物位点可以用于通过使用寡核苷酸引物扩增编辑盒；例如，如果引物位点位于编辑盒的一个或更多个其他组分的侧翼。
此外，编辑盒可以包含条形码。条形码是对应于供体DNA序列的独特DNA序列，使得条形码可以鉴定对对应靶序列进行的编辑。条形码通常包含四个或更多个核苷酸。在一些实施方案中，编辑盒包含代表例如供体核酸的全基因或全基因组文库的供体核酸的集合。编辑盒的文库被克隆到载体骨架中，其中，例如，每个不同的供体核酸与不同的条形码缔合。
此外，在一些实施方案中，编码核酸引导的核酸酶系统的组分的表达载体或盒还编码包含一个或更多个细胞核定位序列(NLS)诸如约或多于约1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个NLS的新颖的MAD系列核酸酶。在一些实施方案中，新颖的核酸酶包含氨基末端处或其附近的NLS、羧基末端处或其附近的NLS，或组合。
引擎和编辑载体包含可操作地连接至待转录的组分序列的控制序列。如以上陈述的，驱动新颖的MAD系列核酸酶编辑系统的一种或更多种组分转录的启动子可以是诱导型的，并且如果要进行选择，可能采用诱导型系统。已经开发了用于在植物、微生物和动物细胞(包括哺乳动物细胞)中控制基因表达的许多基因调控控制系统，包括pL启动子(通过CI857阻遏物的热失活诱导)、pBAD启动子(通过将阿拉伯糖添加至细胞生长培养基中诱导)和鼠李糖诱导型启动子(通过将鼠李糖添加至细胞生长培养基中诱导)。其他系统包括四环素控制的转录激活系统(Tet-On/Tet-Off，Clontech,Inc.(Palo Alto,CA)；Bujard和Gossen,PNAS,89(12):5547-5551(1992))、Lac开关诱导型系统(Wyborski等人，EnvironMol Mutagen,28(4):447-58(1996)；DuCoeur等人，Strategies 5(3):70-72(1992)；美国专利第4,833,080号)、蜕皮素诱导型基因表达系统(No等人，PNAS,93(8):3346-3351(1996))、cumate基因开关系统(Mullick等人，BMC Biotechnology,6:43(2006))、以及他莫昔芬诱导型基因表达(Zhang等人，Nucleic Acids Research,24:543-548(1996))以及其他。
通常，在活细胞中进行基因组编辑要求用进行核酸引导的核酸酶编辑所必需的组分转化细胞。例如，细胞可以用分开的引擎载体和编辑载体同时转化；细胞可能已经表达新颖的MAD系列核酸酶(例如，细胞可能已经用引擎载体转化，或者新颖的MAD系列核酸酶的编码序列可以稳定地整合到细胞基因组中)，使得仅需要将编辑载体转化到细胞中；或者细胞可以用包含进行核酸引导的核酸酶基因组编辑所需的所有组分的单个载体转化。
可以使用各种递送系统将核酸引导的核酸酶编辑系统组分引入(例如，转化或转染)宿主细胞。这些递送系统包括酵母系统、脂质体转染系统、显微注射系统、基因枪系统、病毒微体(virosome)、脂质体、免疫脂质体、聚阳离子、脂质:核酸缀合物、病毒粒子(virion)、人工病毒粒子、病毒载体、电穿孔、细胞可渗透肽、纳米粒子、纳米线、外泌体(exosome)的使用。可选地，可以使用分子特洛伊木马(trojan horse)脂质体跨越血脑屏障递送核酸引导的核酸酶组分。特别感兴趣的是电穿孔的使用，特别地流通式电穿孔(作为独立的仪器或作为自动化多模块系统中的模块)的使用，如以下中描述的：USSN 16/024,831；62/566,375；62/566,688；和62/567,697。
在将细胞用进行核酸引导的核酸酶编辑所必需的组分转化后，使细胞在促进编辑的条件下培养。例如，使用如果组成型启动子驱动新颖的MAD系列核酸酶和/或gRNA的转录，转化的细胞仅需要在典型条件下(例如，温度、CO2气氛等)在典型培养基中培养。可选地，如果编辑是诱导型的——例如通过激活诱导型启动子，该诱导型启动子控制核酸引导的核酸酶编辑所需的一种或更多种组分的转录，诸如，例如gRNA、供体DNA、核酸酶的转录，或者，在细菌的情况下，编辑是重组工程系统的——则细胞经历诱导条件。
实施例
提出以下实施例，以便为本领域技术人员提供如何制备和使用本发明的完整公开和描述，且以下实施例并不意图限制发明人视作其发明的范围，它们也不意图代表或暗示下文的实验是进行的所有实验或仅有的实验。本领域的技术人员应该明白，可以在对具体方面中所示的发明作出许多变化和/或修饰而不脱离本发明所广泛描述的精神或范畴。因此，本文的方面在各个方面中被认为是说明性的而非限制性的。
实施例1：示例性工作流程概述
图2示出了用于创建和用于体外筛选新颖的MAD系列酶的示例性工作流程200。在第一步骤201，进行使用MAD7作为查询序列的计算序列同源性搜索，并选择一组推定的RNA引导的核酸酶。在步骤203，选择与MAD7具有不同水平同源性的序列进行具有大肠杆菌优化的密码子偏倚的DNA合成。所选序列包括MAD7的四个非常接近的直系同源物，命名为MAD7v1、MAD7v2、MAD7v3和MAD7v4。与MAD7差异较大的序列被命名为MAD2至MAD110。在步骤205，将这些合成基因克隆到载体骨架中，并且得到正确序列的单个菌落通过Sanger DNA测序证实。
用新颖的MAD系列酶转化的细胞排列在96孔板中207，用于储存。在步骤209，取来自每个孔的细胞的等份，并从每个等份扩增MAD系列序列。在另一步骤211，构建表达gRNA的质粒，并将其与扩增的MAD系列核酸酶组合进行体外转录和翻译，以制备活性核糖核酸酶蛋白复合体213。构建合成的靶文库215，其中测试每一个MAD系列变体的靶耗尽217。在靶耗尽之后，使用下一代测序产生用于分析的扩增子219，并进行测序数据分析221以确定靶耗尽。
实施例2：载体克隆及新颖的MAD系列酶PCR用于模板产生
将新颖的MAD系列酶编码序列克隆到pUC57载体中，其中T7启动子序列附接到编码序列的5’末端，并且T7终止子序列附接到编码序列的3’末端。
首先，使用pUC57质粒作为MAD系列模板的来源，使用Q5热启动2x主混合物(mastermix)试剂(NEB,Ipswich,MA)扩增新颖的MAD系列序列。正向引物5’-TTGGGTAACGCCAGGGTTTT[SEQ ID No.27]和反向引物5’-TGTGTGGAATTGTGAGCGGA[SEQ ID No.28]扩增了pUC57载体中新颖的MAD系列变体侧翼的序列，包括分别在新颖的MAD系列变体5’-末端和3’-末端被附接至MAD7变体序列的T7-启动子和T7-终止子组分。PCR反应中使用1μM引物和5ng/uL pUC57模板来产生编码新颖的MAD系列变体的线性dsDNA产物。使用表1所示的PCR条件：
表1：
步骤 温度 时间
变性 98℃ 30SEC
30个循环 98℃ 10SEC
66℃ 30SEC
72℃ 2.5MIN
最终延伸 72℃ 2MIN
保持 12℃
实施例3：用于在单孔中产生MAD系列核酸酶和gRNA的体外转录和翻译
使用
Figure BDA0003691580560000161
体外蛋白合成试剂盒(NEB,Ipswich,MA)从PCR扩增的线性dsDNA模板产生新颖的MAD70系列变体蛋白，并且还产生gRNA。在96孔板的每个孔中，混合表2中列出的试剂以开始产生MAD7变体和gRNA：
表2：
试剂 体积(μl)
1 SolA(NEB试剂盒) 3.3
2 SolB(NEB试剂盒) 2.5
3 gRNA混合物(4ng/μl储备液) 0.8
4 鼠RNA酶抑制剂(NEB) 0.2
5 水 0.5
6 PCR扩增的T7 MAD系列变体 1.0
制备具有除了PCR扩增的T7-MAD系列变体之外的所有试剂的主混合物，并保持在冰上。在96孔板的每个孔中分配7.3μL的主混合物后，添加1μL在T7启动子控制下的PCR扩增的MAD系列变体。将96孔板密封，并且在热循环仪中在37℃孵育4小时。将板保持在室温直至添加靶池以进行靶耗尽反应。
实施例4：进行靶耗尽、PCR和NGS
孵育4小时以允许产生新颖的MAD系列变体和gRNA后，将4μL的靶文库池(10ng/μL)添加至体外转录/翻译反应混合物中。添加靶文库后，将反应混合物在37℃孵育过夜。将靶耗尽反应混合物稀释到包含RNA酶A的PCR级水中，并且然后在95℃煮沸5min。然后扩增混合物并测序。PCR条件示于表3：
表3
步骤 温度 时间
变性 98℃ 30SEC
6个循环 98℃ 10SEC
61℃ 30SEC
72℃ 10SEC
22个循环 98℃ 10SEC
72℃ 10SEC
最终延伸 72℃ 2分钟
保持 12℃
表4显示了体外测定的结果。
表4：
Figure BDA0003691580560000171
Figure BDA0003691580560000181
实施例5：大肠杆菌基因组编辑
文库扩增：用来自芯片的5μL稀释的合成寡核苷酸编辑盒运行50μL反应。PCR条件为95℃1分钟，然后18轮的95℃30秒/60℃30秒/72℃2分钟30秒，并最后在72℃保持5分钟。将产物在琼脂糖凝胶上运行以检查均匀性。为了扩增骨架，对pL骨架进行10倍的连续稀释，该骨架具有pL诱导型启动子，该启动子被定位为驱动galK编辑盒的转录。PCR条件为95℃1分钟，然后30轮的95℃1分钟/60℃1分钟30秒/72℃2分钟30秒，并最后在72℃保持5分钟。再次，将产物在琼脂糖凝胶上运行以检查均匀性。将扩增子汇集、小提(miniprep)，并加入6μL
Figure BDA0003691580560000191
(NEB,Ipswich,MA)酶，并且允许消化在37℃继续进行1小时。线性化的骨架在与纯化的盒文库等温组装前进行定量。
Gibson反应用150ng骨架、100ng插入物和
Figure BDA0003691580560000192
(NEB,Ipswich MA)主混合物进行。将反应在50℃孵育45分钟。将反应物透析30分钟。将5μL透析的Gibson反应物转化到E.cloni感受态细胞中。使E.
Figure BDA0003691580560000193
SUPREME电感受态细胞(Lucigen,Middleton WI)在25ML SOB+100μg/mL Carb中长出(outgrown)，并进行中提(midiprep)。在2mm比色皿中，在2400V将100ng克隆的文库转化到50μL感受态细胞中。允许细胞在SOB中恢复，并布点铺板10倍稀释液。为了诱导编辑，将50μL的长出物转移到孔板中的SOB/chlor/carb/1％阿拉伯糖中。允许细胞达到对数中期，并且然后在42℃孵育2-2.5小时。进行连续稀释，并将细胞铺板以确定编辑效率。
图3显示了具有所示的可变环序列的所示蛋白对大肠杆菌的体内编辑的结果，如通过比色筛选galK基因座中的精确编辑评估的。表5显示了体内大肠杆菌编辑的结果：
表5：
核酸酶 大肠杆菌中的活性 crRNA环 SEQ ID No.
MAD7 活性 UGUU,UCUU SEQ ID No.1
MAD7v2 活性 UGUU SEQ ID No.4
MAD2 活性 UCUU SEQ ID No.7
MAD3 无活性 SEQ ID No.8
MAD4 无活性 SEQ ID No.9
MAD6 弱活性 UAUU SEQ ID No.11
MAD41 活性 UGUGU SEQ ID No.15
MAD44 活性 UAUU SEQ ID No.16
MAD50 活性 UGUU SEQ ID No.17
MAD54 活性 UCUU SEQ ID No.19
MAD82 活性 UGUU SEQ ID No.21
MAD89 活性 UAUU SEQ ID No.22
MAD92 活性 UAUU SEQ ID No.24
实施例6：酿酒酵母基因组编辑
对于在体外显示活性的酶，在酿酒酵母中体内测试了基因组编辑活性。构建了具有KanMX抗性基因的两微米(two micron)质粒，用于顺序引入包含具有SNR52启动子驱动的crRNA和CYC1启动子驱动的核酸酶蛋白的编辑盒的DNA。编辑盒由引导核酸酶在特定DNA序列处切割的crRNA、短pentaT接头和修复模板组成，所述修复模板包含侧翼为与基因组同源的区域的感兴趣突变。筛选质粒通过StuI限制性内切核酸酶线性化，并且通过等温组装将编辑盒引入SNR52p启动子的下游。编辑盒(参见下表6)全部靶向CAN1基因座中的TTTV PAM序列，并引入过早终止密码子来敲除功能性Can1蛋白。
表6：测试的酵母编辑盒序列的列表
Figure BDA0003691580560000201
Figure BDA0003691580560000211
Figure BDA0003691580560000221
Figure BDA0003691580560000231
Figure BDA0003691580560000241
核酸酶蛋白用寡核苷酸引物通过聚合酶链式反应扩增以在N末端引入SV40核定位序列，该序列由对应于MAPKKKRKVL[SEQ ID NO.26]的蛋白质序列的DNA序列ATGGCACCCAAGAAGAAGAGGAAGGTGTTA[SEQ ID No.25]组成。然后将所得的扩增的DNA片段(400ng，纯化的)与已经包含以上编辑盒之一的PsiI线性化筛选质粒(250ng)一起共转化，来通过体内缺口修复组装完整的编辑质粒。在包含200mg/L遗传霉素的酵母蛋白胨-右旋糖(YPD)中在加湿振荡培养箱中在30℃持续3天，选择包含修复的质粒的细胞。将所得饱和培养物以1:100至1:200稀释到不含精氨酸且包含50mg/L刀豆氨酸的合成的完全酵母培养基中，并在加湿振荡培养箱中在30℃生长过夜。由于Can1蛋白的敲除允许酵母在存在原本毒性的类似物刀豆氨酸的情况下生长，过夜培养物的相对OD600与由转化的核酸酶蛋白诱导的基因组突变率成比例。表7显示了体内酿酒酵母编辑的结果：
表7：
Figure BDA0003691580560000242
Figure BDA0003691580560000251
图4显示了使用具有所示可变环序列的所示的核酸酶的酿酒酵母的体内编辑结果，如通过Can1基因座中的精确编辑诱导的在含刀豆氨酸的培养基中的生长评估的。图5显示了使用另外的可变环支架，由MAD7和MAD41对酿酒酵母的体内编辑的结果。
实施例7：哺乳动物细胞系基因组编辑
将HEK293T细胞在96孔板中用2μL polyfect和各200ng的引擎质粒和编辑质粒转染。48小时后，抽吸培养基，并加入100μL含蛋白酶K(最终1mg/mL)的Taq裂解缓冲液(10XTaq裂解缓冲液：100mM Tris pH8、500mM NaCl、15mM MgCl2、1％Triton X-100)。将细胞在室温孵育5分钟，并且然后转移到新的96孔板。将细胞在56℃再孵育30分钟，和在98℃再孵育10分钟。用5μL裂解液进行PCR分析。
图6显示了由具有所示可变环的所示的核酸酶诱导的HEK293T人类细胞中的位点特异性核酸酶裂解诱导的插入缺失的比率。hsMAD7是人类密码子优化的核苷酸序列[SEQID No.53]，而MAD7表示用于大肠杆菌和酿酒酵母研究的广谱密码子使用核苷酸序列[SEQID No.1]。
表8：MAD7序列
Figure BDA0003691580560000261
Figure BDA0003691580560000271
Figure BDA0003691580560000281
Figure BDA0003691580560000291
虽然本发明通过许多不同形式的实施方案来满足，但是如结合本发明的优选的实施方案详细描述的，应理解本公开内容应被认为是对本发明的原理的示例而不意在将本发明局限于本文说明和描述的具体实施方案。本领域的技术人员可以作出许多变化而不脱离本发明的精神。本发明的范围将通过附加的权利要求和它们的等同物判断。摘要和标题不应被解释为限制本发明的范围，因为它们的目的是使适当的机构以及一般公众能够迅速确定本发明的一般性质。在以下的权利要求书中，除非使用术语“手段(means)”，否则其中列举的特征或要素都不应该被解释为根据35U.S.C.§112,
Figure BDA0003691580560000292
的手段加功能的限定。
序列表
<110> 因思科瑞普特公司
<120> 工程化的酶
<130> INSC059PCT
<140> USPCT/US20/64727
<141> 2020-12-12
<140> 16/714,320
<141> 2019-12-13
<160> 52
<170> PatentIn version 3.5
<210> 1
<211> 3792
<212> DNA
<213> 直肠真杆菌(Eubacterium rectale)
<400> 1
atgaacaacg gcacaaataa ttttcagaac ttcatcggga tctcaagttt gcagaaaacg 60
ctgcgcaatg ctctgatccc cacggaaacc acgcaacagt tcatcgtcaa gaacggaata 120
attaaagaag atgagttacg tggcgagaac cgccagattc tgaaagatat catggatgac 180
tactaccgcg gattcatctc tgagactctg agttctattg atgacataga ttggactagc 240
ctgttcgaaa aaatggaaat tcagctgaaa aatggtgata ataaagatac cttaattaag 300
gaacagacag agtatcggaa agcaatccat aaaaaatttg cgaacgacga tcggtttaag 360
aacatgttta gcgccaaact gattagtgac atattacctg aatttgtcat ccacaacaat 420
aattattcgg catcagagaa agaggaaaaa acccaggtga taaaattgtt ttcgcgcttt 480
gcgactagct ttaaagatta cttcaagaac cgtgcaaatt gcttttcagc ggacgatatt 540
tcatcaagca gctgccatcg catcgtcaac gacaatgcag agatattctt ttcaaatgcg 600
ctggtctacc gccggatcgt aaaatcgctg agcaatgacg atatcaacaa aatttcgggc 660
gatatgaaag attcattaaa agaaatgagt ctggaagaaa tatattctta cgagaagtat 720
ggggaattta ttacccagga aggcattagc ttctataatg atatctgtgg gaaagtgaat 780
tcttttatga acctgtattg tcagaaaaat aaagaaaaca aaaatttata caaacttcag 840
aaacttcaca aacagattct atgcattgcg gacactagct atgaggtccc gtataaattt 900
gaaagtgacg aggaagtgta ccaatcagtt aacggcttcc ttgataacat tagcagcaaa 960
catatagtcg aaagattacg caaaatcggc gataactata acggctacaa cctggataaa 1020
atttatatcg tgtccaaatt ttacgagagc gttagccaaa aaacctaccg cgactgggaa 1080
acaattaata ccgccctcga aattcattac aataatatct tgccgggtaa cggtaaaagt 1140
aaagccgaca aagtaaaaaa agcggttaag aatgatttac agaaatccat caccgaaata 1200
aatgaactag tgtcaaacta taagctgtgc agtgacgaca acatcaaagc ggagacttat 1260
atacatgaga ttagccatat cttgaataac tttgaagcac aggaattgaa atacaatccg 1320
gaaattcacc tagttgaatc cgagctcaaa gcgagtgagc ttaaaaacgt gctggacgtg 1380
atcatgaatg cgtttcattg gtgttcggtt tttatgactg aggaacttgt tgataaagac 1440
aacaattttt atgcggaact ggaggagatt tacgatgaaa tttatccagt aattagtctg 1500
tacaacctgg ttcgtaacta cgttacccag aaaccgtaca gcacgaaaaa gattaaattg 1560
aactttggaa taccgacgtt agcagacggt tggtcaaagt ccaaagagta ttctaataac 1620
gctatcatac tgatgcgcga caatctgtat tatctgggca tctttaatgc gaagaataaa 1680
ccggacaaga agattatcga gggtaatacg tcagaaaata agggtgacta caaaaagatg 1740
atttataatt tgctcccggg tcccaacaaa atgatcccga aagttttctt gagcagcaag 1800
acgggggtgg aaacgtataa accgagcgcc tatatcctag aggggtataa acagaataaa 1860
catatcaagt cttcaaaaga ctttgatatc actttctgtc atgatctgat cgactacttc 1920
aaaaactgta ttgcaattca tcccgagtgg aaaaacttcg gttttgattt tagcgacacc 1980
agtacttatg aagacatttc cgggttttat cgtgaggtag agttacaagg ttacaagatt 2040
gattggacat acattagcga aaaagacatt gatctgctgc aggaaaaagg tcaactgtat 2100
ctgttccaga tatataacaa agatttttcg aaaaaatcaa ccgggaatga caaccttcac 2160
accatgtacc tgaaaaatct tttctcagaa gaaaatctta aggatatcgt cctgaaactt 2220
aacggcgaag cggaaatctt cttcaggaag agcagcataa agaacccaat cattcataaa 2280
aaaggctcga ttttagtcaa ccgtacctac gaagcagaag aaaaagacca gtttggcaac 2340
attcaaattg tgcgtaaaaa tattccggaa aacatttatc aggagctgta caaatacttc 2400
aacgataaaa gcgacaaaga gctgtctgat gaagcagcca aactgaagaa tgtagtggga 2460
caccacgagg cagcgacgaa tatagtcaag gactatcgct acacgtatga taaatacttc 2520
cttcatatgc ctattacgat caatttcaaa gccaataaaa cgggttttat taatgatagg 2580
atcttacagt atatcgctaa agaaaaagac ttacatgtga tcggcattga tcggggcgag 2640
cgtaacctga tctacgtgtc cgtgattgat acttgtggta atatagttga acagaaaagc 2700
tttaacattg taaacggcta cgactatcag ataaaactga aacaacagga gggcgctaga 2760
cagattgcgc ggaaagaatg gaaagaaatt ggtaaaatta aagagatcaa agagggctac 2820
ctgagcttag taatccacga gatctctaaa atggtaatca aatacaatgc aattatagcg 2880
atggaggatt tgtcttatgg ttttaaaaaa gggcgcttta aggtcgaacg gcaagtttac 2940
cagaaatttg aaaccatgct catcaataaa ctcaactatc tggtatttaa agatatttcg 3000
attaccgaga atggcggtct cctgaaaggt tatcagctga catacattcc tgataaactt 3060
aaaaacgtgg gtcatcagtg cggctgcatt ttttatgtgc ctgctgcata cacgagcaaa 3120
attgatccga ccaccggctt tgtgaatatc tttaaattta aagacctgac agtggacgca 3180
aaacgtgaat tcattaaaaa atttgactca attcgttatg acagtgaaaa aaatctgttc 3240
tgctttacat ttgactacaa taactttatt acgcaaaaca cggtcatgag caaatcatcg 3300
tggagtgtgt atacatacgg cgtgcgcatc aaacgtcgct ttgtgaacgg ccgcttctca 3360
aacgaaagtg ataccattga cataaccaaa gatatggaga aaacgttgga aatgacggac 3420
attaactggc gcgatggcca cgatcttcgt caagacatta tagattatga aattgttcag 3480
cacatattcg aaattttccg tttaacagtg caaatgcgta actccttgtc tgaactggag 3540
gaccgtgatt acgatcgtct catttcacct gtactgaacg aaaataacat tttttatgac 3600
agcgcgaaag cgggggatgc acttcctaag gatgccgatg caaatggtgc gtattgtatt 3660
gcattaaaag ggttatatga aattaaacaa attaccgaaa attggaaaga agatggtaaa 3720
ttttcgcgcg ataaactcaa aatcagcaat aaagattggt tcgactttat ccagaataag 3780
cgctatctct aa 3792
<210> 2
<211> 3792
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 人类密码子优化的MAD7
<400> 2
atgaataatg gcactaacaa ctttcagaat ttcataggca tcagtagtct ccaaaagacg 60
ttgcgcaacg cacttattcc aaccgagaca actcaacagt tcatcgtgaa gaatgggatt 120
attaaagagg acgaactccg aggagagaac cggcaaattc ttaaggacat catggacgat 180
tattacagag ggtttatttc tgagacatta tcaagtattg acgacatcga ctggacctca 240
ctgttcgaga agatggaaat tcagttgaag aacggagaca acaaggacac tctaatcaag 300
gaacaaacag agtaccggaa agctatacat aagaagtttg ccaatgatga ccggtttaag 360
aacatgttct ccgcgaaact catcagcgac attctgccag aattcgtgat ccacaacaat 420
aactattcag cctctgagaa ggaggaaaag acccaggtca tcaagctttt ctctagattc 480
gccactagct tcaaggacta tttcaagaac cgcgccaatt gtttctctgc tgacgatatc 540
tccagcagca gttgccatag gatcgtgaac gacaatgctg aaatcttctt ctctaatgcc 600
cttgtataca gacggatcgt gaagtcactg agcaatgatg acattaacaa gataagcggt 660
gatatgaaag atagtctcaa ggaaatgtca ctcgaagaaa tttatagcta cgagaaatac 720
ggagagttca tcacccagga gggaatcagt ttctacaacg atatttgtgg caaggtaaac 780
tccttcatga atctatattg ccagaaaaac aaggagaata agaatcttta taagctgcag 840
aagttacata agcagatcct gtgtattgca gatacctcct atgaagtgcc atataagttt 900
gagtctgacg aggaagtgta tcaatccgta aatgggttcc tcgacaacat cagctctaag 960
catatagttg aacgacttag aaagataggc gacaactata atggctacaa cctcgacaag 1020
atttatatag tgtccaaatt ctacgagtcc gtatcccaaa agacatacag agattgggaa 1080
acaatcaata cagccctcga aatccactac aataatatcc tacccggcaa tgggaaatcc 1140
aaggcagata aggtaaagaa ggcagtcaag aacgacctcc agaagtccat caccgagatt 1200
aacgaactgg tgagcaatta caaactctgt agtgacgata atatcaaggc tgagacgtac 1260
atccatgaga tttcacacat attgaacaac ttcgaagcac aggaactgaa gtacaatccg 1320
gaaattcatc tcgtagaatc cgagcttaaa gccagcgagc ttaagaacgt gctcgatgtg 1380
attatgaacg cgtttcactg gtgtagtgtc ttcatgactg aagaattagt tgacaaggac 1440
aacaatttct atgccgaact ggaagaaatt tacgatgaga tctatcctgt tatcagtctg 1500
tataacctcg tacggaacta tgtgacccag aagccctact cgaccaaaaa gatcaaactg 1560
aacttcggca ttccaaccct ggccgatgga tggagcaaat ccaaagagta ctctaataac 1620
gctatcattc tcatgcgaga caatctctac tatctcggaa tattcaatgc aaagaataaa 1680
ccagacaaaa agattattga agggaacaca tccgagaaca aaggtgatta taagaaaatg 1740
atttacaacc tgcttccagg gcccaataag atgattccca aggtctttct ttcaagcaag 1800
actggagttg agacttacaa gccgtccgca tacattctcg agggctataa gcagaacaag 1860
cacattaaga gcagtaaaga cttcgatatc actttctgcc atgatctcat tgactacttt 1920
aagaattgta tcgctattca tccggaatgg aagaactttg gatttgactt cagcgataca 1980
agtacctacg aggatatctc tgggttctac cgggaagtgg aacttcaggg atacaagatc 2040
gactggacat atatctctga gaaagacatc gatctgctgc aggagaaagg ccagctgtac 2100
ctgttccaga tttataataa agatttctca aagaagagca caggaaacga taatcttcat 2160
actatgtatc tgaagaatct cttctccgaa gagaacctga aggatatcgt cctcaaactg 2220
aacggagaag ccgagatttt cttcaggaag agtagtatta agaatcccat tattcataag 2280
aaaggctcca tcttggttaa ccgcacttac gaggctgaag agaaggacca gtttggaaat 2340
atccaaatcg tgaggaagaa tattccagag aatatctacc aggaactgta taagtacttt 2400
aatgataaga gcgataaaga actgagcgac gaggcagcga agttgaagaa tgtggtgggc 2460
catcacgaag ctgccacaaa cattgtgaaa gactataggt acacatatga taaatacttt 2520
ctgcatatgc ctataaccat aaatttcaag gccaataaga ctgggttcat taatgaccgc 2580
atcctgcagt acatcgctaa ggagaaggac ctgcacgtca tagggatcga ccgcggtgaa 2640
cggaatttga tttatgtgtc cgttatcgat acctgcggga atatcgtgga gcaaaagagc 2700
tttaatatcg tcaatggata cgactaccag atcaagttaa agcagcaaga aggcgccagg 2760
caaatcgcca ggaaagagtg gaaagagatc ggcaagataa aggaaattaa ggaaggctac 2820
ctttccctgg tcatccatga aattagtaag atggtcatta agtacaatgc catcatagca 2880
atggaagact taagttacgg atttaagaag ggaagattca aagtggaaag gcaggtgtat 2940
cagaagtttg aaacgatgct aataaacaaa cttaattatc ttgtgttcaa agacattagc 3000
atcacagaga atggagggct tctcaaggga taccaactga cctacatccc agataagctt 3060
aagaacgttg gccaccaatg cggctgcata ttctacgtcc cggctgctta cacttctaag 3120
atcgatccaa ccaccggctt tgtgaatatc tttaagttta aagacttgac cgtggatgct 3180
aagcgcgagt tcatcaagaa gtttgacagc atcaggtacg actcagaaaa gaacctcttc 3240
tgtttcacat tcgattataa caactttatt actcagaata ctgtcatgag taagtcatcc 3300
tggtcagtgt atacctacgg agtgaggatc aagcgaaggt ttgtgaacgg caggtttagt 3360
aatgagtctg acacaatcga tattaccaaa gacatggaga aaacactgga gatgacagac 3420
atcaactgga gggatggaca tgacctgcgc caggatatca tagattacga gatcgtgcaa 3480
catatattcg aaatctttag gctgacagtg cagatgcgca actccctgag cgagctcgaa 3540
gacagagatt atgatagact aatcagtccg gttctgaacg agaacaatat cttctacgat 3600
agtgctaagg caggagacgc gctgcccaag gacgcagatg ccaatggcgc gtattgcatt 3660
gcacttaaag gactgtacga aattaagcag attaccgaga actggaagga ggacggcaag 3720
tttagcaggg ataagctgaa gattagtaac aaagattggt ttgactttat acagaataag 3780
cgttatctgt aa 3792
<210> 3
<211> 3792
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 变体MAD7核酸序列
<400> 3
atgaacaacg gcacaaataa ttttcagaac ttcatcggga tctcaagttt gcagaaaacg 60
ctgcgcaatg ctctgatccc cacggaaacc acgcaacagt tcatcgtcaa gaacggaata 120
attaaagaag atgagttacg tggcaaaaac cgccagattc tgaaagatat catggatgac 180
tactaccgcg gattcatctc tgagactctg agttctattg atgacataga ttggactagc 240
ctgttcgaaa aaatggaaat tcagctgaaa aatggtgata ataaagatac cttaattaag 300
gaacaggcgg agaaacggaa agcaatctat aaaaaatttg cggatgacga tcggtttaag 360
aacatgttta gcgccaaact gattagtgac atattacctg aatttgtcat ccacaacaat 420
aattattcgg catcagagaa aaaagaaaaa acccaggtga taaaattgtt ttcgcgcttt 480
gcgactagct ttaaagatta cttcaagaac cgtgcaaatt gcttttcagc ggacgatatt 540
tcatcaagca gctgccatcg catcgtcaac gacaatgcag agatattctt ttcaaatgcg 600
ctggtctacc gccggatcgt aaaaaacctg agcaatgacg atatcaacaa aatttcgggc 660
gatatgaaag attcattaaa agaaatgagt ctggaagaaa tatattctta cgagaagtat 720
ggggaattta ttacccagga aggcattagc ttctataatg atatctgtgg gaaagtgaat 780
tcttttatga acctgtattg tcagaaaaat aaagaaaaca aaaatttata caaacttcgt 840
aaacttcaca aacagattct atgcattgcg gacactagct atgaggtccc gtataaattt 900
gaaagtgacg aggaagtgta ccaatcagtt aacggcttcc ttgataacat tagcagcaaa 960
catatagtcg aaagattacg caaaatcggc gataactata acgattacaa cctggataaa 1020
atttatatcg tgtccaaatt ttacgagagc gttagccaaa aaacctaccg cgactgggaa 1080
acaattaata ccgccctcga aattcattac aataatatct tgccgggtaa cggtaaaagt 1140
aaagccgaca aagtaaaaaa agcggttaag aatgatttac agaaatccat caccgaaata 1200
aatgaactag tgtcaaacta taagctgtgc agtgacgaca acatcaaagc ggagacttat 1260
atacatgaga ttagccatat cttgaataac tttgaagcac atgaattgaa atacaatccg 1320
gaaattcacc tagttgaatc cgagctcaaa gcgagtgagc ttaaaaacgt gctggacatt 1380
atcatgaatg cgtttcattg gtgttcggtt tttatgactg aggaacttgt tgataaagac 1440
aacaattttt atgcggaact ggaggagatt tacgatgaaa tttatccagt aattagtctg 1500
tacaacctgg ttcgtaacta cgttacccag aaaccgtaca gcacgaaaaa gattaaattg 1560
aactttggaa taccgacgtt agcagacggt tggtcaaagt ccaaagagta ttctaataac 1620
gctatcatac tgatgcgcga caatctgtat tatctgggca tctttaatgc gaagaataaa 1680
ccggacaaga agattatcga gggtaatacg tcagaaaata agggtgacta caaaaagatg 1740
atttataatt tgctcccggg tcccaacaaa atgatcccga aagttttctt gagcagcaag 1800
acgggggtgg aaacgtataa accgagcgcc tatatcctag aggggtataa acagaataaa 1860
catctgaagt cttcaaaaga ctttgatatc actttctgtc atgatctgat cgactacttc 1920
aaaaactgta ttgcaattca tcccgagtgg aaaaacttcg gttttgattt tagcgacacc 1980
agtgcgtatg aagacatttc cgggttttat cgtgaggtag agttacaagg ttacaagatt 2040
gattggacat acattagcga aaaagacatt gatctgctgc aggaaaaagg tcaactgtat 2100
ctgttccaga tatataacaa agatttttcg aaaaaatcaa ccgggaatga caaccttcac 2160
accatgtacc tgaaaaatct tttctcagaa gaaaatctta aggatatcgt cctgaaactt 2220
aacggcgaag cggaaatctt cttcaggaag agcagcataa agaacccaat cattcataaa 2280
aaaggctcga ttttagtcaa ccgtacctac gaagcagaag aaaaagacca gtttggcaac 2340
attcaaattg tgcgtaaaac cattccggaa aacatttatc aggagctgta caaatacttc 2400
aacgataaaa gcgacaaaga gctgtctgat gaagcagcca aactgaagaa tgtagtggga 2460
caccacgagg cagcgacgaa tatagtcaag gactatcgct acacgtatga taaatacttc 2520
cttcatatgc ctattacgat caatttcaaa gccaataaaa cgagctttat taatgatagg 2580
atcttacagt atatcgctaa agaaaaagac ttacatgtga tcggcattga tcggggcgag 2640
cgtaacctga tctacgtgtc cgtgattgat acttgtggta atatagttga acagaaaagc 2700
tttaacattg taaacggcta cgactatcag ataaaactga aacaacagga gggcgctaga 2760
cagattgcgc ggaaagaatg gaaagaaatt ggtaaaatta aagagatcaa agagggctac 2820
ctgagcttag taatccacga gatctctaaa atggtaatca aatacaatgc aattatagcg 2880
atggaggatt tgtcttatgg ttttaaaaaa gggcgcttta aggtcgaacg gcaagtttac 2940
cagaaatttg aaaccatgct catcaataaa ctcaactatc tggtatttaa agatatttcg 3000
attaccgaga atggcggtct cctgaaaggt tatcagctga catacattcc tgataaactt 3060
aaaaacgtgg gtcatcagtg cggctgcatt ttttatgtgc ctgctgcata cacgagcaaa 3120
attgatccga ccaccggctt tgtgaatatc tttaaattta aagacctgac agtggacgca 3180
aaacgtgaat tcattaaaaa atttgactca attcgttatg acagtgaaaa aaatctgttc 3240
tgctttacat ttgactacaa taactttatt acgcaaaaca cggtcatgag caaatcatcg 3300
tggagtgtgt atacatacgg cgtgcgcatc aaacgtcgct ttgtgaacgg ccgcttctca 3360
aacgaaagtg ataccattga cataaccaaa gatatggaga aaacgttgga aatgacggac 3420
attaactggc gcgatggcca cgatcttcgt caagacatta tagattatga aattgttcag 3480
cacatattcg aaattttcaa attaacagtg caaatgcgta actccttgtc tgaactggag 3540
gaccgtgatt acgatcgtct catttcacct gtactgaacg aaaataacat tttttatgac 3600
agcgcgaaag cggggtatgc acttcctaag gatgccgatg caaatggtgc gtattgtatt 3660
gcattaaaag ggttatatga aattaaacaa attaccgaaa attggaaaga agatggtaaa 3720
ttttcgcgcg ataaactcaa aatcagcaat aaagattggt tcgactttat ccagaataag 3780
cgctatctct aa 3792
<210> 4
<211> 3792
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD7核酸序列
<400> 4
atgaacaacg gcacaaataa ttttcagaac ttcatcggga tctcaagttt gcagaaaacg 60
ctgcgcaatg ctctgacccc cacggaaacc acgcaacagt tcatcgtcaa gaacggaata 120
attaaagaag atgagttacg tggcgagaac cgccagattc tgaaagatat catggatgac 180
tactaccgcg gattcatctc tgagactctg agttctattg atgacataga ttggactagc 240
ctgttcgaaa aaatggaaat tcagctgaaa aatggtgata ataaagatac cttaattaag 300
gaacaggcgg agaaacggaa agcaatctat aaaaaatttg cggatgacga tcggtttaag 360
aacatgttta gcgccaaact gattagtgac atattacctg aatttgtcat ccacaacaat 420
aattattcgg catcagagaa agaggaaaaa acccaggtga taaaattgtt ttcgcgcttt 480
gcgactagct ttaaagatta cttcaagaac cgtgcaaatt gcttttcagc ggacgatatt 540
tcatcaagca gctgccatcg catcgtcaac gacaatgcag agatattctt ttcaaatgcg 600
ctggtctacc gccggatcgt aaaaaacctg agcaatgacg atatcaacaa aatttcgggc 660
gatatgaaag attcattaaa aaaaatgagt ctggaaaaaa tatattctta cgagaagtat 720
ggggaattta ttacccagga aggcattagc ttctataatg atatctgtgg gaaagtgaat 780
tcttttatga acctgtattg tcagaaaaat aaagaaaaca aaaatttata caaacttcgt 840
aaacttcaca aacagattct atgcattgcg gacactagct atgaggtccc gtataaattt 900
gaaagtgacg aggaagtgta ccaatcagtt aacggcttcc ttgataacat tagcagcaaa 960
catatagtcg aaagattacg caaaatcggc gataactata acggctacaa cctggataaa 1020
atttatatcg tgtccaaatt ttacgagagc gttagccaaa aaacctaccg cgactgggaa 1080
acaattaata ccgccctcga aattcattac aataatatct tgccgggtaa cggtaaaagt 1140
aaagccgaca aagtaaaaaa agcggttaag aatgatttac agaaatccat caccgaaata 1200
aatgaactag tgtcaaacta taagctgtgc ccggacgaca acatcaaagc ggagacttat 1260
atacatgaga ttagccatat cttgaataac tttgaagcac aggaattgaa atacaatccg 1320
gaaattcacc tagttgaatc cgagctcaaa gcgagtgagc ttaaaaacgt gctggacgtg 1380
atcatgaatg cgtttcattg gtgttcggtt tttatgactg aggaacttgt tgataaagac 1440
aacaattttt atgcggaact ggaggagatt tacgatgaaa tttatccagt aattagtctg 1500
tacaacctgg ttcgtaacta cgttacccag aaaccgtaca gcacgaaaaa gattaaattg 1560
aactttggaa taccgacgtt agcagacggt tggtcaaagt ccaaagagta ttctaataac 1620
gctatcatac tgatgcgcga caatctgtat tatctgggca tctttaatgc gaagaataaa 1680
ccggaaaaga agattatcga gggtaatacg tcagaaaata agggtgacta caaaaagatg 1740
atttataatt tgctcccggg tcccaacaaa atgatcccga aagttttctt gagcagcaag 1800
acgggggtgg aaacgtataa accgagcgcc tatatcctag aggggtataa acagaataaa 1860
catctgaagt cttcaaaaga ctttgatatc actttctgtc gtgatctgat cgactacttc 1920
aaaaactgta ttgcaattca tcccgagtgg aaaaacttcg gttttgattt tagcgacacc 1980
agtacttatg aagacatttc cgggttttat cgtgaggtag agttacaagg ttacaagatt 2040
gattggacat acattagcga aaaagacatt gatctgctgc aggaaaaagg tcaactgtat 2100
ctgttccaga tatataacaa agatttttcg aaaaaatcaa ccgggaatga caaccttcac 2160
accatgtacc tgaaaaatct tttctcagaa gaaaatctta aggatgtggt cctgaaactt 2220
aacggcgaag cggaaatctt cttcaggaag agcagcataa agaacccaat cattcataaa 2280
aaaggctcga ttttagtcaa ccgtacctac gaagcagaag aaaaagacca gtttggcaac 2340
attcaaattg tgcgtaaaac cattccggaa aacatttatc aggagctgta caaatacttc 2400
aacgataaaa gcgacaaaga gctgtctgat gaagcagcca aactgaagaa tgcggtggga 2460
caccacgagg cagcgacgaa tatagtcaag gactatcgct acacgtatga taaatacttc 2520
cttcatatgc ctattacgat caatttcaaa gccaataaaa cgagctttat taatgatagg 2580
atcttacagt atatcgctaa agaaaaagac ttacatgtga tcggcattga tcggggcgag 2640
cgtaacctga tctacgtgtc cgtgattgat acttgtggta atatagttga acagaaaagc 2700
tttaacattg taaacggcta cgactatcag ataaaactga aacaacagga gggcgctaga 2760
cagattgcgc ggaaagaatg gaaagaaatt ggtaaaatta aagagatcaa agagggctac 2820
ctgagcttag taatccacga gatctctaaa atggtaatca aatacaatgc aattatagcg 2880
atggaggatt tgtcttatgg ttttaaaaaa gggcgcttta aggtcgaacg gcaagtttac 2940
cagaaatttg aaaccatgct catcaataaa ctcaactatc tggtatttaa agatatttcg 3000
attaccgaga atggcggtct cctgaaaggt tatcagctga catacattcc tgaaaaactt 3060
aaaaacgtgg gtcatcagtg cggctgcatt ttttatgtgc ctgctgcata cacgagcaaa 3120
attgatccga ccaccggctt tgtgaatatc tttaaattta aagacctgac agtggacgca 3180
aaacgtgaat tcattaaaaa atttgactca attcgttatg acagtgataa aaatctgttc 3240
tgctttacat ttgactacaa taactttatt acgcaaaaca cggtcatgag caaatcatcg 3300
tggagtgtgt atacatacgg cgtgcgcatc aaacgtcgct ttgtgaacgg ccgcttctca 3360
aacgaaagtg ataccattga cataaccaaa gatatggaga aaacgttgga aatgacggac 3420
attaactggc gcgatggcca cgatcttcgt caagacatta tagattatga aattgttcag 3480
cacatattcg aaattttcaa attaacagtg caaatgcgta actccttgtc tgaactggag 3540
gaccgtaact acgatcgtct catttcacct gtactgaacg aaaataacat tttttatgac 3600
agcgcgaaag cgggggatgc acttcctaag gatgccgatg caaatggtgc gtattgtatt 3660
gcattaaaag ggttatatga aattaaacaa attaccgaaa attggaaaga agatggtaaa 3720
ttttcgcgcg ataaactcaa aatcagcaat aaagattggt tcgactttat ccagaataag 3780
cgctatctct aa 3792
<210> 5
<211> 3792
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD7核酸序列
<400> 5
atgaacaacg gcacaaataa ttttcagaac ttcatcggga tctcaagttt gcagaaaacg 60
ctgcgcaatg ctctgatccc cacggaaacc acgcaacagt tcatcgtcaa gaacggaata 120
attaaagaag atgagttacg tggcgagaac cgccagattc tgaaagatat catggatgac 180
tactaccgcg gattcatctc tgagactctg agttctattg atgacataga ttggactagc 240
ctgttcgaaa aaatggaaat tcagctgaaa aatggtgata ataaagatac cttaattaag 300
gaacaggcgg agaaacggaa agcaatctat aaaaaatttg cggatgacga tcggtttaag 360
aacatgttta gcgccaaact gattagtgac atattacctg aatttgtcat ccacaacaat 420
aattattcgg catcagagaa agaggaaaaa acccaggtga taaaattgtt ttcgcgcttt 480
gcgactagct ttaaagatta cttcaagaac cgtgcaaatt gcttttcagc ggacgatatt 540
tcatcaagca gctgccatcg catcgtcaac gacaatgcag agatattctt ttcaaatgcg 600
ctggtctacc gccggatcgt aaaaaacctg agcaatgacg atatcaacaa aatttcgggc 660
gatatgaaag attcattaaa agaaatgagt ctggatgaaa tatattctta cgagaagtat 720
ggggaattta ttacccagga aggcattagc ttctataatg atatctgtgg gaaagtgaat 780
tcttttatga acctgtattg tcagaaaaat aaagaaaaca aaaatttata caaacttcgt 840
aaacttcaca aacagattct atgcattgcg gacactagct atgaggtccc gtataaattt 900
gaaagtgacg aggaagtgta ccaatcagtt aacggcttcc ttgataacat tagcagcaaa 960
catatagtcg aaagattacg caaaatcggc gataactata acggctacaa cctggataaa 1020
atttatatcg tgtcccgttt ttacgagagc gttagccaaa aaacctaccg cgactgggaa 1080
acaattaata ccgccctcga aattcattac aataatatct tgccgggtaa cggtaaaagt 1140
aaagccgaca aagtaaaaaa agcggttaag aatgatttac agaaatccat caccgaaata 1200
aatgaactag tgtcaaacta taagctgtgc ccggacgaca acatcaaagc ggagacttat 1260
atacatgaga ttagccatat cttgaataac tttgaagcac aggaattgaa atacaatccg 1320
gaaattcacc tagttgaatc cgagctcaaa gcgagtgagc ttaaaaacgt gctggacgtg 1380
atcatgaatg cgtttcattg gtgttcggtt tttatgactg aggaacttgt tgataaagac 1440
aacaattttt atgcggaact ggaggagatt tacgatgaaa tttatccagt aattagtctg 1500
tacaacctgg ttcgtaacta cgttacccag aaaccgtaca gcacgaaaaa gattaaattg 1560
aactttggaa taccgacgtt agcagacggt tggtcaaagt ccaaagagta ttctaataac 1620
gctatcatac tgatgcgcga caatctgtat tatctgggca tctttaatgc gaagaataaa 1680
ccggacaaga agattatcga gggtaatacg tcagaaaata agggtgacta caaaaagatg 1740
atttataatt tgctcccggg tcccaacaaa atgatcccga aagttttctt gagcagcaag 1800
acgggggtgg aaacgtataa accgagcgcc tatatcctag aggggtataa acagaataaa 1860
catctgaagt cttcaaaaga ctttgatatc actttctgtc gtgatctgat cgactacttc 1920
aaaaactgta ttgcaattca tcccgagtgg aaaaacttcg gttttgattt tagcgacacc 1980
agtacttatg aagacatttc cgggttttat cgtgaggtag agttacaagg ttacaagatt 2040
gattggacat acattagcga aaaagacatt gatctgctgc aggaaaaagg tcaactgtat 2100
ctgttccaga tatataacaa agatttttcg aaaaaatcaa ccgggaatga caaccttcac 2160
accatgtacc tgaaaaatct tttctcagaa gaaaatctta aggatatcgt cctgaaactt 2220
aacggcgaag cggaaatctt cttcaggaag agcagcataa agaacccaat cattcataaa 2280
aaaggctcga ttttagtcaa ccgtacctac gaagcagaag aaaaagacca gtttggcaac 2340
attcaaattg tgcgtaaaac cattccggaa aacatttatc aggagctgta caaatacttc 2400
aacgataaaa gcgacaaaga gctgtctgat gaagcagcca aactgaagaa tgtagtggga 2460
caccacgagg cagcgacgaa tatagtcaag gactatcgct acacgtatga taaatacttc 2520
cttcatatgc ctattacgat caatttcaaa gccaataaaa cgagctttat taatgatagg 2580
atcttacagt atatcgctaa agaaaacgac ttacatgtga tcggcattga tcggggcgag 2640
cgtaacctga tctacgtgtc cgtgattgat acttgtggta atatagttga acagaaaagc 2700
tttaacattg taaacggcta cgactatcag ataaaactga aacaacagga gggcgctaga 2760
cagattgcgc ggaaagaatg gaaagaaatt ggtaaaatta aagagatcaa agagggctac 2820
ctgagcttag taatccacga gatctctaaa atggtaatca aatacaatgc aattatagcg 2880
atggaggatt tgtcttatgg ttttaaaaaa gggcgcttta aggtcgaacg gcaagtttac 2940
cagaaatttg aaaccatgct catcaataaa ctcaactatc tggtatttaa agatatttcg 3000
attaccgaga atggcggtct cctgaaaggt tatcagctga catacattcc tgaaaaactt 3060
aaaaacgtgg gtcatcagtg cggctgcatt ttttatgtgc ctgctgcata cacgagcaaa 3120
attgatccga ccaccggctt tgcgaatatc tttaaattta aagacctgac agtggacgca 3180
aaacgtgaat tcattaaaaa atttgactca attcgttatg acagtgaaaa aaatctgttc 3240
tgctttacat ttgactacaa taactttatt acgcaaaaca cggtcatgag caaatcatcg 3300
tggagtgtgt atacatacgg cgtgcgcatc aaacgtcgct ttgtgaacgg ccgcttctca 3360
aacgaaagtg ataccattga cataaccaaa gatatggaga aaacgttgga aatgacggac 3420
attaactggc gcgatggcca cgatcttcgt caagacatta tagattatga aattgttcag 3480
cacatattcg aaattttcaa attaacagtg caaatgcgta actccttgtc tgaactggag 3540
gaccgtgatt acgatcgtct catttcacct gtactgaacg aaaataacat tttttatgac 3600
agcgcgaaag cgggggatgc acttcctaag gatgccgatg caaatggtgc gtattgtatt 3660
gcattaaaag ggttatatga aattaaacaa attaccgaaa attggaaaga agatggtaaa 3720
ttttcgcgcg ataaactcaa aatcagcaat aaagattggt tcgactttat ccagaataag 3780
cgctatctct aa 3792
<210> 6
<211> 3792
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD7核酸序列
<400> 6
atgaacaacg gcacaaataa ttttcagaac ttcatcggga tctcaagttt gcagaaaacg 60
ctgcgcaatg ctctgatccc cacggaaacc acgcaacagt tcatcgtcaa gaacggaata 120
attaaagaag atgagttacg tggcgagaac cgccagattc tgaaagatat catggatgac 180
tactaccgcg gattcatctc tgagactctg agttctattg atgacataga ttggactagc 240
ctgttcgaaa aaatggaaat tcagctgaaa aatggtgata ataaagatac cttaattaag 300
gaacaggcgg agaaacggaa agcaatctat aaaaaatttg cggatgacga tcggtttaag 360
aacatgttta gcgccaaact gattagtgac atattacctg aatttgtcat ccacaacaat 420
aattattcgg catcagagaa agaggaaaaa acccaggtga taaaattgtt ttcgcgcttt 480
gcgactagct ttaaagatta cttcaagaac cgtgcaaatt gcttttcagc ggacgatatt 540
tcatcaagca gctgccatcg catcgtcaac gacaatgcag agatattctt ttcaaatgcg 600
ctggtctacc gccggatcgt aaaaaacctg agcaatgacg atatcaacaa aatttcgggc 660
gatatgaaag attcattaaa agaaatgagt ctggaagaaa tatattctta cgagaagtat 720
ggggaattta ttacccagga aggcattagc ttctataatg atatctgtgg gaaagtgaat 780
tcttttatga acctgtattg tcagaaaaat aaagaaaaca aaaatttata caaacttcgt 840
aaacttcaca aacagattct atgcattgcg gacactagct atgaggtccc gtataaattt 900
gaaagtgacg aggaagtgta ccaatcagtt aacggcttcc ttgataacat tagcagcaaa 960
catatagtcg aaagattacg caaaatcggc gataactata acggctacaa cctggataaa 1020
atttatatcg tgtccaaatt ttacgagagc gttagccaaa aaacctaccg cgactgggaa 1080
acaattaata ccgccctcga aattcattac aataatatct tgccgggtaa cggtaaaagt 1140
aaagccgaca aagtaaaaaa agcggttaag aatgatttac agaaatccat caccgaaata 1200
aatgaactag tgtcaaacta taagctgtgc agtgacgaca acatcaaagc ggagacttat 1260
atacatgaga ttagccatat cttgaataac tttgaagcac aggaattgaa atacaatccg 1320
gaaattcacc tagttgaatc cgagctcaaa gcgagtgagc ttaaaaacgt gctggacgtg 1380
atcatgaatg cgtttcattg gtgttcggtt tttatgactg aggaacttgt tgataaagac 1440
aacaattttt atgcggaact ggaggagatt tacgatgaaa tttatccagt aattagtctg 1500
tacaacctgg ttcgtaacta cgttacccag aaaccgtaca gcacgaaaaa gattaaattg 1560
aactttggaa taccgacgtt agcagacggt tggtcaaagt ccaaagagta ttctaataac 1620
gctatcatac tgatgcgcga caatctgtat tatctgggca tctttaatgc gaagaataaa 1680
ccggacaaga agattatcga gggtaatacg tcagaaaata agggtgacta caaaaagatg 1740
atttataatt tgctcccggg tcccaacaaa atgatcccga aagttttctt gagcagcaag 1800
acgggggtgg aaacgtataa accgagcgcc tatatcctag aggggtataa acagaataaa 1860
catctgaagt cttcaaaaga ctttgatatc actttctgtc atgatctgat cgactacttc 1920
aaaaactgta ttgcaattca tcccgagtgg aaaaacttcg gttttgattt tagcgacacc 1980
agtacttatg aagacatttc cgggttttat cgtgaggtag agttacaagg ttacaagatt 2040
gattggacat acattagcga aaaagacatt gatctgctgc aggaaaaagg tcaactgtat 2100
ctgttccaga tatataacaa agatttttcg aaaaaatcaa ccgggaatga caaccttcac 2160
accatgtacc tgaaaaatct tttctcagaa gaaaatctta aggatatcgt cctgaaactt 2220
aacggcgaag cggaaatctt cttcaggaag agcagcataa agaacccaat cattcataaa 2280
aaaggctcga ttttagtcaa ccgtacctac gaagcagaag aaaaagacca gtttggcaac 2340
attcaaattg tgcgtaaaac cattccggaa aacatttatc aggagctgta caaatacttc 2400
aacgataaaa gcgacaaaga gctgtctgat gaagcagcca aactgaagaa tgtagtggga 2460
caccacgagg cagcgacgaa tatagtcaag gactatcgct acacgtatga taaatacttc 2520
cttcatatgc ctattacgat caatttcaaa gccaataaaa cgagctttat taatgatagg 2580
atcttacagt atatcgctaa agaaaaagac ttacatgtga tcggcattga tcggggcgag 2640
cgtaacctga tctacgtgtc cgtgattgat acttgtggta atatagttga acagaaaagc 2700
tttaacattg taaacggcta cgactatcag ataaaactga aacaacagga gggcgctaga 2760
cagattgcgc ggaaagaatg gaaagaaatt ggtaaaatta aagagatcaa agagggctac 2820
ctgagcttag taatccacga gatctctaaa atggtaatca aatacaatgc aattatagcg 2880
atggaggatt tgtcttatgg ttttaaaaaa gggcgcttta aggtcgaacg gcaagtttac 2940
cagaaatttg aaaccatgct catcaataaa ctcaactatc tggtatttaa agatatttcg 3000
attaccgaga atggcggtct cctgaaaggt tatcagctga catacattcc tgataaactt 3060
aaaaacgtgg gtcatcagtg cggctgcatt ttttatgtgc ctgctgcata cacgagcaaa 3120
attgatccga ccaccggctt tgtgaatatc tttaaattta aagacctgac agtggacgca 3180
aaacgtgaat tcattaaaaa atttgactca attcgttatg acagtgaaaa aaatctgttc 3240
tgctttacat ttgactacaa taactttatt acgcaaaaca cggtcatgag caaatcatcg 3300
tggagtgtgt atacatacgg cgtgcgcatc aaacgtcgct ttgtgaacgg ccgcttctca 3360
aacgaaagtg ataccattga cataaccaaa gatatggaga aaacgttgga aatgacggac 3420
attaactggc gcgatggcca cgatcttcgt caagacatta tagattatga aattgttcag 3480
cacatattcg aaattttcaa attaacagtg caaatgcgta actccttgtc tgaactggag 3540
gaccgtgatt acgatcgtct catttcacct gtactgaacg aaaataacat tttttatgac 3600
agcgcgaaag cgggggatgc acttcctaag gatgccgatg caaatggtgc gtattgtatt 3660
gcattaaaag ggttatatga aattaaacaa attaccgaaa attggaaaga agatggtaaa 3720
ttttcgcgcg ataaactcaa aatcagcaat aaagattggt tcgactttat ccagaataag 3780
cgctatctct aa 3792
<210> 7
<211> 4005
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD7核酸序列
<400> 7
atgtcatcgc tcacgaaatt cactaacaaa tactctaaac agctcaccat taagaatgaa 60
ctcatcccag ttggcaaaac actggagaac atcaaagaga atggtctgat agatggcgac 120
gaacagctga atgagaatta tcagaaggcg aaaattattg tggatgattt tctgcgggac 180
ttcattaata aagcactgaa taatacgcag atcgggaact ggcgcgaact ggcggatgcc 240
cttaataaag aggatgaaga taacatcgag aaattgcagg ataaaattcg gggaatcatt 300
gtatccaaat ttgaaacgtt tgatctgttt agcagctatt ctattaagaa agatgaaaag 360
attattgacg acgacaatga tgttgaagaa gaggaactgg atctgggcaa gaagaccagc 420
tcatttaaat acatatttaa aaaaaacctg tttaagttag tgttgccatc ctacctgaaa 480
accacaaacc aggacaagct gaagattatt agctcgtttg ataatttttc aacgtacttc 540
cgcgggttct ttgaaaaccg gaaaaacatt tttaccaaga aaccgatctc cacaagtatt 600
gcgtatcgca ttgttcatga taacttcccg aaattccttg ataacattcg ttgttttaat 660
gtgtggcaga cggaatgccc gcaactaatc gtgaaagcag ataactatct gaaaagcaaa 720
aatgttatag cgaaagataa aagtttggca aactatttta ccgtgggcgc gtatgactat 780
ttcctgtctc agaatggtat agatttttac aacaatatta taggtggact gccagcgttc 840
gccggccatg agaaaatcca aggtctcaat gaattcatca atcaagagtg ccaaaaagac 900
agcgagctga aaagtaagct gaaaaaccgt cacgcgttca aaatggcggt actgttcaaa 960
cagatactca gcgatcgtga aaaaagtttt gtaattgatg agttcgagtc ggatgctcaa 1020
gttattgacg ccgttaaaaa cttttacgcc gaacagtgca aagataacaa tgttattttt 1080
aacttattaa atcttatcaa gaatatcgct ttcttaagtg atgacgaact ggacggcata 1140
ttcattgaag ggaaatacct gtcgagcgtt agtcaaaaac tctatagcga ttggtcaaaa 1200
ttacgtaacg acattgagga ttcggctaac tctaaacaag gcaataaaga gctggccaag 1260
aagatcaaaa ccaacaaagg ggatgtagaa aaagcgatct cgaaatatga gttctcgctg 1320
tcggaactga actcgattgt acatgataac accaagtttt ctgacctcct tagttgtaca 1380
ctgcataagg tggcttctga gaaactggtg aaggtcaatg aaggcgactg gccgaaacat 1440
ctcaagaata atgaagagaa acaaaaaatc aaagagccgc ttgatgctct gctggagatc 1500
tataatacac ttctgatttt taactgcaaa agcttcaata aaaacggcaa cttctatgtc 1560
gactatgatc gttgcatcaa tgaactgagt tcggtcgtgt atctgtataa taaaacacgt 1620
aactattgca ctaaaaaacc ctataacacg gacaagttca aactcaattt taacagtccg 1680
cagctcggtg aaggcttttc caagtcgaaa gaaaatgact gtctgactct tttgtttaaa 1740
aaagacgaca actattatgt aggcattatc cgcaaaggtg caaaaatcaa ttttgatgat 1800
acacaagcaa tcgccgataa caccgacaat tgcatcttta aaatgaatta tttcctactt 1860
aaagacgcaa aaaaatttat cccgaaatgt agcattcagc tgaaagaagt caaggcccat 1920
tttaagaaat ctgaagatga ttacattttg tctgataaag agaaatttgc tagcccgctg 1980
gtcattaaaa agagcacatt tttgctggca actgcacatg tgaaagggaa aaaaggcaat 2040
atcaagaaat ttcagaaaga atattcgaaa gaaaacccca ctgagtatcg caattcttta 2100
aacgaatgga ttgctttttg taaagagttc ttaaaaactt ataaagcggc taccattttt 2160
gatataacca cattgaaaaa ggcagaggaa tatgctgata ttgtagaatt ctacaaggat 2220
gtcgataatc tgtgctacaa actggagttc tgcccgatta aaacctcgtt tatagaaaac 2280
ctgatagata acggcgacct gtatctgttt cgcatcaata acaaagactt cagcagtaaa 2340
tcgaccggca ccaagaacct tcatacgtta tatttacaag ctatattcga tgaacgtaat 2400
ctgaacaatc cgacaattat gctgaatggg ggagcagaac tgttctatcg taaagaaagt 2460
attgagcaga aaaaccgtat cacacacaaa gccggttcaa ttctcgtgaa taaggtgtgt 2520
aaagacggta caagcctgga tgataagata cgtaatgaaa tttatcaata tgagaataaa 2580
tttattgata ccctgtctga tgaagctaaa aaggtgttac cgaatgtcat taaaaaggaa 2640
gctacccatg acattacaaa agataaacgt ttcactagtg acaaattctt ctttcactgc 2700
cccctgacaa ttaattataa ggaaggcgat accaagcagt tcaataacga agtgctgagt 2760
tttctgcgtg gaaatcctga catcaacatt atcggcattg accgcggaga gcgtaattta 2820
atctatgtaa cggttataaa ccagaaaggc gagattctgg attcggtttc attcaatacc 2880
gtgaccaaca agagttcaaa aatcgagcag acagtcgatt atgaagagaa attggcagtc 2940
cgcgagaaag agaggattga agcaaaacgt tcctgggact ctatctcaaa aattgcgaca 3000
ctaaaggaag gttatctgag cgcaatagtt cacgagatct gtctgttaat gattaaacac 3060
aacgcgatcg ttgtcttaga gaatcttaat gcaggcttta agcgtattcg tggcggttta 3120
tcagaaaaaa gtgtttatca aaaattcgaa aaaatgttga ttaacaaact gaactatttt 3180
gtcagcaaga aggaatccga ctggaataaa ccgtctggtc tgctgaatgg actgcagctt 3240
tcggatcagt ttgaaagctt cgaaaaactg ggtattcagt ctggttttat tttttacgtg 3300
ccggctgcat atacctcaaa gattgatccg accacgggct tcgccaatgt tctgaatctg 3360
tcgaaggtac gcaatgttga tgcgatcaaa agcttttttt ctaacttcaa cgaaattagt 3420
tatagcaaga aagaagccct tttcaaattc tcattcgatc tggattcact gagtaagaaa 3480
ggctttagta gctttgtgaa atttagtaag agtaaatgga acgtctacac ctttggagaa 3540
cgtatcataa agccaaagaa taagcaaggt tatcgggagg acaaaagaat caacttgacc 3600
ttcgagatga agaagttact taacgagtat aaggtttctt ttgatcttga aaataacttg 3660
attccgaatc tcacgagtgc caacctgaag gatacttttt ggaaagagct attctttatc 3720
ttcaagacta cgctgcagct ccgtaacagc gttactaacg gtaaagaaga tgtgctcatc 3780
tctccggtca aaaatgcgaa gggtgaattc ttcgtttcgg gaacgcataa caagactctt 3840
ccgcaagatt gcgatgcgaa cggtgcatac catattgcgt tgaaaggtct gatgatactc 3900
gaacgtaaca accttgtacg tgaggagaaa gatacgaaaa agattatggc gatttcaaac 3960
gtggattggt tcgagtacgt gcagaaacgt agaggcgttc tgtaa 4005
<210> 8
<211> 3717
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 8
atgaacaact acgacgaatt caccaaactg tacccgatcc agaaaaccat ccgtttcgaa 60
ctgaaaccgc agggtcgtac catggaacac ctggaaacct tcaacttctt cgaagaagac 120
cgtgaccgtg cggaaaaata caaaatcctg aaagaagcga tcgacgaata ccacaaaaaa 180
ttcatcgacg aacacctgac caacatgtct ctggactgga actctctgaa acagatctct 240
gaaaaatact acaaatctcg tgaagaaaaa gacaaaaaag ttttcctgtc tgaacagaaa 300
cgtatgcgtc aggaaatcgt ttctgaattc aaaaaagacg accgtttcaa agacctgttc 360
tctaaaaaac tgttctctga actgctgaaa gaagaaatct acaaaaaagg taaccaccag 420
gaaatcgacg cgctgaaatc tttcgacaaa ttctctggtt acttcatcgg tctgcacgaa 480
aaccgtaaaa acatgtactc tgacggtgac gaaatcaccg cgatctctaa ccgtatcgtt 540
aacgaaaact tcccgaaatt cctggacaac ctgcagaaat accaggaagc gcgtaaaaaa 600
tacccggaat ggatcatcaa agcggaatct gcgctggttg cgcacaacat caaaatggac 660
gaagttttct ctctggaata cttcaacaaa gttctgaacc aggaaggtat ccagcgttac 720
aacctggcgc tgggtggtta cgttaccaaa tctggtgaaa aaatgatggg tctgaacgac 780
gcgctgaacc tggcgcacca gtctgaaaaa tcttctaaag gtcgtatcca catgaccccg 840
ctgttcaaac agatcctgtc tgaaaaagaa tctttctctt acatcccgga cgttttcacc 900
gaagactctc agctgctgcc gtctatcggt ggtttcttcg cgcagatcga aaacgacaaa 960
gacggtaaca tcttcgaccg tgcgctggaa ctgatctctt cttacgcgga atacgacacc 1020
gaacgtatct acatccgtca ggcggacatc aaccgtgttt ctaacgttat cttcggtgaa 1080
tggggtaccc tgggtggtct gatgcgtgaa tacaaagcgg actctatcaa cgacatcaac 1140
ctggaacgta cctgcaaaaa agttgacaaa tggctggact ctaaagaatt cgcgctgtct 1200
gacgttctgg aagcgatcaa acgtaccggt aacaacgacg cgttcaacga atacatctct 1260
aaaatgcgta ccgcgcgtga aaaaatcgac gcggcgcgta aagaaatgaa attcatctct 1320
gaaaaaatct ctggtgacga agaatctatc cacatcatca aaaccctgct ggactctgtt 1380
cagcagttcc tgcacttctt caacctgttc aaagcgcgtc aggacatccc gctggacggt 1440
gcgttctacg cggaattcga cgaagttcac tctaaactgt tcgcgatcgt tccgctgtac 1500
aacaaagttc gtaactacct gaccaaaaac aacctgaaca ccaaaaaaat caaactgaac 1560
ttcaaaaacc cgaccctggc gaacggttgg gaccagaaca aagtttacga ctacgcgtct 1620
ctgatcttcc tgcgtgacgg taactactac ctgggtatca tcaacccgaa acgtaaaaaa 1680
aacatcaaat tcgaacaggg ttctggtaac ggtccgttct accgtaaaat ggtttacaaa 1740
cagatcccgg gtccgaacaa aaacctgccg cgtgttttcc tgacctctac caaaggtaaa 1800
aaagaataca aaccgtctaa agaaatcatc gaaggttacg aagcggacaa acacatccgt 1860
ggtgacaaat tcgacctgga cttctgccac aaactgatcg acttcttcaa agaatctatc 1920
gaaaaacaca aagactggtc taaattcaac ttctacttct ctccgaccga atcttacggt 1980
gacatctctg aattctacct ggacgttgaa aaacagggtt accgtatgca cttcgaaaac 2040
atctctgcgg aaaccatcga cgaatacgtt gaaaaaggtg acctgttcct gttccagatc 2100
tacaacaaag acttcgttaa agcggcgacc ggtaaaaaag acatgcacac catctactgg 2160
aacgcggcgt tctctccgga aaacctgcag gacgttgttg ttaaactgaa cggtgaagcg 2220
gaactgttct accgtgacaa atctgacatc aaagaaatcg ttcaccgtga aggtgaaatc 2280
ctggttaacc gtacctacaa cggtcgtacc ccggttccgg acaaaatcca caaaaaactg 2340
accgactacc acaacggtcg taccaaagac ctgggtgaag cgaaagaata cctggacaaa 2400
gttcgttact tcaaagcgca ctacgacatc accaaagacc gtcgttacct gaacgacaaa 2460
atctacttcc acgttccgct gaccctgaac ttcaaagcga acggtaaaaa aaacctgaac 2520
aaaatggtta tcgaaaaatt cctgtctgac gaaaaagcgc acatcatcgg tatcgaccgt 2580
ggtgaacgta acctgctgta ctactctatc atcgaccgtt ctggtaaaat catcgaccag 2640
cagtctctga acgttatcga cggtttcgac taccgtgaaa aactgaacca gcgtgaaatc 2700
gaaatgaaag acgcgcgtca gtcttggaac gcgatcggta aaatcaaaga cctgaaagaa 2760
ggttacctgt ctaaagcggt tcacgaaatc accaaaatgg cgatccagta caacgcgatc 2820
gttgttatgg aagaactgaa ctacggtttc aaacgtggtc gtttcaaagt tgaaaaacag 2880
atctaccaga aattcgaaaa catgctgatc gacaaaatga actacctggt tttcaaagac 2940
gcgccggacg aatctccggg tggtgttctg aacgcgtacc agctgaccaa cccgctggaa 3000
tctttcgcga aactgggtaa acagaccggt atcctgttct acgttccggc ggcgtacacc 3060
tctaaaatcg acccgaccac cggtttcgtt aacctgttca acacctcttc taaaaccaac 3120
gcgcaggaac gtaaagaatt cctgcagaaa ttcgaatcta tctcttactc tgcgaaagac 3180
ggtggtatct tcgcgttcgc gttcgactac cgtaaattcg gtacctctaa aaccgaccac 3240
aaaaacgttt ggaccgcgta caccaacggt gaacgtatgc gttacatcaa agaaaaaaaa 3300
cgtaacgaac tgttcgaccc gtctaaagaa atcaaagaag cgctgacctc ttctggtatc 3360
aaatacgacg gtggtcagaa catcctgccg gacatcctgc gttctaacaa caacggtctg 3420
atctacacca tgtactcttc tttcatcgcg gcgatccaga tgcgtgttta cgacggtaaa 3480
gaagactaca tcatctctcc gatcaaaaac tctaaaggtg aattcttccg taccgacccg 3540
aaacgtcgtg aactgccgat cgacgcggac gcgaacggtg cgtacaacat cgcgctgcgt 3600
ggtgaactga ccatgcgtgc gatcgcggaa aaattcgacc cggactctga aaaaatggcg 3660
aaactggaac tgaaacacaa agactggttc gaattcatgc agacccgtgg tgactaa 3717
<210> 9
<211> 3897
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 9
atgactaaaa catttgattc agagtttttt aatttgtact cgctgcaaaa aacggtacgc 60
tttgagttaa aacccgtggg agaaaccgcg tcatttgtgg aagactttaa aaacgagggc 120
ttgaaacgtg ttgtgagcga agatgaaagg cgagccgtcg attaccagaa agttaaggaa 180
ataattgacg attaccatcg ggatttcatt gaagaaagtt taaattattt tccggaacag 240
gtgagtaaag atgctcttga gcaggcgttt catctttatc agaaactgaa ggcagcaaaa 300
gttgaggaaa gggaaaaagc gctgaaagaa tgggaagcgc tgcagaaaaa gctacgtgaa 360
aaagtggtga aatgcttctc ggactcgaat aaagcccgct tctcaaggat tgataaaaag 420
gaactgatta aggaagacct gataaattgg ttggtcgccc agaatcgcga ggatgatatc 480
cctacggtcg aaacgtttaa caacttcacc acatatttta ccggcttcca tgagaatcgt 540
aaaaatattt actccaaaga tgatcacgcc accgctatta gctttcgcct tattcatgaa 600
aatcttccaa agttttttga caacgtgatt agcttcaata agttgaaaga gggtttccct 660
gaattaaaat ttgataaagt gaaagaggat ttagaagtag attatgatct gaagcatgcg 720
tttgaaatag aatatttcgt taacttcgtg acccaagcgg gcatagatca gtataattat 780
ctgttaggag ggaaaaccct ggaggacggg acgaaaaaac aagggatgaa tgagcaaatt 840
aatctgttca aacaacagca aacgcgagat aaagcgcgtc agattcccaa actgatcccc 900
ctgttcaaac agattcttag cgaaaggact gaaagccagt cctttattcc taaacaattt 960
gaaagtgatc aggagttgtt cgattcactg cagaagttac ataataactg ccaggataaa 1020
ttcaccgtgc tgcaacaagc cattctcggt ctggcagagg cggatcttaa gaaggtcttc 1080
atcaaaacct ctgatttaaa tgccttatct aacaccattt tcgggaatta cagcgtcttt 1140
tccgatgcac tgaacctgta taaagaaagc ctgaaaacga aaaaagcgca ggaggctttt 1200
gagaaactac cggcccattc tattcacgac ctcattcaat acttggaaca gttcaattcc 1260
agcctggacg cggaaaaaca acagagcacc gacaccgtcc tgaactactt catcaagacc 1320
gatgaattat attctcgctt cattaaatcc actagcgagg ctttcactca ggtgcagcct 1380
ttgttcgaac tggaagccct gtcatctaag cgccgcccac cggaatcgga agatgaaggg 1440
gcaaaagggc aggaaggctt cgagcagatc aagcgtatta aagcttacct ggatacgctt 1500
atggaagcgg tacactttgc aaagccgttg tatcttgtta agggtcgtaa aatgatcgaa 1560
gggctcgata aagaccagtc cttttatgaa gcgtttgaaa tggcgtacca agaacttgaa 1620
tcgttaatca ttcctatcta taacaaagcg cggagctatc tgtcgcggaa acctttcaag 1680
gccgataaat tcaagattaa ttttgacaac aacacgctac tgagcggatg ggatgcgaac 1740
aaggaaactg ctaacgcgtc cattctgttt aagaaagacg ggttatatta ccttggaatt 1800
atgccgaaag gtaagacctt tctctttgac tactttgtat cgagcgagga ttcagagaaa 1860
ctgaaacagc gtcgccagaa gaccgccgaa gaagctctgg cgcaggatgg tgaaagttac 1920
ttcgaaaaaa ttcgttataa actgttacca ggggcttcaa agatgttacc gaaagtcttt 1980
tttagcaaca aaaatattgg cttttacaac ccgtcggatg acattttacg cattcgcaac 2040
acagcctctc acaccaaaaa cgggacccct cagaaaggcc actcaaaagt tgagtttaac 2100
ctgaatgatt gtcataagat gattgatttc ttcaaatcat caattcagaa acacccggaa 2160
tgggggtctt ttggctttac gttttctgat accagtgatt ttgaagacat gagtgccttc 2220
taccgggaag tagaaaacca gggttacgta attagctttg acaaaatcaa agagacctat 2280
atacagagcc aggtggaaca gggtaatctc tacttattcc agatttataa caaggatttc 2340
tcgccctaca gcaaaggcaa accaaacctg catactctgt actggaaagc cctgtttgaa 2400
gaagcgaacc tgaataacgt agtggcgaag ttgaacggtg aagcggaaat cttcttccgt 2460
cgtcactcca ttaaggcctc tgataaagtt gtccatccgg caaatcaggc cattgataat 2520
aagaatccac acacggaaaa aacgcagtca acctttgaat atgacctcgt taaagacaaa 2580
cgctacacgc aagataagtt ctttttccac gtcccaatca gcctcaactt taaagcacaa 2640
ggggtttcaa agtttaatga taaagtcaat gggttcctca agggcaaccc ggatgtcaac 2700
attataggta tagacagggg cgaacgccat ctgctttact ttaccgtagt gaatcagaaa 2760
ggtgaaatac tggttcagga atcattaaat accttgatgt cggacaaagg gcacgttaat 2820
gattaccagc agaaactgga taaaaaagaa caggaacgtg atgctgcgcg taaatcgtgg 2880
accacggttg agaacattaa agagctgaaa gaggggtatc taagccatgt ggtacacaaa 2940
ctggcgcacc tcatcattaa atataacgca atagtctgcc tagaagactt gaattttggc 3000
tttaaacgcg gccgcttcaa agtggaaaaa caagtttatc aaaaatttga aaaggcgctt 3060
atagataaac tgaattatct ggtttttaaa gaaaaggaac ttggtgaggt agggcactac 3120
ttgacagctt atcaactgac ggccccgttc gaatcattca aaaaactggg caaacagtct 3180
ggcattctgt tttacgtgcc ggcagattat acttcaaaaa tcgatccaac aactggcttt 3240
gtgaacttcc tggacctgag atatcagtct gtagaaaaag ctaaacaact tcttagcgat 3300
tttaatgcca ttcgttttaa cagcgttcag aattactttg aattcgaaat tgactataaa 3360
aaacttactc cgaaacgtaa agtcggaacc caaagtaaat gggtaatttg tacgtatggc 3420
gatgtcaggt atcagaaccg tcggaatcaa aaaggtcatt gggagaccga agaagtgaac 3480
gtgaccgaaa agctgaaggc tctgttcgcc agcgattcaa aaactacaac tgtgatcgat 3540
tacgcaaatg atgataacct gatagatgtg attttagagc aggataaagc cagctttttt 3600
aaagaactgt tgtggctcct gaaacttacg atgaccttac gacattccaa gatcaaatcg 3660
gaagatgatt ttattctgtc accggtcaag aatgagcagg gtgaattcta tgatagtagg 3720
aaagccggcg aagtgtggcc gaaagacgcc gacgccaatg gcgcctatca tatcgcgctc 3780
aaagggcttt ggaatttgca gcagattaac cagtgggaaa aaggtaaaac cctgaatctg 3840
gctatcaaaa accaggattg gtttagcttt atccaagaga aaccgtatca ggaatga 3897
<210> 10
<211> 3708
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 10
atgcatacag gcggtcttct tagtatggac gcgaaagagt tcacaggtca gtatccgttg 60
tcgaaaacat tacgattcga acttcggccc atcggccgca cgtgggataa cctggaggcc 120
tcaggctact tagcggaaga ccgccatcgt gccgaatgtt atcctcgtgc gaaagagtta 180
ttggatgaca accatcgtgc cttcctgaat cgtgtgttgc cacaaatcga tatggattgg 240
cacccgattg cggaggcctt ttgtaaggta cataaaaacc ctggtaataa agaacttgcc 300
caggattaca accttcagtt gtcaaagcgc cgtaaggaga tcagcgcata tcttcaggat 360
gcagatggct ataaaggcct gttcgcgaag cccgccttag acgaagctat gaaaattgcg 420
aaagaaaacg ggaacgaaag tgatattgag gttctcgaag cgtttaacgg ttttagcgta 480
tacttcaccg gttatcatga gtcacgcgag aacatttata gcgatgagga tatggtgagc 540
gtagcctacc gaattactga ggataatttc ccgcgctttg tctcaaacgc tttgatcttt 600
gataaattaa acgaaagcca tccggatatt atctctgaag tatcgggcaa tcttggagtt 660
gatgacattg gtaagtactt tgacgtgtcg aactataaca attttctttc ccaggccggt 720
atagatgact acaatcacat tattggcggc catacaaccg aagacggact gatacaagcg 780
tttaatgtcg tattgaactt acgtcaccaa aaagaccctg gctttgaaaa aattcagttc 840
aaacagctct acaaacaaat cctgagcgtg cgtaccagca aaagctacat cccgaaacag 900
tttgacaact ctaaggagat ggttgactgc atttgcgatt atgtcagcaa aatagagaaa 960
tccgaaacag tagaacgggc cctgaaacta gtccgtaata tcagttcttt cgacttgcgc 1020
gggatctttg tcaataaaaa gaacttgcgc atactgagca acaaactgat aggagattgg 1080
gacgcgatcg aaaccgcatt gatgcatagt tcttcatcag aaaacgataa gaaaagcgta 1140
tatgatagcg cggaggcttt tacgttggat gacatctttt caagcgtgaa aaaattttct 1200
gatgcctctg ccgaagatat tggcaacagg gcggaagaca tctgtagagt gataagtgag 1260
acggcccctt ttatcaacga tctgcgagcg gtggacctgg atagcctgaa cgacgatggt 1320
tatgaagcgg ccgtctcaaa aattcgggag tcgctggagc cttatatgga tcttttccat 1380
gaactggaaa ttttctcggt tggcgatgag ttcccaaaat gcgcagcatt ttacagcgaa 1440
ctggaggaag tcagcgaaca gctgatcgaa attattccgt tattcaacaa ggcgcgttcg 1500
ttctgcaccc ggaaacgcta tagcaccgat aagattaaag tgaacttaaa attcccgacc 1560
ttggcggacg ggtgggacct gaacaaagag agagacaaca aagccgcgat tctgcggaaa 1620
gacggtaagt attatctggc aattctggat atgaagaaag atctgtcaag cattaggacc 1680
agcgacgaag atgaatccag cttcgaaaag atggagtata aactgttacc gagtccagta 1740
aaaatgctgc caaagatatt cgtaaaatcg aaagccgcta aggaaaaata tggcctgaca 1800
gatcgtatgc ttgaatgcta cgataaaggt atgcataagt cgggtagtgc gtttgatctt 1860
ggcttttgcc atgaactcat tgattattac aagcgttgta tcgcggagta cccaggctgg 1920
gatgtgttcg atttcaagtt tcgcgaaact tccgattatg ggtccatgaa agagttcaat 1980
gaagatgtgg ccggagccgg ttactatatg agtctgagaa aaattccgtg cagcgaagtg 2040
taccgtctgt tagacgagaa atcgatttat ctatttcaaa tttataacaa agattactct 2100
gaaaatgcac atggtaataa gaacatgcat accatgtact gggagggtct cttttccccg 2160
caaaacctgg agtcgcccgt tttcaagttg tcgggtgggg cagaactttt ctttcgaaaa 2220
tcctcaatcc ctaacgatgc caaaacagta cacccgaaag gctcagtgct ggttccacgt 2280
aatgatgtta acggtcggcg tattccagat tcaatctacc gcgaactgac acgctatttt 2340
aaccgtggcg attgccgaat cagtgacgaa gccaaaagtt atcttgacaa ggttaagact 2400
aaaaaagcgg accatgacat tgtgaaagat cgccgcttta ccgtggataa aatgatgttc 2460
cacgtcccga ttgcgatgaa ctttaaggcg atcagtaaac cgaacttaaa caaaaaagtc 2520
attgatggca tcattgatga tcaggatctg aaaatcattg gtattgatcg tggcgagcgg 2580
aacttaattt acgtcacgat ggttgacaga aaagggaata tcttatatca ggattctctt 2640
aacatcctca atggctacga ctatcgtaaa gctctggatg tgcgcgaata tgacaacaag 2700
gaagcgcgtc gtaactggac taaagtggag ggcattcgca aaatgaagga aggctatctg 2760
tcattagcgg tctcgaaatt agcggatatg attatcgaaa ataacgccat catcgttatg 2820
gaggacctga accacggatt caaagcgggc cgctcaaaga ttgaaaaaca agtttatcag 2880
aaatttgaga gtatgctgat taacaaactg ggctatatgg tgttaaaaga caagtcaatt 2940
gaccaatcag gtggcgcgct gcatggatac cagctggcga accatgttac caccttagca 3000
tcagttggaa agcagtgtgg ggttatcttt tatataccgg cagcgttcac tagtaaaata 3060
gatccgacca ctggtttcgc cgatctcttt gccctgagta acgttaaaaa cgtagcgagc 3120
atgcgtgaat tcttttccaa aatgaaatct gtcatttatg ataaagctga aggcaaattc 3180
gcattcacct ttgattactt ggattacaac gtgaagagcg aatgtggtcg tacgctgtgg 3240
accgtttaca ccgttggtga gcgcttcacc tattcccgtg tgaaccgcga atatgtacgt 3300
aaagtcccca ccgatattat ctatgatgcc ctccagaaag caggcattag cgtcgaagga 3360
gacttaaggg acagaattgc cgaaagcgat ggcgatacgc tgaagtctat tttttacgca 3420
ttcaaatacg cgctagatat gcgcgttgag aatcgcgagg aagactacat tcaatcacct 3480
gtgaaaaatg cctctgggga atttttttgt tcaaaaaatg ctggtaaaag cctcccacaa 3540
gatagcgatg caaacggtgc atataacatt gccctgaaag gtattcttca attacgcatg 3600
ctgtctgagc agtacgaccc caacgcggaa tctattagac ttccgctgat aaccaataaa 3660
gcctggctga cattcatgca gtctggcatg aagacctgga aaaattag 3708
<210> 11
<211> 3783
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 11
atggatagtt taaaagattt tacgaatcta tatcccgtaa gcaaaactct tcgttttgaa 60
ctgaaacctg ttggaaaaac gttggagaat atcgagaaag cgggcatcct gaaagaagac 120
gagcaccgtg ccgaaagcta caggcgtgtc aaaaagatta tcgatactta tcacaaagtg 180
ttcattgata gcagtctgga gaacatggca aaaatgggca tagaaaatga aatcaaagca 240
atgctgcaga gcttttgcga gctctacaag aaagatcacc gaacggaagg tgaagataaa 300
gcactggaca aaattcgcgc cgttcttcgc ggtctgattg ttggcgcgtt caccggcgtg 360
tgcggccgcc gtgaaaacac cgtgcagaac gaaaagtacg agtcgctgtt caaagaaaaa 420
ctgataaaag aaattttgcc tgactttgtg ctttcgaccg aagcggaatc cctgccattt 480
tctgtcgaag aagcgacccg cagcctgaaa gaatttgact cattcacaag ttactttgca 540
ggcttctacg aaaaccgtaa aaacatctac agcacgaagc cacagagcac ggctattgct 600
tatcgcctga ttcatgagaa cctgccgaag ttcatcgata acatccttgt ttttcaaaaa 660
attaaagagc cgattgcgaa agagttagaa catattcgag ctgacttttc tgcgggtggg 720
tacattaaaa aagatgagcg gctggaagac atcttcagtc taaactatta tatccacgtt 780
ctgtcgcagg caggcattga gaaatataat gcgctgattg gtaagattgt cacagaaggc 840
gatggtgaga tgaaaggtct taatgaacat atcaatctgt ataaccagca gcgtggtcgc 900
gaagaccgtc ttccactgtt ccgcccactg tataaacaga tcctgtctga ccgggaacag 960
ctgtcctacc tgccggaaag ctttgaaaag gatgaagagc tacttcgcgc attaaaggag 1020
ttttacgacc atattgcgga agacattttg ggtagaacgc agcaactgat gacgtcaatt 1080
tctgaatacg atctgagtag aatctacgtt aggaatgata gccagctgac cgatattagc 1140
aaaaaaatgc tgggcgactg gaacgctatc tatatggcac gtgaacgtgc atatgatcat 1200
gaacaagcac cgaaacgtat aaccgcgaaa tatgagcgtg atcgcattaa ggcgctaaag 1260
ggagaagaaa gcatctcact cgcaaacctg aactcctgta tcgctttctt agataacgtg 1320
cgcgattgtc gcgtcgacac gtatctgtca acccttgggc agaaagaggg tccacatggt 1380
ctgtctaacc tggtggaaaa tgtctttgcg agttaccatg aagcggaaca actgctgtct 1440
tttccatacc ccgaagaaaa caatctaata caggataaag ataacgtggt gttaatcaaa 1500
aacctgctgg acaacatcag cgatctgcaa cgtttcctga aacctttgtg gggtatgggt 1560
gacgagccag acaaagacga acgtttttat ggtgagtata attatatacg tggcgccctt 1620
gaccaagtta ttccgctgta taacaaagta cggaactatc tgacccgtaa gccatattct 1680
acccgtaaag tgaaactgaa ctttggcaac tcgcaactgc tgtcgggttg ggatcgtaac 1740
aaagaaaaag ataatagttg tgttatcctg cgtaagggac aaaattttta cctcgcgatt 1800
atgaacaaca gacacaagcg ttcatttgaa aataaggttc tgccggagta taaagagggc 1860
gaaccgtact tcgagaaaat ggattataag ttcttaccag accctaataa gatgttaccg 1920
aaagtctttc tttcgaaaaa aggcatagaa atctataagc cgtccccgaa attactcgaa 1980
cagtatgggc acgggaccca caagaaaggg gatactttta gcatggacga tctgcacgaa 2040
ctgatcgatt tttttaaaca ctccatcgaa gcccatgaag actggaaaca gtttgggttc 2100
aagttctctg atacagccac atacgagaat gtgtctagtt tttatcggga agtggaggat 2160
cagggctaca aacttagttt tcgtaaagtt tcagagagtt atgtttatag tttaattgat 2220
cagggaaaac tttacctgtt ccagatctac aacaaagatt tctcgccatg tagtaagggt 2280
accccgaatc tgcatacact ctattggaga atgttattcg atgagcgtaa cttagcggat 2340
gtcatttata aattggacgg gaaagcagag atcttttttc gtgaaaaatc actgaagaat 2400
gaccacccga ctcatccggc cgggaaaccg atcaaaaaaa aatcccgcca gaaaaaagga 2460
gaagagtctc tgtttgaata tgatctggtg aaagaccgtc attacactat ggataaattt 2520
caatttcatg ttccaattac aatgaacttc aaatgttcgg cgggttccaa agtaaatgat 2580
atggtaaacg cccatattcg cgaagcgaaa gatatgcatg ttattggcat cgatagaggc 2640
gaaagaaacc tgctttatat ttgcgtaatt gacagccgtg gtaccattct ggaccagatc 2700
tctttaaaca ccatcaatga catcgattat cacgacctgt tggagtctcg ggacaaggac 2760
cgccagcagg agcgccgtaa ttggcagaca attgaaggca taaaagaatt aaaacagggt 2820
tacctttccc aggccgtaca ccgcatagcg gaactgatgg tggcctacaa agccgtagtt 2880
gccctggaag acttgaatat ggggtttaaa cgtggccgtc aaaaagtcga gagcagcgtg 2940
tatcagcaat ttgaaaaaca gttgattgac aagttgaatt atttggttga taaaaagaaa 3000
cgtccagaag atattggtgg cttactgcgt gcataccagt ttacggcacc ttttaagtcc 3060
ttcaaagaaa tgggtaaaca gaacgggttt ctgttttaca tcccggcctg gaatacatcc 3120
aacatcgatc ctaccaccgg gtttgtcaac ctgtttcatg cacaatatga aaacgtggat 3180
aaagcgaaga gttttttcca aaaattcgat agtatttcgt ataacccaaa aaaagattgg 3240
tttgagtttg cgttcgatta taaaaatttt actaaaaagg ctgagggatc ccgcagtatg 3300
tggatcctct gcacccatgg cagtcgtatt aaaaattttc gtaattcgca aaagaatggc 3360
cagtgggact cggaagagtt tgccctgacc gaagcgttca aatcgctgtt tgtacgctac 3420
gaaattgact acacagcaga tctgaaaaca gccatcgtcg atgaaaaaca gaaagatttt 3480
tttgtagatc tcctaaaact gttcaaactg actgttcaga tgcgcaattc ctggaaagag 3540
aaagacctgg attatctgat tagcccggta gccggtgctg atggacgatt tttcgatact 3600
cgtgaaggta acaaaagtct cccgaaagat gctgatgcca atggtgcata caatattgca 3660
ttaaaggggc tatgggcctt gcgacagatc cgccagacca gcgaaggcgg caagctgaaa 3720
ttggccatat cgaataagga atggttacaa tttgttcagg aacgtagcta tgaaaaagat 3780
tga 3783
<210> 12
<211> 3923
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 12
tgactcagtt tgagggtttt accaatctgt accaagtgtc aaaaaccctg cgttttgagt 60
taatacctca gggtaaaaca ctcaagcaca ttcaggagca aggtttcatc gaagaggaca 120
aagcgcgcaa tgaccattat aaagaactga aacccatcat tgaccgtatt tataaaacct 180
atgcagatca gtgcctgcag ctggtgcaac ttgattggga gaaccttagc gccgccatcg 240
acagttatcg taaagagaaa acagaagaaa cgcgtaacgc gttaatcgaa gaacaagcga 300
catatcgtaa cgctatccac gattacttta ttggtcgaac tgataatctg actgacgcta 360
tcaataaacg gcatgcggaa atctacaaag gtctattcaa agcggaacta tttaacggta 420
aggtcctgaa acaactgggg accgtaacga ccacagagca tgagaacgcc ctcctgcgct 480
ctttcgataa atttacgacc tatttttctg gtttttacga aaatcgtaag aacgtcttct 540
cagcggaaga cattagcacc gcgatcccgc atcggattgt tcaggataat tttccgaaat 600
ttaaggaaaa ctgccacatt tttacacgtt tgatcacagc cgtcccgagc ctgcgtgaac 660
acttcgaaaa cgttaaaaag gcgatcggca ttttcgtgtc aacgagcatc gaagaagtct 720
tcagcttccc tttttataac cagcttctga cacagacaca gattgacttg tacaaccaat 780
tgttaggagg catttccagg gaagctggca cagaaaaaat taaagggctg aatgaagtcc 840
ttaatttagc gattcagaaa aatgatgaga cggctcatat tattgcgtct ctgccgcacc 900
gatttatccc attattcaag caaattcttt ccgatcgcaa caccttatca ttcattttgg 960
aggaatttaa aagcgacgaa gaagtcatcc agtctttctg caaatacaaa acactgctgc 1020
gcaacgaaaa cgtgttggag accgccgaag ctctgttcaa cgagctcaac tctattgatc 1080
tgacccatat ctttatcagc cataagaaac tggaaacgat ttcatcagcc ctgtgcgatc 1140
actgggatac actgcgtaat gctctttatg agcgtagaat ctcagagctg acgggcaaga 1200
ttacgaaaag tgcaaaagaa aaagtgcagc gctctctgaa gcacgaagat attaacctgc 1260
aggaaatcat cagtgctgca ggcaaggaac tctctgaagc gtttaaacag aagaccagcg 1320
aaattcttag tcatgctcac gctgcattag accagccgct gccgacgaca ctcaagaagc 1380
aagaagaaaa agaaatcctg aagagtcagc tggattctct tctgggattg tatcacttgc 1440
tcgattggtt tgcagttgat gagtccaatg aggtagatcc tgaatttagt gcgcgtctga 1500
ccggcattaa acttgaaatg gaaccgagcc tgagtttcta caataaagcg cgtaattacg 1560
cgaccaaaaa accttatagc gtggagaaat ttaaactgaa tttccagatg ccgaccctag 1620
cgtccgggtg ggacgtaaat aaagaaaaaa acaacggcgc cattctcttc gtgaaaaacg 1680
gtttatacta tcttggaatt atgccgaaac agaaaggacg ttacaaggca ctgagcttcg 1740
aaccaacaga gaagacgtcc gaggggtttg ataagatgta ttacgattac tttccagatg 1800
cagccaaaat gatacctaaa tgctcaacac aattaaaagc ggttacagcg cattttcaaa 1860
cacataccac cccaattctt ctgtcgaata atttcattga gccccttgaa attacaaaag 1920
aaatttatga cttaaataat ccggaaaaag aaccgaaaaa gtttcaaacc gcctatgcga 1980
aaaaaaccgg cgaccagaaa ggataccgtg aagcgctgtg caaatggatc gactttaccc 2040
gcgatttcct tagtaaatat acgaaaacca cgtcaatcga tttgagctca cttcgtcctt 2100
caagtcagta taaagattta ggcgaatact acgcagaatt aaatcccctg ttatatcaca 2160
tctcttttca acgtatcgcg gaaaaagaaa tcatggacgc tgttgaaacg ggaaaactgt 2220
atctgtttca gatatacaat aaggattttg cgaaaggcca tcacggtaaa ccgaaccttc 2280
atacacttta ctggacagga ttattcagcc ctgagaattt ggcgaaaact tcgattaaat 2340
taaacggcca agcagaatta ttttatcggc cgaagagccg catgaagagg atggcccatc 2400
gcctgggaga aaaaatgctt aacaaaaaat tgaaagacca gaagacaccc attccggaca 2460
ccctgtacca ggagctgtat gactatgtaa atcatcgctt gagccatgat ctgtctgacg 2520
aagcgcgtgc actgctccct aacgtcatca ccaaggaagt ttcacacgag atcatcaaag 2580
accgccgttt taccagcgat aaattctttt ttcacgtgcc gatcacatta aactaccagg 2640
cagctaactc tccgtctaaa ttcaaccaac gcgttaacgc gtatcttaaa gaacatccag 2700
agaccccgat tattggcatc gaccgtgggg agcgtaacct gatttatatt accgtgatag 2760
acagcacggg aaagatttta gagcagcgaa gccttaacac cattcagcag ttcgactatc 2820
aaaaaaaatt ggacaaccgt gaaaaggagc gtgttgcggc ccgtcaagct tggagtgtcg 2880
ttggaaccat taaagacctg aaacagggct atttatccca ggtaattcat gaaatagttg 2940
atttaatgat tcactatcag gcagtggttg tgctggagaa cctgaacttt ggctttaaat 3000
cgaagcgcac tggcatagct gaaaaggcgg tgtatcagca gttcgagaag atgctgatcg 3060
ataagctgaa ttgtctcgtc ctgaaagact acccagcaga aaaggtcggc ggtgtcctga 3120
acccttatca actgaccgac cagttcacct catttgcgaa gatgggcacc caatccggat 3180
ttctcttcta tgtgccggcc ccatatacct cgaagattga cccgttaaca ggctttgtgg 3240
atccgttcgt gtggaaaact atcaaaaacc acgaaagccg gaaacacttc ctggagggat 3300
tcgattttct gcactacgac gtaaaaaccg gggatttcat tctgcatttc aaaatgaatc 3360
gtaacctgtc attccagcgc gggcttcctg gctttatgcc ggcatgggat attgtgtttg 3420
aaaaaaacga aactcagttc gatgctaaag gcactccgtt catagctgga aagagaatcg 3480
tcccagtcat agaaaaccat cgcttcaccg gtcgctatcg ggatttgtat ccggccaacg 3540
agctcattgc actgctggaa gaaaaaggca tcgtgtttag agatgggagt aacattcttc 3600
cgaaactcct ggaaaacgat gactcacacg ccattgacac tatggtggcc ctgattcgct 3660
ccgttttaca gatgcgcaat tccaacgcag cgacgggtga agattatatc aatagccctg 3720
tccgagactt gaacggcgtt tgctttgata gcaggttcca aaatccagaa tggccgatgg 3780
atgcggacgc caatggagcg tatcacatcg cgctgaaggg acaattactg ctgaaccacc 3840
tgaaagagtc aaaagactta aaattgcaga acggtatcag caatcaagat tggctggctt 3900
acattcaaga attacggaat taa 3923
<210> 13
<211> 4212
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 13
atgctatttt ttatgtccac agatattacc aacaaaccga gagagaaagg cgtctttgat 60
aatttcacaa acttatacga gttcagtaag acgctaacct tcggcctcat tccacttaaa 120
tgggatgaca acaagaaaat gatcgtcgaa gacgaagatt tctcggtcct gcgcaaatat 180
ggtgttattg aggaagataa acgcatcgcg gagagtatta agattgccaa gttctacctg 240
aacatcctgc atcgtgaact gattggcaaa gtcctgggta gcctgaaatt tgaaaagaag 300
aacctggaga attacgaccg tttgctgggc gaaatagaga agaataataa gaatgagaat 360
atatcggaag acaagaagaa ggagataagg aagaacttca agaaggagtt gtctatcgcg 420
caggatatcc tgttaaagaa ggtgggtgaa gtgttcgaga gcaacggcag cggcattctg 480
agctccaaga attgtcttga tgagttgacc aaacgattta ctaggcaaga agtagataaa 540
ctgagaagag agaacaaaga tattggcgtt gaatacccag acgtagcata cagggagaag 600
gatgggaaag aggaaactaa atctttcttc gcgatggatg tgggttactt ggacgatttc 660
cataagaatc ggaaacagct atactctgtg aaaggaaaga agaatagcct gggcagacga 720
attctggaca acttcgagat cttctgcaag aataagaagt tgtacgagaa atacaagaat 780
ttggacatcg atttcagcga aatcgaacgg aacttcaatc tcacgctgga gaaagtgttc 840
gactttgaca attacaacga acgcctgact caagagggtt tagatgagta tgctaagatc 900
ctcggaggcg agagcaacaa acaggaacgc acggccaaca ttcacggcct aaaccaaatc 960
attaacttat acatccagaa gaaacagagt gaacagaaag ccgaacagaa ggaaactgga 1020
aagaagaaga tcaagtttaa taagaaagat tatccgacct tcacgtgctt acagaagcag 1080
atcctatcac aggtattccg caaggagatc atcattgaat cggaccgcga tttaattcgt 1140
gaactgaagt tctttgttga agagtctaaa gagaaggttg ataaagctag aggaattatc 1200
gaatttctgc tgaatcacga agagaatgat atcgatctgg ccatggtgta tctaccaaag 1260
tctaagatca acagctttgt gtataaagta ttcaaagagc ctcaggattt cttatctgtg 1320
tttcaggatg gcgcttccaa tctagacttc gtttcgttcg acaagatcaa gacccacctg 1380
gagaacaaca aacttactta caagatattc ttcaagaccc tgattaaaga gaaccatgat 1440
ttcgaatcgt tcttgatctt attacagcaa gaaatcgatc tgcttattga cggcggcgaa 1500
actgttactc ttggtgggaa gaaggagtcg attactagtc tggacgagaa gaagaataga 1560
ctgaaggaga agcttggctg gttcgaaggc aaagtccgcg agaatgagaa gatgaaagat 1620
gaagaggagg gcgagttctg cagcacggtt cttgcttatt cacaggcggt cctgaacata 1680
accaagcgtg ccgaaatatt ctggttgaat gagaagcaag acgcgaaagt tggcgaagat 1740
aacaaagata tgatattcta caagaaattt gacgagtttg ccgacgatgg cttcgcaccg 1800
ttcttctact ttgataaatt cggcaactac ctgaaacgcc gctccagaaa tacgaccaaa 1860
gaaatcaagt tacacttcgg caatgatgac ctgcttgaag gctgggatat gaacaaagaa 1920
cccgagtact ggtcattcat tctgagggat cgcaaccagt attatttagg tattgggaag 1980
aaagatggtg agatcttcca caagaagctt ggtaattctg tggaagcggt taaggaggca 2040
tatgagcttg agaatgaagc cgacttctac gaaaagatag actataaaca gttgaatatt 2100
gaccgattcg aaggtattgc ttttccgaag aagactaaga cagaggaagc gttcagacaa 2160
gtctgcaaga agagagcgga cgagttctta ggaggagata catacgagtt taagattctg 2220
ctggcgataa agaaagaata tgatgacttc aaagctcgcc gccagaaaga gaaggattgg 2280
gactctaaat ttagcaaaga gaagatgagc aaattaattg aatattacat tacttgcctt 2340
ggcaagcgcg atgattggaa gagatttaac cttaactttc gacagccgaa agaatatgaa 2400
gaccgctccg acttcgtgcg gcacattcaa cgtcaggcat attggattga ccctcgtaaa 2460
gtaagtaaag attacgtgga caagaaagtc gccgaaggtg aaatgttcct cttcaaagtg 2520
cataataaag acttctatga cttcgaaaga aagagcgaag acaagaagaa tcacactgca 2580
aatttgttta cacagtatct gctggagctc ttctcttgcg agaatattaa gaacatcaaa 2640
tcgaaagact tgatcgaatc tatcttcgaa ctggatggta aggcggagat ccgtttcagg 2700
cccaagaccg atgacgtgaa attaaagata taccagaaga agggtaagga tgttacgtac 2760
gctgacaaac gtgatggcaa caaggagaag gaggtgattc agcacaggcg gttcgcgaaa 2820
gacgcattaa ccctccacct caagattagg ttaaactttg ggaagcacgt gaatctgttc 2880
gacttcaaca aactggttaa tacagaactg tttgccaaag tgccagtaaa gatccttggc 2940
atggatcgcg gtgagaataa cctgatctac tattgtttcc tggacgaaca tggtgagatt 3000
gagaatggga agtgcggaag tctgaaccgc gtcggagagc aaattattac gctggaagat 3060
gacaagaaag ttaaggagcc ggtcgattac ttccagcttc tggtagatcg tgaaggtcag 3120
cgagattggg aacaaaagaa ttggcagaag atgacccgta tcaaagactt aaagaaagcg 3180
tatttgggta atgttgtcag ctggatctct aaagaaatgc tgagcggtat taaagaaggc 3240
gtggttacca tcggtgtact ggaggattta aactcgaact tcaagcggac gcgtttcttt 3300
cgagaacggc aggtctatca gggctttgag aaggcactag ttaataaatt gggttactta 3360
gtggataaga aatacgataa ctaccgtaat gtgtatcagt ttgctccaat cgttgatagc 3420
gttgaggaaa tggagaagaa caaacagatc ggcacccttg tgtatgtccc agcctcttac 3480
acctcaaaga tttgccctca tcctaaatgc ggttggcgcg agcgtctcta tatgaagaac 3540
tcagccagta aagagaagat cgtaggcctg ttaaagagcg acgggataaa gatctcctat 3600
gatcaaaaga atgaccgctt ctactttgaa tatcaatggg aacaggaaca taagagtgat 3660
ggaaagaaaa agaaatactc aggcgtagac aaagtcttct ctaatgtgag tcggatgcgc 3720
tgggatgtgg aacagaagaa atctattgac tttgtagatg gcaccgacgg cagcattacc 3780
aacaaactaa agagcctgtt gaaaggcaaa ggtattgagt tagacaacat caatcaacag 3840
attgttaatc agcagaaaga actgggagtg gagttctttc agagcatcat tttctacttc 3900
aatctgatta tgcagatccg taactacgac aaagagaagt caggctccga agcggactat 3960
atccagtgcc caagttgttt attcgattca cgcaaaccgg aaatgaacgg caaactgtca 4020
gcgatcacga acggagacgc aaacggcgcc tacaatattg cccgtaaagg cttcatgcag 4080
ctgtgtagga ttagagagaa tcctcaggaa cctatgaaac tgattaccaa ccgggagtgg 4140
gatgaagcag tgcgcgaatg ggacatctac tcagctgctc aaaagatccc ggttctttct 4200
gaggagaatt aa 4212
<210> 14
<211> 3741
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 14
atgttgaaga acgtaggcat cgatcgctta gatgtagaga aaggacgtaa gaatatgtct 60
aaactggaga aattcaccaa ttgttattca ctgagcaaga cactgcgatt taaagcgatc 120
ccggtcggca agacccagga gaacatcgat aataaacgcc tgctggtgga agatgagaag 180
cgagctgagg attataaggg tgtaaagaaa ctgctggatc gctactatct cagtttcatt 240
aatgacgtgc tgcacagtat caagctgaag aacttgaaca actatatcag cttatttcga 300
aagaagaccc gtaccgagaa agagaataag gaattggaga atctggagat taatctacgt 360
aaagagatcg ctaaggcgtt caaagggaat gagggatata aatcgctgtt caagaaagat 420
attattgaaa caatcctgcc ggaattcctt gacgataaag acgagatcgc gctcgttaac 480
tcgttcaacg gctttaccac tgcattcacc ggtttctttg ataatcggga gaatatgttc 540
tcagaggaag ccaagagtac ctctattgcg ttcaggtgca ttaatgagaa tctgaccagg 600
tatatttcta atatggatat ctttgagaaa gtggatgcca tatttgacaa gcacgaagtg 660
caggaaatca aagagaagat actgaattca gattacgatg tggaagattt ctttgaaggc 720
gagttcttca actttgttct aacacaggaa ggaatcgatg tatacaacgc gatcatcggc 780
ggtttcgtca cggagtcagg agaaaagatt aagggtttga atgaatacat taatttgtac 840
aatcaaaaga ccaaacagaa actgccgaaa tttaaaccac tgtacaaaca ggtgctgtcc 900
gaccgcgaat cattaagttt ctatggcgaa gggtacacct ctgacgaaga agtcctagaa 960
gtatttcgta acacgctcaa caagaattct gaaatattct cgtctatcaa gaagctggag 1020
aaattattca agaactttga tgagtattcc agcgccggaa tcttcgtcaa gaacggccca 1080
gccatctcta caattagcaa agatatattt ggtgaatgga atgtcatccg cgataagtgg 1140
aatgccgagt acgatgatat ccacctcaag aagaaggcag ttgttactga gaaatacgaa 1200
gacgatcgcc gcaagtcctt caagaagatc ggtagcttct cgctggaaca gctgcaggaa 1260
tatgcagacg cggatttatc tgtagttgag aagcttaaag agattattat tcagaaggtc 1320
gatgaaatct ataaagtgta tggtagtagt gagaagctgt ttgatgccga cttcgtcctc 1380
gagaagtcac taaagaagaa cgatgcggtg gtggctatta tgaaagatct gctggattcc 1440
gtgaaatctt tcgagaacta tattaaggcg ttctttggcg aaggcaaaga gaccaatcgt 1500
gacgaaagtt tctatggcga tttcgtactc gcctatgata ttcttcttaa ggttgatcac 1560
atttacgatg cgattcgcaa ctacgtaact cagaaaccgt attctaaaga taagttcaaa 1620
ctgtacttcc agaatccgca gtttatgggc ggctgggata aagacaaaga aaccgattac 1680
cgcgccacca tattgcgtta cggttccaaa tattatctgg cgattatgga caagaaatat 1740
gccaagtgcc tgcagaagat tgacaaggat gatgtaaacg gtaactacga aaagattaac 1800
tacaaactcc taccgggacc gaataagatg cttcccaaag tgttcttttc taagaagtgg 1860
atggcatatt ataacccaag tgaagatatt caaaagatct acaagaatgg cacgttcaag 1920
aaaggcgaca tgtttaattt gaatgattgt cacaaactga tagatttctt taaagactca 1980
atcagtcgct atcccaagtg gagtaacgca tacgatttca acttcagcga aaccgagaag 2040
tataaggata ttgcgggttt ctatcgcgag gtcgaagaac aaggctacaa agtttcattc 2100
gaatctgcgt caaagaagga ggtcgataaa ttggtggagg aagggaaact atatatgttt 2160
cagatctata ataaggactt ctctgacaag agccatggta ctccgaattt acacaccatg 2220
tacttcaaac tgctgttcga cgagaataac catggccaga ttcgactgag tggcggtgct 2280
gaattgttca tgcgtcgagc ttctctaaag aaagaagagc tggttgttca tcctgcgaat 2340
agtccgattg ccaacaagaa cccagataac ccgaaaaaga ctacaacttt atcttatgat 2400
gtgtacaagg acaaacgttt cagcgaagat cagtacgaac tgcatattcc aattgccatt 2460
aacaaatgtc ctaagaacat attcaagata aataccgagg tccgtgtact gctgaaacac 2520
gatgacaatc cgtatgtcat tggtattgac cgcggcgaac ggaacctgtt gtatattgtg 2580
gtagtggatg gtaaaggaaa tatcgtcgaa cagtattctc tgaatgaaat cataaataac 2640
ttcaacggca tccgcatcaa gaccgattac cattcactgc tggacaagaa ggagaaagaa 2700
agatttgagg cccgtcagaa ctggaccagc attgagaaca ttaaggaatt gaaagcaggt 2760
tatatctctc aagtggtcca taagatttgc gagttggtgg agaaatacga tgcggtgata 2820
gcgttagaag acctgaatag cggatttaag aactcaagag ttaaagtcga gaaacaagta 2880
tatcagaagt ttgagaaaat gcttatcgac aaattaaact acatggttga taagaaaagc 2940
aatccttgcg ccactggcgg tgcgcttaaa ggataccaga ttaccaataa attcgagtcg 3000
tttaagagta tgagcacgca gaacggcttc attttctaca tcccggcatg gttgacatcg 3060
aagattgatc catcaacggg attcgtgaat cttcttaaga ccaaatacac ttctatagct 3120
gattcgaaga aattcatctc ttcgttcgat cgtatcatgt acgtgcccga agaagatctg 3180
tttgaatttg ccctggatta taagaacttc tctcgcaccg atgccgatta catcaagaaa 3240
tggaaactgt acagttatgg taaccgcatc cgcatcttca gaaatcccaa gaagaacaat 3300
gtctttgatt gggaagaagt gtgtctgacc agtgcataca aagagttatt taataaatac 3360
ggcatcaact atcagcaggg cgatatccgt gctttactgt gcgaacagtc tgacaaagcc 3420
ttctacagtt ccttcatggc gttaatgagc ttaatgcttc agatgcggaa ttcgatcacg 3480
ggacgcaccg acgtggactt cctgatcagc ccagtaaaga atagtgacgg gatcttctac 3540
gatagccgga actacgaagc acaagagaac gcaatcttac cgaagaacgc cgatgcgaac 3600
ggtgcttata atattgcccg gaaagtcctt tgggccattg gccagttcaa gaaagcggag 3660
gacgagaaac ttgacaaagt taagattgcg attagcaata aagaatggct ggaatatgcg 3720
cagacgagtg tgaagcacta a 3741
<210> 15
<211> 3918
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 15
atgaataaag cggccgataa ttacacgggc ggcaactatg atgagtttat cgccctttct 60
aaagttcaga agactctacg caatgagctg aaaccaactc cctttactgc cgagcacatc 120
aagcagcgtg gcattataag cgaagatgaa tatcgtgccc agcaatcatt ggagctcaag 180
aagatcgcgg atgaatatta ccgtaattat atcacacata agttaaacga tattaataat 240
ctggatttct acaacttgtt cgacgctatc gaagagaaat acaagaagaa tgacaaggat 300
aatagggaca aactggacct ggtggagaag agcaaacgtg gtgaaatcgc caagatgctg 360
agcgctgacg ataactttaa atccatgttt gaagcgaaac tgattactaa actgcttcct 420
gattatgtgg agcggaacta taccggcgaa gataaagaga aggctctgga aacactggcg 480
ctatttaaag ggttcacgac atacttcaaa ggatacttca agactaggaa gaacatgttc 540
tcgggcgagg gtggagcaag ttctatctgc catcgtatag tgaacgtgaa cgcctccatc 600
ttctacgata acctgaagac attcatgcgc atccaagaga aagcgggcga tgaaatcgca 660
ttaatcgaag aggaactgac ggagaagttg gatggctggc gtctggaaca tattttctcg 720
cgtgactatt acaatgaagt ccttgcgcag aaaggaattg actactataa ccagatctgc 780
ggcgacatta ataaacacat gaacctgtat tgccagcaga acaaatttaa agcgaatata 840
ttcaagatga tgaaattaca gaagcaaatt atgggtatca gcgagaaggt cttcgagatt 900
ccgccaatgt accagaacga tgaagaggtg tatgcttcgt ttaatgaatt tatttcccgc 960
cttgaggaag tcaaactgac cgatcgcctg cgtaatattc ttcagaacat caacatctac 1020
aacactgcta agatctatat caacgcgcgc tattacacca acgtcagtac ctatgtgtat 1080
ggcggttggg gggtgattga aagcgcaatc gaacgctatc tgtgtaacac tattgcaggt 1140
aaaggccaat cgaaggtgaa gaaaatcgag aatgcaaaga aggataacaa attcatgagc 1200
gtcaaggagt tggattcaat tgtggccgaa tatgagccgg attactttaa tgctccttat 1260
attgacgacg atgataacgc agtgaaagtc ttcggtggtc agggtgtgtt aggatacttt 1320
aataagatga gtgagctgct tgctgacgtt agtttgtata ccatcgacta taactcagat 1380
gacagcctga tagagaacaa agaaagcgct ctccgcatta agaaacaatt ggatgacatc 1440
atgagtttat atcattggct acagacgttc attatcgatg aggttgttga gaaagacaat 1500
gccttctacg ccgaactgga ggatatttgc tgcgaactag agaacgtggt caccttgtat 1560
gataggattc gaaactacgt gacccgtaaa ccgtactcga cccagaaatt taagcttaac 1620
ttcgctagtc cgaccctggc atccggctgg agccgctcta aggaattcga taacaatgct 1680
atcattctgc tgcgtaataa taaatattac atcgcgatat tcaatgttaa caataaacca 1740
gataaacaga tcatcaaggg cagcgaagaa caacgcttgt caacagatta taagaagatg 1800
gtttacaacc tactgcccgg tccaaataag atgttgccga aggtgtttat caaatccgac 1860
acgggcaaac gtgattataa cccgtcgtca tacatcctag aaggttacga aaagaaccgc 1920
cacattaaga gtagcggcaa cttcgatatt aactactgcc acgaccttat tgattattat 1980
aaagcttgca ttaacaaaca tcccgagtgg aagaattatg gatttaagtt taaggaaact 2040
aaccagtaca atgatatagg tcagttctat aaagatgttg agaagcaggg ctattccatc 2100
agctgggcgt atatcagcga agaggatata aacaagctgg atgaggaagg gaagatctac 2160
ctgtttgaaa tctacaataa agatttgtca gctcattcaa caggtcgtga taacctgcat 2220
accatgtacc tcaagaatat attttctgaa gacaacctaa agaacatctg tattgaactt 2280
aacggcgaag ccgagttatt ctatcgtaag agttcaatga aatcgaacat aactcacaag 2340
aaagatacca tcctggttaa taagacctat atcaacgaaa ctggcgttcg cgtgtctctt 2400
tctgatgaag actatatgaa agtatataac tattacaaca ataactacgt tatcgacacc 2460
gagaatgata agaacctgat tgacatcatt gagaagatag ggcacaggaa gtcaaagata 2520
gacatagtga aagataaacg ctacacagaa gataaatact tcctttattt accgattacg 2580
attaattatg gcattgagga tgagaatgtc aacagtaaga tcatcgaata tatcgccaaa 2640
caggacaaca tgaacgttat cggtatagat cgtggagaac gcaacttaat ttatatatct 2700
gtgattgaca ataaaggtaa catcatcgaa cagaagtctt tcaatttggt gaacaactac 2760
gactacaaga ataaacttaa gaacatggag aaaacccgcg ataatgctag aaagaactgg 2820
caggaaattg gaaagatcaa agatgttaag agcggctatc ttagtggcgt catatccaag 2880
atcgctcgta tggtaattga ttataacgcc atcattgtta tggaagatct gaataaaggc 2940
tttaagaggg gacggtttaa agtagaacgc caggtatacc agaagttcga gaatatgctg 3000
atcagtaagc tgaactacct ggtatttaaa gaacgtaagg ctgatgagaa tggtggtatc 3060
ctccgtggtt atcaattaac ttacattcct aagagtatta agaacgtcgg taaacaatgc 3120
ggttgcatct tctatgttcc tgctgcatat acttctaaga tcgacccggc aacagggttt 3180
atcaatatct tcgattttaa gaaatattca ggttcaggta tcaacgcgaa ggtgaaagat 3240
aagaaggaat tcctcatgtc aatgaattct atccgctata ttaatgaagg cagcgaagaa 3300
tatgagaaga taggccatag agaactgttt gcctttagct ttgattataa caactttaag 3360
acttataacg tttctagtcc ggttaacgag tggaccgcct acacctacgg cgaacggatc 3420
aagaaactgt acaaggatgg tagatggctg cgtagcgaag tgctgaacct gactgagaat 3480
cttatcaaac tgatggaaca gtataacatc gaatataagg atggccatga tattcgtgaa 3540
gacattagtc atatggatga aacacgcaac gcagacttca tttgcagcct attcgaagag 3600
ctgaaatata ctgttcagtt gcgtaatagt aaatccgagg ctgaagacga gaattatgac 3660
cgactggtta gtcccatact gaatagctcg aacggcttct atgattcgag cgactatatg 3720
gagaatgaga ataacacgac gcatacgatg ccaaaggacg cagatgccaa cggtgcctat 3780
tgtattgcgt tgaaagggct ctatgagatt aataagatta agcagaattg gagcgacgac 3840
aagaagttca aagagaacga gctgtacatt aacgttacgg aatggttaga ttacattcag 3900
aatcgtcgct tcgaataa 3918
<210> 16
<211> 3819
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 16
atggaagata aacagtttct cgaacgttac aaggaattta ttggtctcaa ttccctgagt 60
aagaccctgc gcaactcgct gatcccagtc ggcagcacac ttaagcacat tcaagaatat 120
ggtattctgg aggaagatag cttacgcgct cagaaacgcg aagagctgaa aggtattatg 180
gatgattact atcggaacta tattgaaatg caccttcgtg atgtccatga cattgattgg 240
aacgagctgt ttgaagcgtt aacggaagta aagaagaacc agacagacga cgcaaagaaa 300
tgcctagaga agatacagga gaagaagagg aaggagatct accagtattt gagcgatgac 360
gcggtattct ccgaaatgtt caaagagaag atgatttcag gtattctacc agactttatt 420
cgttgtaacg aagagtatag cgaagaagag aaagaagaga aactaaagac agttgccctg 480
tttcaccggt tcacgagttc cttcaacgat ttcttcctga accgtaagaa cgtcttcacg 540
aaagaggcca ttgctacagc tattggttat cgcgtagtgc atgagaatgc tgaaatcttt 600
cttgagaaca tggttgcctt tcagaacatt cagaagtctg ctgagagtca aattagcatc 660
attgaacgaa agaacgaaca ctacttcatg gaatggaaac tgtcccatat cttcacagcg 720
gattactata tgatgcttat gacgcagaag gcgatcgagc actataacga gatgtgtggc 780
gtcgtaaatc agcacatgaa agaatactgt cagaaggaaa agaagaattg gaatctttac 840
cgtatgaaac gcttgcacaa acagattctg tcaaacgcga gcacctcttt taagattccc 900
gagaagtacg agaatgatgc ggaggtgtac gaaagcgtga actccttctt acagaatgtg 960
atggaaaaga ccgttatgga acgtatcgct gtactgaaga acaacaccga caactttgac 1020
ctttccaaga tctacataac cgcgccctac tacgagaaaa tttctaacta tctgtgtggt 1080
tcgtggaaca ccatcgccga ctgtctgact cactattacg aacaacagat cgcgggcaaa 1140
ggcgctcgca aagaccagaa agtgaaagct gcggtgaagg cggataagtg gaagtcgctg 1200
tcggaaatcg agcagttact taaagaatac gcccgggctg aagaggtcaa acgtaaacct 1260
gaagagtaca tcgcagaaat agagaacatt gtctctttga aggaagtcca cttgctggaa 1320
tatcatccgg aagttaacct gatcgagaac gagaagtatg ctacagaaat caaagatgta 1380
ctggacaact atatggaatt atttcattgg atgaaatggt tctatatcga agaagctgtg 1440
gagaaagaag ttaatttcta cggtgaattg gatgatctct atgaagaaat tcgtgatatt 1500
gtcccgttat ataacaaagt gcgcaattat gtgacccaga aaccgtatag tgataccaag 1560
attaaactaa actttggtac gccgacccta gccaatgggt ggtccaagtc gaaagaatac 1620
gattataacg cgattctgct tcagaaagac ggcaagtact atatgggtat cttcaatccg 1680
gtgcagaaac cggagaaaga aatcattgaa ggacattcgc atcctttgga aggcaatgaa 1740
tacaagaaaa tggtttatta ttacttaccg tccgcgaaca agatgctgcc caaggttctt 1800
ctttctaaga aagggatgga aatataccag ccgagcgagt acatcattaa tggttataaa 1860
gagcgtcgcc atatcaaatc ggaggagaaa tttgatttac agttctgtca tgacttgatt 1920
gattatttca aatcaggcat tgaacgcaac ccggattgga aagtgtttgg ctttcacttc 1980
tcggacaccg acacgtatca agacatatct ggcttctata gggaagtgga ggatcagggc 2040
tacaagatcg attggactta tatcaaagaa gccgatatag atcgtttaaa cgaagaaggc 2100
aaattatatc tcttccagat ctataacaaa gacttcagtg agaaatcgac aggacgcgag 2160
aaccttcaca caatgtatct taagaatcta ttttccgaag agaacatacg cgaacaagtt 2220
cttaagttaa acggtgaagc ggagatattc tttcggaaga gcagtgtgaa gaaaccaata 2280
atccacaaga aaggtacgat gttagtgaac aggacgtaca tggaagagat gcatggcgag 2340
agtgtaaaga agaatatacc ggagaaagag taccaagaaa tttataacta catgaaccat 2400
cggtggaaag gtgagcttag cgctgaagcg aaagagtatc tgaagaaagc agtttgtcac 2460
gaaacgaaga aagatattgt taaagattat cgttatagcg tcgataagtt cttcattcac 2520
cttccgatca cgattaacta tcgtgcaagt ggcaaagaag cgttgaattc agtagctcag 2580
cgctatatcg cgcaccagaa tgatatgcat gtgattggta ttgaccgtgg agagagaaat 2640
cttatttatg ttagcgttat caacatgcag ggagaaatca ttgagcagaa atctttcaac 2700
gttgtgaata aatataatta caaagagaag ctgaaagaac gcgaacagaa tcgtgacgag 2760
gctcggaaga attggaaaga gattggccag attaaagatc tcaaggaagg ttatctaagc 2820
ggcgtaatcc atgaaattgc caagatgatg attaaatacc atgcaatcgt ggcgatggaa 2880
gaccttaatt acgggttcaa gagggggaga ttcaaagttg aacgacaggt atatcagaag 2940
ttcgagaaca tgctgattca gaaattgaat tatctggtat ttaaggatcg tagcgccgat 3000
gaggatggcg gtgttctgcg tggataccag ctggcctaca ttcctgatag tgtaaagaaa 3060
ttaggacgcc aatgcggaat gattttctat gtgccggcag cattcacgag caagattgat 3120
ccagctacgg gcttcgtcga tatcttcaac cacaaggcat acacgacaga ccaagcgaag 3180
cgtgagttta tattaagctt tgatgaaata tgttatgatg tggaacgtca actgttccgc 3240
tttacattcg actacgccaa ctttgcgaca cacaacgtga cattagcacg taataattgg 3300
actatctata ccaacggtac gcgtacccag aaggaatttg tgaaccgtcg tgtccgcgac 3360
aagaaagaag tatttgaccc taccgagaag atgttaaagt tgttagaact ggagggtgtt 3420
gagtaccaga gtggcgcgaa tcttcttcca aagttggaga agatcagtga tcctcacctg 3480
tttcatgagc tgcagcgcat tgtacgcttc acggtacagc tgcgcaattc gaagaacgaa 3540
gagaatgatg tggattacga ccatgttata tctcccgtac tgaatgaaga gggcaaattc 3600
tttgactcaa gtaagtacga gaacaaagaa gaaaagaagg agtcattact gcctgtagat 3660
gcggacgcta acggcgccta ttgcatagct ttgaaaggcc tttacattat gcaggcaata 3720
cagaagaatt ggtcggaaga gaaagccctg agtcccgatg tcttacgcct gaataataac 3780
gactggttcg attacattca gaacaaacgc tatcggtaa 3819
<210> 17
<211> 3864
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 17
atgaataata acactaataa ttctttcgaa ccgttcatcg gcggaaattc agttagtaag 60
accttgagaa atgagttacg ggttggaagc gagtatacag gtaaacacat taaagaatgc 120
gcgatcattg cggaggatgc cgtcaaagct gagaatcagt atatcgttaa agaaatgatg 180
gatgacttct atcgggactt catcaaccgc aaactggatg cgttacaagg tatcaactgg 240
gagcaattgt ttgacattat gaagaaagcg aaactggata agagcaacaa agtgtctaaa 300
gaattagata agattcagga atcaacgcgc aaagagattg tcaagatatt tagtagtgat 360
cccatttata aggacatgct aaaggcagat atgattagta agattctgcc tgaatacatc 420
gttgataaat acggcgatgc tgcaagccgc attgaagctg tgaaggtctt ttacggattc 480
tcaggctact tcatcgattt ctgggcttct cgtaagaacg tgtttagcga taagaatatt 540
gcttctgcga taccgcaccg tattgtcaat gttaatgctc gtatccatct cgacaacatc 600
acggcgttta atcgtattgc ggagattgcg ggagatgagg tagccggaat tgccgaggac 660
gcgtgcgcat atttgcagaa tatgagcctg gaagacgtgt ttactggtgc atgttatggt 720
gaattcattt gccagaagga tattgatcgc tataataata tttgcggcgt gattaatcaa 780
catatgaacc agtattgtca gaataaaaag atcagtcgtt ccaaattcaa gatggagcgt 840
cttcacaaac agattctgtg tcgcagtgaa tcaggtttcg aaatcccgat tggattccaa 900
accgacggcg aagttattga tgcaattaat agcttctcaa ctattcttga agagaaggac 960
attctggatc gcctgcggac tttgagccaa gaagtaactg ggtacgacat ggagcgcatt 1020
tatgtgtcgt ctaaagcctt cgaatcggtg agcaagtaca ttgatcacaa gtgggatgtt 1080
atcgcaagca gcatgtacaa ttacttctca ggtgcggttc gcggcaaaga cgataagaaa 1140
gatgcgaaga tccagactga aattaaaaag atcaagagct gttctctgtt agatttaaag 1200
aaattagtgg acatgtatta caagatggat gggatgtgtc tggaacacga agccacagag 1260
tacgtggcgg gtattacgga gatcctggtg gacttcaact acaagacctt cgatatggac 1320
gatagtgtaa agatgatcca gaatgaacat atgataaatg agatcaaaga atatctcgac 1380
acgtacatgt caatttatca ttgggcgaaa gactttatga tcgacgagct agtcgaccgc 1440
gatatggagt tctactccga attagatgag atttactacg acctttcaga tattgtcccg 1500
ctgtacaaca aggtacgcaa ttatgttact cagaaaccgt acagccaaga caagatcaaa 1560
ctgaactttg gctctccgac cctggctaac ggatggagca aatccaaaga atttgacaac 1620
aatgttgtgg tgctgctgcg tgatgaaaag atctacttag cgatactaaa tgtcggtaac 1680
aagccttcca aagacatcat ggcaggcgag gaccgccgtc gcagtgatac ggattacaag 1740
aagatgaatt actatttact gcccggtgcg tcaaagaccc tcccacacgt gttcatctcg 1800
tctaacgcgt ggaagaaaag ccatggcatc ccggatgaga ttatgtacgg atataaccag 1860
aataagcacc tgaaatcttc tccgaacttt gatctggaat tctgccgaaa gcttattgat 1920
tattacaagg aatgcataga tagctatcct aactaccaga tcttcaactt taaattcgct 1980
gccaccgaga cctataatga tatttcagag ttctataaag atgttgaacg ccagggttat 2040
aagatcgaat ggagttatat atcagaggat gacattaatc agatggaccg cgatggtcag 2100
atctacctct tccagattta taacaaagac ttcgcgccga actcgaaggg tatgcagaac 2160
ctccacactc tgtatttgaa gaatatattc agtgaggaga atctgagcga cgtcgttatt 2220
aagctcaacg gcgaagccga gcttttcttt cgtaaatcat caatccaaca caaacgtggg 2280
cataagaaag gttccgttct cgttaataag acctacaaga ccacagagaa gacagagaac 2340
ggtcagggcg aaatcgaagt aattgagagc gtcccggatc agtgctatct tgaactcgtg 2400
aaatactggt ctgagggtgg cgtgggtcag ctgagcgagg aagcctctaa atacaaggac 2460
aaagtgtctc actatgcagc gaccatggat attgttaaag atcgccgtta tactgaagac 2520
aaattcttta ttcacatgcc gatcaccatt aatttcaaag ccgataaccg caacaacgta 2580
aacgagaagg tgctgaaatt tattgcggag aacgacgacc tccacgtaat tgggattgac 2640
cgtggtgaac gtaatttgtt gtatgtaagc gtcattgact cccgcggacg tattgtagaa 2700
cagaagtcct ttaacatcgt tgagaactac gagagcagca agaacgtcat tcgaaggcat 2760
gattataagg gcaaacttgt caataaagaa cactaccgaa acgaggccag gaagtcctgg 2820
aaagaaatag gcaagataaa ggagatcaaa gaaggctatc tgtcacaggt tatccatgaa 2880
atctcgaaac ttgtgctgaa gtacaacgca atcatcgtca tggaagacct aaactatggg 2940
tttaaacgtg gcaggtttaa agtggaacgt caggtgtatc agaaatttga aaccatgctg 3000
attaataaac tggcgtacct tgtagataaa tcacgcgccg tagatgaacc gggcggacta 3060
ctgaaaggtt atcagctgac ctatgttccg gataacctgg gtgaactggg aagccaatgc 3120
ggcattattt tctatgttcc agcagcttac acctccaaga ttgatccagt gaccgggttc 3180
gtcgatgtat ttgactttaa agcatatagt aatgccgaag cccgattaga cttcattaac 3240
aaattagact gcatccgtta tgatgcctca cgcaataaat ttgagatcgc cttcgattat 3300
ggtaatttcc gcacccatca tactacatta gcaaagacgt cttggacaat ctttattcat 3360
ggcgatcgca tcaagaagga acgtgggtcc tatggctgga aggacgaaat aattgacatt 3420
gaagcccgaa tccgtaaact atttgaagac accgacatcg agtatgccga tggccacaac 3480
ttaattggcg atattaatga actggaatca cccattcaga aaaaattcgt tggagaatta 3540
ttcgacataa tccgcttcac ggtccagcta cgcaactcga agagcgagaa atatgatgga 3600
accgagaagg aatatgataa gattatctcg ccggtgatgg atgaagaggg tgtgtttttc 3660
accaccgatt cctatatccg cgcggacggc acagaactac ctaaagatgc agatgcaaat 3720
ggcgcatatt gtatagccct gaaaggtctg tatgacgtct tagccgtgaa gaaatactgg 3780
aaggaaggcg agaaattcga tcggaagctg ctcgcgatca caaattataa ttggtttgac 3840
ttcatacaga accgtcggtt ctaa 3864
<210> 18
<211> 3693
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 18
atgcatgaga acaatgggaa gattgctgac aactttattg gtatctaccc ggtatctaag 60
acattgcgct tcgaactgaa acccgttggt aagacacagg aatacatcga gaaacacggc 120
attctggacg aagatctgaa acgtgcaggc gactacaaga gcgtaaaaaa gataattgac 180
gcgtatcata aatacttcat agatgaggcg ctgaatggca ttcaactgga cggattaaag 240
aactactatg aattatacga aaagaaaaga gataacaatg aggagaaaga attccagaaa 300
atccagatgt cgctgcggaa acaaatagtt aaacgtttct cagaacatcc gcagtataag 360
tatttattca agaaagaact gatcaagaac gtcctcccag aatttactaa ggataatgcg 420
gaagagcaaa cgctggtgaa gagcttccag gaattcacaa cttacttcga aggcttccac 480
cagaatcgta agaacatgta ttcggatgaa gagaagtcga ccgcgattgc gtatcgtgtc 540
gtgcaccaga acctccctaa atatatcgac aacatgcgca tcttctcaat gattctgaac 600
acagacatta gaagcgactt aaccgaatta ttcaataacc taaagactaa gatggatatt 660
acgatcgttg aagaatactt cgcgattgat gggttcaata aagtggtaaa tcagaaggga 720
atagacgttt acaatacaat tctaggcgcc ttctcaactg atgacaatac gaagattaaa 780
ggcctgaacg agtatatcaa cctgtacaat cagaagaaca aagcgaagct gccgaagctg 840
aaaccgttgt ttaaacagat tctcagcgat cgtgataaga taagcttcat tccggaacag 900
tttgatagtg ataccgaagt gctagaagcg gtagatatgt tctacaatag attactgcag 960
ttcgtgatcg agaacgaagg tcagatcacg attagtaagc tcttgaccaa cttctctgcc 1020
tacgatctta acaagatcta cgtcaagaac gatactacta ttagcgctat cagcaatgac 1080
ttattcgatg actggagcta cattagcaaa gccgtacgtg agaactacga tagcgagaac 1140
gttgacaaga acaagcgcgc ggcagcgtat gaggagaaga aagagaaagc tctgagcaag 1200
atcaagatgt attcaattga agaactgaat ttctttgtca agaagtatag ttgtaacgaa 1260
tgtcacatag aaggctattt cgaacgcagg atcttggaaa tcctcgataa gatgcgctac 1320
gcgtacgaat cctgcaagat cttgcatgat aaaggcctga ttaacaacat tagtctgtgc 1380
caggaccgtc aagccatttc ggagcttaag gacttcctcg atagtatcaa agaggtccaa 1440
tggttactga aacctctgat gattggccag gaacaggcag ataaggaaga agccttctat 1500
acggaactct tacggatctg ggaagaatta gaaccgatta cgctgctgta taataaagta 1560
cgtaattacg taacaaagaa accgtacacc ctcgagaagg tcaagttaaa cttctataag 1620
agcactctgc ttgacggttg ggataagaat aaagagaaag acaacctggg cattattctg 1680
ctgaaagatg ggcagtatta tttgggaatt atgaatcgtc gtaacaacaa gattgccgat 1740
gatgcgccat tagctaagac agataatgta tataggaaga tggaatataa attacttacg 1800
aaagtgtctg caaacctgcc tcgcatattt cttaaagata aatataatcc gtcggaggaa 1860
atgctggaga agtacgagaa agggacccat ctcaagggtg agaatttctg catagatgat 1920
tgtcgcgaac tgatcgactt cttcaagaaa gggattaaac agtatgaaga ttggggccag 1980
tttgacttca aatttagcga tacagaaagc tatgatgata tttcagcctt ctataaagaa 2040
gtggagcatc aaggctacaa gatcaccttt agagacatag atgaaacgta catcgatagt 2100
ctggtcaacg aaggcaaact ttatttattt caaatctaca acaaggattt ctcaccgtac 2160
tctaaaggaa cgaagaacct ccatacctta tactgggaaa tgctctttag tcaacagaat 2220
ctgcagaata tcgtgtacaa actgaatgga aacgcggaaa tattctaccg taaagcaagc 2280
attaatcaga aagacgttgt cgtacacaag gcggacctcc caataaagaa taaagaccct 2340
cagaacagca agaaggagag tatgtttgat tatgatatca ttaaggacaa gcgattcacg 2400
tgcgataaat atcaatttca tgttcctatt accatgaact tcaaagccct tggtgagaat 2460
cactttaatc gcaaggtgaa ccgcttaatc cacgatgccg agaatatgca cattattggg 2520
attgatcgtg gagaacgtaa tcttatctat ctgtgtatga ttgatatgaa aggtaacatt 2580
gtaaagcaga ttagtcttaa cgagatcatc agctacgata agaataaatt agaacacaag 2640
cgtaactatc accagctgct caagacacgg gaagacgaga ataaatctgc ccgccagtca 2700
tggcagacca ttcataccat taaagaatta aaggagggct acttatcgca ggttattcat 2760
gtcatcacgg atctaatggt agaatataat gctattgttg ttctggaaga tcttaacttc 2820
ggcttcaaac agggtcgcca gaagtttgaa cgccaggtgt accagaagtt tgagaagatg 2880
ctgattgata aactgaatta ccttgtggac aagagcaaag ggatggatga agacggaggt 2940
cttctacacg cttatcagct cacggatgaa tttaagagct ttaagcagtt aggcaaacaa 3000
agcggcttcc tttactatat tcccgcatgg aatacttcta aattagatcc cactactggt 3060
ttcgtaaatt tattctatac gaaatacgaa tcggtggaga agagtaagga atttatcaat 3120
aacttcacca gcattctcta taaccaggag cgggaatact tcgaatttct ctttgattac 3180
tcggccttca caagcaaagc tgaaggaagc cgtctgaaat ggacagtgtg ttctaaaggc 3240
gagcgtgttg agacctatcg caatccgaaa aagaacaacg agtgggacac gcaaaagatt 3300
gatcttacct ttgagctaaa gaaattattt aatgactatt caattagcct gttggacggt 3360
gatttaagag aacagatggg taagatcgat aaagcagact tctacaagaa atttatgaaa 3420
ttattcgccc tgattgtcca gatgcgaaat tccgatgagc gtgaagacaa actgatttca 3480
ccggttctga ataaatatgg tgctttcttt gaaactggaa agaacgagcg gatgccgctg 3540
gacgcggacg cgaacggagc gtacaatatt gcgcgtaaag gcctttggat tattgagaag 3600
attaagaata ccgatgttga acagcttgat aaggtgaaac tcaccattag taacaaagag 3660
tggcttcagt atgcgcagga gcatatctta taa 3693
<210> 19
<211> 3708
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 19
atggttgcct ttatcgatga attcgtaggt cagtacccag tttcaaagac ccttcgcttc 60
gaagcacgtc cggttccaga gacgaagaaa tggttggaat cggatcaatg ttccgtcctc 120
tttaacgacc agaagcgcaa cgaatactac ggtgtactta aggaactgct ggacgattac 180
tatcgcgcgt atattgaaga tgccctgacc tccttcacgc tagataaagc cttgctcgag 240
aacgcgtatg atctgtattg taaccgtgat acgaacgcct tctcttcatg ctgcgagaag 300
ctacgtaaag acctggtcaa ggcatttgga aacttgaagg actacctgtt aggctcggat 360
cagttgaagg atctggttaa gctgaaagca aaggttgatg cacctgcggg caagggaaaa 420
aagaaaattg aagtggactc tcgtttaatt aattggttaa acaataacgc gaaatactct 480
gcagaagacc gtgagaagta cattaaggcg attgaatctt tcgaaggctt cgttacctat 540
ctgactaatt ataaacaggc tcgcgagaat atgtttagca gtgaagacaa gagcaccgcg 600
atcgcgttta gagtgattga ccagaacatg gtgacctatt tcggcaatat cagaatatat 660
gagaagatca aggcgaagta tcccgaatta tatagcgcgc tgaagggctt cgagaagttt 720
ttctcaccca ccgcgtatag tgaaatcctc tcccaaagta agattgatga atataactac 780
caatgtattg gccgcccgat tgacgatgcc gactttaagg gcgtgaacag ccttataaat 840
gaatatcgcc agaagaacgg catcaaagca cgcgaactgc cggttatgtc tatgctttat 900
aaacagatcc tatcagacag agataactcg tttatgtccg aggtcataaa tcgtaacgag 960
gaggcgattg agtgcgctaa gaatggatac aaggtatcat acgcgctgtt taacgagctg 1020
ctgcagctgt ataagaaaat attcacagaa gacaactacg gcaatatcta tgttaagact 1080
caacctctta ccgaacttag tcaggcgctc ttcggcgatt ggagcatcct gcgcaatgcc 1140
ttggacaacg gtaaatatga caaagacatc attaatttag cggagttgga gaaatacttc 1200
agcgaatact gcaaggttct ggacgcagat gacgcagcga agattcagga caagttcaac 1260
cttaaagatt atttcatcca gaaaaacgcc ctggatgcga cactcccgga tctggataag 1320
attacgcagt acaagccgca tttagacgcc atgctacagg cgatccgcaa atacaagcta 1380
ttctcgatgt acaacggcag gaagaaaatg gacgttccgg agaacggtat cgatttcagt 1440
aacgaattta acgccatata tgataagctt tctgaattct caatcttgta tgaccgtatc 1500
cgcaatttcg cgaccaagaa accttactcc gatgagaaga tgaaactgtc ctttaatatg 1560
cctaccatgc tggcaggctg ggattacaac aatgagaccg caaatgggtg ctttctcttc 1620
atcaaggacg gcaaatactt cttaggtgtt gcggacagta aaagtaagaa tatcttcgac 1680
tttaagaaga atccgcatct attagacaaa tattcctcta aggatattta ctacaaagtg 1740
aagtataaac aggtatctgg gtccgccaag atgctgccga aagtcgtctt tgctggttcg 1800
aacgagaaga tctttggtca tttgattagc aaacgcattc tggaaatccg tgagaaaaaa 1860
ctatacactg ccgctgccgg tgatcgcaag gccgttgcag agtggattga cttcatgaaa 1920
tctgcgattg ctattcaccc ggagtggaac gaatacttca agttcaagtt taagaacacc 1980
gcagaatatg ataacgcgaa taaattctat gaagacattg ataaacaaac ctatagtcta 2040
gagaaagtcg aaatacctac ggaatatatc gacgaaatgg tgtcccaaca taagctctac 2100
ctgtttcagc tttatacgaa agatttctcg gacaagaaaa agaagaaggg tacagacaat 2160
cttcatacaa tgtactggca cggtgtcttt agcgatgaga atctgaaagc cgtgactgaa 2220
ggtacgcaac ccatcattaa actgaatgga gaggccgaga tgttcatgcg caacccgagc 2280
atcgaatttc aggttacaca tgagcacaac aaacccatag cgaacaagaa cccgttaaac 2340
acgaagaagg aatcggtatt taattacgat ttaatcaaag ataaacgcta cactgaacgt 2400
aagttctact ttcattgtcc tatcactctg aacttccgcg ccgataaacc cattaaatac 2460
aatgagaaga tcaatcggtt cgtggagaac aacccggacg tctgcattat aggtatcgat 2520
cgtggagagc gtcacctgct gtattataca gtgatcaatc agaccggcga tattcttgag 2580
caaggaagtt tgaacaagat cagcggcagc tatacgaacg ataaaggtga gaaggtgaac 2640
aaagaaaccg attaccatga cctgctggat cggaaggaga aaggaaagca tgttgcgcag 2700
caggcatggg aaacaattga gaacatcaaa gaactcaagg cgggttattt aagccaggta 2760
gtgtataaac tgacccagtt aatgttgcag tacaacgcgg tgattgttct ggagaatctc 2820
aatgttggat tcaaacgtgg ccgtacgaaa gtcgagaagc aggtctatca gaaattcgag 2880
aaggcgatga tcgacaagtt aaattacttg gtctttaaag atcgtggtta tgagatgaac 2940
ggtagctacg ctaagggtct gcagctaact gataaatttg aatcgtttga caagattggt 3000
aagcagacgg gatgtattta ttatgttata ccgtcttata cgagccatat tgaccctaag 3060
acgggattcg tgaacctgct aaatgcgaaa ctacgctatg agaatataac gaaagcacaa 3120
gataccattc gtaaatttga ttcgattagc tacaacgcta aagcggatta tttcgagttt 3180
gcattcgatt accgttcatt tggcgtcgat atggcccgta atgaatgggt ggtatgcacg 3240
tgcggtgact tacgctggga atattccgcc aagacacgtg aaaccaaagc gtattcggtg 3300
accgaccgtc ttaaagaact cttcaaggcg cacggtattg attacgtcgg aggcgagaat 3360
ttagtatcgc acattaccga ggtcgcagat aaacatttcc tgtcgactct gctgttctat 3420
ttacggttgg ttcttaagat gcgttatacc gtcagcggca ccgagaacga gaatgacttt 3480
atactctcgc cggttgagta cgcaccaggg aagttctttg actcacgcga ggccactagc 3540
accgaaccga tgaatgcaga cgcaaatggt gcttatcata ttgcgcttaa gggattgatg 3600
acaattcgtg gaattgaaga cggcaagtta cacaactatg gtaaaggagg cgagaacgct 3660
gcctggttca aatttatgca gaaccaagaa tacaagaata atggttaa 3708
<210> 20
<211> 3780
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 20
atgaattata agaccggcct ggaagatttc atcggcaaag aatctttaag taagacgctg 60
cgcaatgcgt tgattccaac agaaagtacg aagattcaca tggaagaaat gggcgtgatt 120
cgtgacgatg aactgagagc ggagaaacag caggaactga aggaaatcat ggacgattat 180
tatcgcgcgt ttatagaaga gaagctcggt cagatacaag gaattcagtg gaacagccta 240
tttcaaaaga tggaggagac catggaggat attagtgtga ggaaagatct ggataagatt 300
cagaacgaga aacgcaaaga gatttgttgc tacttcacta gcgataagcg attcaaagac 360
ctgtttaatg cgaaattaat caccgatatc ctgccaaact tcattaaaga taacaaagaa 420
tatacggaag aagagaaggc agagaaagaa caaactcgcg tattgttcca gcgctttgct 480
accgcattca ctaactactt taaccagcga cgtaataact ttagtgaaga caatatttcg 540
accgcaatct catttcgcat cgtgaatgag aattctgaga ttcatctgca gaatatgcgt 600
gccttccagc gcattgagca gcagtacccg gaagaagtct gtggcatgga ggaagaatat 660
aaagatatgc ttcaagaatg gcaaatgaag catatttact ctgtggattt ctatgatcgc 720
gaacttactc agccaggaat agagtactat aacggcattt gcggaaagat taatgagcac 780
atgaatcaat tctgtcagaa aaaccgcatt aataagaatg acttcagaat gaagaaattg 840
cacaaacaaa tattatgcaa gaaatctagt tactatgaaa taccattccg ctttgaatcc 900
gaccaagaag tatatgacgc attgaatgag tttataaaga caatgaagaa gaaagaaatt 960
attcgccgtt gtgttcactt gggtcaggaa tgcgacgact acgacttagg aaagatctac 1020
attagcagca ataaatatga gcagataagc aatgctttgt atggatcttg ggacaccatt 1080
cgtaaatgca tcaaagaaga atacatggat gcgttaccgg gcaaaggcga gaagaaggaa 1140
gagaaggcag aagctgccgc caagaaggag gaatatcgca gtatagctga tattgacaag 1200
attattagcc tctacggaag tgagatggac cggaccataa gcgccaagaa atgcattaca 1260
gagatctgcg atatggcggg ccaaattagc atcgacccgc ttgtgtgtaa ctccgacatt 1320
aaactgctgc agaataagga gaagaccacg gagattaaga cgattctgga ctcgtttctg 1380
catgtttatc aatggggcca gacatttatc gtaagcgata ttattgagaa ggacagctat 1440
ttctacagtg aacttgaaga tgttctagaa gactttgaag gtattactac cctgtataac 1500
cacgtgcgta gctatgtgac ccagaagccg tatagtaccg tcaaattcaa actccacttt 1560
gggtcgccga cgctggcaaa cggttggagt cagtccaagg aatatgataa taatgccatc 1620
ctgctgatgc gcgaccagaa attctacctg ggcatattca acgttcgtaa taaaccagac 1680
aaacaaataa ttaaaggaca cgagaaagaa gagaagggcg actacaaaaa gatgatctat 1740
aacctgctgc ctggtccgtc gaagatgctg cctaaggtgt tcataaccag ccgctccggc 1800
caggagacct ataagcctag caaacatatc ttggatgggt ataatgagaa acgtcacatc 1860
aaatcatctc ccaagtttga tctgggctat tgttgggatt tgatagatta ttataaggaa 1920
tgcattcaca agcacccgga ttggaagaat tatgactttc acttctccga caccaaagat 1980
tacgaggata ttagcggatt ctatagagaa gtagaaatgc agggctacca gattaagtgg 2040
acgtatatct cagcagatga aatccagaag cttgacgaga aaggccaaat attcctgttt 2100
cagatctata acaaagactt ctcggtacat tcaactggca aggacaacct ccataccatg 2160
tatttgaaga acctgttctc agaagagaac cttaaggata tagtactcaa attaaatggc 2220
gaggccgaac tgttctttcg taaagcgtct atcaagactc caattgttca caagaaaggg 2280
tcggttctgg tcaaccgttc gtatactcaa accgtgggta acaaagagat aagagttagc 2340
attcctgaag aatactatac agaaatttat aactacctga atcacattgg caaaggcaaa 2400
ttatctagcg aagcccagcg ttacctggac gaaggaaaga taaagagttt cacggcgacc 2460
aaagacattg ttaagaacta tcgttattgc tgcgatcatt atttcttaca cttaccgatt 2520
actattaact ttaaagctaa gagcgacatc gcggttaacg aacgtacact ggcgtatatc 2580
gcgaagaagg aagatatcca tatcataggc atagaccgag gtgagagaaa cctgctctat 2640
ataagcgtaa tcgatgtgca cggcaacatt cgtgaacagc gcagcttcaa tattgtaaat 2700
ggttacgact accagcagaa acttaaagac cgggagaaga gtcgcgacgc agcacgaaag 2760
aactgggaag aaatcgagaa gatcaaagaa ctcaaggagg gctacttatc tatggttatc 2820
cactatatcg cgcgcttggt tgtcaagtac aatgcagtgg tggcgatgga ggacctgaac 2880
tatgggttta agaccggacg gtttaaagtg gaacgtcagg tttatcagaa atttgaaacg 2940
atgctgattg agaagttgca ttaccttgta tttaaagacc gtgaagtgtg tgaggaaggt 3000
ggagtactgc gcgggtatca actgacttat atcccagaat cactcaagaa ggtaggcaaa 3060
cagtgcgggt tcatcttcta cgttccggca ggctatacta gtaagatcga cccaactact 3120
ggctttgtta atctgttcag ctttaagaac ttgaccaacc gggaatcacg tcaggacttc 3180
gttggtgagt tcgatgaaat ccgttatgat cgtgacaaga acatgtttga attctccttc 3240
gactataata attatataaa gaagggcacc atgctggcta gcacgaaatg gaaggtttac 3300
accaacggta cacgtttaaa gagaatagtt gttaatggca aatataccag tcagtccatg 3360
gaagtagaac taactgatgc catggagaag atgttacaac gtgctggtat cgaataccac 3420
gacggcaaag acctgaaagg gcaaatcgtt gagaagggca tcgaagccga gattattgat 3480
atcttccgtc taaccgtcca gatgaggaac tcgcgttcgg aatctgagga tcgtgaatat 3540
gatagactaa tttctcccgt gcttaatgat aaaggtgagt tctttgatac agccactgcc 3600
gacaagacgt taccgcaaga tgccgacgca aatggtgcgt actgtattgc gctgaaaggt 3660
ctgtatgaag tgaagcagat caaagagaac tggaaagaga acgaacaatt cccgcgaaat 3720
aagcttgtgc aggacaacaa gacgtggttt gacttcatgc aaaagaagcg atatctgtaa 3780
<210> 21
<211> 3903
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 21
atgagcattt accaggaatt tgtgaacaag tatagtctgt ccaagacact gcggttcgaa 60
ctaatcccac agggtaagac cctggagaat atcaaagccc gcggccttat tctggatgac 120
gagaagcgcg cgaaggacta caagaaagcg aaacagatca tcgataaata ccaccaattc 180
ttcattgagg agatcctgtc atcggtatgt atttcagaag atttattaca gaattatagt 240
gacgtttatt tcaaactcaa aaagagtgac gatgataatc tgcagaaaga ctttaagagt 300
gcgaaagaca ccataaagaa acagatttct gaatacatca aggatagtga gaaatttaag 360
aacctgttca atcagaatct gatcgatgcg aagaaagggc aagaatcaga tttaatcctg 420
tggctcaagc agtcgaaaga taatggtatt gaattattta aagccaattc tgacatcacg 480
gatattgatg aagcgctgga aatcataaag agcttcaagg gttggacaac gtacttcaag 540
ggcttccatg agaatcgcaa gaatgtatat agttctaacg acatcccaac ctccatcatt 600
tatcgtatcg tagatgataa ccttcccaag tttctggaga ataaagcgaa atacgagtct 660
ttgaaagata aagcgccaga ggccattaac tacgaacaga ttaagaagga tctggcagaa 720
gaattgacat tcgatattga ttacaagaca tccgaagtga accaaagggt gttcagctta 780
gatgaagtct ttgaaattgc taatttcaat aattatttaa atcaatccgg cattaccaaa 840
tttaacacca taataggtgg caaattcgtg aatggcgaga acactaagcg caaaggtatt 900
aacgagtaca tcaatctgta ttcacagcag attaacgaca agaccctgaa gaagtataag 960
atgtcagtct tgtttaaaca gatcctcagt gatacagaga gcaaatcgtt cgtaatagat 1020
aaactggaag atgactctga cgtcgtaacc actatgcagt cgttctatga gcagatcgcg 1080
gcctttaaga ccgttgaaga gaagagcatt aaggaaacgt tatcactcct gtttgacgac 1140
cttaaagcac agaaactgga cctttcgaag atttacttta agaatgataa atctctgact 1200
gatctgtctc aacaggtatt tgatgattac tcggtgattg gcactgctgt gttagaatat 1260
attacccagc aaattgcacc taagaatttg gataatccct ccaagaagga acaggagctc 1320
atagctaaga agacggagaa agctaagtac ctgtcactgg aaacaattaa gctggcctta 1380
gaagagttta acaaacatcg cgatatcgat aagcagtgtc ggtttgaaga aatcttagct 1440
aacttcgccg ctatacctat gatcttcgat gaaattgccc agaacaagga taatctggct 1500
caaattagca tcaaatatca gaatcaaggg aagaaagact tgttacaggc tagcgcggag 1560
gatgatgtta aagcgattaa ggacttactg gaccagacga ataacttatt acataaactt 1620
aagatctttc acatctcaca gtctgaagat aaggccaaca tcctggataa agatgaacat 1680
ttctatcttg tgtttgaaga atgctatttc gagttagcta atatagtacc tttatacaac 1740
aagattcgta attacatcac acagaaacca tacagcgacg agaagtttaa gttgaacttt 1800
gagaactcca cccttgctaa tggctgggac aagaataaag aaccagataa taccgcaatc 1860
ctctttatca aagatgacaa atactacctg ggtgttatga acaagaagaa taacaagatc 1920
tttgacgata aggccattaa agagaacaaa ggagaaggtt acaagaagat cgtttataaa 1980
ttgttgcccg gcgcgaacaa gatgctccct aaggtcttct ttagtgctaa gagcattaag 2040
ttctataacc cgtcagaaga tattctgcgc atccgaaatc acagcaccca cacgaagaac 2100
ggatctccac agaaaggcta tgagaaattc gagtttaaca tagaggattg tcgcaagttt 2160
attgacttct ataagcagag catttcaaag catcctgaat ggaaagattt cggattccgc 2220
ttcagtgata cccagcgcta taatagcatt gatgaattct accgagaagt cgagaaccaa 2280
ggctacaaac tgacgtttga gaacatctct gaatcctata ttgattcggt ggttaatcag 2340
ggcaagctgt acttatttca aatttataat aaggatttct ccgcctacag taaaggtcga 2400
cctaacctgc acaccctgta ttggaaagcg ttatttgatg agcgtaatct ccaagacgtt 2460
gtgtacaaac tcaacggtga agccgaatta ttctatcgca aacagtcgat tcccaagaaa 2520
atcacccatc cggcgaagga ggctattgcg aacaagaaca aagataatcc taagaaggaa 2580
tctgtgttcg aatacgatct aattaaagac aagagattca cggaggacaa gttcttcttc 2640
cactgcccga tcaccattaa cttcaaatcc agcggcgcca ataagtttaa cgacgaaatc 2700
aacctgttgc tcaaagagaa ggctaacgac gtgcacatac tgagtataga tcgaggcgaa 2760
cggcacttag cgtattatac cttagtggat ggcaagggta atattatcaa gcaagacaca 2820
tttaatatta tcggtaatga ccgcatgaag acaaattacc acgacaagct ggccgccatc 2880
gagaaggatc gtgatagtgc tcgtaaagat tggaagaaga ttaacaatat caaagagatg 2940
aaagaaggtt atttgagcca ggtagttcat gaaatcgcca aattagttat tgaatataat 3000
gcaatcgttg tatttgaaga cctgaacttc ggctttaaac gcggtcgatt caaagttgag 3060
aagcaggtgt atcagaagct ggaaaagatg ctgattgaga aattgaacta ccttgtgttt 3120
aaagacaatg agttcgacaa gacgggcggc gtgctgaggg cctatcagct aaccgcgccg 3180
tttgagacat ttaagaaaat gggtaaacaa acaggcatca tttactacgt tccagcgggc 3240
ttcaccagca agatatgtcc tgtcacaggc ttcgtgaatc agctgtaccc aaagtacgaa 3300
agtgttagta aatctcagga atttttctct aaatttgata agatttgcta caatttggat 3360
aaaggctatt tcgaatttag ctttgattac aagaacttcg gtgacaaggc tgcgaaaggc 3420
aaatggacaa ttgcatcgtt cgggagccgt ctgattaact ttcgtaatag tgacaagaat 3480
cataactggg ataccaggga agtgtatcca accaaagaac tggagaaact tctcaaagac 3540
tattccatcg aatacggcca tggtgaatgt attaaagcgg cgatctgcgg agagagtgac 3600
aagaaattct tcgccaaact gacctcagtg ttaaacacca ttctgcagat gcgaaacagt 3660
aagactggta cagagctgga ctatttaatt tcaccggttg cagatgtaaa tggcaacttc 3720
tttgatagcc gtcaggcacc gaagaatatg ccacaggatg cagatgcaaa cggtgcatac 3780
catattggtt tgaaaggtct gatgctcctg ggtcgcataa agaacaacca agagggcaag 3840
aagctgaacc tggttataaa gaacgaagaa tacttcgaat tcgttcagaa tcgtaacaac 3900
taa 3903
<210> 22
<211> 3624
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 22
atgtattatc agaatttaac caagatgtat ccgattagta agacccttcg taacgaacta 60
attccggtag gaaagactct ggagaacata cggaagaatg gtatcttgga agcagatatc 120
caacgtaaag ccgactatga acatgtcaag aaattgatgg acaattacca caaacaacta 180
atcaacgaag cgctgcaggg agtgcatctg tcggatctga gcgacgctta tgacctgtac 240
tttaatcttt ctaaagagaa gaactcagta gatgccttct ccaaatgcca ggataaactt 300
cggaaagaga tcgtgtcttt cctgaagaat cacgagaatt ttccgaagat cggaaataaa 360
gaaattatca aactgatcca gagcctgaat gacaacgacg cagacaataa cgcgctcgat 420
tccttctcga atttctatac ctacttcagc agctataacg aggttagaaa gaatctctac 480
agcgatgagg agaagagtag cacagtagca tataggttaa taaacgagaa cttgccgaaa 540
tcgttagata atattaaagc gtacgccatc gctaagaaag ccggtgtccg tgcggaaggc 600
ctctcggaag aggaacagga ttgtttattc attattgaaa cctttgaacg taccctgaca 660
caggacggca tcgataatta caatgctgat atcggcaagc ttaacaccgc aatcaatctg 720
tacaatcaac agaacaagaa gcaggaaggt ttccgcaaag taccgcagat gaaatgcctg 780
tacaaacaga ttctgagcga ccgggaagag gcattcatcg atgaatttag tgatgacgag 840
gacctgataa ccaacattga aagcttcgct gagaatatga atgtattcct aaactccgaa 900
ataatcaccg actttaagaa tgcgctcgta gaatctgacg gctccctggt ctatataaag 960
aatgatgtgt ccaagacctt attctcaaat attgtattcg gaagctggaa cgcaattgat 1020
gagaagttat cggatgaata cgatctggcg aattcaaaga agaaaaaaga cgagaagtat 1080
tatgagaagc gtcagaagga actaaagaag aataagagct atgatctgga aactattatt 1140
gggctgtttg atgactctat cgacgtcatc ggtaaataca tagagaagct cgagtcagac 1200
attaccgcca ttgctgaagc caagaacgac ttcgatgaga tcgtccttcg taagcatgat 1260
aagaacaaat cacttcgtaa gaacacaaac gcggttgaag ccataaagag ttacctggac 1320
accgttaaag atttcgaacg ggatattaaa ctgattaacg ggtctggcca ggaggtggag 1380
aagaatctgg ttgtatatgc agagcaggag aacatactcg cagagatcaa gaacgtggac 1440
agtctctata acatgtcacg taactatctg acacagaaac cattctcgac ggagaaattt 1500
aaactgaact ttgagaatcc cacgttacta aatggttggg accgtaacaa agagaaagac 1560
tatctaggaa tactgttcga gaaagagggt atgtattatc ttggcatcat caataacaat 1620
caccgtaaga tcttcgagaa cgagaaactg tgcaccggta aagaaagttg cttcaataag 1680
atcgtgtata aacagatctc gaatgcggcc aaatacctgt ctagcaaaca gattaacccg 1740
cagaacccgc ctaaggaaat tgcagagatc ctgctgaaac gcaaagcaga tagcagttcc 1800
ttaagtcgta aagaaacgga actgttcatc gattatttga aagacgattt cttagtaaat 1860
tatccaatga tcatcaacag tgacggcgag aatttcttta actttcactt taaacaggct 1920
aaggactacg gctcgttaca ggagttcttc aaggaagtgg aacatcaagc gtattccttg 1980
aagacacgtc cgattgacga ttcttacatt tatcggatga ttgacgaagg taagctgtac 2040
ctgtttcaga ttcataataa agacttcagc ccgtactcga aaggaaatct caacctgcat 2100
actatatatc tccagatgtt attcgatcag cgtaatctga ataacgttgt atataaactg 2160
aacggcgaag cagaagtgtt ttatcgccca gcgtccatta acgatgagga agttattatc 2220
cacaaagcag gtgaagaaat taagaacaag aatagcaaac gggccgttga caaacctacg 2280
agcaaattcg gctatgatat tattaaagac cgccggtatt cgaaagataa gtttatgctt 2340
catatccctg tgaccatgaa cttcggcgtt gacgagaccc gccgcttcaa tgatgtcgta 2400
aatgatgcct tacgcaatga tgagaaggtt cgcgtgattg gcattgatag aggtgaacgc 2460
aacctgttat acgtcgtagt ggtcgatacg gatggaacta tccttgaaca gattagtctc 2520
aacagtatta ttaataacga gtatagcatt gaaactgatt atcacaagct gctggacgag 2580
aaagagggtg atcgcgaccg cgccagaaag aactggacca caattgagaa tattaaggaa 2640
ctgaaagagg gctatctgtc acaagttgta aatgttatcg cgaagttggt gttaaagtac 2700
aatgcgatta tttgcctgga agatttaaat ttcggtttca aacgtgggcg ccagaaggtc 2760
gagaagcagg tgtatcagaa gtttgaaaag atgctgatcg ataaactgaa ttatttagta 2820
attgataaat cgcgtaaaca ggagaagccg gaagaattcg gtggtgcttt gaacgcattg 2880
cagttaacaa gcaaatttac ttctttcaaa gatatgggta aacagacagg aattatttat 2940
tatgtccctg cgtatcttac ctctaagatt gacccaacca cgggctttgc gaacctgttc 3000
tatgtgaaat atgagaatgt cgagaaagcc aaggaattct tttctagatt cgactctatc 3060
agctataaca acgagagcgg atactttgaa tttgcctttg attataagaa attcactgat 3120
cgcgcctgtg gcgctcggag ccagtggaca gtttgcacct atggcgagcg aattattaag 3180
taccgtaacg cggacaagaa taacagcttt gatgacaaga ccatcgtact gtcggaagaa 3240
ttcaaagagt tgtttagcat ctatggtatc agctacgaag atggcgcgga attaaagaac 3300
aagatcatga gcgtagatga ggcggatttc tttcgttgtc tgaccggctt attacagaag 3360
acattacaaa tgcgtaacag cagtaatgat ggcacacggg attacattat aagcccaatt 3420
atgaacgata gaggcgagtt cttcaattct gaggcgtgtg atgcttcgaa accgaaagat 3480
gccgatgcca acggcgcctt caacattgcg cgcaaaggcc tgtgggtgtt agagcagatt 3540
cgcaatactc ccagcggcga taaattgaat ctggcgatga gcaacgctga atggctggag 3600
tacgcacaga ggaatcagat ctaa 3624
<210> 23
<211> 3621
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 23
atgtattacc agaatttaac gaagaaatac ccggtgagca agactatacg gaatgaactg 60
attcctattg gtaagactct ggagaacatt cgtaagaata atatcctcga atccgatgtc 120
aagcgcaagc aagattatga acatgtgaaa gggattatgg acgaatatca taaacaactg 180
attaacgaag cgctggataa ctacatgctg ccgagtctga atcaagccgc agagatctat 240
ctaaagaaac atgttgacgt cgaggacaga gaggaattta agaagaccca ggatctgttg 300
cgcagagagg ttacgggtcg cttgaaggaa cacgagaatt atacgaagat cggaaagaaa 360
gatatccttg atcttctgga gaagctgccg tctatttcgg aagaagatta taatgccctg 420
gagagcttcc gcaatttcta cacatacttc acctcttata acaaggtgcg tgagaacctg 480
tattcggatg aagagaagtc aagcacagtg gcctacagat taatcaacga gaaccttccg 540
aaatttcttg ataatattaa gagttacgcg tttgtcaaag ccgcaggcgt cctggcagat 600
tgcattgaag aagaagagca agatgcactg tttatggttg agaccttcaa tatgactctg 660
actcaagaag gcatcgatat gtataattat caaatcggga aggtgaactc cgcgattaat 720
ctgtataatc agaagaatca caaagttgaa gaatttaaga agatcccgaa gatgaaagtt 780
ctatacaaac agatcctgag tgatagggag gaggtattca taggagagtt caaagacgat 840
gaaacgttgc tcagctcaat cggcgcgtat ggcaatgtct taatgacata tcttaaatcc 900
gagaagatta acatcttctt cgatgcactc cgggaatctg aagggaagaa cgtgtacgta 960
aagaacgacc tttcaaagac caccatgtcg aatatcgtct tcggaagctg gagcgcattc 1020
gatgaattgt tgaaccagga gtatgatctt gccaacgaga acaagaagaa ggacgacaaa 1080
tactttgaga agcgccagaa ggagctaaag aagaataaga gttatacgct ggagcaaatg 1140
tctaatctga gtaaggaaga cattagccct attgagaatt acatcgaacg gatttcagaa 1200
gacatcgaga agatatgcat atataatggc gaattcgaga agattgtggt gaacgaacat 1260
gacagctctc gtaaactgag taagaacatc aaagcggtta aagtcatcaa ggattacttg 1320
gattcgatca aagaactgga acacgacatt aaattgatca acggtagtgg ccaggaattg 1380
gagaagaact tggttgtcta tgtgggtcaa gaagaagccc tggagcagct ccgtccagtg 1440
gatagtttat acaaccttac tcgaaactat ttaacaaaga agcccttctc aactgagaaa 1500
gtgaaactta acttcaacaa gagtacgctg ttaaatggtt gggacaagaa caaagaaacg 1560
gataatctcg gtatcttgtt cttcaaagac gggaagtatt atcttggcat catgaataca 1620
actgctaaca aagcctttgt gaatccgccc gccgccaaga ccgagaatgt ctttaagaaa 1680
gttgattata agttactgcc gggcagtaat aagatgctgc caaaggtctt tttcgctaag 1740
agcaacattg gatactataa cccatctacg gaactgtact ctaattataa gaaaggcacc 1800
cacaagaaag gcccgtcatt ctctatcgat gattgtcata acttaattga tttcttcaaa 1860
gaaagcatta agaaacatga ggactggtcg aaatttggtt tcgaattctc tgacaccgca 1920
gactaccgcg atatttcaga gttctaccgc gaagtagaga aacagggcta taaacttacg 1980
tttacggaca tagacgaaag ctatattaac gatctgattg aaaagaatga actgtattta 2040
ttccaaattt ataacaaaga tttcagtgaa tatagcaaag gtaaactcaa cctgcatacc 2100
ctgtacttca tgatgttgtt cgatcagcgc aacttggaca atgtggtcta caaactgaac 2160
ggtgaggcag aagttttcta ccgcccggca tcgatcgccg agaatgaact ggttattcat 2220
aaagcaggtg agggtataaa gaacaagaat ccgaaccgtg caaaggtcaa agaaactagc 2280
acgttctctt acgatattgt gaaagataaa cgatatagca aatacaaatt taccctgcat 2340
attcctatta ccatgaactt cggagtcgac gaagtgcggc gtttcaatga cgtgatcaac 2400
aacgccctgc gtacggacga taatgtcaat gttattggca tcgatcgtgg tgaacgcaat 2460
ctgctttacg tcgttgtaat aaacagtgaa ggaaagattc tcgaacagat ttctttaaat 2520
tctatcatca acaaagaata tgatatcgaa accaactacc atgctctgtt ggatgaacgt 2580
gaggacgatc ggaacaaagc gcgtaaagat tggaatacga tcgagaatat taaagaattg 2640
aagaccggct atctttcaca ggttgtcaat gttgttgcta aattagtgct gaaatataac 2700
gcgatcattt gcctggaaga tttaaacttt gggttcaaac gaggccgtca gaaagtggag 2760
aagcaagttt accagaagtt cgagaagatg cttattgaga aactaaacta cctcgtgatt 2820
gacaagagcc gcgaacaggt gtcaccggag aaaatgggtg gcgcgttgaa tgcattgcag 2880
ttaacttcta aatttaagtc gttcgctgaa ctaggcaagc aaagcggtat tatctattac 2940
gtaccggcct acttaactag taagattgat cccacgaccg gctttgtaaa cctcttctat 3000
attaaatacg agaacatcga gaaagccaag cagttcttcg atggatttga cttcattcgt 3060
ttcaacaaga aagacgacat gtttgagttc tcgtttgatt ataagtcatt cacccagaaa 3120
gcttgtggaa tccgtagcaa atggattgtg tacacgaatg gagaacgtat tattaaatat 3180
ccgaacccgg agaagaataa tttgtttgat gagaaagtga ttaacgtgac cgacgagatt 3240
aagggtttgt tcaaacaata ccgcatcccg tacgagaacg gtgaagacat taaggaaatt 3300
ataatcagca aagcagaggc tgacttctat aaacgcctat tccgcctgtt gcatcagact 3360
ttgcagatgc gcaactccac cagcgatggc actcgtgact acataatttc tccggtgaag 3420
aacgatagag gtgagttttt ctgttccgaa ttctcagaag ggaccatgcc gaaagatgcg 3480
gatgccaatg gagcgtacaa tatcgcgcgc aagggtctgt gggtactgga acagataaga 3540
cagaaggatg aaggagagaa ggtaaactta tcgatgacaa atgcagaatg gctgaagtat 3600
gcccaactgc acctgctgta a 3621
<210> 24
<211> 3756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 修饰的MAD核酸酶核酸序列
<400> 24
atgaatcaca tgaaacagtt cactaatcaa ttctcgttat cgaagacact tagattcgaa 60
ctcatcccac agggaaagac gaaagaattt attgaaataa atggcctgat cgagaaggat 120
aacgaacgtg ccgtgagcta caagaaagtc aagaagatca tcgatgaata tcacaagtac 180
tttattgaaa tggttctgtg cgactttaaa ctgcacggtc tggagaccta tgaaacgatc 240
ttcaataaga aggagaaaga tgacaccgac aagaaggagt ttgacaacat tcgtaattct 300
ctgcgcaagc aaatcgcgga cgccttcgca aagaatccga acgatgaaat caaagaacgt 360
tttaagaatc tgttcgctaa ggaactgatt aaacaggacc ttcttaactt cgtggatgac 420
gagcagaagg agctggtgaa cgaatttaag gacttcacta cttactttac cggcttccat 480
cagaatcgtc gtaacatgta cgttgcagat gagaaggcaa ccgcgatcgc ataccgtctc 540
gttaacgaga acctgcccaa gttcatcgat aatcttaaga tctatgagaa gatcaagaag 600
gacgctccgg aactgatctc cgatcttaac aagacactgg ttgagatgga agaaatcgtg 660
cagggcaaga cactggatga aatatttagc ttaagcttct tcaaccagac cttaacgcaa 720
actggcattg aactgtataa tattgttatt ggtgggcgca ccgcggacga agggaagaca 780
aagattaaag gactgaatga atatatcaac acagactaca accagaaaca aacggacaag 840
aagaagaaac aagccaagtt taaacagctc tataaacaaa ttctgagtga ccgtcattct 900
gtgagcttcg ttgcggagac ctttgagacc gatgcacaat tactggagaa tattgaacag 960
ttctactcat ccgtgctgtg taactatgaa gatgatggtc acaccacaaa tatattcgaa 1020
gcgataaaga atctgataat aggtctcaag acgttcgacc tatcaaagat ctatctccga 1080
aacgatacgt ccttaaccga tattagtcag aaactgtttg gcgactggag catcatcagc 1140
agcgcactca acgactatta tgagaagcag aacccgatct cgtctaagga gaagcaggag 1200
aagtatgatg agaggaaagc gaaatggttg aaacaggact ttaatatcga aactattcaa 1260
acggcgctca atgaatgcga ctcagaaatc attaaagaga aaaacaacaa gaatattgtt 1320
agcgagtatt tcgcgaaatt aggcttagat aaagacaaca agattgacct cttgcaaaag 1380
atccaccata attacgttgt aattaaggac ttgctgaatg agccgtatcc agagaatatc 1440
aaactgggaa atcagaagga acaagtgtct cagattaagg actttctgga tagcatccta 1500
aaccttatac acttcttgaa accgctcagt ctgaaagata aagataaaga gaaggatgag 1560
ttattttatt ctttgttcac cgcgctgttc gagcacctgt cgcagaccat atcgatctat 1620
aacaaggttc gcaactactt gacgcagaag gcttacagta ccgaaaagat caagttgaac 1680
tttgagaata gtacattgct gaacggatgg gacgtgaaca aagagccggt gaatactagc 1740
gtcatattcc gtaagaatgg tttgttctac ctgggaatca tgtctaaatc caataaccgc 1800
atctttgaac gtaatgtacc ggtgtgtaag aatgaagaaa ccgcctttga gaaaatgaat 1860
tataaattac tgccgggcgc taacaagatg ctcccgaagg tattcctgag cgctaagggg 1920
atagaaagct ttcagccgtc agcagaaatc cagagcaaat atcagaagga gacccataag 1980
aaaggtgatg cgttcgtgcg caaagatatg gagaacctta tcgacttctt taaacaaagt 2040
attgccaaac ataccgattg gaagcacttc aaccaccagt tctcgaagac ggaaacttac 2100
aacgatttaa gtgaattcta taaggaggtt gagaagcaag gatataaatt aacctttacc 2160
aagttggacg agacttatat taaccaactg gtggatgagg gtaaactgta tctgttccaa 2220
atctataaca aggacttcag tcccttcagt aagggcaagc cgaacatgca taccctgtat 2280
tggaagatgt tatttgacga acagaatctg cagaatgttg tatataaact gaatggtgaa 2340
gccgaagtct tcttccggca gagttccatc aaacagaccg accgtatcat tcacaaagca 2400
aaccaagcca ttgacaacaa gaatccactg aacaataaga agcagtcgtc tttcaattac 2460
gacttaatta aggacaaacg gtttaccctg gataaatttc agttccacgt tccgattacg 2520
ctgaacttca aagccgaagg gaatgaatac ctgaacacta aagtgaacga ataccttaag 2580
agcaacagtg atgtgaagat cattggcttg gacagaggtg agcgacattt gatctatctg 2640
actttaatca atcagaaggg tgaactactc aaacagcaaa gtcttaacgt cattgctact 2700
agccaagaac atgagactga ctataagaac ttactggtta acaaggagaa cgaaagagca 2760
aatgccaggc aagattggaa gaccatcgag actattaaag aattgaaaga aggttactta 2820
tcgcaggtcg tacatcaaat agcaaccatg atggtggacg agaacgcgat cgtggttatg 2880
gaagatctga atgccggatt catgcgtggc agacagaagg ttgaacggca ggtgtatcag 2940
aagctggaga aaatgcttat tgagaagtta aactacctgg tgttcaagaa taatgatgtg 3000
aatgaaaccg ccggtgtatt aaatgcgtta cagctcacga ataaatttga aagtttcgag 3060
aagatgggca agcagagtgg ctttctgttc tatgtgcccg cgtggaacac gagtaagatc 3120
gacccggcca caggatttgt cgactttctt aaacccaaat acgaaagcgt cgagaaagct 3180
aagctcttct ttgagaagtt tgaatccatt aaatttaacg cggacaagaa ttacttcgaa 3240
tttgaatttg attacaagaa gttcaccgag aaggcggaag gcagtcaaac caaatggacg 3300
gtctgcacgc atagtgacgt ccgctaccgc tataatccgc agaccaaagc tagcgatgaa 3360
gtcaatgtaa ctaacgaact taaactgata tttgacaaat ttaagattga atacaagaat 3420
gggaagaact taaagaccga attgcttctc caagatgata agcagctgtt ctccaaactc 3480
ctccattatc tggcgctgac ccttatgctc agacaaagta agagtggcac ggatatcgat 3540
ttcattctta gcccggtcgc caagaacggt gtgttctatg actcgaggaa tgccatgcca 3600
aacttaccta aggatgccga tgcgaacgga gccttccaca ttgctctgaa aggcctgtgg 3660
tgtgtgcagc aaataaagaa ggcggatgac ctgaagaaaa ttaagctggc aatttcgaat 3720
aaagaatggc tctcatttgt ccagaatctg aaataa 3756
<210> 25
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物序列
<400> 25
atggcaccca agaagaagag gaaggtgtta 30
<210> 26
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 人工肽
<400> 26
Met Ala Pro Lys Lys Lys Arg Lys Val Leu
1 5 10
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物序列
<400> 27
ttgggtaacg ccagggtttt 20
<210> 28
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物序列
<400> 28
tgtgtggaat tgtgagcgga 20
<210> 29
<211> 199
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒
<400> 29
ggccccaaat tctaatttct actgttgtag atacgacgtt gaagcttcac aatttttacg 60
ccgacataga ggagaagcat atgtacaatg agccggtcac aaccctcgag acacgacgtt 120
gaagcttaac aaacacacca cagacgtggg tcaataccat tgaaagatga gaaaagtaac 180
aatatacgcg ctcctgccc 199
<210> 30
<211> 199
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 30
ggccccaaat tctaatttct actcttgtag atacgacgtt gaagcttcac aatttttacg 60
ccgacataga ggagaagcat atgtacaatg agccggtcac aaccctcgag acacgacgtt 120
gaagcttaac aaacacacca cagacgtggg tcaataccat tgaaagatga gaaaagtaac 180
aatatacgcg ctcctgccc 199
<210> 31
<211> 199
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 31
ggccccaaat tctaatttct actattgtag atacgacgtt gaagcttcac aatttttacg 60
ccgacataga ggagaagcat atgtacaatg agccggtcac aaccctcgag acacgacgtt 120
gaagcttaac aaacacacca cagacgtggg tcaataccat tgaaagatga gaaaagtaac 180
aatatacgcg ctcctgccc 199
<210> 32
<211> 200
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 32
ggccccaaat tctaatttct actgtgtgta gatacgacgt tgaagcttca caatttttac 60
gccgacatag aggagaagca tatgtacaat gagccggtca caaccctcga gacacgacgt 120
tgaagcttaa caaacacacc acagacgtgg gtcaatacca ttgaaagatg agaaaagtaa 180
caatatacgc gctcctgccc 200
<210> 33
<211> 195
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 33
ggccccaaat tctaatttct actgttgtag atcttttctc atctttcaat ggtttttgta 60
tcctcgccat ttactctcgt cgggaaagag cgcaatggat acaattcccc acttttctca 120
tcttacaatg gtattgaccc acgtctgtgg tgtgtttgtg aagcttcaac gtcgtcaata 180
tacgcgctcc tgccc 195
<210> 34
<211> 195
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 34
ggccccaaat tctaatttct actcttgtag atcttttctc atctttcaat ggtttttgta 60
tcctcgccat ttactctcgt cgggaaagag cgcaatggat acaattcccc acttttctca 120
tcttacaatg gtattgaccc acgtctgtgg tgtgtttgtg aagcttcaac gtcgtcaata 180
tacgcgctcc tgccc 195
<210> 35
<211> 195
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 35
ggccccaaat tctaatttct actattgtag atcttttctc atctttcaat ggtttttgta 60
tcctcgccat ttactctcgt cgggaaagag cgcaatggat acaattcccc acttttctca 120
tcttacaatg gtattgaccc acgtctgtgg tgtgtttgtg aagcttcaac gtcgtcaata 180
tacgcgctcc tgccc 195
<210> 36
<211> 196
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 36
ggccccaaat tctaatttct actgtgtgta gatcttttct catctttcaa tggtttttgt 60
atcctcgcca tttactctcg tcgggaaaga gcgcaatgga tacaattccc cacttttctc 120
atcttacaat ggtattgacc cacgtctgtg gtgtgtttgt gaagcttcaa cgtcgtcaat 180
atacgcgctc ctgccc 196
<210> 37
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 37
ggccccaaat tctaatttct actgttgtag atccgacgag agtaaatggc gattttttca 60
ataccattga aagatgagaa aagtaaagaa ttgtatccat tgcgctcgtt cccgacgaga 120
gtataaggcg aggatacgtt ctctatggag gatggcatag gtgatgaaga tgaaggagaa 180
gcaatatacg cgctcctgcc c 201
<210> 38
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 38
ggccccaaat tctaatttct actcttgtag atccgacgag agtaaatggc gattttttca 60
ataccattga aagatgagaa aagtaaagaa ttgtatccat tgcgctcgtt cccgacgaga 120
gtataaggcg aggatacgtt ctctatggag gatggcatag gtgatgaaga tgaaggagaa 180
gcaatatacg cgctcctgcc c 201
<210> 39
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 39
ggccccaaat tctaatttct actattgtag atccgacgag agtaaatggc gattttttca 60
ataccattga aagatgagaa aagtaaagaa ttgtatccat tgcgctcgtt cccgacgaga 120
gtataaggcg aggatacgtt ctctatggag gatggcatag gtgatgaaga tgaaggagaa 180
gcaatatacg cgctcctgcc c 201
<210> 40
<211> 202
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 40
ggccccaaat tctaatttct actgtgtgta gatccgacga gagtaaatgg cgattttttc 60
aataccattg aaagatgaga aaagtaaaga attgtatcca ttgcgctcgt tcccgacgag 120
agtataaggc gaggatacgt tctctatgga ggatggcata ggtgatgaag atgaaggaga 180
agcaatatac gcgctcctgc cc 202
<210> 41
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 41
ggccccaaat tctaatttct actgttgtag attccacacc tctgaccaac gctttttatt 60
ggtatgattg cccttggtgg tactattggt acaggtcttt tcattggatt atccacacct 120
ctgtaaaacg ccggcccagt gggcgctctt atatcatatt tatttatggg ttctttggca 180
tcaatatacg cgctcctgcc c 201
<210> 42
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 42
ggccccaaat tctaatttct actcttgtag attccacacc tctgaccaac gctttttatt 60
ggtatgattg cccttggtgg tactattggt acaggtcttt tcattggatt atccacacct 120
ctgtaaaacg ccggcccagt gggcgctctt atatcatatt tatttatggg ttctttggca 180
tcaatatacg cgctcctgcc c 201
<210> 43
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 43
ggccccaaat tctaatttct actattgtag attccacacc tctgaccaac gctttttatt 60
ggtatgattg cccttggtgg tactattggt acaggtcttt tcattggatt atccacacct 120
ctgtaaaacg ccggcccagt gggcgctctt atatcatatt tatttatggg ttctttggca 180
tcaatatacg cgctcctgcc c 201
<210> 44
<211> 202
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 44
ggccccaaat tctaatttct actgtgtgta gattccacac ctctgaccaa cgctttttat 60
tggtatgatt gcccttggtg gtactattgg tacaggtctt ttcattggat tatccacacc 120
tctgtaaaac gccggcccag tgggcgctct tatatcatat ttatttatgg gttctttggc 180
atcaatatac gcgctcctgc cc 202
<210> 45
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 45
ggccccaaat tctaatttct actgttgtag atacagtttt ctcacaaaga ttttttttct 60
gtcacgcagt ccttgggtga aatggctaca ttcatccctg ttacatcctc gttcacagtt 120
ttctcataaa gattcctttc tccagcattt ggtgcggcca atggttacat gtattggttt 180
tcaatatacg cgctcctgcc c 201
<210> 46
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 46
ggccccaaat tctaatttct actcttgtag atacagtttt ctcacaaaga ttttttttct 60
gtcacgcagt ccttgggtga aatggctaca ttcatccctg ttacatcctc gttcacagtt 120
ttctcataaa gattcctttc tccagcattt ggtgcggcca atggttacat gtattggttt 180
tcaatatacg cgctcctgcc c 201
<210> 47
<211> 201
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 47
ggccccaaat tctaatttct actattgtag atacagtttt ctcacaaaga ttttttttct 60
gtcacgcagt ccttgggtga aatggctaca ttcatccctg ttacatcctc gttcacagtt 120
ttctcataaa gattcctttc tccagcattt ggtgcggcca atggttacat gtattggttt 180
tcaatatacg cgctcctgcc c 201
<210> 48
<211> 202
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 48
ggccccaaat tctaatttct actgtgtgta gatacagttt tctcacaaag attttttttc 60
tgtcacgcag tccttgggtg aaatggctac attcatccct gttacatcct cgttcacagt 120
tttctcataa agattccttt ctccagcatt tggtgcggcc aatggttaca tgtattggtt 180
ttcaatatac gcgctcctgc cc 202
<210> 49
<211> 197
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 49
ggccccaaat tctaatttct actgttgtag atggtaatta tcacaataat gatttttcat 60
tcaattttgg acgtacaaag ttccactggc ggcatggatt agtatttgga aggtaattat 120
cacataaatg aacttgttcc ctgtcaaata ttacggtgaa ttcgagttct gggtcgccaa 180
tatacgcgct cctgccc 197
<210> 50
<211> 197
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 50
ggccccaaat tctaatttct actcttgtag atggtaatta tcacaataat gatttttcat 60
tcaattttgg acgtacaaag ttccactggc ggcatggatt agtatttgga aggtaattat 120
cacataaatg aacttgttcc ctgtcaaata ttacggtgaa ttcgagttct gggtcgccaa 180
tatacgcgct cctgccc 197
<210> 51
<211> 197
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 51
ggccccaaat tctaatttct actattgtag atggtaatta tcacaataat gatttttcat 60
tcaattttgg acgtacaaag ttccactggc ggcatggatt agtatttgga aggtaattat 120
cacataaatg aacttgttcc ctgtcaaata ttacggtgaa ttcgagttct gggtcgccaa 180
tatacgcgct cctgccc 197
<210> 52
<211> 198
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 酵母编辑盒序列
<400> 52
ggccccaaat tctaatttct actgtgtgta gatggtaatt atcacaataa tgatttttca 60
ttcaattttg gacgtacaaa gttccactgg cggcatggat tagtatttgg aaggtaatta 120
tcacataaat gaacttgttc cctgtcaaat attacggtga attcgagttc tgggtcgcca 180
atatacgcgc tcctgccc 198

Claims (20)

1.一种新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶具有密码子优化的核酸序列，所述核酸序列包含与SEQ ID No.3-7、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15-22和SEQ ID No.24中任一项的至少80％同源性。
2.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3-7、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15-22和SEQ ID No.24中任一项的至少85％同源性。
3.根据权利要求2所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3-7、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15-22和SEQ ID No.24中任一项的至少90％同源性。
4.根据权利要求3所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3-7、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15-22和SEQ ID No.24中任一项的至少92％同源性。
5.根据权利要求4所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3-7、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15-22和SEQ ID No.24中任一项的至少95％同源性。
6.根据权利要求5所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3-7、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15-22和SEQ ID No.24中任一项的至少97％同源性。
7.根据权利要求6所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3-7、SEQ ID No.11、SEQ ID No.13、SEQ ID No.15-22和SEQ ID No.24中任一项的至少99％同源性。
8.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3、SEQ ID No.4或SEQ ID No.5中任一项的至少80％同源性。
9.根据权利要求8所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQID No.3、SEQ ID No.4或SEQ ID No.5中任一项的至少95％同源性。
10.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.6、SEQ ID No.11或SEQ ID No.13中任一项的至少80％同源性。
11.根据权利要求10所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.6、SEQ ID No.11或SEQ ID No.13中任一项的至少95％同源性。
12.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.15、SEQ ID No.16或SEQ ID No.17中任一项的至少80％同源性。
13.根据权利要求12所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.15、SEQ ID No.16或SEQ ID No.17中任一项的至少95％同源性。
14.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.18、SEQ ID No.19或SEQ ID No.20中任一项的至少80％同源性。
15.根据权利要求14所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.18、SEQ ID No.19或SEQ ID No.20中任一项的至少95％同源性。
16.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.21、SEQ ID No.22或SEQ ID No.24中任一项的至少80％同源性。
17.根据权利要求16所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶包含与SEQ ID No.21、SEQ ID No.22或SEQ ID No.24中任一项的至少95％同源性。
18.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶在包含具有最佳crRNA可变环的gRNA的系统中，所述最佳crRNA可变环包含UGUU、UCUU或UAUU。
19.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶用于在细菌中编辑，所述新颖的MAD系列核酸酶包含与SEQ ID No.4、SEQ ID No.11、SEQ ID No.15、SEQ ID No.16、SEQ ID No.17、SEQ ID No.19、SEQ ID No.21、SEQ ID No.22或SEQ IDNo.24中任一项的至少80％同源性。
20.根据权利要求1所述的新颖的MAD系列核酸酶，所述新颖的MAD系列核酸酶用于在酵母中编辑，所述新颖的MAD系列核酸酶包含与SEQ ID No.3-6、SEQ ID No.13、SEQ IDNo.15-22或SEQ ID No.24中任一项的至少80％同源性。
CN202080086562.6A 2019-12-13 2020-12-12 新颖的酶 Pending CN114828875A (zh)

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