CN116940689A - Cas3蛋白质的制造方法 - Google Patents

Abstract

发现了通过将导入了Cas3基因的昆虫细胞在比较低温培养，能够在维持活性的状态下有效地表达重组Cas3蛋白质，通过纯化该细胞的可溶化级分，能够高纯度且高收量地回收重组Cas3蛋白质的活性型。

Description

Cas3蛋白质的制造方法

技术领域

本发明涉及制造Cas3蛋白质的方法，更具体地涉及在维持活性的状态下高纯度且高收率地制造重组Cas3蛋白质的方法。

背景技术

基因组编辑技术是通过在动物、植物的细胞中特异性地切割基因组DNA序列，通过利用内在的修复机制，自由地重写成任意序列的技术。在世界中其应用不仅在生物科学研究中，还推广到农作物/畜产动物的品种改良、再生医疗、基因治疗等。

细菌、古细菌所具有的CRISPR-Cas系统被分为通过多种蛋白质的复合体而切割靶标序列的1类、和通过单种蛋白质而切割的2类。到目前为止作为基因组编辑工具而开发出的CRISPR-Cas9、CRISPR-Cas12(Cpf1)、CRISPR-Cas13等全部被分类为2类，但最近发现了作为属于1类的I型CRISPR的CRISPR-Cas3能够作为真核细胞的基因组编辑工具利用(专利文献1)。其中，判明了大肠杆菌K株来源的I型-E CRISPR-Cas3除了3碱基的PAM序列，还识别并结合27碱基作为靶标，在人培养细胞中能够在靶标序列的上游高效率地导入达数百～数kb的大范围的缺失突变。另外，与CRISPR-Cas9相比，认为由于指导RNA中的靶标识别序列长、不易产生非特异性的切割，因而安全性高。

CRISPR-Cas3一般作为表达质粒导入细胞使其表达而被利用，但根据细胞的种类有时其导入和表达难、另外得不到充分的基因组编辑的效率。因此，期望将CRISPR-Cas3以指导RNA(crRNA)和蛋白质(Cas3蛋白质和级联反应蛋白质)的形态导入细胞。

然而，构成CRISPR-Cas3的蛋白质群中，特别对于Cas3蛋白质，到目前为止难以以充分的质和量制备能够经受实用的活性型。例如，作为已有的Cas3蛋白质的纯化法，有关于嗜热菌褐色嗜热裂孢菌(Thermobifida fusca)来源的Cas3蛋白质(TfuCas3)、大肠杆菌K株来源的Cas3蛋白质(EcoCss3)的报告(非专利文献1～3)，TfuCas3由于是嗜热菌来源的，因而存在不适合在大量细胞的生长发育温度下利用这样的问题。另一方面，关于EcoCas3，由于是大肠杆菌来源的，能够在与包含动物细胞在内的大量细胞的生长发育温度接近的温度范围发挥活性，因而具有适合可预计产业应用的大范围的生物的基因组编辑的性质，但即使要用现有的方法制造，也存在在维持活性的状态下高纯度且高浓度地纯化困难这样的问题(请参照后述的比较例1)。

现有技术文献

专利文献

专利文献1：国际公开2018/225858号公报

非专利文献

非专利文献1：Huo Y.et al.,Nat.Struct.Mol.Biol.2014Sep；21(9):771-777

非专利文献2：Mulepati S.&Bailey S.,J.Biol.Chem.2013Aug 2；288(31):22184-22192

非专利文献3：Robert P.Hayes et al.,Nature 2016 530:499-503

发明内容

发明要解决的课题

本发明是鉴于上述现有技术所具有的问题而做出的，其目的是提供高纯度且高收率地制造能够在各种生物的基因组编辑中利用的Cas3蛋白质的活性型的方法。

用于解决课题的手段

本发明者们为了解决上述课题而进行了深入研究，结果发现，大肠杆菌来源的Cas3蛋白质热稳定性低，即使在通常的大肠杆菌的培养温度下培养而表达重组蛋白质的情况下，也变性而活性下降。

基于该意外的知识，本发明者们作为能够在比较低温培养的细胞选择了昆虫细胞，导入Cas3基因并在各种温度条件进行了培养，结果发现，通过在20～28℃培养，从而Cas3蛋白质有效地表达，并且其活性被维持。

另外，本发明者们进行了在昆虫细胞中表达的重组Cas3蛋白质的纯化条件的研究，结果发现，通过在磷酸缓冲液中进行纯化，从而能够高纯度且高收量地回收重组Cas3蛋白质的活性型。

进而，本发明者们发现，这样回收的重组Cas3蛋白质在基因组编辑处理所需的比较短时间内，即使在动物细胞等的培养温度下也发挥高的活性，至此完成了本发明。

即，本发明涉及在维持活性的状态下高纯度且高浓度地制造能够在各种细胞的基因组编辑中利用的重组Cas3蛋白质的方法，更详细地，提供以下的发明。

(1)Cas3蛋白质的制造方法，该方法包括：

(a)将导入有Cas3基因的昆虫细胞在20～28℃培养，在该昆虫细胞内使Cas3蛋白质表达的工序，和

(b)回收所表达的Cas3蛋白质的工序。

(2)根据(1)所述的方法，Cas3蛋白质是大肠杆菌来源的。

(3)根据(1)或(2)所述的方法，昆虫细胞是Sf9细胞。

(4)根据(1)～(3)的任一项所述的方法，所表达的Cas3蛋白质的回收包括Cas3蛋白质的纯化。

(5)根据(4)所述的方法，Cas3蛋白质添加有标签，Cas3蛋白质的纯化包括针对该标签的亲和纯化。

(6)根据(5)所述的方法，标签包含HN标签。

(7)根据(4)～(6)的任一项所述的方法，Cas3蛋白质的纯化包括利用凝胶过滤层析的纯化。

(8)根据(4)～(7)的任一项所述的方法，纯化中使用的缓冲液是磷酸缓冲液。

发明的效果

根据本发明，能够高纯度且高收率地制造重组Cas3蛋白质的活性型。本发明的方法能够在各种重组Cas3蛋白质的制造中利用，特别是在对热稳定性低的Cas3蛋白质应用中有用。

另外，根据本发明，在进行基因组编辑之前，可以以能够使用CRISPR-Cas3系统的状态进行准备。进而，即使在难以将Cas3作为基因导入使其表达的细胞、在以Cas3作为重组蛋白质表达时得不到充分的基因组编辑的效率的细胞中，也能够有效地进行基因组编辑。因此，能够简便且通用地利用CRISPR-Cas3系统。

附图说明

图1是显示在大肠杆菌中表达的重组EcoCas3蛋白质的利用凝胶过滤层析的纯化的图。

图2是显示在各温度培养的昆虫细胞中的重组EcoCas3蛋白质的表达的电泳照片。

图3显示将在昆虫细胞中表达的重组EcoCas3蛋白质通过利用磷酸缓冲液的凝胶过滤层析进行纯化而得的结果的图(上)和SDS-PAGE的照片(下)。SDS-PAGE中的各道如下。a.上清、b.穿流(flow through)、c.TEV消化、d.洗涤、e.回溯(backtrack)的穿流(flowthrough)、2-20.SEC级分、f.浓缩后的13-16级分。

图4是显示将在昆虫细胞中表达的重组EcoCas3蛋白质通过利用Hepes缓冲液的凝胶过滤层析进行纯化而得的结果的图(上)和SDS-PAGE的照片(下)。

图5是显示重组EcoCas3蛋白质的由热变性产生的拐点温度的图。拐点温度通过TychoNT6测定。

图6是显示EcoCas3、Cas9、Cas12、和TfuCas3在37℃的稳定性的图。这些蛋白质的稳定性是通过Sypro_orange与随着蛋白质的变性而向溶剂面露出的疏水性区域结合而产生的荧光强度的变化而测定。

图7是显示Multi-NLS Eco级联反应的制造用质粒的图。

图8是显示使用纯化后的Cas3蛋白质、级联反应蛋白质、和crRNA的复合体在体外(in vitro)测定基因组编辑活性而得的结果的毛细管电泳的照片。红箭头显示检测到的DNA的分解。左显示使用了EMX1靶标级联反应的结果，右显示使用了Tyr靶标级联反应的结果。

图9是显示使用纯化后的Cas3蛋白质、级联反应蛋白质、和crRNA的复合体在人培养细胞中测定基因组编辑活性而得的结果的图。上显示通过FACS分析而得的代表性图，下是显示由GFP阴性细胞数计算出的GFP敲除效率的分析结果的图(n＝3)。

具体实施方式

本发明提供Cas3蛋白质的制造方法。

本发明中，“Cas3蛋白质”是构成CRISPR-Cas3系统的蛋白质，具有核酸酶活性和解旋酶活性。Cas3蛋白质通过与构成CRISPR-Cas3系统的级联反应(Cascade)和crRNA一起作用，能够切割靶标DNA。

在I型的CRISPR-Cas3系统中一般的I-E型的CRISPR-Cas3系统通过crRNA与Cas3和级联反应(Cse1(Cas8)、Cse2(Cas11)、Cas5、Cas6、和Cas7)协同，来切割DNA。

I-A型的系统中，作为级联反应包含Cas8a1、Csa5(Cas11)、Cas5、Cas6、和Cas7作为构成要素，I-B型中，作为级联反应包含Cas8b1、Cas5、Cas6、和Cas7作为构成要素，I-C型中，作为级联反应包含Cas8c、Cas5、和Cas7作为构成要素，I-D型中，作为级联反应包含Cas10d、Csc1(Cas5)、Cas6、和Csc2(Cas7)作为构成要素，I-F型中，作为级联反应包含Csy1(Cas8f)、Csy2(Cas5)、Cas6、和Csy3(Cas7)作为构成要素，I-G型的系统中，作为级联反应包含Cst1(Cas8a1)、Cas5、Cas6、和Cst2(Cas7)作为构成要素。

本发明的Cas3蛋白质不限制其来源，但从适合包括动物细胞在内的广泛的细胞中的基因组编辑这样的观点考虑，优选大肠杆菌来源的Cas3蛋白质。典型的大肠杆菌来源的Cas3蛋白质的氨基酸酸序列示于序列号2，编码该蛋白质的DNA的碱基序列示于序列号1。本发明的Cas3蛋白质包括自然界生成的，或人工地改变而得的突变体。

本发明的Cas3蛋白质可以是由与序列号1所记载的大肠杆菌来源的Cas3蛋白质的氨基酸序列具有高的同一性的氨基酸序列组成的蛋白质。所谓高的同一性，例如是80％以上、优选为85％以上、更优选为90％以上(例如，91％以上、92％以上、93％以上、94％以上)、进一步优选为95％以上(例如，96％以上、97％以上、98％以上、99％以上)的序列同一性。序列同一性可以利用BLAST(Basic Local Alignment Search Tool at the NationalCenter for Biological Information(美国国家生物技术信息中心的基本局部比对搜索工具))等(使用例如，默认、即初始设定的参数)来确定。另外，本发明的Cas3蛋白质可以是由在序列号1所记载的大肠杆菌来源的Cas3蛋白质的氨基酸酸序列中替换、缺失、添加、和/或插入了1个或多个氨基酸的氨基酸序列组成的蛋白质。这里所谓“多个”，通常为50个氨基酸以内、优选为30个氨基酸以内、更优选为20个氨基酸以内、特别优选为10个氨基酸以内(例如，5个氨基酸以内、3个氨基酸以内、2个氨基酸以内、1个氨基酸)。

Cas3蛋白质根据需要可以进一步添加功能性分子。作为功能性分子，可列举例如，用于促进向真核细胞的核内的转运的核定位信号、用于使纯化容易的标签、用于使检测容易的报告物蛋白质等，但不限于这些。这些功能性分子可以添加在例如，构成Cas蛋白质的各蛋白质的N末端侧和/或C末端侧。

作为核定位信号，可列举例如，PKKKRKV(序列号3)、KRTADGSEFESPKKKRKV(序列号4)等。作为标签，可列举例如，HN标签、His标签、FLAG标签、谷胱甘肽-S-转移酶(GST)等。另外，作为报告物蛋白质，可列举例如，绿色荧光蛋白(GFP)等荧光蛋白质、萤光素酶等化学发光蛋白质等。

在本发明的制造方法中，将导入有Cas3基因的昆虫细胞在20～28℃培养，在该昆虫细胞内使Cas3蛋白质表达(工序(a))。

作为在昆虫细胞中使重组Cas3蛋白质表达的方法，例如，可以利用公知的杆状病毒表达系统。在利用杆状病毒表达系统的方法的一例中，首先，将Cas3基因克隆到pFastBac1等Bac-to-Bac用载体中，将其导入具有杆状病毒DNA的大肠杆菌中，制备导入有Cas3基因的杆状病毒DNA。此外，除了这样在大肠杆菌中制备重组杆状病毒DNA的方法之外，还可以利用在昆虫细胞中制备的方法。在利用昆虫细胞的情况下，只要将包含Cas3基因的载体和杆状病毒DNA导入昆虫细胞，在两者之间使同源重组发生即可。接着，将所制备的重组杆状病毒DNA转染昆虫细胞，制备包含Cas3基因的重组杆状病毒。接着，使所制备得重组杆状病毒传代感染昆虫细胞，获得高效价杆状病毒，使该病毒感染昆虫细胞，使重组Cas3蛋白质表达。作为昆虫细胞，Sf9细胞是适合的，但不限于此。

用于使重组Cas3蛋白质表达的昆虫细胞的培养优选为20～28℃。如果小于20℃，则重组Cas3蛋白质的表达效率倾向于降低，另一方面，如果超过28℃，则在培养过程中表达的重组Cas3蛋白质倾向于变性。从进一步抑制变性的观点考虑，更优选为20～24℃，进一步优选为20～22℃，特别优选为20℃。培养时间只要是对重组Cas3蛋白质的表达充分的时间就不特别限制，通常为24小时以上，优选为60～72小时。

在本发明的制造方法中，接着，回收所表达的Cas3蛋白质(工序(b))。

在所表达的Cas3蛋白质的回收中，可以利用各种蛋白质的分离/纯化方法。在从细胞的重组Cas3蛋白质的分离中，可以利用细胞的破碎处理和离心处理。例如，将细胞用超声波破碎，然后以100,000g离心，回收其上清，从而能够获得包含重组Cas3蛋白质的可溶性级分。

在Cas3蛋白质添加有标签的情况下，在重组Cas3蛋白质的纯化中，可以利用针对该标签的亲和纯化。例如，在标签为HN标签、His标签的情况下，可以利用镍柱进行亲和纯化，在标签为FLAG标签的情况下，可以利用结合有针对FLAG标签的抗体的珠进行亲和纯化，在标签为谷胱甘肽-S-转移酶(GST)标签的情况下，可以利用谷胱甘肽Sepharose进行亲和纯化。从将Cas3蛋白质电中和、抑制凝集的观点考虑，作为添加于Cas3蛋白质的标签，优选HN标签。

另外，本发明中的重组Cas3蛋白质的纯化优选包括利用凝胶过滤层析的纯化。因为Cas3蛋白质是分子量100kDa左右的球状蛋白质，所以优选选择具有适合该分子量的球状蛋白质的级分范围的柱。作为这样的柱，例如，可以利用Superdex 200Increase(Cytiva社)等市售品。

从抑制由Cas3蛋白质的变性造成的凝集的观点考虑，纯化中使用的缓冲液优选为磷酸缓冲液。

这样制备的重组Cas3蛋白质具有优异的活性，只要是在基因组编辑所需的数小时以内的比较短时间，则例如在37℃的温度条件下也能够不变性地发挥其活性。实际上，在本实施例中，也在37℃确认了优异的DNA切割活性和高的基因组编辑效率。因此，本发明的方法中获得的重组Cas3蛋白质通过与级联反应蛋白质和crRNA组合，从而能够在广泛的细胞中有效地进行基因组编辑。

实施例

以下，基于实施例和比较例更具体地说明本发明，但本发明不限于以下的实施例。

[比较例1]利用了大肠杆菌的EcoCas3蛋白质的制备

现有的EcoCas3的制造方法(Mulepati S.&Bailey S.，J Biol Chem.2013Aug 2；288(31)：22184-22192)中，存在以下大量问题：(i)需要将导入了编码EcoCas3的质粒的大肠杆菌在20℃这样的低温进行培养的问题；(ii)为了保持EcoCas3的溶解度需要对EcoCas3融合maltose-binding protein(MBP)、small ubiquitin-like modifier(SUMO)的问题；(iii)还需要使作为伴侣分子的HtpG蛋白质共表达的问题；(iv)收量每1L培养最大为1mg的问题；(v)作为电泳图谱确认了EcoCas3以外的蛋白质的条带，因而难以制造高纯度的EcoCas3的问题；等等。实际上按照上述文献利用大肠杆菌进行了EcoCas3蛋白质的制备，结果为低纯度且低收量(图1)，活性也极低。

[实施例1]利用了昆虫细胞Sf9的EcoCas3蛋白质的制备

使原核生物来源的蛋白质在更高级的真核生物中作为重组蛋白质表达的情况下，可能发生在原核生物内不会发生的翻译后修饰。因而，在真核生物中制造EcoCas3蛋白质没有进行，本发明者鉴于比较例1所记载的课题，勇于尝试了使用昆虫细胞Sf9的EcoCas3蛋白质的制备。

(1)重组EcoCas3蛋白质的表达用质粒的构建

合成在EcoCas3的N末端融合了8HN标签(夹着GS接头融合有His标签和HN标签的标签/序列号5)和NLS(序列号3)，进而在C末端融合了NLS(序列号3)的基因(序列号6、7)。使用HN标签(组氨酸和天冬酰胺的重复序列)是因为，His标签的正电荷的偏倚较强，可能使目的蛋白质凝集因而要中和该正电荷。另外，为了表达确认，也制成了在8HN标签的3’末端融合了作为报告物的EGFP的基因(序列号8、9)。

将上述的融合基因克隆到pFastbac-1质粒(ThermoFishers社制)中。所得的EcoCas3/pFastbac-1质粒向DH10bac转化，通过同源重组向DH10bac内的杆状病毒基因组整合之后，提取包含EcoCas3基因的杆状病毒基因组。

(2)重组EcoCas3蛋白质的表达

将包含EcoCas3基因或EGFP融合EcoCas3基因的杆状病毒基因组转染至Sf9细胞，在Sf9细胞内制成包含EcoCas3基因的杆状病毒。使该杆状病毒向Sf9细胞传代感染，获得EcoCas3表达用的高效价病毒。使该高效价病毒感染Sf9细胞，使EcoCas3作为重组蛋白质表达。

在重组EcoCas3蛋白质的表达的研究中，使用包含EGFP融合EcoCas3基因的杆状病毒，杆状病毒对Sf9细胞的感染在28℃进行24小时，然后将Sf9细胞在各培养温度(12℃～28℃)培养60小时，使重组EcoCas3蛋白质表达。将细胞进行超声波破碎，将以100,000g离心而回收得的上清(可溶性级分)电泳，进行荧光检测。

其结果在16℃以上的温度条件确认了向可溶性级分中的重组EcoCas3蛋白质的表达，但特别在20℃以上的温度条件下向可溶性级分中的表达效率高(图2)。在以下的实验中，使用在20℃的温度条件下在Sf9细胞中表达的重组EcoCas3蛋白质。

(3)重组EcoCas3蛋白质的制备

将添加有His标签样的8HN标签的重组EcoCas3蛋白质用镍柱亲和纯化之后，通过凝胶过滤层析进行最终纯化。

将表达重组EcoCas3蛋白质的昆虫细胞通过超声波破碎，将以100,000g离心而回收的上清(可溶性级分)与镍琼脂糖树脂(キアゲン社)混合，从而使重组EcoCas3蛋白质与树脂结合后，使用洗涤缓冲液(20mM HEPES或20mM KH₂PO₄，350mM NaCl，40mM咪唑，0.5mMDTT，pH7.0)。接着，用溶出缓冲液(20mM Hepes或20mM KH₂PO₄，350mM NaCl，200mM咪唑，0.5mM DTT，pH7.0)使重组EcoCas3蛋白质从树脂溶出。

通过对溶出级分进行TEV消化处理，从而切下添加于重组EcoCas3蛋白质的N末端的8HN标签。最后通过凝胶过滤层析进行重组EcoCas3蛋白质的最终纯化。纯化使用Superdex200increase(Cytiva社)柱，移动相缓冲液使用“20mM HEPES或20mM KH₂PO₄，200mMNaCl，1.0mM DTT，pH7.0”。

其结果是在使用HEPES缓冲液进行纯化的情况下，确认了大量的包含被认为在凝胶过滤层析中凝集了的EcoCas3的排除级分(void fraction)(图3)。另一方面，在使用磷酸缓冲液进行纯化的情况下，拐点温度Ti改善了约2℃，最终成功地获得了每1L培养最大2mg的纯化EcoCas3蛋白质(图4)。认为通过使用磷酸缓冲液，被分离到排除级分的EcoCas3被抑制。用SDS-PAGE确认了纯度，结果没有检测到目的EcoCas3蛋白质以外的蛋白质的条带，由此判明，与利用了大肠杆菌表达系统的现有的EcoCas3纯化蛋白质相比，显著以高纯度被纯化。

[实施例2]重组EcoCas3蛋白质的热稳定性的测定

通过使用TychoNT6(NanoTempar社)测定热变性图谱的拐点温度Ti，从而进行重组EcoCas3蛋白质的热稳定性的评价。在330nm和350nm二波长检测伴随由热产生的蛋白质分子的解折叠的分子内色氨酸残基来源的自身荧光的峰移位，将其荧光强度的比相对于温度作图来确定热变性图谱的拐点温度Ti。

另外，也进行了利用SYPRO Orange荧光试剂的重组EcoCas3蛋白质的热稳定性的测定。SYPRO Orange通过与蛋白质的变性了的疏水区域结合而呈现荧光。通过实时PCR装置来检测依赖于由伴随热变性的结构变化产生的蛋白质的疏水性区域向溶剂的露出的SYPROOrange的结合和荧光强度的增加。荧光检测在激发波长473nm、荧光波长520nm进行。

以上的结果表明，虽然在使用TychoNT6的测定中，重组EcoCas3蛋白质的拐点温度与Cas9基本为相同程度(图5)，但是在使用SYPRO Orange荧光试剂的37℃恒温的稳定性测定中，重组EcoCas3蛋白质在约8小时后完全变性(图6)。由该结果考虑，重组EcoCas3蛋白质处于显示分子的杂乱度的熵高的状态，涨落较大，因此作为热力学参数的吉布斯自由能变化量变小，与Cas9、Cas12、TfuCas3相比处于不稳定。关于该重组EcoCas3蛋白质的稳定性的结果良好地说明，在Sf9细胞中，能够在低于通常的昆虫细胞培养温度(28℃)的温度下高效率地表达该蛋白质(实施例1(2)的结果)。另一方面，判明了纯化所得的重组Cas3蛋白质即使在37℃也在数小时左右具有高活性，该事实良好地说明，在体外(in vitro)、各种细胞中靶标DNA的切割处理中能够无特别问题地使用(后述的实施例3和4的结果)。

[实施例3]Eco级联反应复合体的制备

为了将重组Eco级联反应与重组EcoCas3蛋白质同时导入细胞进行基因组编辑，使其在细胞内高效地向核内转运是重要的。然而，Eco级联反应的分子量大到约0.4MDa，核定位率令人担心。于是，本发明者们将构成Eco级联反应的5个基因的操纵子分割成2段，在各自的3’末端添加NLS，从而将更多的NLS导入级联反应，尝试了向细胞核内的高效率转运。

Eco级联反应是由Cas8-Cas11-Cas7-Cas5-Cas6组成的超分子复合体。构成数是Cas8为1分子、Cas11为2分子、Cas7为5分子、Cas5为2分子、Cas6为1分子。本发明者们构建了3个质粒：将融合了His标签的Cas11-NLS装入pCDFuet-1质粒；将在3’末端添加了NLS的“Cas8-Cas11-Cas7操纵子”、和在3’末端添加了NLS的“Cas5-Cas6操纵子”装入pRSFDuet-1质粒；将crRNA整合到pACYCDuet-1(图7、序列号10～24)。

将这3个质粒装入大肠杆菌JM109(DE3)(保持有装入了在lacUV5启动子控制下的T7 RNA聚合酶基因的噬菌体λDE3的溶原性大肠杆菌)，通过IPTG使重组蛋白质或crRNA。介由被导入Cas11中的His标签，进行利用镍柱的Multi-NLS-Eco级联反应的亲和纯化，接着通过凝胶过滤层析进行纯化。其结果成功地从2L培养制造约1mg的Multi-NLS-Eco级联反应。

其中，crRNA的靶标序列为人EMX1基因内序列和小鼠Tyr基因内的序列和东海墨绿多管水母GFP基因内的序列。

[实施例4]体外(in vitro)的DNA切割测定

使用双链DNA，在体外(in vitro)研究由纯化后的EcoCas3和Eco级联反应蛋白质产生的靶标DNA切割活性。反应缓冲液(5mM HEPES-K pH7.5、60mM KCl、10mM MgCl₂、10μMCoCl₂、2.5mM ATP)中，混合Cas3蛋白质(20nM)、crRNA与级联反应的复合体(20nM)、包含靶标序列的双链DNA(60ng/μL)。将该反应溶液在37℃孵育1小时，使用MultiNa(岛津制作所)进行毛细管电泳。靶标序列为EMX1基因区域和Tyr基因区域。此外，对于EMX1，也制作了将双链DNA的PAM序列由大肠杆菌来源的I型-ECRISPR能够识别的“AAG”变更为不能识别的“CCA”的序列，进行了研究。

其结果是，在混合了包含PAM序列能够识别的AAG的靶标序列的双链的供体DNA的情况下，观察到供体DNA的分解(图8)。另一方面，在混合了变更为PAM序列不能识别的CCA的供体DNA的情况下，观察不到DNA的分解。在Cas3蛋白质单独、级联反应复合体单独的情况下，没观察到DNA的切割活性。另一方面级联反应复合体单独的情况下，在通常更上检测到条带。这考虑由于级联反应复合体识别并结合DNA，因而在非变性条件下，尺寸比通常变大，发生了凝胶位移。

[实施例5]人培养细胞HEK293T中的活性测定

使用具有mCherry-P2A-EGFP的报告物HEK293T细胞，研究了纯化后的EcoCas3和Eco级联反应蛋白质在人细胞内的突变导入效率。通过使用Neon Transfection System(Thermo Fisher Scientific社)的电穿孔法将Cas3蛋白质(30μM或45μM)、GFP靶标crRNA与级联反应的复合体(30μM或45μM)导入报告物细胞。将细胞在37℃、5％CO₂培养5天后，回收全细胞，使用SH800(SONY社)计数GFP阴性细胞数，计算出突变导入效率。

其结果是，在以30μM导入的情况下计数到20％左右的GFP阴性细胞，在以45μM导入的情况下计数到40％左右的GFP阴性细胞(图9)。即，显示了使用CRISPR-Cas3蛋白质的基因组编辑能够以最大约40％左右的高效率实施。

产业可利用性

通过本发明的方法制造的Cas3蛋白质能够在各种细胞的基因组编辑中利用，因此不仅在基础研究中，也能够在医疗、农业、工业等各种基因组编辑技术的应用领域利用。

序列表

<110> C4U株式会社

国立大学法人东京大学

国立研究开发法人理化学研究所

<120> Cas3蛋白质的制造方法

<130> IBPF22-507WO

<150> JP 2021-031907

<151> 2021-03-01

<160> 24

<170> PatentIn 版本 3.5

<210> 1

<211> 2667

<212> DNA

<213> 大肠杆菌(Escherichia coli)

<220>

<221> CDS

<222> (1)..(2667)

<223> EcoCas3

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<210> 2

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<212> PRT

<213> 大肠杆菌(Escherichia coli)

<400> 2

Met Glu Pro Phe Lys Tyr Ile Cys His Tyr Trp Gly Lys Ser Ser Lys

1 5 10 15

Ser Leu Thr Lys Gly Asn Asp Ile His Leu Leu Ile Tyr His Cys Leu

20 25 30

Asp Val Ala Ala Val Ala Asp Cys Trp Trp Asp Gln Ser Val Val Leu

35 40 45

Gln Asn Thr Phe Cys Arg Asn Glu Met Leu Ser Lys Gln Arg Val Lys

50 55 60

Ala Trp Leu Leu Phe Phe Ile Ala Leu His Asp Ile Gly Lys Phe Asp

65 70 75 80

Ile Arg Phe Gln Tyr Lys Ser Ala Glu Ser Trp Leu Lys Leu Asn Pro

85 90 95

Ala Thr Pro Ser Leu Asn Gly Pro Ser Thr Gln Met Cys Arg Lys Phe

100 105 110

Asn His Gly Ala Ala Gly Leu Tyr Trp Phe Asn Gln Asp Ser Leu Ser

115 120 125

Glu Gln Ser Leu Gly Asp Phe Phe Ser Phe Phe Asp Ala Ala Pro His

130 135 140

Pro Tyr Glu Ser Trp Phe Pro Trp Val Glu Ala Val Thr Gly His His

145 150 155 160

Gly Phe Ile Leu His Ser Gln Asp Gln Asp Lys Ser Arg Trp Glu Met

165 170 175

Pro Ala Ser Leu Ala Ser Tyr Ala Ala Gln Asp Lys Gln Ala Arg Glu

180 185 190

Glu Trp Ile Ser Val Leu Glu Ala Leu Phe Leu Thr Pro Ala Gly Leu

195 200 205

Ser Ile Asn Asp Ile Pro Pro Asp Cys Ser Ser Leu Leu Ala Gly Phe

210 215 220

Cys Ser Leu Ala Asp Trp Leu Gly Ser Trp Thr Thr Thr Asn Thr Phe

225 230 235 240

Leu Phe Asn Glu Asp Ala Pro Ser Asp Ile Asn Ala Leu Arg Thr Tyr

245 250 255

Phe Gln Asp Arg Gln Gln Asp Ala Ser Arg Val Leu Glu Leu Ser Gly

260 265 270

Leu Val Ser Asn Lys Arg Cys Tyr Glu Gly Val His Ala Leu Leu Asp

275 280 285

Asn Gly Tyr Gln Pro Arg Gln Leu Gln Val Leu Val Asp Ala Leu Pro

290 295 300

Val Ala Pro Gly Leu Thr Val Ile Glu Ala Pro Thr Gly Ser Gly Lys

305 310 315 320

Thr Glu Thr Ala Leu Ala Tyr Ala Trp Lys Leu Ile Asp Gln Gln Ile

325 330 335

Ala Asp Ser Val Ile Phe Ala Leu Pro Thr Gln Ala Thr Ala Asn Ala

340 345 350

Met Leu Thr Arg Met Glu Ala Ser Ala Ser His Leu Phe Ser Ser Pro

355 360 365

Asn Leu Ile Leu Ala His Gly Asn Ser Arg Phe Asn His Leu Phe Gln

370 375 380

Ser Ile Lys Ser Arg Ala Ile Thr Glu Gln Gly Gln Glu Glu Ala Trp

385 390 395 400

Val Gln Cys Cys Gln Trp Leu Ser Gln Ser Asn Lys Lys Val Phe Leu

405 410 415

Gly Gln Ile Gly Val Cys Thr Ile Asp Gln Val Leu Ile Ser Val Leu

420 425 430

Pro Val Lys His Arg Phe Ile Arg Gly Leu Gly Ile Gly Arg Ser Val

435 440 445

Leu Ile Val Asp Glu Val His Ala Tyr Asp Thr Tyr Met Asn Gly Leu

450 455 460

Leu Glu Ala Val Leu Lys Ala Gln Ala Asp Val Gly Gly Ser Val Ile

465 470 475 480

Leu Leu Ser Ala Thr Leu Pro Met Lys Gln Lys Gln Lys Leu Leu Asp

485 490 495

Thr Tyr Gly Leu His Thr Asp Pro Val Glu Asn Asn Ser Ala Tyr Pro

500 505 510

Leu Ile Asn Trp Arg Gly Val Asn Gly Ala Gln Arg Phe Asp Leu Leu

515 520 525

Ala His Pro Glu Gln Leu Pro Pro Arg Phe Ser Ile Gln Pro Glu Pro

530 535 540

Ile Cys Leu Ala Asp Met Leu Pro Asp Leu Thr Met Leu Glu Arg Met

545 550 555 560

Ile Ala Ala Ala Asn Ala Gly Ala Gln Val Cys Leu Ile Cys Asn Leu

565 570 575

Val Asp Val Ala Gln Val Cys Tyr Gln Arg Leu Lys Glu Leu Asn Asn

580 585 590

Thr Gln Val Asp Ile Asp Leu Phe His Ala Arg Phe Thr Leu Asn Asp

595 600 605

Arg Arg Glu Lys Glu Asn Arg Val Ile Ser Asn Phe Gly Lys Asn Gly

610 615 620

Lys Arg Asn Val Gly Arg Ile Leu Val Ala Thr Gln Val Val Glu Gln

625 630 635 640

Ser Leu Asp Val Asp Phe Asp Trp Leu Ile Thr Gln His Cys Pro Ala

645 650 655

Asp Leu Leu Phe Gln Arg Leu Gly Arg Leu His Arg His His Arg Lys

660 665 670

Tyr Arg Pro Ala Gly Phe Glu Ile Pro Val Ala Thr Ile Leu Leu Pro

675 680 685

Asp Gly Glu Gly Tyr Gly Arg His Glu His Ile Tyr Ser Asn Val Arg

690 695 700

Val Met Trp Arg Thr Gln Gln His Ile Glu Glu Leu Asn Gly Ala Ser

705 710 715 720

Leu Phe Phe Pro Asp Ala Tyr Arg Gln Trp Leu Asp Ser Ile Tyr Asp

725 730 735

Asp Ala Glu Met Asp Glu Pro Glu Trp Val Gly Asn Gly Met Asp Lys

740 745 750

Phe Glu Ser Ala Glu Cys Glu Lys Arg Phe Lys Ala Arg Lys Val Leu

755 760 765

Gln Trp Ala Glu Glu Tyr Ser Leu Gln Asp Asn Asp Glu Thr Ile Leu

770 775 780

Ala Val Thr Arg Asp Gly Glu Met Ser Leu Pro Leu Leu Pro Tyr Val

785 790 795 800

Gln Thr Ser Ser Gly Lys Gln Leu Leu Asp Gly Gln Val Tyr Glu Asp

805 810 815

Leu Ser His Glu Gln Gln Tyr Glu Ala Leu Ala Leu Asn Arg Val Asn

820 825 830

Val Pro Phe Thr Trp Lys Arg Ser Phe Ser Glu Val Val Asp Glu Asp

835 840 845

Gly Leu Leu Trp Leu Glu Gly Lys Gln Asn Leu Asp Gly Trp Val Trp

850 855 860

Gln Gly Asn Ser Ile Val Ile Thr Tyr Thr Gly Asp Glu Gly Met Thr

865 870 875 880

Arg Val Ile Pro Ala Asn Pro Lys

885

<210> 3

<211> 7

<212> PRT

<213> 人工序列

<220>

<223> NLS

<400> 3

Pro Lys Lys Lys Arg Lys Val

1 5

<210> 4

<211> 18

<212> PRT

<213> 人工序列

<220>

<223> NLS

<400> 4

Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val

<210> 5

<211> 21

<212> PRT

<213> 人工序列

<220>

<223> HN标签

<400> 5

His His His His His His His His Gly Ser His Asn His Asn His Asn

1 5 10 15

His Asn His Asn His

20

<210> 6

<211> 7554

<212> DNA

<213> 人工序列

<220>

<223> EcoCas3质粒

<220>

<221> CDS

<222> (4059)..(6908)

<400> 6

gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 60

gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 120

acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 180

agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240

ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300

ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360

taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420

aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840

gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980

tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400

cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460

ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520

caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580

acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640

tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700

ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760

aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820

tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880

ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940

tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000

gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060

gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120

cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180

ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240

agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300

ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360

tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420

acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480

ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540

ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600

cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660

ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720

cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780

ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840

gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900

tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960

ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020

ccatcgggcg cggatctaac tcctaaaaaa ccgccacc atg cat cat cac cat cac 4076

Met His His His His His

1 5

cac cat cac ggt tct cat aac cat aac cac aac cat aac cac aac cac 4124

His His His Gly Ser His Asn His Asn His Asn His Asn His Asn His

10 15 20

ggt acc ggt tct gag aat ctg tac ttc caa ggt gga tcc ccc aag aag 4172

Gly Thr Gly Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Pro Lys Lys

25 30 35

aag cgg aag gtc gga tcc gaa cct ttt aaa tat ata tgc cat tac tgg 4220

Lys Arg Lys Val Gly Ser Glu Pro Phe Lys Tyr Ile Cys His Tyr Trp

40 45 50

gga aaa tcc tca aaa agc ttg acg aaa gga aat gat att cat ctg tta 4268

Gly Lys Ser Ser Lys Ser Leu Thr Lys Gly Asn Asp Ile His Leu Leu

55 60 65 70

att tat cat tgc ctt gat gtt gct gct gtt gca gat tgc tgg tgg gat 4316

Ile Tyr His Cys Leu Asp Val Ala Ala Val Ala Asp Cys Trp Trp Asp

75 80 85

caa tca gtc gta ctg caa aat act ttt tgc cga aat gaa atg cta tca 4364

Gln Ser Val Val Leu Gln Asn Thr Phe Cys Arg Asn Glu Met Leu Ser

90 95 100

aaa cag agg gtg aag gcc tgg ctg tta ttt ttc att gct ctt cat gat 4412

Lys Gln Arg Val Lys Ala Trp Leu Leu Phe Phe Ile Ala Leu His Asp

105 110 115

att gga aag ttt gat ata cga ttc caa tat aaa tca gca gaa agt tgg 4460

Ile Gly Lys Phe Asp Ile Arg Phe Gln Tyr Lys Ser Ala Glu Ser Trp

120 125 130

ctg aaa tta aat cct gca acg cca tca ctt aat ggt cca tca aca caa 4508

Leu Lys Leu Asn Pro Ala Thr Pro Ser Leu Asn Gly Pro Ser Thr Gln

135 140 145 150

atg tgc cgt aaa ttt aat cat ggt gca gcc ggt ctg tat tgg ttt aac 4556

Met Cys Arg Lys Phe Asn His Gly Ala Ala Gly Leu Tyr Trp Phe Asn

155 160 165

cag gat tca ctt tca gag caa tct ctc ggg gat ttt ttc agt ttt ttt 4604

Gln Asp Ser Leu Ser Glu Gln Ser Leu Gly Asp Phe Phe Ser Phe Phe

170 175 180

gat gcc gct cct cat cct tat gag tcc tgg ttt cca tgg gta gag gcc 4652

Asp Ala Ala Pro His Pro Tyr Glu Ser Trp Phe Pro Trp Val Glu Ala

185 190 195

gtt aca gga cat cat ggt ttt ata tta cat tcc cag gat caa gat aag 4700

Val Thr Gly His His Gly Phe Ile Leu His Ser Gln Asp Gln Asp Lys

200 205 210

tcg cgt tgg gaa atg cca gct tct ctg gca tct tat gct gcg caa gat 4748

Ser Arg Trp Glu Met Pro Ala Ser Leu Ala Ser Tyr Ala Ala Gln Asp

215 220 225 230

aaa cag gct cgt gag gag tgg ata tct gta ctg gaa gca tta ttt tta 4796

Lys Gln Ala Arg Glu Glu Trp Ile Ser Val Leu Glu Ala Leu Phe Leu

235 240 245

acg cca gcg ggg tta tct ata aac gat ata cca cct gat tgt tca tca 4844

Thr Pro Ala Gly Leu Ser Ile Asn Asp Ile Pro Pro Asp Cys Ser Ser

250 255 260

ctg tta gca ggt ttt tgc tcg ctt gct gac tgg tta ggc tcc tgg act 4892

Leu Leu Ala Gly Phe Cys Ser Leu Ala Asp Trp Leu Gly Ser Trp Thr

265 270 275

aca acg aat acc ttt ctg ttt aat gag gat gcg cct tcc gac ata aat 4940

Thr Thr Asn Thr Phe Leu Phe Asn Glu Asp Ala Pro Ser Asp Ile Asn

280 285 290

gct ctg aga acg tat ttc cag gac cga cag cag gat gcg agc cgg gta 4988

Ala Leu Arg Thr Tyr Phe Gln Asp Arg Gln Gln Asp Ala Ser Arg Val

295 300 305 310

ttg gag ttg agt gga ctt gta tca aat aag cga tgt tat gaa ggt gtt 5036

Leu Glu Leu Ser Gly Leu Val Ser Asn Lys Arg Cys Tyr Glu Gly Val

315 320 325

cat gca cta ctg gac aat ggc tat caa ccc aga caa tta cag gtg tta 5084

His Ala Leu Leu Asp Asn Gly Tyr Gln Pro Arg Gln Leu Gln Val Leu

330 335 340

gtt gat gct ctt cca gta gct ccc ggg ctg acg gta ata gag gca cct 5132

Val Asp Ala Leu Pro Val Ala Pro Gly Leu Thr Val Ile Glu Ala Pro

345 350 355

aca ggc tcc ggt aaa acg gaa aca gcg ctg gcc tat gct tgg aaa ctt 5180

Thr Gly Ser Gly Lys Thr Glu Thr Ala Leu Ala Tyr Ala Trp Lys Leu

360 365 370

att gat caa caa att gcg gat agt gtt att ttt gcc ctc cca aca caa 5228

Ile Asp Gln Gln Ile Ala Asp Ser Val Ile Phe Ala Leu Pro Thr Gln

375 380 385 390

gct acc gcg aat gct atg ctt acg aga atg gaa gcg agc gcg agc cac 5276

Ala Thr Ala Asn Ala Met Leu Thr Arg Met Glu Ala Ser Ala Ser His

395 400 405

tta ttt tca tcc cca aat ctt att ctt gct cat ggc aat tca cgg ttt 5324

Leu Phe Ser Ser Pro Asn Leu Ile Leu Ala His Gly Asn Ser Arg Phe

410 415 420

aac cac ctc ttt caa tca ata aaa tca cgc gcg att act gaa cag ggg 5372

Asn His Leu Phe Gln Ser Ile Lys Ser Arg Ala Ile Thr Glu Gln Gly

425 430 435

caa gaa gaa gcg tgg gtt cag tgt tgt cag tgg ttg tca caa agc aat 5420

Gln Glu Glu Ala Trp Val Gln Cys Cys Gln Trp Leu Ser Gln Ser Asn

440 445 450

aag aaa gtg ttt ctt ggg caa atc ggc gtt tgc acg att gat cag gtg 5468

Lys Lys Val Phe Leu Gly Gln Ile Gly Val Cys Thr Ile Asp Gln Val

455 460 465 470

ttg ata tcg gta ttg cca gtt aaa cac cgc ttt atc cgt ggt ttg gga 5516

Leu Ile Ser Val Leu Pro Val Lys His Arg Phe Ile Arg Gly Leu Gly

475 480 485

att ggt cga agt gtt tta att gtt gat gaa gtt cat gct tac gac acc 5564

Ile Gly Arg Ser Val Leu Ile Val Asp Glu Val His Ala Tyr Asp Thr

490 495 500

tat atg aac ggc ttg ctg gag gca gtg ctc aag gct cag gct gat gtg 5612

Tyr Met Asn Gly Leu Leu Glu Ala Val Leu Lys Ala Gln Ala Asp Val

505 510 515

gga ggg agt gtt att ctt ctt tcc gca acc cta cca atg aaa caa aaa 5660

Gly Gly Ser Val Ile Leu Leu Ser Ala Thr Leu Pro Met Lys Gln Lys

520 525 530

cag aaa ctt ctg gat act tat ggt ctg cat aca gat cca gtg gaa aat 5708

Gln Lys Leu Leu Asp Thr Tyr Gly Leu His Thr Asp Pro Val Glu Asn

535 540 545 550

aac tcc gca tat cca ctc att aac tgg cga ggt gtg aat ggt gcg caa 5756

Asn Ser Ala Tyr Pro Leu Ile Asn Trp Arg Gly Val Asn Gly Ala Gln

555 560 565

cgt ttt gat ctg cta gct cat cca gaa caa ctc ccg ccc cgc ttt tcg 5804

Arg Phe Asp Leu Leu Ala His Pro Glu Gln Leu Pro Pro Arg Phe Ser

570 575 580

att cag cca gaa cct att tgt tta gct gac atg tta cct gac ctt acg 5852

Ile Gln Pro Glu Pro Ile Cys Leu Ala Asp Met Leu Pro Asp Leu Thr

585 590 595

atg tta gag cga atg atc gca gcg gca aac gcg ggt gca cag gtc tgt 5900

Met Leu Glu Arg Met Ile Ala Ala Ala Asn Ala Gly Ala Gln Val Cys

600 605 610

ctt att tgc aat ttg gtt gac gtt gca caa gta tgc tac caa cgg cta 5948

Leu Ile Cys Asn Leu Val Asp Val Ala Gln Val Cys Tyr Gln Arg Leu

615 620 625 630

aag gag cta aat aac acg caa gta gat ata gat ttg ttt cat gcg cgc 5996

Lys Glu Leu Asn Asn Thr Gln Val Asp Ile Asp Leu Phe His Ala Arg

635 640 645

ttt acg ctg aac gat cgt cgt gaa aaa gag aat cga gtt att agc aat 6044

Phe Thr Leu Asn Asp Arg Arg Glu Lys Glu Asn Arg Val Ile Ser Asn

650 655 660

ttc ggc aaa aat ggg aag cga aat gtt gga cgg ata ctt gtc gca acc 6092

Phe Gly Lys Asn Gly Lys Arg Asn Val Gly Arg Ile Leu Val Ala Thr

665 670 675

cag gtc gtg gaa caa tca ctc gac gtt gat ttt gat tgg tta att act 6140

Gln Val Val Glu Gln Ser Leu Asp Val Asp Phe Asp Trp Leu Ile Thr

680 685 690

cag cat tgt cct gca gat ttg ctt ttc caa cga ttg ggc cgt tta cat 6188

Gln His Cys Pro Ala Asp Leu Leu Phe Gln Arg Leu Gly Arg Leu His

695 700 705 710

cgc cat cat cgc aaa tat cgt ccc gct ggt ttt gag att cct gtt gcc 6236

Arg His His Arg Lys Tyr Arg Pro Ala Gly Phe Glu Ile Pro Val Ala

715 720 725

acc att ttg ctg cct gat ggc gag ggt tac gga cga cat gag cat att 6284

Thr Ile Leu Leu Pro Asp Gly Glu Gly Tyr Gly Arg His Glu His Ile

730 735 740

tat agc aac gtt aga gtc atg tgg cgg acg cag caa cat att gag gag 6332

Tyr Ser Asn Val Arg Val Met Trp Arg Thr Gln Gln His Ile Glu Glu

745 750 755

ctt aat gga gca tcc tta ttt ttc cct gat gct tac cgg caa tgg ctg 6380

Leu Asn Gly Ala Ser Leu Phe Phe Pro Asp Ala Tyr Arg Gln Trp Leu

760 765 770

gat agc att tac gat gat gcg gaa atg gat gag cca gaa tgg gtc ggc 6428

Asp Ser Ile Tyr Asp Asp Ala Glu Met Asp Glu Pro Glu Trp Val Gly

775 780 785 790

aat ggc atg gat aaa ttt gaa agc gcc gag tgt gaa aaa agg ttc aag 6476

Asn Gly Met Asp Lys Phe Glu Ser Ala Glu Cys Glu Lys Arg Phe Lys

795 800 805

gct cgc aag gtc ctg cag tgg gct gaa gaa tat agc ttg cag gat aac 6524

Ala Arg Lys Val Leu Gln Trp Ala Glu Glu Tyr Ser Leu Gln Asp Asn

810 815 820

gat gaa acc att ctt gcg gta acg agg gat ggg gaa atg agc ctg cca 6572

Asp Glu Thr Ile Leu Ala Val Thr Arg Asp Gly Glu Met Ser Leu Pro

825 830 835

tta ttg cct tat gta caa acg tct tca ggt aaa caa ctg ctc gat ggc 6620

Leu Leu Pro Tyr Val Gln Thr Ser Ser Gly Lys Gln Leu Leu Asp Gly

840 845 850

cag gtc tac gag gac cta agt cat gaa cag cag tat gag gcg ctt gca 6668

Gln Val Tyr Glu Asp Leu Ser His Glu Gln Gln Tyr Glu Ala Leu Ala

855 860 865 870

ctt aat cgc gtc aat gta ccc ttc acc tgg aaa cgt agt ttt tct gaa 6716

Leu Asn Arg Val Asn Val Pro Phe Thr Trp Lys Arg Ser Phe Ser Glu

875 880 885

gta gta gat gaa gat ggg tta ctt tgg ctg gaa ggg aaa cag aat ctg 6764

Val Val Asp Glu Asp Gly Leu Leu Trp Leu Glu Gly Lys Gln Asn Leu

890 895 900

gat gga tgg gtc tgg cag ggt aac agt att gtt att acc tat aca ggg 6812

Asp Gly Trp Val Trp Gln Gly Asn Ser Ile Val Ile Thr Tyr Thr Gly

905 910 915

gat gaa ggg atg acc aga gtc atc cct gca aat ccc aaa aag aga aca 6860

Asp Glu Gly Met Thr Arg Val Ile Pro Ala Asn Pro Lys Lys Arg Thr

920 925 930

gcc gat ggc agc gag ttc gag agc ccc aag aag aag cgg aag gtc taa 6908

Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val

935 940 945

ctcgagaagc ttgtcgagaa gtactagagg atcataatca gccataccac atttgtagag 6968

gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat 7028

gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc 7088

atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 7148

ctcatcaatg tatcttatca tgtctggatc tgatcactgc ttgagcctag gagatccgaa 7208

ccagataagt gaaatctagt tccaaactat tttgtcattt ttaattttcg tattagctta 7268

cgacgctaca cccagttccc atctattttg tcactcttcc ctaaataatc cttaaaaact 7328

ccatttccac ccctcccagt tcccaactat tttgtccgcc cacagcgggg catttttctt 7388

cctgttatgt ttttaatcaa acatcctgcc aactccatgt gacaaaccgt catcttcggc 7448

tactttttct ctgtcacaga atgaaaattt ttctgtcatc tcttcgttat taatgtttgt 7508

aattgactga atatcaacgc ttatttgcag cctgaatggc gaatgg 7554

<210> 7

<211> 949

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 7

Met His His His His His His His His Gly Ser His Asn His Asn His

1 5 10 15

Asn His Asn His Asn His Gly Thr Gly Ser Glu Asn Leu Tyr Phe Gln

20 25 30

Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Ser Glu Pro Phe Lys

35 40 45

Tyr Ile Cys His Tyr Trp Gly Lys Ser Ser Lys Ser Leu Thr Lys Gly

50 55 60

Asn Asp Ile His Leu Leu Ile Tyr His Cys Leu Asp Val Ala Ala Val

65 70 75 80

Ala Asp Cys Trp Trp Asp Gln Ser Val Val Leu Gln Asn Thr Phe Cys

85 90 95

Arg Asn Glu Met Leu Ser Lys Gln Arg Val Lys Ala Trp Leu Leu Phe

100 105 110

Phe Ile Ala Leu His Asp Ile Gly Lys Phe Asp Ile Arg Phe Gln Tyr

115 120 125

Lys Ser Ala Glu Ser Trp Leu Lys Leu Asn Pro Ala Thr Pro Ser Leu

130 135 140

Asn Gly Pro Ser Thr Gln Met Cys Arg Lys Phe Asn His Gly Ala Ala

145 150 155 160

Gly Leu Tyr Trp Phe Asn Gln Asp Ser Leu Ser Glu Gln Ser Leu Gly

165 170 175

Asp Phe Phe Ser Phe Phe Asp Ala Ala Pro His Pro Tyr Glu Ser Trp

180 185 190

Phe Pro Trp Val Glu Ala Val Thr Gly His His Gly Phe Ile Leu His

195 200 205

Ser Gln Asp Gln Asp Lys Ser Arg Trp Glu Met Pro Ala Ser Leu Ala

210 215 220

Ser Tyr Ala Ala Gln Asp Lys Gln Ala Arg Glu Glu Trp Ile Ser Val

225 230 235 240

Leu Glu Ala Leu Phe Leu Thr Pro Ala Gly Leu Ser Ile Asn Asp Ile

245 250 255

Pro Pro Asp Cys Ser Ser Leu Leu Ala Gly Phe Cys Ser Leu Ala Asp

260 265 270

Trp Leu Gly Ser Trp Thr Thr Thr Asn Thr Phe Leu Phe Asn Glu Asp

275 280 285

Ala Pro Ser Asp Ile Asn Ala Leu Arg Thr Tyr Phe Gln Asp Arg Gln

290 295 300

Gln Asp Ala Ser Arg Val Leu Glu Leu Ser Gly Leu Val Ser Asn Lys

305 310 315 320

Arg Cys Tyr Glu Gly Val His Ala Leu Leu Asp Asn Gly Tyr Gln Pro

325 330 335

Arg Gln Leu Gln Val Leu Val Asp Ala Leu Pro Val Ala Pro Gly Leu

340 345 350

Thr Val Ile Glu Ala Pro Thr Gly Ser Gly Lys Thr Glu Thr Ala Leu

355 360 365

Ala Tyr Ala Trp Lys Leu Ile Asp Gln Gln Ile Ala Asp Ser Val Ile

370 375 380

Phe Ala Leu Pro Thr Gln Ala Thr Ala Asn Ala Met Leu Thr Arg Met

385 390 395 400

Glu Ala Ser Ala Ser His Leu Phe Ser Ser Pro Asn Leu Ile Leu Ala

405 410 415

His Gly Asn Ser Arg Phe Asn His Leu Phe Gln Ser Ile Lys Ser Arg

420 425 430

Ala Ile Thr Glu Gln Gly Gln Glu Glu Ala Trp Val Gln Cys Cys Gln

435 440 445

Trp Leu Ser Gln Ser Asn Lys Lys Val Phe Leu Gly Gln Ile Gly Val

450 455 460

Cys Thr Ile Asp Gln Val Leu Ile Ser Val Leu Pro Val Lys His Arg

465 470 475 480

Phe Ile Arg Gly Leu Gly Ile Gly Arg Ser Val Leu Ile Val Asp Glu

485 490 495

Val His Ala Tyr Asp Thr Tyr Met Asn Gly Leu Leu Glu Ala Val Leu

500 505 510

Lys Ala Gln Ala Asp Val Gly Gly Ser Val Ile Leu Leu Ser Ala Thr

515 520 525

Leu Pro Met Lys Gln Lys Gln Lys Leu Leu Asp Thr Tyr Gly Leu His

530 535 540

Thr Asp Pro Val Glu Asn Asn Ser Ala Tyr Pro Leu Ile Asn Trp Arg

545 550 555 560

Gly Val Asn Gly Ala Gln Arg Phe Asp Leu Leu Ala His Pro Glu Gln

565 570 575

Leu Pro Pro Arg Phe Ser Ile Gln Pro Glu Pro Ile Cys Leu Ala Asp

580 585 590

Met Leu Pro Asp Leu Thr Met Leu Glu Arg Met Ile Ala Ala Ala Asn

595 600 605

Ala Gly Ala Gln Val Cys Leu Ile Cys Asn Leu Val Asp Val Ala Gln

610 615 620

Val Cys Tyr Gln Arg Leu Lys Glu Leu Asn Asn Thr Gln Val Asp Ile

625 630 635 640

Asp Leu Phe His Ala Arg Phe Thr Leu Asn Asp Arg Arg Glu Lys Glu

645 650 655

Asn Arg Val Ile Ser Asn Phe Gly Lys Asn Gly Lys Arg Asn Val Gly

660 665 670

Arg Ile Leu Val Ala Thr Gln Val Val Glu Gln Ser Leu Asp Val Asp

675 680 685

Phe Asp Trp Leu Ile Thr Gln His Cys Pro Ala Asp Leu Leu Phe Gln

690 695 700

Arg Leu Gly Arg Leu His Arg His His Arg Lys Tyr Arg Pro Ala Gly

705 710 715 720

Phe Glu Ile Pro Val Ala Thr Ile Leu Leu Pro Asp Gly Glu Gly Tyr

725 730 735

Gly Arg His Glu His Ile Tyr Ser Asn Val Arg Val Met Trp Arg Thr

740 745 750

Gln Gln His Ile Glu Glu Leu Asn Gly Ala Ser Leu Phe Phe Pro Asp

755 760 765

Ala Tyr Arg Gln Trp Leu Asp Ser Ile Tyr Asp Asp Ala Glu Met Asp

770 775 780

Glu Pro Glu Trp Val Gly Asn Gly Met Asp Lys Phe Glu Ser Ala Glu

785 790 795 800

Cys Glu Lys Arg Phe Lys Ala Arg Lys Val Leu Gln Trp Ala Glu Glu

805 810 815

Tyr Ser Leu Gln Asp Asn Asp Glu Thr Ile Leu Ala Val Thr Arg Asp

820 825 830

Gly Glu Met Ser Leu Pro Leu Leu Pro Tyr Val Gln Thr Ser Ser Gly

835 840 845

Lys Gln Leu Leu Asp Gly Gln Val Tyr Glu Asp Leu Ser His Glu Gln

850 855 860

Gln Tyr Glu Ala Leu Ala Leu Asn Arg Val Asn Val Pro Phe Thr Trp

865 870 875 880

Lys Arg Ser Phe Ser Glu Val Val Asp Glu Asp Gly Leu Leu Trp Leu

885 890 895

Glu Gly Lys Gln Asn Leu Asp Gly Trp Val Trp Gln Gly Asn Ser Ile

900 905 910

Val Ile Thr Tyr Thr Gly Asp Glu Gly Met Thr Arg Val Ile Pro Ala

915 920 925

Asn Pro Lys Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys

930 935 940

Lys Lys Arg Lys Val

945

<210> 8

<211> 8274

<212> DNA

<213> 人工序列

<220>

<223> EGFP-EcoCas3质粒

<220>

<221> CDS

<222> (4059)..(7628)

<400> 8

gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 60

gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 120

acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 180

agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240

ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300

ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360

taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420

aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840

gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980

tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400

cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460

ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520

caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580

acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640

tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700

ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760

aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820

tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880

ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940

tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000

gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060

gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120

cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180

ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240

agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300

ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360

tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420

acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480

ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540

ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600

cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660

ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720

cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780

ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840

gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900

tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960

ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020

ccatcgggcg cggatctaac tcctaaaaaa ccgccacc atg cat cat cac cat cac 4076

Met His His His His His

1 5

cac cat cac ggt tct cat aac cat aac cac aac cat aac cac aac cac 4124

His His His Gly Ser His Asn His Asn His Asn His Asn His Asn His

10 15 20

ggt acc gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc 4172

Gly Thr Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile

25 30 35

ctg gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc 4220

Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser

40 45 50

ggc gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc 4268

Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe

55 60 65 70

atc tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg acc 4316

Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr

75 80 85

acc ctg acc tac ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg 4364

Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met

90 95 100

aag cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag 4412

Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln

105 110 115

gag cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc 4460

Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala

120 125 130

gag gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag 4508

Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys

135 140 145 150

ggc atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag 4556

Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu

155 160 165

tac aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aag 4604

Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys

170 175 180

aac ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gac ggc 4652

Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly

185 190 195

agc gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gac 4700

Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp

200 205 210

ggc ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc aaa 4748

Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys

215 220 225 230

ctg agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctg ctg gag 4796

Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu

235 240 245

ttc gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag 4844

Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

250 255 260

ggt acc ggt tct gag aat ctg tac ttc caa ggt gga tcc ccc aag aag 4892

Gly Thr Gly Ser Glu Asn Leu Tyr Phe Gln Gly Gly Ser Pro Lys Lys

265 270 275

aag cgg aag gtc gga tcc gaa cct ttt aaa tat ata tgc cat tac tgg 4940

Lys Arg Lys Val Gly Ser Glu Pro Phe Lys Tyr Ile Cys His Tyr Trp

280 285 290

gga aaa tcc tca aaa agc ttg acg aaa gga aat gat att cat ctg tta 4988

Gly Lys Ser Ser Lys Ser Leu Thr Lys Gly Asn Asp Ile His Leu Leu

295 300 305 310

att tat cat tgc ctt gat gtt gct gct gtt gca gat tgc tgg tgg gat 5036

Ile Tyr His Cys Leu Asp Val Ala Ala Val Ala Asp Cys Trp Trp Asp

315 320 325

caa tca gtc gta ctg caa aat act ttt tgc cga aat gaa atg cta tca 5084

Gln Ser Val Val Leu Gln Asn Thr Phe Cys Arg Asn Glu Met Leu Ser

330 335 340

aaa cag agg gtg aag gcc tgg ctg tta ttt ttc att gct ctt cat gat 5132

Lys Gln Arg Val Lys Ala Trp Leu Leu Phe Phe Ile Ala Leu His Asp

345 350 355

att gga aag ttt gat ata cga ttc caa tat aaa tca gca gaa agt tgg 5180

Ile Gly Lys Phe Asp Ile Arg Phe Gln Tyr Lys Ser Ala Glu Ser Trp

360 365 370

ctg aaa tta aat cct gca acg cca tca ctt aat ggt cca tca aca caa 5228

Leu Lys Leu Asn Pro Ala Thr Pro Ser Leu Asn Gly Pro Ser Thr Gln

375 380 385 390

atg tgc cgt aaa ttt aat cat ggt gca gcc ggt ctg tat tgg ttt aac 5276

Met Cys Arg Lys Phe Asn His Gly Ala Ala Gly Leu Tyr Trp Phe Asn

395 400 405

cag gat tca ctt tca gag caa tct ctc ggg gat ttt ttc agt ttt ttt 5324

Gln Asp Ser Leu Ser Glu Gln Ser Leu Gly Asp Phe Phe Ser Phe Phe

410 415 420

gat gcc gct cct cat cct tat gag tcc tgg ttt cca tgg gta gag gcc 5372

Asp Ala Ala Pro His Pro Tyr Glu Ser Trp Phe Pro Trp Val Glu Ala

425 430 435

gtt aca gga cat cat ggt ttt ata tta cat tcc cag gat caa gat aag 5420

Val Thr Gly His His Gly Phe Ile Leu His Ser Gln Asp Gln Asp Lys

440 445 450

tcg cgt tgg gaa atg cca gct tct ctg gca tct tat gct gcg caa gat 5468

Ser Arg Trp Glu Met Pro Ala Ser Leu Ala Ser Tyr Ala Ala Gln Asp

455 460 465 470

aaa cag gct cgt gag gag tgg ata tct gta ctg gaa gca tta ttt tta 5516

Lys Gln Ala Arg Glu Glu Trp Ile Ser Val Leu Glu Ala Leu Phe Leu

475 480 485

acg cca gcg ggg tta tct ata aac gat ata cca cct gat tgt tca tca 5564

Thr Pro Ala Gly Leu Ser Ile Asn Asp Ile Pro Pro Asp Cys Ser Ser

490 495 500

ctg tta gca ggt ttt tgc tcg ctt gct gac tgg tta ggc tcc tgg act 5612

Leu Leu Ala Gly Phe Cys Ser Leu Ala Asp Trp Leu Gly Ser Trp Thr

505 510 515

aca acg aat acc ttt ctg ttt aat gag gat gcg cct tcc gac ata aat 5660

Thr Thr Asn Thr Phe Leu Phe Asn Glu Asp Ala Pro Ser Asp Ile Asn

520 525 530

gct ctg aga acg tat ttc cag gac cga cag cag gat gcg agc cgg gta 5708

Ala Leu Arg Thr Tyr Phe Gln Asp Arg Gln Gln Asp Ala Ser Arg Val

535 540 545 550

ttg gag ttg agt gga ctt gta tca aat aag cga tgt tat gaa ggt gtt 5756

Leu Glu Leu Ser Gly Leu Val Ser Asn Lys Arg Cys Tyr Glu Gly Val

555 560 565

cat gca cta ctg gac aat ggc tat caa ccc aga caa tta cag gtg tta 5804

His Ala Leu Leu Asp Asn Gly Tyr Gln Pro Arg Gln Leu Gln Val Leu

570 575 580

gtt gat gct ctt cca gta gct ccc ggg ctg acg gta ata gag gca cct 5852

Val Asp Ala Leu Pro Val Ala Pro Gly Leu Thr Val Ile Glu Ala Pro

585 590 595

aca ggc tcc ggt aaa acg gaa aca gcg ctg gcc tat gct tgg aaa ctt 5900

Thr Gly Ser Gly Lys Thr Glu Thr Ala Leu Ala Tyr Ala Trp Lys Leu

600 605 610

att gat caa caa att gcg gat agt gtt att ttt gcc ctc cca aca caa 5948

Ile Asp Gln Gln Ile Ala Asp Ser Val Ile Phe Ala Leu Pro Thr Gln

615 620 625 630

gct acc gcg aat gct atg ctt acg aga atg gaa gcg agc gcg agc cac 5996

Ala Thr Ala Asn Ala Met Leu Thr Arg Met Glu Ala Ser Ala Ser His

635 640 645

tta ttt tca tcc cca aat ctt att ctt gct cat ggc aat tca cgg ttt 6044

Leu Phe Ser Ser Pro Asn Leu Ile Leu Ala His Gly Asn Ser Arg Phe

650 655 660

aac cac ctc ttt caa tca ata aaa tca cgc gcg att act gaa cag ggg 6092

Asn His Leu Phe Gln Ser Ile Lys Ser Arg Ala Ile Thr Glu Gln Gly

665 670 675

caa gaa gaa gcg tgg gtt cag tgt tgt cag tgg ttg tca caa agc aat 6140

Gln Glu Glu Ala Trp Val Gln Cys Cys Gln Trp Leu Ser Gln Ser Asn

680 685 690

aag aaa gtg ttt ctt ggg caa atc ggc gtt tgc acg att gat cag gtg 6188

Lys Lys Val Phe Leu Gly Gln Ile Gly Val Cys Thr Ile Asp Gln Val

695 700 705 710

ttg ata tcg gta ttg cca gtt aaa cac cgc ttt atc cgt ggt ttg gga 6236

Leu Ile Ser Val Leu Pro Val Lys His Arg Phe Ile Arg Gly Leu Gly

715 720 725

att ggt cga agt gtt tta att gtt gat gaa gtt cat gct tac gac acc 6284

Ile Gly Arg Ser Val Leu Ile Val Asp Glu Val His Ala Tyr Asp Thr

730 735 740

tat atg aac ggc ttg ctg gag gca gtg ctc aag gct cag gct gat gtg 6332

Tyr Met Asn Gly Leu Leu Glu Ala Val Leu Lys Ala Gln Ala Asp Val

745 750 755

gga ggg agt gtt att ctt ctt tcc gca acc cta cca atg aaa caa aaa 6380

Gly Gly Ser Val Ile Leu Leu Ser Ala Thr Leu Pro Met Lys Gln Lys

760 765 770

cag aaa ctt ctg gat act tat ggt ctg cat aca gat cca gtg gaa aat 6428

Gln Lys Leu Leu Asp Thr Tyr Gly Leu His Thr Asp Pro Val Glu Asn

775 780 785 790

aac tcc gca tat cca ctc att aac tgg cga ggt gtg aat ggt gcg caa 6476

Asn Ser Ala Tyr Pro Leu Ile Asn Trp Arg Gly Val Asn Gly Ala Gln

795 800 805

cgt ttt gat ctg cta gct cat cca gaa caa ctc ccg ccc cgc ttt tcg 6524

Arg Phe Asp Leu Leu Ala His Pro Glu Gln Leu Pro Pro Arg Phe Ser

810 815 820

att cag cca gaa cct att tgt tta gct gac atg tta cct gac ctt acg 6572

Ile Gln Pro Glu Pro Ile Cys Leu Ala Asp Met Leu Pro Asp Leu Thr

825 830 835

atg tta gag cga atg atc gca gcg gca aac gcg ggt gca cag gtc tgt 6620

Met Leu Glu Arg Met Ile Ala Ala Ala Asn Ala Gly Ala Gln Val Cys

840 845 850

ctt att tgc aat ttg gtt gac gtt gca caa gta tgc tac caa cgg cta 6668

Leu Ile Cys Asn Leu Val Asp Val Ala Gln Val Cys Tyr Gln Arg Leu

855 860 865 870

aag gag cta aat aac acg caa gta gat ata gat ttg ttt cat gcg cgc 6716

Lys Glu Leu Asn Asn Thr Gln Val Asp Ile Asp Leu Phe His Ala Arg

875 880 885

ttt acg ctg aac gat cgt cgt gaa aaa gag aat cga gtt att agc aat 6764

Phe Thr Leu Asn Asp Arg Arg Glu Lys Glu Asn Arg Val Ile Ser Asn

890 895 900

ttc ggc aaa aat ggg aag cga aat gtt gga cgg ata ctt gtc gca acc 6812

Phe Gly Lys Asn Gly Lys Arg Asn Val Gly Arg Ile Leu Val Ala Thr

905 910 915

cag gtc gtg gaa caa tca ctc gac gtt gat ttt gat tgg tta att act 6860

Gln Val Val Glu Gln Ser Leu Asp Val Asp Phe Asp Trp Leu Ile Thr

920 925 930

cag cat tgt cct gca gat ttg ctt ttc caa cga ttg ggc cgt tta cat 6908

Gln His Cys Pro Ala Asp Leu Leu Phe Gln Arg Leu Gly Arg Leu His

935 940 945 950

cgc cat cat cgc aaa tat cgt ccc gct ggt ttt gag att cct gtt gcc 6956

Arg His His Arg Lys Tyr Arg Pro Ala Gly Phe Glu Ile Pro Val Ala

955 960 965

acc att ttg ctg cct gat ggc gag ggt tac gga cga cat gag cat att 7004

Thr Ile Leu Leu Pro Asp Gly Glu Gly Tyr Gly Arg His Glu His Ile

970 975 980

tat agc aac gtt aga gtc atg tgg cgg acg cag caa cat att gag gag 7052

Tyr Ser Asn Val Arg Val Met Trp Arg Thr Gln Gln His Ile Glu Glu

985 990 995

ctt aat gga gca tcc tta ttt ttc cct gat gct tac cgg caa tgg 7097

Leu Asn Gly Ala Ser Leu Phe Phe Pro Asp Ala Tyr Arg Gln Trp

1000 1005 1010

ctg gat agc att tac gat gat gcg gaa atg gat gag cca gaa tgg 7142

Leu Asp Ser Ile Tyr Asp Asp Ala Glu Met Asp Glu Pro Glu Trp

1015 1020 1025

gtc ggc aat ggc atg gat aaa ttt gaa agc gcc gag tgt gaa aaa 7187

Val Gly Asn Gly Met Asp Lys Phe Glu Ser Ala Glu Cys Glu Lys

1030 1035 1040

agg ttc aag gct cgc aag gtc ctg cag tgg gct gaa gaa tat agc 7232

Arg Phe Lys Ala Arg Lys Val Leu Gln Trp Ala Glu Glu Tyr Ser

1045 1050 1055

ttg cag gat aac gat gaa acc att ctt gcg gta acg agg gat ggg 7277

Leu Gln Asp Asn Asp Glu Thr Ile Leu Ala Val Thr Arg Asp Gly

1060 1065 1070

gaa atg agc ctg cca tta ttg cct tat gta caa acg tct tca ggt 7322

Glu Met Ser Leu Pro Leu Leu Pro Tyr Val Gln Thr Ser Ser Gly

1075 1080 1085

aaa caa ctg ctc gat ggc cag gtc tac gag gac cta agt cat gaa 7367

Lys Gln Leu Leu Asp Gly Gln Val Tyr Glu Asp Leu Ser His Glu

1090 1095 1100

cag cag tat gag gcg ctt gca ctt aat cgc gtc aat gta ccc ttc 7412

Gln Gln Tyr Glu Ala Leu Ala Leu Asn Arg Val Asn Val Pro Phe

1105 1110 1115

acc tgg aaa cgt agt ttt tct gaa gta gta gat gaa gat ggg tta 7457

Thr Trp Lys Arg Ser Phe Ser Glu Val Val Asp Glu Asp Gly Leu

1120 1125 1130

ctt tgg ctg gaa ggg aaa cag aat ctg gat gga tgg gtc tgg cag 7502

Leu Trp Leu Glu Gly Lys Gln Asn Leu Asp Gly Trp Val Trp Gln

1135 1140 1145

ggt aac agt att gtt att acc tat aca ggg gat gaa ggg atg acc 7547

Gly Asn Ser Ile Val Ile Thr Tyr Thr Gly Asp Glu Gly Met Thr

1150 1155 1160

aga gtc atc cct gca aat ccc aaa aag aga aca gcc gat ggc agc 7592

Arg Val Ile Pro Ala Asn Pro Lys Lys Arg Thr Ala Asp Gly Ser

1165 1170 1175

gag ttc gag agc ccc aag aag aag cgg aag gtc taa ctcgagaagc 7638

Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val

1180 1185

ttgtcgagaa gtactagagg atcataatca gccataccac atttgtagag gttttacttg 7698

ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg 7758

ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 7818

tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 7878

tatcttatca tgtctggatc tgatcactgc ttgagcctag gagatccgaa ccagataagt 7938

gaaatctagt tccaaactat tttgtcattt ttaattttcg tattagctta cgacgctaca 7998

cccagttccc atctattttg tcactcttcc ctaaataatc cttaaaaact ccatttccac 8058

ccctcccagt tcccaactat tttgtccgcc cacagcgggg catttttctt cctgttatgt 8118

ttttaatcaa acatcctgcc aactccatgt gacaaaccgt catcttcggc tactttttct 8178

ctgtcacaga atgaaaattt ttctgtcatc tcttcgttat taatgtttgt aattgactga 8238

atatcaacgc ttatttgcag cctgaatggc gaatgg 8274

<210> 9

<211> 1189

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 9

Met His His His His His His His His Gly Ser His Asn His Asn His

1 5 10 15

Asn His Asn His Asn His Gly Thr Val Ser Lys Gly Glu Glu Leu Phe

20 25 30

Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly

35 40 45

His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly

50 55 60

Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro

65 70 75 80

Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser

85 90 95

Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met

100 105 110

Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly

115 120 125

Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val

130 135 140

Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile

145 150 155 160

Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile

165 170 175

Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg

180 185 190

His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln

195 200 205

Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr

210 215 220

Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp

225 230 235 240

His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly

245 250 255

Met Asp Glu Leu Tyr Lys Gly Thr Gly Ser Glu Asn Leu Tyr Phe Gln

260 265 270

Gly Gly Ser Pro Lys Lys Lys Arg Lys Val Gly Ser Glu Pro Phe Lys

275 280 285

Tyr Ile Cys His Tyr Trp Gly Lys Ser Ser Lys Ser Leu Thr Lys Gly

290 295 300

Asn Asp Ile His Leu Leu Ile Tyr His Cys Leu Asp Val Ala Ala Val

305 310 315 320

Ala Asp Cys Trp Trp Asp Gln Ser Val Val Leu Gln Asn Thr Phe Cys

325 330 335

Arg Asn Glu Met Leu Ser Lys Gln Arg Val Lys Ala Trp Leu Leu Phe

340 345 350

Phe Ile Ala Leu His Asp Ile Gly Lys Phe Asp Ile Arg Phe Gln Tyr

355 360 365

Lys Ser Ala Glu Ser Trp Leu Lys Leu Asn Pro Ala Thr Pro Ser Leu

370 375 380

Asn Gly Pro Ser Thr Gln Met Cys Arg Lys Phe Asn His Gly Ala Ala

385 390 395 400

Gly Leu Tyr Trp Phe Asn Gln Asp Ser Leu Ser Glu Gln Ser Leu Gly

405 410 415

Asp Phe Phe Ser Phe Phe Asp Ala Ala Pro His Pro Tyr Glu Ser Trp

420 425 430

Phe Pro Trp Val Glu Ala Val Thr Gly His His Gly Phe Ile Leu His

435 440 445

Ser Gln Asp Gln Asp Lys Ser Arg Trp Glu Met Pro Ala Ser Leu Ala

450 455 460

Ser Tyr Ala Ala Gln Asp Lys Gln Ala Arg Glu Glu Trp Ile Ser Val

465 470 475 480

Leu Glu Ala Leu Phe Leu Thr Pro Ala Gly Leu Ser Ile Asn Asp Ile

485 490 495

Pro Pro Asp Cys Ser Ser Leu Leu Ala Gly Phe Cys Ser Leu Ala Asp

500 505 510

Trp Leu Gly Ser Trp Thr Thr Thr Asn Thr Phe Leu Phe Asn Glu Asp

515 520 525

Ala Pro Ser Asp Ile Asn Ala Leu Arg Thr Tyr Phe Gln Asp Arg Gln

530 535 540

Gln Asp Ala Ser Arg Val Leu Glu Leu Ser Gly Leu Val Ser Asn Lys

545 550 555 560

Arg Cys Tyr Glu Gly Val His Ala Leu Leu Asp Asn Gly Tyr Gln Pro

565 570 575

Arg Gln Leu Gln Val Leu Val Asp Ala Leu Pro Val Ala Pro Gly Leu

580 585 590

Thr Val Ile Glu Ala Pro Thr Gly Ser Gly Lys Thr Glu Thr Ala Leu

595 600 605

Ala Tyr Ala Trp Lys Leu Ile Asp Gln Gln Ile Ala Asp Ser Val Ile

610 615 620

Phe Ala Leu Pro Thr Gln Ala Thr Ala Asn Ala Met Leu Thr Arg Met

625 630 635 640

Glu Ala Ser Ala Ser His Leu Phe Ser Ser Pro Asn Leu Ile Leu Ala

645 650 655

His Gly Asn Ser Arg Phe Asn His Leu Phe Gln Ser Ile Lys Ser Arg

660 665 670

Ala Ile Thr Glu Gln Gly Gln Glu Glu Ala Trp Val Gln Cys Cys Gln

675 680 685

Trp Leu Ser Gln Ser Asn Lys Lys Val Phe Leu Gly Gln Ile Gly Val

690 695 700

Cys Thr Ile Asp Gln Val Leu Ile Ser Val Leu Pro Val Lys His Arg

705 710 715 720

Phe Ile Arg Gly Leu Gly Ile Gly Arg Ser Val Leu Ile Val Asp Glu

725 730 735

Val His Ala Tyr Asp Thr Tyr Met Asn Gly Leu Leu Glu Ala Val Leu

740 745 750

Lys Ala Gln Ala Asp Val Gly Gly Ser Val Ile Leu Leu Ser Ala Thr

755 760 765

Leu Pro Met Lys Gln Lys Gln Lys Leu Leu Asp Thr Tyr Gly Leu His

770 775 780

Thr Asp Pro Val Glu Asn Asn Ser Ala Tyr Pro Leu Ile Asn Trp Arg

785 790 795 800

Gly Val Asn Gly Ala Gln Arg Phe Asp Leu Leu Ala His Pro Glu Gln

805 810 815

Leu Pro Pro Arg Phe Ser Ile Gln Pro Glu Pro Ile Cys Leu Ala Asp

820 825 830

Met Leu Pro Asp Leu Thr Met Leu Glu Arg Met Ile Ala Ala Ala Asn

835 840 845

Ala Gly Ala Gln Val Cys Leu Ile Cys Asn Leu Val Asp Val Ala Gln

850 855 860

Val Cys Tyr Gln Arg Leu Lys Glu Leu Asn Asn Thr Gln Val Asp Ile

865 870 875 880

Asp Leu Phe His Ala Arg Phe Thr Leu Asn Asp Arg Arg Glu Lys Glu

885 890 895

Asn Arg Val Ile Ser Asn Phe Gly Lys Asn Gly Lys Arg Asn Val Gly

900 905 910

Arg Ile Leu Val Ala Thr Gln Val Val Glu Gln Ser Leu Asp Val Asp

915 920 925

Phe Asp Trp Leu Ile Thr Gln His Cys Pro Ala Asp Leu Leu Phe Gln

930 935 940

Arg Leu Gly Arg Leu His Arg His His Arg Lys Tyr Arg Pro Ala Gly

945 950 955 960

Phe Glu Ile Pro Val Ala Thr Ile Leu Leu Pro Asp Gly Glu Gly Tyr

965 970 975

Gly Arg His Glu His Ile Tyr Ser Asn Val Arg Val Met Trp Arg Thr

980 985 990

Gln Gln His Ile Glu Glu Leu Asn Gly Ala Ser Leu Phe Phe Pro Asp

995 1000 1005

Ala Tyr Arg Gln Trp Leu Asp Ser Ile Tyr Asp Asp Ala Glu Met

1010 1015 1020

Asp Glu Pro Glu Trp Val Gly Asn Gly Met Asp Lys Phe Glu Ser

1025 1030 1035

Ala Glu Cys Glu Lys Arg Phe Lys Ala Arg Lys Val Leu Gln Trp

1040 1045 1050

Ala Glu Glu Tyr Ser Leu Gln Asp Asn Asp Glu Thr Ile Leu Ala

1055 1060 1065

Val Thr Arg Asp Gly Glu Met Ser Leu Pro Leu Leu Pro Tyr Val

1070 1075 1080

Gln Thr Ser Ser Gly Lys Gln Leu Leu Asp Gly Gln Val Tyr Glu

1085 1090 1095

Asp Leu Ser His Glu Gln Gln Tyr Glu Ala Leu Ala Leu Asn Arg

1100 1105 1110

Val Asn Val Pro Phe Thr Trp Lys Arg Ser Phe Ser Glu Val Val

1115 1120 1125

Asp Glu Asp Gly Leu Leu Trp Leu Glu Gly Lys Gln Asn Leu Asp

1130 1135 1140

Gly Trp Val Trp Gln Gly Asn Ser Ile Val Ile Thr Tyr Thr Gly

1145 1150 1155

Asp Glu Gly Met Thr Arg Val Ile Pro Ala Asn Pro Lys Lys Arg

1160 1165 1170

Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys

1175 1180 1185

Val

<210> 10

<211> 4299

<212> DNA

<213> 人工序列

<220>

<223> HisTag-Cas11-bpNLS/pCDFDuet-1

<220>

<221> CDS

<222> (71)..(667)

<400> 10

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atg gca agc cat cat cac cat cac cat cac cat ggt tct 109

Met Ala Ser His His His His His His His His Gly Ser

1 5 10

ctg gaa gtt ctg ttc cag ggg ccc gct gat gaa att gat gca atg gct 157

Leu Glu Val Leu Phe Gln Gly Pro Ala Asp Glu Ile Asp Ala Met Ala

15 20 25

tta tat cga gcc tgg caa caa ctg gat aat gga tca tgt gcg caa att 205

Leu Tyr Arg Ala Trp Gln Gln Leu Asp Asn Gly Ser Cys Ala Gln Ile

30 35 40 45

aga cgt gtt tca gaa cct gat gaa tta cgc gat atc cct gcg ttt tat 253

Arg Arg Val Ser Glu Pro Asp Glu Leu Arg Asp Ile Pro Ala Phe Tyr

50 55 60

agg ctg gtg caa cct ttt ggt tgg gaa aac cca cgt cac cag cag gct 301

Arg Leu Val Gln Pro Phe Gly Trp Glu Asn Pro Arg His Gln Gln Ala

65 70 75

ctt ttg cgc atg gtg ttt tgc ctg agc gca gga aag aat gtc atc cga 349

Leu Leu Arg Met Val Phe Cys Leu Ser Ala Gly Lys Asn Val Ile Arg

80 85 90

cat cag gac aaa aaa tcg gag caa aca aca ggt atc tcg ttg gga aga 397

His Gln Asp Lys Lys Ser Glu Gln Thr Thr Gly Ile Ser Leu Gly Arg

95 100 105

gct tta gcc aat agt gga aga att aac gag cgc cgt atc ttt caa tta 445

Ala Leu Ala Asn Ser Gly Arg Ile Asn Glu Arg Arg Ile Phe Gln Leu

110 115 120 125

att cgg gct gac aga aca gcc gat atg gtc cag tta cgt cga tta ctt 493

Ile Arg Ala Asp Arg Thr Ala Asp Met Val Gln Leu Arg Arg Leu Leu

130 135 140

act cac gcc gaa ccc gta ctt gac tgg cca tta atg gcc agg atg ttg 541

Thr His Ala Glu Pro Val Leu Asp Trp Pro Leu Met Ala Arg Met Leu

145 150 155

acc tgg tgg gga aag cgc gaa cgc cag caa ctt ctg gaa gat ttt gta 589

Thr Trp Trp Gly Lys Arg Glu Arg Gln Gln Leu Leu Glu Asp Phe Val

160 165 170

ttg acc aca aac aaa aat gcg aag aga aca gcc gat ggc agc gag ttc 637

Leu Thr Thr Asn Lys Asn Ala Lys Arg Thr Ala Asp Gly Ser Glu Phe

175 180 185

gag agc ccc aag aag aag cgg aag gtc taa gcggccgcat aatgcttaag 687

Glu Ser Pro Lys Lys Lys Arg Lys Val

190 195

tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc gaaattaata cgactcacta 747

taggggaatt gtgagcggat aacaattccc catcttagta tattagttaa gtataagaag 807

gagatataca tatggcagat ctcaattgga tatcggccgg ccacgcgatc gctgacgtcg 867

gtaccctcga gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact 927

cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat 987

aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaacct caggcatttg 1047

agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat 1107

aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcatcgtgg ccggatcttg 1167

cggcccctcg gcttgaacga attgttagac attatttgcc gactaccttg gtgatctcgc 1227

ctttcacgta gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt 1287

cttgtccaag ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc 1347

gctccattgc ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt 1407

accaaatgcg ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg 1467

agttccatag cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat 1527

caaagagttc ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca 1587

gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa gatacctgca agaatgtcat 1647

tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt 1707

cgtcgtgcac aacaatggtg acttctacag cgcggagaat ctcctctctc caggggaagc 1767

cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt 1827

caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc 1887

gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc 1947

tgatagttga gtcgatactt cggcgatcac cgcttccctc atactcttcc tttttcaata 2007

ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 2067

gaaaaataaa caaatagcta gctcactcgg tcgctacgct ccgggcgtga gactgcggcg 2127

ggcgctgcgg acacatacaa agttacccac agattccgtg gataagcagg ggactaacat 2187

gtgaggcaaa acagcagggc cgcgccggtg gcgtttttcc ataggctccg ccctcctgcc 2247

agagttcaca taaacagacg cttttccggt gcatctgtgg gagccgtgag gctcaaccat 2307

gaatctgaca gtacgggcga aacccgacag gacttaaaga tccccaccgt ttccggcggg 2367

tcgctccctc ttgcgctctc ctgttccgac cctgccgttt accggatacc tgttccgcct 2427

ttctccctta cgggaagtgt ggcgctttct catagctcac acactggtat ctcggctcgg 2487

tgtaggtcgt tcgctccaag ctgggctgta agcaagaact ccccgttcag cccgactgct 2547

gcgccttatc cggtaactgt tcacttgagt ccaacccgga aaagcacggt aaaacgccac 2607

tggcagcagc cattggtaac tgggagttcg cagaggattt gtttagctaa acacgcggtt 2667

gctcttgaag tgtgcgccaa agtccggcta cactggaagg acagatttgg ttgctgtgct 2727

ctgcgaaagc cagttaccac ggttaagcag ttccccaact gacttaacct tcgatcaaac 2787

cacctcccca ggtggttttt tcgtttacag ggcaaaagat tacgcgcaga aaaaaaggat 2847

ctcaagaaga tcctttgatc ttttctactg aaccgctcta gatttcagtg caatttatct 2907

cttcaaatgt agcacctgaa gtcagcccca tacgatataa gttgtaattc tcatgttagt 2967

catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg 3027

agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc 3087

tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 3147

aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca 3207

gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt 3267

gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt 3327

cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg 3387

taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa 3447

cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc 3507

cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca 3567

gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac 3627

ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac 3687

tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag 3747

cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc 3807

gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca 3867

tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt 3927

gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc 3987

ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca 4047

ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct 4107

gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca 4167

ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt 4227

cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaaattaata 4287

cgactcacta ta 4299

<210> 11

<211> 198

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 11

Met Ala Ser His His His His His His His His Gly Ser Leu Glu Val

1 5 10 15

Leu Phe Gln Gly Pro Ala Asp Glu Ile Asp Ala Met Ala Leu Tyr Arg

20 25 30

Ala Trp Gln Gln Leu Asp Asn Gly Ser Cys Ala Gln Ile Arg Arg Val

35 40 45

Ser Glu Pro Asp Glu Leu Arg Asp Ile Pro Ala Phe Tyr Arg Leu Val

50 55 60

Gln Pro Phe Gly Trp Glu Asn Pro Arg His Gln Gln Ala Leu Leu Arg

65 70 75 80

Met Val Phe Cys Leu Ser Ala Gly Lys Asn Val Ile Arg His Gln Asp

85 90 95

Lys Lys Ser Glu Gln Thr Thr Gly Ile Ser Leu Gly Arg Ala Leu Ala

100 105 110

Asn Ser Gly Arg Ile Asn Glu Arg Arg Ile Phe Gln Leu Ile Arg Ala

115 120 125

Asp Arg Thr Ala Asp Met Val Gln Leu Arg Arg Leu Leu Thr His Ala

130 135 140

Glu Pro Val Leu Asp Trp Pro Leu Met Ala Arg Met Leu Thr Trp Trp

145 150 155 160

Gly Lys Arg Glu Arg Gln Gln Leu Leu Glu Asp Phe Val Leu Thr Thr

165 170 175

Asn Lys Asn Ala Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro

180 185 190

Lys Lys Lys Arg Lys Val

195

<210> 12

<211> 8166

<212> DNA

<213> 人工序列

<220>

<223> (Cas8-Cas11-Cas7-bpNLS)+(Cas5-Cas6-bpNLS)/pRSFDuet-1

<220>

<221> CDS

<222> (71)..(1585)

<223> Cas8

<400> 12

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atg gaa atg aat ttg ctt att gat aac tgg atc cct gta 109

Met Glu Met Asn Leu Leu Ile Asp Asn Trp Ile Pro Val

1 5 10

cgc ccg cga aac ggg ggg aaa gtc caa atc ata aat ctg caa tcg cta 157

Arg Pro Arg Asn Gly Gly Lys Val Gln Ile Ile Asn Leu Gln Ser Leu

15 20 25

tac tgc agt aga gat cag tgg cga tta agt ttg ccc cgt gac gat atg 205

Tyr Cys Ser Arg Asp Gln Trp Arg Leu Ser Leu Pro Arg Asp Asp Met

30 35 40 45

gaa ctg gcc gct tta gca ctg ctg gtt tgc att ggg caa att atc gcc 253

Glu Leu Ala Ala Leu Ala Leu Leu Val Cys Ile Gly Gln Ile Ile Ala

50 55 60

ccg gca aaa gat gac gtt gaa ttt cga cat cgc ata atg aat ccg ctc 301

Pro Ala Lys Asp Asp Val Glu Phe Arg His Arg Ile Met Asn Pro Leu

65 70 75

act gaa gat gag ttt caa caa ctc atc gcg ccg tgg ata gat atg ttc 349

Thr Glu Asp Glu Phe Gln Gln Leu Ile Ala Pro Trp Ile Asp Met Phe

80 85 90

tac ctt aat cac gca gaa cat ccc ttt atg cag acc aaa ggt gtc aaa 397

Tyr Leu Asn His Ala Glu His Pro Phe Met Gln Thr Lys Gly Val Lys

95 100 105

gca aat gat gtg act cca atg gaa aaa ctg ttg gct ggg gta agc ggc 445

Ala Asn Asp Val Thr Pro Met Glu Lys Leu Leu Ala Gly Val Ser Gly

110 115 120 125

gcg acg aat tgt gca ttt gtc aat caa ccg ggg cag ggt gaa gca tta 493

Ala Thr Asn Cys Ala Phe Val Asn Gln Pro Gly Gln Gly Glu Ala Leu

130 135 140

tgt ggt gga tgc act gcg att gcg tta ttc aac cag gcg aat cag gca 541

Cys Gly Gly Cys Thr Ala Ile Ala Leu Phe Asn Gln Ala Asn Gln Ala

145 150 155

cca ggt ttt ggt ggt ggt ttt aaa agc ggt tta cgt gga gga aca cct 589

Pro Gly Phe Gly Gly Gly Phe Lys Ser Gly Leu Arg Gly Gly Thr Pro

160 165 170

gta aca acg ttc gta cgt ggg atc gat ctt cgt tca acg gtg tta ctc 637

Val Thr Thr Phe Val Arg Gly Ile Asp Leu Arg Ser Thr Val Leu Leu

175 180 185

aat gtc ctc aca tta cct cgt ctt caa aaa caa ttt cct aat gaa tca 685

Asn Val Leu Thr Leu Pro Arg Leu Gln Lys Gln Phe Pro Asn Glu Ser

190 195 200 205

cat acg gaa aac caa cct acc tgg att aaa cct atc aag tcc aat gag 733

His Thr Glu Asn Gln Pro Thr Trp Ile Lys Pro Ile Lys Ser Asn Glu

210 215 220

tct ata cct gct tcg tca att ggg ttt gtc cgt ggt cta ttc tgg caa 781

Ser Ile Pro Ala Ser Ser Ile Gly Phe Val Arg Gly Leu Phe Trp Gln

225 230 235

cca gcg cat att gaa tta tgc gat ccc att ggg att ggt aaa tgt tct 829

Pro Ala His Ile Glu Leu Cys Asp Pro Ile Gly Ile Gly Lys Cys Ser

240 245 250

tgc tgt gga cag gaa agc aat ttg cgt tat acc ggt ttt ctt aag gaa 877

Cys Cys Gly Gln Glu Ser Asn Leu Arg Tyr Thr Gly Phe Leu Lys Glu

255 260 265

aaa ttt acc ttt aca gtt aat ggg cta tgg ccc cat ccg cat tcc cct 925

Lys Phe Thr Phe Thr Val Asn Gly Leu Trp Pro His Pro His Ser Pro

270 275 280 285

tgt ctg gta aca gtc aag aaa ggg gag gtt gag gaa aaa ttt ctt gct 973

Cys Leu Val Thr Val Lys Lys Gly Glu Val Glu Glu Lys Phe Leu Ala

290 295 300

ttc acc acc tcc gca cca tca tgg aca caa atc agc cga gtt gtg gta 1021

Phe Thr Thr Ser Ala Pro Ser Trp Thr Gln Ile Ser Arg Val Val Val

305 310 315

gat aag att att caa aat gaa aat gga aat cgc gtg gcg gcg gtt gtg 1069

Asp Lys Ile Ile Gln Asn Glu Asn Gly Asn Arg Val Ala Ala Val Val

320 325 330

aat caa ttc aga aat att gcg ccg caa agt cct ctt gaa ttg att atg 1117

Asn Gln Phe Arg Asn Ile Ala Pro Gln Ser Pro Leu Glu Leu Ile Met

335 340 345

ggg gga tat cgt aat aat caa gca tct att ctt gaa cgg cgt cat gat 1165

Gly Gly Tyr Arg Asn Asn Gln Ala Ser Ile Leu Glu Arg Arg His Asp

350 355 360 365

gtg ttg atg ttt aat cag ggg tgg caa caa tac ggc aat gtg ata aac 1213

Val Leu Met Phe Asn Gln Gly Trp Gln Gln Tyr Gly Asn Val Ile Asn

370 375 380

gaa ata gtg act gtt ggt ttg gga tat aaa aca gcc tta cgc aag gcg 1261

Glu Ile Val Thr Val Gly Leu Gly Tyr Lys Thr Ala Leu Arg Lys Ala

385 390 395

tta tat acc ttt gca gaa ggg ttt aaa aat aaa gac ttc aaa ggg gcc 1309

Leu Tyr Thr Phe Ala Glu Gly Phe Lys Asn Lys Asp Phe Lys Gly Ala

400 405 410

gga gtc tct gtt cat gag act gca gaa agg cat ttc tat cga cag agt 1357

Gly Val Ser Val His Glu Thr Ala Glu Arg His Phe Tyr Arg Gln Ser

415 420 425

gaa tta tta att ccc gat gta ctg gcg aat gtt aat ttt tcc cag gct 1405

Glu Leu Leu Ile Pro Asp Val Leu Ala Asn Val Asn Phe Ser Gln Ala

430 435 440 445

gat gag gta ata gct gat tta cga gac aaa ctt cat caa ttg tgt gaa 1453

Asp Glu Val Ile Ala Asp Leu Arg Asp Lys Leu His Gln Leu Cys Glu

450 455 460

atg cta ttt aat caa tct gta gct ccc tat gca cat cat cct aaa tta 1501

Met Leu Phe Asn Gln Ser Val Ala Pro Tyr Ala His His Pro Lys Leu

465 470 475

ata agc aca tta gcg ctt gcc cgc gcc acg cta tac aaa cat tta cgg 1549

Ile Ser Thr Leu Ala Leu Ala Arg Ala Thr Leu Tyr Lys His Leu Arg

480 485 490

gag tta aaa ccg caa gga ggg cca tca aat ggc tga tgaaattgat 1595

Glu Leu Lys Pro Gln Gly Gly Pro Ser Asn Gly

495 500

gcaatggctt tatatcgagc ctggcaacaa ctggataatg gatcatgtgc gcaaattaga 1655

cgtgtttcag aacctgatga attacgcgat atccctgcgt tttataggct ggtgcaacct 1715

tttggttggg aaaacccacg tcaccagcag gctcttttgc gcatggtgtt ttgcctgagc 1775

gcaggaaaga atgtcatccg acatcaggac aaaaaatcgg agcaaacaac aggtatctcg 1835

ttgggaagag ctttagccaa tagtggaaga attaacgagc gccgtatctt tcaattaatt 1895

cgggctgaca gaacagccga tatggtccag ttacgtcgat tacttactca cgccgaaccc 1955

gtacttgact ggccattaat ggccaggatg ttgacctggt ggggaaagcg cgaacgccag 2015

caacttctgg aagattttgt attgaccaca aacaaaaatg cgtaaggaaa cctttctatg 2075

tctaacttta tcaatattca tgttctgatc tctcacagcc cttcatgtct gaaccgcgac 2135

gatatgaaca tgcagaaaga cgctattttc ggcggcaaaa gacgagtaag aatttcaagt 2195

caaagcctta aacgtgcgat gcgtaaaagt ggttattacg cacaaaatat tggtgaatcc 2255

agtctcagaa ccattcatct tgcacaatta cgtgatgttc ttcggcaaaa acttggtgaa 2315

cgttttgacc aaaaaatcat cgataagaca ttagcgctgc tctccggtaa atcagttgat 2375

gaagccgaaa agatttctgc cgatgcggtt actccctggg ttgtgggaga aatagcctgg 2435

ttctgtgagc aggttgcaaa agcagaggct gataatctgg atgataaaaa gctgctcaaa 2495

gttcttaagg aagatattgc cgccatacgt gtgaatttac agcagggtgt tgatattgcg 2555

cttagtggaa gaatggcaac cagcggcatg atgactgagt tgggaaaagt tgatggtgca 2615

atgtccattg cgcatgcgat cactactcat caggttgatt ctgatattga ctggttcacc 2675

gctgtagatg atttacagga acaaggttct gcacatctgg gaactcagga attttcatcg 2735

ggtgtttttt atcgttatgc caacattaac ctcgctcaac ttcaggaaaa tttaggtggt 2795

gcctccaggg agcaggctct ggaaattgca acccatgttg ttcatatgct ggcaacagag 2855

gtccctggag caaaacagcg tacttatgcc gcttttaacc ctgcggatat ggtaatggtt 2915

aatttctccg atatgccact ttctatggca aatgcttttg aaaaagcggt taaagcgaaa 2975

gatggctttt tgcaaccgtc tatacaggcg tttaatcaat attgggatcg cgttgccaat 3035

ggatatggtc tgaacggagc tgctgcgcaa ttcagcttat ctgatgtaga cccaattact 3095

gctcaagtta aacaaatgcc tactttagaa cagttaaaat cctgggttcg taataatggc 3155

gaggcgaaga gaacagccga tggcagcgag ttcgagagcc ccaagaagaa gcggaaggtc 3215

tgaaagcttg cggccgcata atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc 3275

cgcataatcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc 3335

atcttagtat attagttaag tataagaagg agatatacat atgagatctt atttgatctt 3395

gcggcttgct gggccaatgc aagcctgggg gcagccgacc tttgaaggaa cgcgacctac 3455

cggaagattt ccgacccgaa gcgggttatt agggctactc ggggcttgtc ttgggatcca 3515

acgtgatgat acttcttcat tacaggcgtt atcagagagt gtgcaatttg cagtgcgctg 3575

cgatgaactc attcttgacg atcgtcgtgt gtctgtaacg gggttgcgtg attaccatac 3635

agtccttgga gcgcgagaag attaccgtgg tttgaaaagt catgaaacga ttcaaacatg 3695

gcgcgaatat ttatgtgatg cctcctttac cgtcgctctc tggttaacac cccatgcaac 3755

gatggttatc tcagaacttg aaaaagcagt attaaagcct cggtatacac cttacctggg 3815

gcggagaagt tgcccactaa cacacccgct ttttttgggg acatgtcagg catcggatcc 3875

tcagaaggcg ctattaaatt atgagcccgt tggcggcgat atatatagtg aggaatcagt 3935

tacagggcat catttaaaat ttacggcgcg cgacgaaccg atgatcacct tgcctcgaca 3995

atttgcttcc cgagaatggt atgtgattaa aggaggtatg gatgtatctc agtaaagtca 4055

tcattgccag ggcctggagc agggatcttt accaacttca ccagggatta tggcatttat 4115

ttccaaacag accggatgct gctcgtgatt ttctttttca tgttgagaag cgaaacacac 4175

cagaaggctg tcatgtttta ttgcagtcag cgcaaatgcc tgtttcaact gccgttgcga 4235

cagtcattaa aactaaacag gttgaatttc aacttcaggt tggtgttcca ctctattttc 4295

ggcttcgggc aaatccgatc aaaactattc tcgacaatca aaagcgcctg gacagtaaag 4355

ggaatattaa acgctgtcgg gttccgttaa taaaagaagc agaacaaatc gcgtggttgc 4415

aacgtaaatt gggcaatgcg gcgcgcgttg aagatgtgca tcccatatcg gaacggccac 4475

agtatttttc tggtgatggt aaaagtggaa agatccaaac ggtttgcttt gaaggtgtgc 4535

tcaccatcaa cgacgcgcca gcgttaatag atcttgtaca gcaaggtatt gggccagcta 4595

aatcgatggg atgtggcttg ctatctttgg ctccactgaa gagaacagcc gatggcagcg 4655

agttcgagag ccccaagaag aagcggaagg tctgactcga gtctggtaaa gaaaccgctg 4715

ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg 4775

ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga 4835

ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag 4895

tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac 4955

cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga 5015

acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc 5075

ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 5135

accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca 5195

taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc 5255

tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac 5315

tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca 5375

gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 5435

cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga 5495

atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata 5555

ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc 5615

atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt 5675

tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa 5735

caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac 5795

attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg 5855

cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg 5915

aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 5975

taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt 6035

cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatccacaga 6095

atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa 6155

cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 6215

caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 6275

gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 6335

tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc agttcggtgt 6395

aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 6455

ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 6515

cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac ctgttaaggc 6575

taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa 6635

gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt ttttctttat 6695

ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc gttactctag 6755

atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag 6815

ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg 6875

ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg 6935

cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 6995

tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc 7055

accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc 7115

aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc 7175

gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca 7235

acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca 7295

accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg 7355

gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga 7415

tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac 7475

agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct 7535

tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc 7595

ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta 7655

atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg 7715

acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat 7775

ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca 7835

atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc 7895

tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc 7955

accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt 8015

actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg 8075

cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc 8135

ctgcattagg aaattaatac gactcactat a 8166

<210> 13

<211> 504

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 13

Met Glu Met Asn Leu Leu Ile Asp Asn Trp Ile Pro Val Arg Pro Arg

1 5 10 15

Asn Gly Gly Lys Val Gln Ile Ile Asn Leu Gln Ser Leu Tyr Cys Ser

20 25 30

Arg Asp Gln Trp Arg Leu Ser Leu Pro Arg Asp Asp Met Glu Leu Ala

35 40 45

Ala Leu Ala Leu Leu Val Cys Ile Gly Gln Ile Ile Ala Pro Ala Lys

50 55 60

Asp Asp Val Glu Phe Arg His Arg Ile Met Asn Pro Leu Thr Glu Asp

65 70 75 80

Glu Phe Gln Gln Leu Ile Ala Pro Trp Ile Asp Met Phe Tyr Leu Asn

85 90 95

His Ala Glu His Pro Phe Met Gln Thr Lys Gly Val Lys Ala Asn Asp

100 105 110

Val Thr Pro Met Glu Lys Leu Leu Ala Gly Val Ser Gly Ala Thr Asn

115 120 125

Cys Ala Phe Val Asn Gln Pro Gly Gln Gly Glu Ala Leu Cys Gly Gly

130 135 140

Cys Thr Ala Ile Ala Leu Phe Asn Gln Ala Asn Gln Ala Pro Gly Phe

145 150 155 160

Gly Gly Gly Phe Lys Ser Gly Leu Arg Gly Gly Thr Pro Val Thr Thr

165 170 175

Phe Val Arg Gly Ile Asp Leu Arg Ser Thr Val Leu Leu Asn Val Leu

180 185 190

Thr Leu Pro Arg Leu Gln Lys Gln Phe Pro Asn Glu Ser His Thr Glu

195 200 205

Asn Gln Pro Thr Trp Ile Lys Pro Ile Lys Ser Asn Glu Ser Ile Pro

210 215 220

Ala Ser Ser Ile Gly Phe Val Arg Gly Leu Phe Trp Gln Pro Ala His

225 230 235 240

Ile Glu Leu Cys Asp Pro Ile Gly Ile Gly Lys Cys Ser Cys Cys Gly

245 250 255

Gln Glu Ser Asn Leu Arg Tyr Thr Gly Phe Leu Lys Glu Lys Phe Thr

260 265 270

Phe Thr Val Asn Gly Leu Trp Pro His Pro His Ser Pro Cys Leu Val

275 280 285

Thr Val Lys Lys Gly Glu Val Glu Glu Lys Phe Leu Ala Phe Thr Thr

290 295 300

Ser Ala Pro Ser Trp Thr Gln Ile Ser Arg Val Val Val Asp Lys Ile

305 310 315 320

Ile Gln Asn Glu Asn Gly Asn Arg Val Ala Ala Val Val Asn Gln Phe

325 330 335

Arg Asn Ile Ala Pro Gln Ser Pro Leu Glu Leu Ile Met Gly Gly Tyr

340 345 350

Arg Asn Asn Gln Ala Ser Ile Leu Glu Arg Arg His Asp Val Leu Met

355 360 365

Phe Asn Gln Gly Trp Gln Gln Tyr Gly Asn Val Ile Asn Glu Ile Val

370 375 380

Thr Val Gly Leu Gly Tyr Lys Thr Ala Leu Arg Lys Ala Leu Tyr Thr

385 390 395 400

Phe Ala Glu Gly Phe Lys Asn Lys Asp Phe Lys Gly Ala Gly Val Ser

405 410 415

Val His Glu Thr Ala Glu Arg His Phe Tyr Arg Gln Ser Glu Leu Leu

420 425 430

Ile Pro Asp Val Leu Ala Asn Val Asn Phe Ser Gln Ala Asp Glu Val

435 440 445

Ile Ala Asp Leu Arg Asp Lys Leu His Gln Leu Cys Glu Met Leu Phe

450 455 460

Asn Gln Ser Val Ala Pro Tyr Ala His His Pro Lys Leu Ile Ser Thr

465 470 475 480

Leu Ala Leu Ala Arg Ala Thr Leu Tyr Lys His Leu Arg Glu Leu Lys

485 490 495

Pro Gln Gly Gly Pro Ser Asn Gly

500

<210> 14

<211> 8166

<212> DNA

<213> 人工序列

<220>

<223> (Cas8-Cas11-Cas7-bpNLS)+(Cas5-Cas6-bpNLS)/pRSFDuet-1

<220>

<221> CDS

<222> (1578)..(2060)

<223> Cas11

<400> 14

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggaaatga atttgcttat tgataactgg atccctgtac gcccgcgaaa 120

cggggggaaa gtccaaatca taaatctgca atcgctatac tgcagtagag atcagtggcg 180

attaagtttg ccccgtgacg atatggaact ggccgcttta gcactgctgg tttgcattgg 240

gcaaattatc gccccggcaa aagatgacgt tgaatttcga catcgcataa tgaatccgct 300

cactgaagat gagtttcaac aactcatcgc gccgtggata gatatgttct accttaatca 360

cgcagaacat ccctttatgc agaccaaagg tgtcaaagca aatgatgtga ctccaatgga 420

aaaactgttg gctggggtaa gcggcgcgac gaattgtgca tttgtcaatc aaccggggca 480

gggtgaagca ttatgtggtg gatgcactgc gattgcgtta ttcaaccagg cgaatcaggc 540

accaggtttt ggtggtggtt ttaaaagcgg tttacgtgga ggaacacctg taacaacgtt 600

cgtacgtggg atcgatcttc gttcaacggt gttactcaat gtcctcacat tacctcgtct 660

tcaaaaacaa tttcctaatg aatcacatac ggaaaaccaa cctacctgga ttaaacctat 720

caagtccaat gagtctatac ctgcttcgtc aattgggttt gtccgtggtc tattctggca 780

accagcgcat attgaattat gcgatcccat tgggattggt aaatgttctt gctgtggaca 840

ggaaagcaat ttgcgttata ccggttttct taaggaaaaa tttaccttta cagttaatgg 900

gctatggccc catccgcatt ccccttgtct ggtaacagtc aagaaagggg aggttgagga 960

aaaatttctt gctttcacca cctccgcacc atcatggaca caaatcagcc gagttgtggt 1020

agataagatt attcaaaatg aaaatggaaa tcgcgtggcg gcggttgtga atcaattcag 1080

aaatattgcg ccgcaaagtc ctcttgaatt gattatgggg ggatatcgta ataatcaagc 1140

atctattctt gaacggcgtc atgatgtgtt gatgtttaat caggggtggc aacaatacgg 1200

caatgtgata aacgaaatag tgactgttgg tttgggatat aaaacagcct tacgcaaggc 1260

gttatatacc tttgcagaag ggtttaaaaa taaagacttc aaaggggccg gagtctctgt 1320

tcatgagact gcagaaaggc atttctatcg acagagtgaa ttattaattc ccgatgtact 1380

ggcgaatgtt aatttttccc aggctgatga ggtaatagct gatttacgag acaaacttca 1440

tcaattgtgt gaaatgctat ttaatcaatc tgtagctccc tatgcacatc atcctaaatt 1500

aataagcaca ttagcgcttg cccgcgccac gctatacaaa catttacggg agttaaaacc 1560

gcaaggaggg ccatcaa atg gct gat gaa att gat gca atg gct tta tat 1610

Met Ala Asp Glu Ile Asp Ala Met Ala Leu Tyr

1 5 10

cga gcc tgg caa caa ctg gat aat gga tca tgt gcg caa att aga cgt 1658

Arg Ala Trp Gln Gln Leu Asp Asn Gly Ser Cys Ala Gln Ile Arg Arg

15 20 25

gtt tca gaa cct gat gaa tta cgc gat atc cct gcg ttt tat agg ctg 1706

Val Ser Glu Pro Asp Glu Leu Arg Asp Ile Pro Ala Phe Tyr Arg Leu

30 35 40

gtg caa cct ttt ggt tgg gaa aac cca cgt cac cag cag gct ctt ttg 1754

Val Gln Pro Phe Gly Trp Glu Asn Pro Arg His Gln Gln Ala Leu Leu

45 50 55

cgc atg gtg ttt tgc ctg agc gca gga aag aat gtc atc cga cat cag 1802

Arg Met Val Phe Cys Leu Ser Ala Gly Lys Asn Val Ile Arg His Gln

60 65 70 75

gac aaa aaa tcg gag caa aca aca ggt atc tcg ttg gga aga gct tta 1850

Asp Lys Lys Ser Glu Gln Thr Thr Gly Ile Ser Leu Gly Arg Ala Leu

80 85 90

gcc aat agt gga aga att aac gag cgc cgt atc ttt caa tta att cgg 1898

Ala Asn Ser Gly Arg Ile Asn Glu Arg Arg Ile Phe Gln Leu Ile Arg

95 100 105

gct gac aga aca gcc gat atg gtc cag tta cgt cga tta ctt act cac 1946

Ala Asp Arg Thr Ala Asp Met Val Gln Leu Arg Arg Leu Leu Thr His

110 115 120

gcc gaa ccc gta ctt gac tgg cca tta atg gcc agg atg ttg acc tgg 1994

Ala Glu Pro Val Leu Asp Trp Pro Leu Met Ala Arg Met Leu Thr Trp

125 130 135

tgg gga aag cgc gaa cgc cag caa ctt ctg gaa gat ttt gta ttg acc 2042

Trp Gly Lys Arg Glu Arg Gln Gln Leu Leu Glu Asp Phe Val Leu Thr

140 145 150 155

aca aac aaa aat gcg taa ggaaaccttt ctatgtctaa ctttatcaat 2090

Thr Asn Lys Asn Ala

160

attcatgttc tgatctctca cagcccttca tgtctgaacc gcgacgatat gaacatgcag 2150

aaagacgcta ttttcggcgg caaaagacga gtaagaattt caagtcaaag ccttaaacgt 2210

gcgatgcgta aaagtggtta ttacgcacaa aatattggtg aatccagtct cagaaccatt 2270

catcttgcac aattacgtga tgttcttcgg caaaaacttg gtgaacgttt tgaccaaaaa 2330

atcatcgata agacattagc gctgctctcc ggtaaatcag ttgatgaagc cgaaaagatt 2390

tctgccgatg cggttactcc ctgggttgtg ggagaaatag cctggttctg tgagcaggtt 2450

gcaaaagcag aggctgataa tctggatgat aaaaagctgc tcaaagttct taaggaagat 2510

attgccgcca tacgtgtgaa tttacagcag ggtgttgata ttgcgcttag tggaagaatg 2570

gcaaccagcg gcatgatgac tgagttggga aaagttgatg gtgcaatgtc cattgcgcat 2630

gcgatcacta ctcatcaggt tgattctgat attgactggt tcaccgctgt agatgattta 2690

caggaacaag gttctgcaca tctgggaact caggaatttt catcgggtgt tttttatcgt 2750

tatgccaaca ttaacctcgc tcaacttcag gaaaatttag gtggtgcctc cagggagcag 2810

gctctggaaa ttgcaaccca tgttgttcat atgctggcaa cagaggtccc tggagcaaaa 2870

cagcgtactt atgccgcttt taaccctgcg gatatggtaa tggttaattt ctccgatatg 2930

ccactttcta tggcaaatgc ttttgaaaaa gcggttaaag cgaaagatgg ctttttgcaa 2990

ccgtctatac aggcgtttaa tcaatattgg gatcgcgttg ccaatggata tggtctgaac 3050

ggagctgctg cgcaattcag cttatctgat gtagacccaa ttactgctca agttaaacaa 3110

atgcctactt tagaacagtt aaaatcctgg gttcgtaata atggcgaggc gaagagaaca 3170

gccgatggca gcgagttcga gagccccaag aagaagcgga aggtctgaaa gcttgcggcc 3230

gcataatgct taagtcgaac agaaagtaat cgtattgtac acggccgcat aatcgaaatt 3290

aatacgactc actatagggg aattgtgagc ggataacaat tccccatctt agtatattag 3350

ttaagtataa gaaggagata tacatatgag atcttatttg atcttgcggc ttgctgggcc 3410

aatgcaagcc tgggggcagc cgacctttga aggaacgcga cctaccggaa gatttccgac 3470

ccgaagcggg ttattagggc tactcggggc ttgtcttggg atccaacgtg atgatacttc 3530

ttcattacag gcgttatcag agagtgtgca atttgcagtg cgctgcgatg aactcattct 3590

tgacgatcgt cgtgtgtctg taacggggtt gcgtgattac catacagtcc ttggagcgcg 3650

agaagattac cgtggtttga aaagtcatga aacgattcaa acatggcgcg aatatttatg 3710

tgatgcctcc tttaccgtcg ctctctggtt aacaccccat gcaacgatgg ttatctcaga 3770

acttgaaaaa gcagtattaa agcctcggta tacaccttac ctggggcgga gaagttgccc 3830

actaacacac ccgctttttt tggggacatg tcaggcatcg gatcctcaga aggcgctatt 3890

aaattatgag cccgttggcg gcgatatata tagtgaggaa tcagttacag ggcatcattt 3950

aaaatttacg gcgcgcgacg aaccgatgat caccttgcct cgacaatttg cttcccgaga 4010

atggtatgtg attaaaggag gtatggatgt atctcagtaa agtcatcatt gccagggcct 4070

ggagcaggga tctttaccaa cttcaccagg gattatggca tttatttcca aacagaccgg 4130

atgctgctcg tgattttctt tttcatgttg agaagcgaaa cacaccagaa ggctgtcatg 4190

ttttattgca gtcagcgcaa atgcctgttt caactgccgt tgcgacagtc attaaaacta 4250

aacaggttga atttcaactt caggttggtg ttccactcta ttttcggctt cgggcaaatc 4310

cgatcaaaac tattctcgac aatcaaaagc gcctggacag taaagggaat attaaacgct 4370

gtcgggttcc gttaataaaa gaagcagaac aaatcgcgtg gttgcaacgt aaattgggca 4430

atgcggcgcg cgttgaagat gtgcatccca tatcggaacg gccacagtat ttttctggtg 4490

atggtaaaag tggaaagatc caaacggttt gctttgaagg tgtgctcacc atcaacgacg 4550

cgccagcgtt aatagatctt gtacagcaag gtattgggcc agctaaatcg atgggatgtg 4610

gcttgctatc tttggctcca ctgaagagaa cagccgatgg cagcgagttc gagagcccca 4670

agaagaagcg gaaggtctga ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac 4730

gccagcacat ggactcgtct actagcgcag cttaattaac ctaggctgct gccaccgctg 4790

agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga 4850

aacctcaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat aaaccggtaa 4910

accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg acaagctgac 4970

gaccgggtct ccgcaagtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 5030

tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc 5090

gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa 5150

aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc 5210

ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc 5270

gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa 5330

tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc 5390

atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg 5450

aaatacgcgg tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag 5510

gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg 5570

gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat 5630

aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc 5690

atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc 5750

gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca 5810

tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt 5870

ttcccgttga atatggctca tactcttcct ttttcaatat tattgaagca tttatcaggg 5930

ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggcat 5990

gcagcgctct tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg 6050

agcggtgtca gctcactcaa aagcggtaat acggttatcc acagaatcag gggataaagc 6110

cggaaagaac atgtgagcaa aaagcaaagc accggaagaa gccaacgccg caggcgtttt 6170

tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagc cagaggtggc 6230

gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 6290

ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 6350

tggcgctttc tcatagctca cgctgttggt atctcagttc ggtgtaggtc gttcgctcca 6410

agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 6470

atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccattggta 6530

actgatttag aggactttgt cttgaagtta tgcacctgtt aaggctaaac tgaaagaaca 6590

gattttggtg agtgcggtcc tccaacccac ttaccttggt tcaaagagtt ggtagctcag 6650

cgaaccttga gaaaaccacc gttggtagcg gtggtttttc tttatttatg agatgatgaa 6710

tcaatcggtc tatcaagtca acgaacagct attccgttac tctagatttc agtgcaattt 6770

atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt 6830

tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 6890

gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc 6950

ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 7010

ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc 7070

aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg 7130

gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag 7190

ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac 7250

tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg 7310

ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag 7370

tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca 7430

gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg 7490

tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata 7550

atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag 7610

gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg 7670

acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct 7730

accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca 7790

atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt 7850

ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct 7910

tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg 7970

gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc 8030

accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc 8090

cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaaatt 8150

aatacgactc actata 8166

<210> 15

<211> 160

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 15

Met Ala Asp Glu Ile Asp Ala Met Ala Leu Tyr Arg Ala Trp Gln Gln

1 5 10 15

Leu Asp Asn Gly Ser Cys Ala Gln Ile Arg Arg Val Ser Glu Pro Asp

20 25 30

Glu Leu Arg Asp Ile Pro Ala Phe Tyr Arg Leu Val Gln Pro Phe Gly

35 40 45

Trp Glu Asn Pro Arg His Gln Gln Ala Leu Leu Arg Met Val Phe Cys

50 55 60

Leu Ser Ala Gly Lys Asn Val Ile Arg His Gln Asp Lys Lys Ser Glu

65 70 75 80

Gln Thr Thr Gly Ile Ser Leu Gly Arg Ala Leu Ala Asn Ser Gly Arg

85 90 95

Ile Asn Glu Arg Arg Ile Phe Gln Leu Ile Arg Ala Asp Arg Thr Ala

100 105 110

Asp Met Val Gln Leu Arg Arg Leu Leu Thr His Ala Glu Pro Val Leu

115 120 125

Asp Trp Pro Leu Met Ala Arg Met Leu Thr Trp Trp Gly Lys Arg Glu

130 135 140

Arg Gln Gln Leu Leu Glu Asp Phe Val Leu Thr Thr Asn Lys Asn Ala

145 150 155 160

<210> 16

<211> 8166

<212> DNA

<213> 人工序列

<220>

<223> (Cas8-Cas11-Cas7-bpNLS)+(Cas5-Cas6-bpNLS)/pRSFDuet-1

<220>

<221> CDS

<222> (2073)..(3218)

<223> Cas7-bpNLS

<400> 16

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggaaatga atttgcttat tgataactgg atccctgtac gcccgcgaaa 120

cggggggaaa gtccaaatca taaatctgca atcgctatac tgcagtagag atcagtggcg 180

attaagtttg ccccgtgacg atatggaact ggccgcttta gcactgctgg tttgcattgg 240

gcaaattatc gccccggcaa aagatgacgt tgaatttcga catcgcataa tgaatccgct 300

cactgaagat gagtttcaac aactcatcgc gccgtggata gatatgttct accttaatca 360

cgcagaacat ccctttatgc agaccaaagg tgtcaaagca aatgatgtga ctccaatgga 420

aaaactgttg gctggggtaa gcggcgcgac gaattgtgca tttgtcaatc aaccggggca 480

gggtgaagca ttatgtggtg gatgcactgc gattgcgtta ttcaaccagg cgaatcaggc 540

accaggtttt ggtggtggtt ttaaaagcgg tttacgtgga ggaacacctg taacaacgtt 600

cgtacgtggg atcgatcttc gttcaacggt gttactcaat gtcctcacat tacctcgtct 660

tcaaaaacaa tttcctaatg aatcacatac ggaaaaccaa cctacctgga ttaaacctat 720

caagtccaat gagtctatac ctgcttcgtc aattgggttt gtccgtggtc tattctggca 780

accagcgcat attgaattat gcgatcccat tgggattggt aaatgttctt gctgtggaca 840

ggaaagcaat ttgcgttata ccggttttct taaggaaaaa tttaccttta cagttaatgg 900

gctatggccc catccgcatt ccccttgtct ggtaacagtc aagaaagggg aggttgagga 960

aaaatttctt gctttcacca cctccgcacc atcatggaca caaatcagcc gagttgtggt 1020

agataagatt attcaaaatg aaaatggaaa tcgcgtggcg gcggttgtga atcaattcag 1080

aaatattgcg ccgcaaagtc ctcttgaatt gattatgggg ggatatcgta ataatcaagc 1140

atctattctt gaacggcgtc atgatgtgtt gatgtttaat caggggtggc aacaatacgg 1200

caatgtgata aacgaaatag tgactgttgg tttgggatat aaaacagcct tacgcaaggc 1260

gttatatacc tttgcagaag ggtttaaaaa taaagacttc aaaggggccg gagtctctgt 1320

tcatgagact gcagaaaggc atttctatcg acagagtgaa ttattaattc ccgatgtact 1380

ggcgaatgtt aatttttccc aggctgatga ggtaatagct gatttacgag acaaacttca 1440

tcaattgtgt gaaatgctat ttaatcaatc tgtagctccc tatgcacatc atcctaaatt 1500

aataagcaca ttagcgcttg cccgcgccac gctatacaaa catttacggg agttaaaacc 1560

gcaaggaggg ccatcaaatg gctgatgaaa ttgatgcaat ggctttatat cgagcctggc 1620

aacaactgga taatggatca tgtgcgcaaa ttagacgtgt ttcagaacct gatgaattac 1680

gcgatatccc tgcgttttat aggctggtgc aaccttttgg ttgggaaaac ccacgtcacc 1740

agcaggctct tttgcgcatg gtgttttgcc tgagcgcagg aaagaatgtc atccgacatc 1800

aggacaaaaa atcggagcaa acaacaggta tctcgttggg aagagcttta gccaatagtg 1860

gaagaattaa cgagcgccgt atctttcaat taattcgggc tgacagaaca gccgatatgg 1920

tccagttacg tcgattactt actcacgccg aacccgtact tgactggcca ttaatggcca 1980

ggatgttgac ctggtgggga aagcgcgaac gccagcaact tctggaagat tttgtattga 2040

ccacaaacaa aaatgcgtaa ggaaaccttt ct atg tct aac ttt atc aat att 2093

Met Ser Asn Phe Ile Asn Ile

1 5

cat gtt ctg atc tct cac agc cct tca tgt ctg aac cgc gac gat atg 2141

His Val Leu Ile Ser His Ser Pro Ser Cys Leu Asn Arg Asp Asp Met

10 15 20

aac atg cag aaa gac gct att ttc ggc ggc aaa aga cga gta aga att 2189

Asn Met Gln Lys Asp Ala Ile Phe Gly Gly Lys Arg Arg Val Arg Ile

25 30 35

tca agt caa agc ctt aaa cgt gcg atg cgt aaa agt ggt tat tac gca 2237

Ser Ser Gln Ser Leu Lys Arg Ala Met Arg Lys Ser Gly Tyr Tyr Ala

40 45 50 55

caa aat att ggt gaa tcc agt ctc aga acc att cat ctt gca caa tta 2285

Gln Asn Ile Gly Glu Ser Ser Leu Arg Thr Ile His Leu Ala Gln Leu

60 65 70

cgt gat gtt ctt cgg caa aaa ctt ggt gaa cgt ttt gac caa aaa atc 2333

Arg Asp Val Leu Arg Gln Lys Leu Gly Glu Arg Phe Asp Gln Lys Ile

75 80 85

atc gat aag aca tta gcg ctg ctc tcc ggt aaa tca gtt gat gaa gcc 2381

Ile Asp Lys Thr Leu Ala Leu Leu Ser Gly Lys Ser Val Asp Glu Ala

90 95 100

gaa aag att tct gcc gat gcg gtt act ccc tgg gtt gtg gga gaa ata 2429

Glu Lys Ile Ser Ala Asp Ala Val Thr Pro Trp Val Val Gly Glu Ile

105 110 115

gcc tgg ttc tgt gag cag gtt gca aaa gca gag gct gat aat ctg gat 2477

Ala Trp Phe Cys Glu Gln Val Ala Lys Ala Glu Ala Asp Asn Leu Asp

120 125 130 135

gat aaa aag ctg ctc aaa gtt ctt aag gaa gat att gcc gcc ata cgt 2525

Asp Lys Lys Leu Leu Lys Val Leu Lys Glu Asp Ile Ala Ala Ile Arg

140 145 150

gtg aat tta cag cag ggt gtt gat att gcg ctt agt gga aga atg gca 2573

Val Asn Leu Gln Gln Gly Val Asp Ile Ala Leu Ser Gly Arg Met Ala

155 160 165

acc agc ggc atg atg act gag ttg gga aaa gtt gat ggt gca atg tcc 2621

Thr Ser Gly Met Met Thr Glu Leu Gly Lys Val Asp Gly Ala Met Ser

170 175 180

att gcg cat gcg atc act act cat cag gtt gat tct gat att gac tgg 2669

Ile Ala His Ala Ile Thr Thr His Gln Val Asp Ser Asp Ile Asp Trp

185 190 195

ttc acc gct gta gat gat tta cag gaa caa ggt tct gca cat ctg gga 2717

Phe Thr Ala Val Asp Asp Leu Gln Glu Gln Gly Ser Ala His Leu Gly

200 205 210 215

act cag gaa ttt tca tcg ggt gtt ttt tat cgt tat gcc aac att aac 2765

Thr Gln Glu Phe Ser Ser Gly Val Phe Tyr Arg Tyr Ala Asn Ile Asn

220 225 230

ctc gct caa ctt cag gaa aat tta ggt ggt gcc tcc agg gag cag gct 2813

Leu Ala Gln Leu Gln Glu Asn Leu Gly Gly Ala Ser Arg Glu Gln Ala

235 240 245

ctg gaa att gca acc cat gtt gtt cat atg ctg gca aca gag gtc cct 2861

Leu Glu Ile Ala Thr His Val Val His Met Leu Ala Thr Glu Val Pro

250 255 260

gga gca aaa cag cgt act tat gcc gct ttt aac cct gcg gat atg gta 2909

Gly Ala Lys Gln Arg Thr Tyr Ala Ala Phe Asn Pro Ala Asp Met Val

265 270 275

atg gtt aat ttc tcc gat atg cca ctt tct atg gca aat gct ttt gaa 2957

Met Val Asn Phe Ser Asp Met Pro Leu Ser Met Ala Asn Ala Phe Glu

280 285 290 295

aaa gcg gtt aaa gcg aaa gat ggc ttt ttg caa ccg tct ata cag gcg 3005

Lys Ala Val Lys Ala Lys Asp Gly Phe Leu Gln Pro Ser Ile Gln Ala

300 305 310

ttt aat caa tat tgg gat cgc gtt gcc aat gga tat ggt ctg aac gga 3053

Phe Asn Gln Tyr Trp Asp Arg Val Ala Asn Gly Tyr Gly Leu Asn Gly

315 320 325

gct gct gcg caa ttc agc tta tct gat gta gac cca att act gct caa 3101

Ala Ala Ala Gln Phe Ser Leu Ser Asp Val Asp Pro Ile Thr Ala Gln

330 335 340

gtt aaa caa atg cct act tta gaa cag tta aaa tcc tgg gtt cgt aat 3149

Val Lys Gln Met Pro Thr Leu Glu Gln Leu Lys Ser Trp Val Arg Asn

345 350 355

aat ggc gag gcg aag aga aca gcc gat ggc agc gag ttc gag agc ccc 3197

Asn Gly Glu Ala Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro

360 365 370 375

aag aag aag cgg aag gtc tga aagcttgcgg ccgcataatg cttaagtcga 3248

Lys Lys Lys Arg Lys Val

380

acagaaagta atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg 3308

ggaattgtga gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga 3368

tatacatatg agatcttatt tgatcttgcg gcttgctggg ccaatgcaag cctgggggca 3428

gccgaccttt gaaggaacgc gacctaccgg aagatttccg acccgaagcg ggttattagg 3488

gctactcggg gcttgtcttg ggatccaacg tgatgatact tcttcattac aggcgttatc 3548

agagagtgtg caatttgcag tgcgctgcga tgaactcatt cttgacgatc gtcgtgtgtc 3608

tgtaacgggg ttgcgtgatt accatacagt ccttggagcg cgagaagatt accgtggttt 3668

gaaaagtcat gaaacgattc aaacatggcg cgaatattta tgtgatgcct cctttaccgt 3728

cgctctctgg ttaacacccc atgcaacgat ggttatctca gaacttgaaa aagcagtatt 3788

aaagcctcgg tatacacctt acctggggcg gagaagttgc ccactaacac acccgctttt 3848

tttggggaca tgtcaggcat cggatcctca gaaggcgcta ttaaattatg agcccgttgg 3908

cggcgatata tatagtgagg aatcagttac agggcatcat ttaaaattta cggcgcgcga 3968

cgaaccgatg atcaccttgc ctcgacaatt tgcttcccga gaatggtatg tgattaaagg 4028

aggtatggat gtatctcagt aaagtcatca ttgccagggc ctggagcagg gatctttacc 4088

aacttcacca gggattatgg catttatttc caaacagacc ggatgctgct cgtgattttc 4148

tttttcatgt tgagaagcga aacacaccag aaggctgtca tgttttattg cagtcagcgc 4208

aaatgcctgt ttcaactgcc gttgcgacag tcattaaaac taaacaggtt gaatttcaac 4268

ttcaggttgg tgttccactc tattttcggc ttcgggcaaa tccgatcaaa actattctcg 4328

acaatcaaaa gcgcctggac agtaaaggga atattaaacg ctgtcgggtt ccgttaataa 4388

aagaagcaga acaaatcgcg tggttgcaac gtaaattggg caatgcggcg cgcgttgaag 4448

atgtgcatcc catatcggaa cggccacagt atttttctgg tgatggtaaa agtggaaaga 4508

tccaaacggt ttgctttgaa ggtgtgctca ccatcaacga cgcgccagcg ttaatagatc 4568

ttgtacagca aggtattggg ccagctaaat cgatgggatg tggcttgcta tctttggctc 4628

cactgaagag aacagccgat ggcagcgagt tcgagagccc caagaagaag cggaaggtct 4688

gactcgagtc tggtaaagaa accgctgctg cgaaatttga acgccagcac atggactcgt 4748

ctactagcgc agcttaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac 4808

cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaacctcag gcatttgaga 4868

agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag 4928

cggctattta acgaccctgc cctgaaccga cgacaagctg acgaccgggt ctccgcaagt 4988

ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 5048

aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg 5108

caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga 5168

aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat 5228

tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc 5288

aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat 5348

ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc 5408

aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc ggtcgctgtt 5468

aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc 5528

aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg 5588

gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg 5648

aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc 5708

aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg 5768

atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc 5828

agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct 5888

catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 5948

atacatattt gaatgtattt agaaaaataa acaaataggc atgcagcgct cttccgcttc 6008

ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggtgt cagctcactc 6068

aaaagcggta atacggttat ccacagaatc aggggataaa gccggaaaga acatgtgagc 6128

aaaaagcaaa gcaccggaag aagccaacgc cgcaggcgtt tttccatagg ctccgccccc 6188

ctgacgagca tcacaaaaat cgacgctcaa gccagaggtg gcgaaacccg acaggactat 6248

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 6308

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 6368

cacgctgttg gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 6428

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 6488

cggtaagaca cgacttatcg ccactggcag cagccattgg taactgattt agaggacttt 6548

gtcttgaagt tatgcacctg ttaaggctaa actgaaagaa cagattttgg tgagtgcggt 6608

cctccaaccc acttaccttg gttcaaagag ttggtagctc agcgaacctt gagaaaacca 6668

ccgttggtag cggtggtttt tctttattta tgagatgatg aatcaatcgg tctatcaagt 6728

caacgaacag ctattccgtt actctagatt tcagtgcaat ttatctcttc aaatgtagca 6788

cctgaagtca gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc 6848

accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct 6908

aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 6968

acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 7028

ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc 7088

accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga 7148

aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg 7208

tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt 7268

gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc 7328

agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct 7388

atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc 7448

gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga 7508

tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc 7568

tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg 7628

gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga 7688

ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg 7748

ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc 7808

agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt 7868

gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt 7928

ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg 7988

gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc 8048

tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg 8108

atctcgacgc tctcccttat gcgactcctg cattaggaaa ttaatacgac tcactata 8166

<210> 17

<211> 381

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 17

Met Ser Asn Phe Ile Asn Ile His Val Leu Ile Ser His Ser Pro Ser

1 5 10 15

Cys Leu Asn Arg Asp Asp Met Asn Met Gln Lys Asp Ala Ile Phe Gly

20 25 30

Gly Lys Arg Arg Val Arg Ile Ser Ser Gln Ser Leu Lys Arg Ala Met

35 40 45

Arg Lys Ser Gly Tyr Tyr Ala Gln Asn Ile Gly Glu Ser Ser Leu Arg

50 55 60

Thr Ile His Leu Ala Gln Leu Arg Asp Val Leu Arg Gln Lys Leu Gly

65 70 75 80

Glu Arg Phe Asp Gln Lys Ile Ile Asp Lys Thr Leu Ala Leu Leu Ser

85 90 95

Gly Lys Ser Val Asp Glu Ala Glu Lys Ile Ser Ala Asp Ala Val Thr

100 105 110

Pro Trp Val Val Gly Glu Ile Ala Trp Phe Cys Glu Gln Val Ala Lys

115 120 125

Ala Glu Ala Asp Asn Leu Asp Asp Lys Lys Leu Leu Lys Val Leu Lys

130 135 140

Glu Asp Ile Ala Ala Ile Arg Val Asn Leu Gln Gln Gly Val Asp Ile

145 150 155 160

Ala Leu Ser Gly Arg Met Ala Thr Ser Gly Met Met Thr Glu Leu Gly

165 170 175

Lys Val Asp Gly Ala Met Ser Ile Ala His Ala Ile Thr Thr His Gln

180 185 190

Val Asp Ser Asp Ile Asp Trp Phe Thr Ala Val Asp Asp Leu Gln Glu

195 200 205

Gln Gly Ser Ala His Leu Gly Thr Gln Glu Phe Ser Ser Gly Val Phe

210 215 220

Tyr Arg Tyr Ala Asn Ile Asn Leu Ala Gln Leu Gln Glu Asn Leu Gly

225 230 235 240

Gly Ala Ser Arg Glu Gln Ala Leu Glu Ile Ala Thr His Val Val His

245 250 255

Met Leu Ala Thr Glu Val Pro Gly Ala Lys Gln Arg Thr Tyr Ala Ala

260 265 270

Phe Asn Pro Ala Asp Met Val Met Val Asn Phe Ser Asp Met Pro Leu

275 280 285

Ser Met Ala Asn Ala Phe Glu Lys Ala Val Lys Ala Lys Asp Gly Phe

290 295 300

Leu Gln Pro Ser Ile Gln Ala Phe Asn Gln Tyr Trp Asp Arg Val Ala

305 310 315 320

Asn Gly Tyr Gly Leu Asn Gly Ala Ala Ala Gln Phe Ser Leu Ser Asp

325 330 335

Val Asp Pro Ile Thr Ala Gln Val Lys Gln Met Pro Thr Leu Glu Gln

340 345 350

Leu Lys Ser Trp Val Arg Asn Asn Gly Glu Ala Lys Arg Thr Ala Asp

355 360 365

Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val

370 375 380

<210> 18

<211> 8166

<212> DNA

<213> 人工序列

<220>

<223> (Cas8-Cas11-Cas7-bpNLS)+(Cas5-Cas6-bpNLS)/pRSFDuet-1

<220>

<221> CDS

<222> (3376)..(4050)

<223> Cas5

<400> 18

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggaaatga atttgcttat tgataactgg atccctgtac gcccgcgaaa 120

cggggggaaa gtccaaatca taaatctgca atcgctatac tgcagtagag atcagtggcg 180

attaagtttg ccccgtgacg atatggaact ggccgcttta gcactgctgg tttgcattgg 240

gcaaattatc gccccggcaa aagatgacgt tgaatttcga catcgcataa tgaatccgct 300

cactgaagat gagtttcaac aactcatcgc gccgtggata gatatgttct accttaatca 360

cgcagaacat ccctttatgc agaccaaagg tgtcaaagca aatgatgtga ctccaatgga 420

aaaactgttg gctggggtaa gcggcgcgac gaattgtgca tttgtcaatc aaccggggca 480

gggtgaagca ttatgtggtg gatgcactgc gattgcgtta ttcaaccagg cgaatcaggc 540

accaggtttt ggtggtggtt ttaaaagcgg tttacgtgga ggaacacctg taacaacgtt 600

cgtacgtggg atcgatcttc gttcaacggt gttactcaat gtcctcacat tacctcgtct 660

tcaaaaacaa tttcctaatg aatcacatac ggaaaaccaa cctacctgga ttaaacctat 720

caagtccaat gagtctatac ctgcttcgtc aattgggttt gtccgtggtc tattctggca 780

accagcgcat attgaattat gcgatcccat tgggattggt aaatgttctt gctgtggaca 840

ggaaagcaat ttgcgttata ccggttttct taaggaaaaa tttaccttta cagttaatgg 900

gctatggccc catccgcatt ccccttgtct ggtaacagtc aagaaagggg aggttgagga 960

aaaatttctt gctttcacca cctccgcacc atcatggaca caaatcagcc gagttgtggt 1020

agataagatt attcaaaatg aaaatggaaa tcgcgtggcg gcggttgtga atcaattcag 1080

aaatattgcg ccgcaaagtc ctcttgaatt gattatgggg ggatatcgta ataatcaagc 1140

atctattctt gaacggcgtc atgatgtgtt gatgtttaat caggggtggc aacaatacgg 1200

caatgtgata aacgaaatag tgactgttgg tttgggatat aaaacagcct tacgcaaggc 1260

gttatatacc tttgcagaag ggtttaaaaa taaagacttc aaaggggccg gagtctctgt 1320

tcatgagact gcagaaaggc atttctatcg acagagtgaa ttattaattc ccgatgtact 1380

ggcgaatgtt aatttttccc aggctgatga ggtaatagct gatttacgag acaaacttca 1440

tcaattgtgt gaaatgctat ttaatcaatc tgtagctccc tatgcacatc atcctaaatt 1500

aataagcaca ttagcgcttg cccgcgccac gctatacaaa catttacggg agttaaaacc 1560

gcaaggaggg ccatcaaatg gctgatgaaa ttgatgcaat ggctttatat cgagcctggc 1620

aacaactgga taatggatca tgtgcgcaaa ttagacgtgt ttcagaacct gatgaattac 1680

gcgatatccc tgcgttttat aggctggtgc aaccttttgg ttgggaaaac ccacgtcacc 1740

agcaggctct tttgcgcatg gtgttttgcc tgagcgcagg aaagaatgtc atccgacatc 1800

aggacaaaaa atcggagcaa acaacaggta tctcgttggg aagagcttta gccaatagtg 1860

gaagaattaa cgagcgccgt atctttcaat taattcgggc tgacagaaca gccgatatgg 1920

tccagttacg tcgattactt actcacgccg aacccgtact tgactggcca ttaatggcca 1980

ggatgttgac ctggtgggga aagcgcgaac gccagcaact tctggaagat tttgtattga 2040

ccacaaacaa aaatgcgtaa ggaaaccttt ctatgtctaa ctttatcaat attcatgttc 2100

tgatctctca cagcccttca tgtctgaacc gcgacgatat gaacatgcag aaagacgcta 2160

ttttcggcgg caaaagacga gtaagaattt caagtcaaag ccttaaacgt gcgatgcgta 2220

aaagtggtta ttacgcacaa aatattggtg aatccagtct cagaaccatt catcttgcac 2280

aattacgtga tgttcttcgg caaaaacttg gtgaacgttt tgaccaaaaa atcatcgata 2340

agacattagc gctgctctcc ggtaaatcag ttgatgaagc cgaaaagatt tctgccgatg 2400

cggttactcc ctgggttgtg ggagaaatag cctggttctg tgagcaggtt gcaaaagcag 2460

aggctgataa tctggatgat aaaaagctgc tcaaagttct taaggaagat attgccgcca 2520

tacgtgtgaa tttacagcag ggtgttgata ttgcgcttag tggaagaatg gcaaccagcg 2580

gcatgatgac tgagttggga aaagttgatg gtgcaatgtc cattgcgcat gcgatcacta 2640

ctcatcaggt tgattctgat attgactggt tcaccgctgt agatgattta caggaacaag 2700

gttctgcaca tctgggaact caggaatttt catcgggtgt tttttatcgt tatgccaaca 2760

ttaacctcgc tcaacttcag gaaaatttag gtggtgcctc cagggagcag gctctggaaa 2820

ttgcaaccca tgttgttcat atgctggcaa cagaggtccc tggagcaaaa cagcgtactt 2880

atgccgcttt taaccctgcg gatatggtaa tggttaattt ctccgatatg ccactttcta 2940

tggcaaatgc ttttgaaaaa gcggttaaag cgaaagatgg ctttttgcaa ccgtctatac 3000

aggcgtttaa tcaatattgg gatcgcgttg ccaatggata tggtctgaac ggagctgctg 3060

cgcaattcag cttatctgat gtagacccaa ttactgctca agttaaacaa atgcctactt 3120

tagaacagtt aaaatcctgg gttcgtaata atggcgaggc gaagagaaca gccgatggca 3180

gcgagttcga gagccccaag aagaagcgga aggtctgaaa gcttgcggcc gcataatgct 3240

taagtcgaac agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc 3300

actatagggg aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa 3360

gaaggagata tacat atg aga tct tat ttg atc ttg cgg ctt gct ggg cca 3411

Met Arg Ser Tyr Leu Ile Leu Arg Leu Ala Gly Pro

1 5 10

atg caa gcc tgg ggg cag ccg acc ttt gaa gga acg cga cct acc gga 3459

Met Gln Ala Trp Gly Gln Pro Thr Phe Glu Gly Thr Arg Pro Thr Gly

15 20 25

aga ttt ccg acc cga agc ggg tta tta ggg cta ctc ggg gct tgt ctt 3507

Arg Phe Pro Thr Arg Ser Gly Leu Leu Gly Leu Leu Gly Ala Cys Leu

30 35 40

ggg atc caa cgt gat gat act tct tca tta cag gcg tta tca gag agt 3555

Gly Ile Gln Arg Asp Asp Thr Ser Ser Leu Gln Ala Leu Ser Glu Ser

45 50 55 60

gtg caa ttt gca gtg cgc tgc gat gaa ctc att ctt gac gat cgt cgt 3603

Val Gln Phe Ala Val Arg Cys Asp Glu Leu Ile Leu Asp Asp Arg Arg

65 70 75

gtg tct gta acg ggg ttg cgt gat tac cat aca gtc ctt gga gcg cga 3651

Val Ser Val Thr Gly Leu Arg Asp Tyr His Thr Val Leu Gly Ala Arg

80 85 90

gaa gat tac cgt ggt ttg aaa agt cat gaa acg att caa aca tgg cgc 3699

Glu Asp Tyr Arg Gly Leu Lys Ser His Glu Thr Ile Gln Thr Trp Arg

95 100 105

gaa tat tta tgt gat gcc tcc ttt acc gtc gct ctc tgg tta aca ccc 3747

Glu Tyr Leu Cys Asp Ala Ser Phe Thr Val Ala Leu Trp Leu Thr Pro

110 115 120

cat gca acg atg gtt atc tca gaa ctt gaa aaa gca gta tta aag cct 3795

His Ala Thr Met Val Ile Ser Glu Leu Glu Lys Ala Val Leu Lys Pro

125 130 135 140

cgg tat aca cct tac ctg ggg cgg aga agt tgc cca cta aca cac ccg 3843

Arg Tyr Thr Pro Tyr Leu Gly Arg Arg Ser Cys Pro Leu Thr His Pro

145 150 155

ctt ttt ttg ggg aca tgt cag gca tcg gat cct cag aag gcg cta tta 3891

Leu Phe Leu Gly Thr Cys Gln Ala Ser Asp Pro Gln Lys Ala Leu Leu

160 165 170

aat tat gag ccc gtt ggc ggc gat ata tat agt gag gaa tca gtt aca 3939

Asn Tyr Glu Pro Val Gly Gly Asp Ile Tyr Ser Glu Glu Ser Val Thr

175 180 185

ggg cat cat tta aaa ttt acg gcg cgc gac gaa ccg atg atc acc ttg 3987

Gly His His Leu Lys Phe Thr Ala Arg Asp Glu Pro Met Ile Thr Leu

190 195 200

cct cga caa ttt gct tcc cga gaa tgg tat gtg att aaa gga ggt atg 4035

Pro Arg Gln Phe Ala Ser Arg Glu Trp Tyr Val Ile Lys Gly Gly Met

205 210 215 220

gat gta tct cag taa agtcatcatt gccagggcct ggagcaggga tctttaccaa 4090

Asp Val Ser Gln

cttcaccagg gattatggca tttatttcca aacagaccgg atgctgctcg tgattttctt 4150

tttcatgttg agaagcgaaa cacaccagaa ggctgtcatg ttttattgca gtcagcgcaa 4210

atgcctgttt caactgccgt tgcgacagtc attaaaacta aacaggttga atttcaactt 4270

caggttggtg ttccactcta ttttcggctt cgggcaaatc cgatcaaaac tattctcgac 4330

aatcaaaagc gcctggacag taaagggaat attaaacgct gtcgggttcc gttaataaaa 4390

gaagcagaac aaatcgcgtg gttgcaacgt aaattgggca atgcggcgcg cgttgaagat 4450

gtgcatccca tatcggaacg gccacagtat ttttctggtg atggtaaaag tggaaagatc 4510

caaacggttt gctttgaagg tgtgctcacc atcaacgacg cgccagcgtt aatagatctt 4570

gtacagcaag gtattgggcc agctaaatcg atgggatgtg gcttgctatc tttggctcca 4630

ctgaagagaa cagccgatgg cagcgagttc gagagcccca agaagaagcg gaaggtctga 4690

ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct 4750

actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc 4810

cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag 4870

cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg 4930

gctatttaac gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg 4990

cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 5050

tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca 5110

atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 5170

gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 5230

cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 5290

gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt 5350

ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 5410

ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa 5470

aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 5530

caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 5590

tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 5650

gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 5710

cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 5770

agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 5830

catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca 5890

tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 5950

acatatttga atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct 6010

cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa 6070

aagcggtaat acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa 6130

aaagcaaagc accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct 6190

gacgagcatc acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa 6250

agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 6310

cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 6370

cgctgttggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 6430

ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 6490

gtaagacacg acttatcgcc actggcagca gccattggta actgatttag aggactttgt 6550

cttgaagtta tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc 6610

tccaacccac ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc 6670

gttggtagcg gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca 6730

acgaacagct attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc 6790

tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac 6850

cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa 6910

tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 6970

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 7030

gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac 7090

cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa 7150

atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta 7210

tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc 7270

gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag 7330

catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat 7390

cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga 7450

gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg 7510

ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg 7570

gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc 7630

atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt 7690

gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct 7750

ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag 7810

ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc 7870

cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt 7930

cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc 7990

atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc 8050

ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat 8110

ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc actata 8166

<210> 19

<211> 224

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 19

Met Arg Ser Tyr Leu Ile Leu Arg Leu Ala Gly Pro Met Gln Ala Trp

1 5 10 15

Gly Gln Pro Thr Phe Glu Gly Thr Arg Pro Thr Gly Arg Phe Pro Thr

20 25 30

Arg Ser Gly Leu Leu Gly Leu Leu Gly Ala Cys Leu Gly Ile Gln Arg

35 40 45

Asp Asp Thr Ser Ser Leu Gln Ala Leu Ser Glu Ser Val Gln Phe Ala

50 55 60

Val Arg Cys Asp Glu Leu Ile Leu Asp Asp Arg Arg Val Ser Val Thr

65 70 75 80

Gly Leu Arg Asp Tyr His Thr Val Leu Gly Ala Arg Glu Asp Tyr Arg

85 90 95

Gly Leu Lys Ser His Glu Thr Ile Gln Thr Trp Arg Glu Tyr Leu Cys

100 105 110

Asp Ala Ser Phe Thr Val Ala Leu Trp Leu Thr Pro His Ala Thr Met

115 120 125

Val Ile Ser Glu Leu Glu Lys Ala Val Leu Lys Pro Arg Tyr Thr Pro

130 135 140

Tyr Leu Gly Arg Arg Ser Cys Pro Leu Thr His Pro Leu Phe Leu Gly

145 150 155 160

Thr Cys Gln Ala Ser Asp Pro Gln Lys Ala Leu Leu Asn Tyr Glu Pro

165 170 175

Val Gly Gly Asp Ile Tyr Ser Glu Glu Ser Val Thr Gly His His Leu

180 185 190

Lys Phe Thr Ala Arg Asp Glu Pro Met Ile Thr Leu Pro Arg Gln Phe

195 200 205

Ala Ser Arg Glu Trp Tyr Val Ile Lys Gly Gly Met Asp Val Ser Gln

210 215 220

<210> 20

<211> 8166

<212> DNA

<213> 人工序列

<220>

<223> (Cas8-Cas11-Cas7-bpNLS)+(Cas5-Cas6-bpNLS)/pRSFDuet-1

<220>

<221> CDS

<222> (4037)..(4690)

<223> Cas6-bpNLS

<400> 20

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggaaatga atttgcttat tgataactgg atccctgtac gcccgcgaaa 120

cggggggaaa gtccaaatca taaatctgca atcgctatac tgcagtagag atcagtggcg 180

attaagtttg ccccgtgacg atatggaact ggccgcttta gcactgctgg tttgcattgg 240

gcaaattatc gccccggcaa aagatgacgt tgaatttcga catcgcataa tgaatccgct 300

cactgaagat gagtttcaac aactcatcgc gccgtggata gatatgttct accttaatca 360

cgcagaacat ccctttatgc agaccaaagg tgtcaaagca aatgatgtga ctccaatgga 420

aaaactgttg gctggggtaa gcggcgcgac gaattgtgca tttgtcaatc aaccggggca 480

gggtgaagca ttatgtggtg gatgcactgc gattgcgtta ttcaaccagg cgaatcaggc 540

accaggtttt ggtggtggtt ttaaaagcgg tttacgtgga ggaacacctg taacaacgtt 600

cgtacgtggg atcgatcttc gttcaacggt gttactcaat gtcctcacat tacctcgtct 660

tcaaaaacaa tttcctaatg aatcacatac ggaaaaccaa cctacctgga ttaaacctat 720

caagtccaat gagtctatac ctgcttcgtc aattgggttt gtccgtggtc tattctggca 780

accagcgcat attgaattat gcgatcccat tgggattggt aaatgttctt gctgtggaca 840

ggaaagcaat ttgcgttata ccggttttct taaggaaaaa tttaccttta cagttaatgg 900

gctatggccc catccgcatt ccccttgtct ggtaacagtc aagaaagggg aggttgagga 960

aaaatttctt gctttcacca cctccgcacc atcatggaca caaatcagcc gagttgtggt 1020

agataagatt attcaaaatg aaaatggaaa tcgcgtggcg gcggttgtga atcaattcag 1080

aaatattgcg ccgcaaagtc ctcttgaatt gattatgggg ggatatcgta ataatcaagc 1140

atctattctt gaacggcgtc atgatgtgtt gatgtttaat caggggtggc aacaatacgg 1200

caatgtgata aacgaaatag tgactgttgg tttgggatat aaaacagcct tacgcaaggc 1260

gttatatacc tttgcagaag ggtttaaaaa taaagacttc aaaggggccg gagtctctgt 1320

tcatgagact gcagaaaggc atttctatcg acagagtgaa ttattaattc ccgatgtact 1380

ggcgaatgtt aatttttccc aggctgatga ggtaatagct gatttacgag acaaacttca 1440

tcaattgtgt gaaatgctat ttaatcaatc tgtagctccc tatgcacatc atcctaaatt 1500

aataagcaca ttagcgcttg cccgcgccac gctatacaaa catttacggg agttaaaacc 1560

gcaaggaggg ccatcaaatg gctgatgaaa ttgatgcaat ggctttatat cgagcctggc 1620

aacaactgga taatggatca tgtgcgcaaa ttagacgtgt ttcagaacct gatgaattac 1680

gcgatatccc tgcgttttat aggctggtgc aaccttttgg ttgggaaaac ccacgtcacc 1740

agcaggctct tttgcgcatg gtgttttgcc tgagcgcagg aaagaatgtc atccgacatc 1800

aggacaaaaa atcggagcaa acaacaggta tctcgttggg aagagcttta gccaatagtg 1860

gaagaattaa cgagcgccgt atctttcaat taattcgggc tgacagaaca gccgatatgg 1920

tccagttacg tcgattactt actcacgccg aacccgtact tgactggcca ttaatggcca 1980

ggatgttgac ctggtgggga aagcgcgaac gccagcaact tctggaagat tttgtattga 2040

ccacaaacaa aaatgcgtaa ggaaaccttt ctatgtctaa ctttatcaat attcatgttc 2100

tgatctctca cagcccttca tgtctgaacc gcgacgatat gaacatgcag aaagacgcta 2160

ttttcggcgg caaaagacga gtaagaattt caagtcaaag ccttaaacgt gcgatgcgta 2220

aaagtggtta ttacgcacaa aatattggtg aatccagtct cagaaccatt catcttgcac 2280

aattacgtga tgttcttcgg caaaaacttg gtgaacgttt tgaccaaaaa atcatcgata 2340

agacattagc gctgctctcc ggtaaatcag ttgatgaagc cgaaaagatt tctgccgatg 2400

cggttactcc ctgggttgtg ggagaaatag cctggttctg tgagcaggtt gcaaaagcag 2460

aggctgataa tctggatgat aaaaagctgc tcaaagttct taaggaagat attgccgcca 2520

tacgtgtgaa tttacagcag ggtgttgata ttgcgcttag tggaagaatg gcaaccagcg 2580

gcatgatgac tgagttggga aaagttgatg gtgcaatgtc cattgcgcat gcgatcacta 2640

ctcatcaggt tgattctgat attgactggt tcaccgctgt agatgattta caggaacaag 2700

gttctgcaca tctgggaact caggaatttt catcgggtgt tttttatcgt tatgccaaca 2760

ttaacctcgc tcaacttcag gaaaatttag gtggtgcctc cagggagcag gctctggaaa 2820

ttgcaaccca tgttgttcat atgctggcaa cagaggtccc tggagcaaaa cagcgtactt 2880

atgccgcttt taaccctgcg gatatggtaa tggttaattt ctccgatatg ccactttcta 2940

tggcaaatgc ttttgaaaaa gcggttaaag cgaaagatgg ctttttgcaa ccgtctatac 3000

aggcgtttaa tcaatattgg gatcgcgttg ccaatggata tggtctgaac ggagctgctg 3060

cgcaattcag cttatctgat gtagacccaa ttactgctca agttaaacaa atgcctactt 3120

tagaacagtt aaaatcctgg gttcgtaata atggcgaggc gaagagaaca gccgatggca 3180

gcgagttcga gagccccaag aagaagcgga aggtctgaaa gcttgcggcc gcataatgct 3240

taagtcgaac agaaagtaat cgtattgtac acggccgcat aatcgaaatt aatacgactc 3300

actatagggg aattgtgagc ggataacaat tccccatctt agtatattag ttaagtataa 3360

gaaggagata tacatatgag atcttatttg atcttgcggc ttgctgggcc aatgcaagcc 3420

tgggggcagc cgacctttga aggaacgcga cctaccggaa gatttccgac ccgaagcggg 3480

ttattagggc tactcggggc ttgtcttggg atccaacgtg atgatacttc ttcattacag 3540

gcgttatcag agagtgtgca atttgcagtg cgctgcgatg aactcattct tgacgatcgt 3600

cgtgtgtctg taacggggtt gcgtgattac catacagtcc ttggagcgcg agaagattac 3660

cgtggtttga aaagtcatga aacgattcaa acatggcgcg aatatttatg tgatgcctcc 3720

tttaccgtcg ctctctggtt aacaccccat gcaacgatgg ttatctcaga acttgaaaaa 3780

gcagtattaa agcctcggta tacaccttac ctggggcgga gaagttgccc actaacacac 3840

ccgctttttt tggggacatg tcaggcatcg gatcctcaga aggcgctatt aaattatgag 3900

cccgttggcg gcgatatata tagtgaggaa tcagttacag ggcatcattt aaaatttacg 3960

gcgcgcgacg aaccgatgat caccttgcct cgacaatttg cttcccgaga atggtatgtg 4020

attaaaggag gtatgg atg tat ctc agt aaa gtc atc att gcc agg gcc tgg 4072

Met Tyr Leu Ser Lys Val Ile Ile Ala Arg Ala Trp

1 5 10

agc agg gat ctt tac caa ctt cac cag gga tta tgg cat tta ttt cca 4120

Ser Arg Asp Leu Tyr Gln Leu His Gln Gly Leu Trp His Leu Phe Pro

15 20 25

aac aga ccg gat gct gct cgt gat ttt ctt ttt cat gtt gag aag cga 4168

Asn Arg Pro Asp Ala Ala Arg Asp Phe Leu Phe His Val Glu Lys Arg

30 35 40

aac aca cca gaa ggc tgt cat gtt tta ttg cag tca gcg caa atg cct 4216

Asn Thr Pro Glu Gly Cys His Val Leu Leu Gln Ser Ala Gln Met Pro

45 50 55 60

gtt tca act gcc gtt gcg aca gtc att aaa act aaa cag gtt gaa ttt 4264

Val Ser Thr Ala Val Ala Thr Val Ile Lys Thr Lys Gln Val Glu Phe

65 70 75

caa ctt cag gtt ggt gtt cca ctc tat ttt cgg ctt cgg gca aat ccg 4312

Gln Leu Gln Val Gly Val Pro Leu Tyr Phe Arg Leu Arg Ala Asn Pro

80 85 90

atc aaa act att ctc gac aat caa aag cgc ctg gac agt aaa ggg aat 4360

Ile Lys Thr Ile Leu Asp Asn Gln Lys Arg Leu Asp Ser Lys Gly Asn

95 100 105

att aaa cgc tgt cgg gtt ccg tta ata aaa gaa gca gaa caa atc gcg 4408

Ile Lys Arg Cys Arg Val Pro Leu Ile Lys Glu Ala Glu Gln Ile Ala

110 115 120

tgg ttg caa cgt aaa ttg ggc aat gcg gcg cgc gtt gaa gat gtg cat 4456

Trp Leu Gln Arg Lys Leu Gly Asn Ala Ala Arg Val Glu Asp Val His

125 130 135 140

ccc ata tcg gaa cgg cca cag tat ttt tct ggt gat ggt aaa agt gga 4504

Pro Ile Ser Glu Arg Pro Gln Tyr Phe Ser Gly Asp Gly Lys Ser Gly

145 150 155

aag atc caa acg gtt tgc ttt gaa ggt gtg ctc acc atc aac gac gcg 4552

Lys Ile Gln Thr Val Cys Phe Glu Gly Val Leu Thr Ile Asn Asp Ala

160 165 170

cca gcg tta ata gat ctt gta cag caa ggt att ggg cca gct aaa tcg 4600

Pro Ala Leu Ile Asp Leu Val Gln Gln Gly Ile Gly Pro Ala Lys Ser

175 180 185

atg gga tgt ggc ttg cta tct ttg gct cca ctg aag aga aca gcc gat 4648

Met Gly Cys Gly Leu Leu Ser Leu Ala Pro Leu Lys Arg Thr Ala Asp

190 195 200

ggc agc gag ttc gag agc ccc aag aag aag cgg aag gtc tga 4690

Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val

205 210 215

ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgtct 4750

actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact agcataaccc 4810

cttggggcct ctaaacgggt cttgaggggt tttttgctga aacctcaggc atttgagaag 4870

cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg 4930

gctatttaac gaccctgccc tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg 4990

cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 5050

tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca 5110

atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 5170

gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 5230

cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 5290

gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt 5350

ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 5410

ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa 5470

aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 5530

caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 5590

tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 5650

gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 5710

cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 5770

agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 5830

catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca 5890

tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 5950

acatatttga atgtatttag aaaaataaac aaataggcat gcagcgctct tccgcttcct 6010

cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggtgtca gctcactcaa 6070

aagcggtaat acggttatcc acagaatcag gggataaagc cggaaagaac atgtgagcaa 6130

aaagcaaagc accggaagaa gccaacgccg caggcgtttt tccataggct ccgcccccct 6190

gacgagcatc acaaaaatcg acgctcaagc cagaggtggc gaaacccgac aggactataa 6250

agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 6310

cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 6370

cgctgttggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 6430

ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 6490

gtaagacacg acttatcgcc actggcagca gccattggta actgatttag aggactttgt 6550

cttgaagtta tgcacctgtt aaggctaaac tgaaagaaca gattttggtg agtgcggtcc 6610

tccaacccac ttaccttggt tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc 6670

gttggtagcg gtggtttttc tttatttatg agatgatgaa tcaatcggtc tatcaagtca 6730

acgaacagct attccgttac tctagatttc agtgcaattt atctcttcaa atgtagcacc 6790

tgaagtcagc cccatacgat ataagttgta attctcatgt tagtcatgcc ccgcgcccac 6850

cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa 6910

tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 6970

ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 7030

gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac 7090

cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa 7150

atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta 7210

tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc 7270

gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag 7330

catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat 7390

cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga 7450

gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg 7510

ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg 7570

gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc 7630

atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt 7690

gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct 7750

ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag 7810

ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc 7870

cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt 7930

cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc 7990

atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc 8050

ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat 8110

ctcgacgctc tcccttatgc gactcctgca ttaggaaatt aatacgactc actata 8166

<210> 21

<211> 217

<212> PRT

<213> 人工序列

<220>

<223> 合成的构建物

<400> 21

Met Tyr Leu Ser Lys Val Ile Ile Ala Arg Ala Trp Ser Arg Asp Leu

1 5 10 15

Tyr Gln Leu His Gln Gly Leu Trp His Leu Phe Pro Asn Arg Pro Asp

20 25 30

Ala Ala Arg Asp Phe Leu Phe His Val Glu Lys Arg Asn Thr Pro Glu

35 40 45

Gly Cys His Val Leu Leu Gln Ser Ala Gln Met Pro Val Ser Thr Ala

50 55 60

Val Ala Thr Val Ile Lys Thr Lys Gln Val Glu Phe Gln Leu Gln Val

65 70 75 80

Gly Val Pro Leu Tyr Phe Arg Leu Arg Ala Asn Pro Ile Lys Thr Ile

85 90 95

Leu Asp Asn Gln Lys Arg Leu Asp Ser Lys Gly Asn Ile Lys Arg Cys

100 105 110

Arg Val Pro Leu Ile Lys Glu Ala Glu Gln Ile Ala Trp Leu Gln Arg

115 120 125

Lys Leu Gly Asn Ala Ala Arg Val Glu Asp Val His Pro Ile Ser Glu

130 135 140

Arg Pro Gln Tyr Phe Ser Gly Asp Gly Lys Ser Gly Lys Ile Gln Thr

145 150 155 160

Val Cys Phe Glu Gly Val Leu Thr Ile Asn Asp Ala Pro Ala Leu Ile

165 170 175

Asp Leu Val Gln Gln Gly Ile Gly Pro Ala Lys Ser Met Gly Cys Gly

180 185 190

Leu Leu Ser Leu Ala Pro Leu Lys Arg Thr Ala Asp Gly Ser Glu Phe

195 200 205

Glu Ser Pro Lys Lys Lys Arg Lys Val

210 215

<210> 22

<211> 4128

<212> DNA

<213> 人工序列

<220>

<223> EMX1-crRNA/pACYCDuet-1

<220>

<221> precursor_RNA

<222> (81)..(262)

<223> pre-crRNA

<400> 22

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggcatatg tggatgtgtt gtttgtgtga tactataaag ttggtagatt 120

gtgactggct taaaaaatca ttaattaata ataggttatg tttagagtgt tccccgcgcc 180

agcggggata aaccgcaggc caatggggag gacatcgatg tcacctcgtg ttccccgcgc 240

cagcggggat aaaccgtttt ttctcgaggc ggccgcataa tgcttaagtc gaacagaaag 300

taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt 360

gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata 420

tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt 480

ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg 540

cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg 600

cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg 660

tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt 720

aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat 780

ccgcttatta tcacttattc aggcgtagca ccaggcgttt aagggcacca ataactgcct 840

taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt 900

ctgccgacat ggaagccatc acagacggca tgatgaacct gaatcgccag cggcatcagc 960

accttgtcgc cttgcgtata atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc 1020

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 1080

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 1140

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 1200

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 1260

accagctcac cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca 1320

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 1380

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 1440

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 1500

ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt 1560

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 1620

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 1680

tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct 1740

gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc 1800

tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc 1860

cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc 1920

agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt 1980

gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc 2040

ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac 2100

ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga 2160

caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag 2220

ataccaggcg tttcccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt 2280

accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc 2340

cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg 2400

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc 2460

actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt 2520

aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt 2580

tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt 2640

tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc 2700

agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca 2760

gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga 2820

gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag 2880

ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 2940

ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca 3000

gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc 3060

cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt 3120

tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta 3180

ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg 3240

ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca 3300

tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa 3360

tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac 3420

ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc 3480

ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga 3540

catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt 3600

catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg 3660

ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca 3720

gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac 3780

tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt 3840

tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa 3900

cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg 3960

cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc 4020

gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc 4080

tctcccttat gcgactcctg cattaggaaa ttaatacgac tcactata 4128

<210> 23

<211> 4128

<212> DNA

<213> 人工序列

<220>

<223> Tyr-crRNA/pACYCDuet-1

<220>

<221> 前体_RNA

<222> (81)..(262)

<223> pre-crRNA

<400> 23

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggcatatg tggatgtgtt gtttgtgtga tactataaag ttggtagatt 120

gtgactggct taaaaaatca ttaattaata ataggttatg tttagagtgt tccccgcgcc 180

agcggggata aaccgggaca cactgcttgg gggctctgaa atatggagtg ttccccgcgc 240

cagcggggat aaaccgtttt ttctcgaggc ggccgcataa tgcttaagtc gaacagaaag 300

taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt 360

gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata 420

tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt 480

ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg 540

cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg 600

cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg 660

tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt 720

aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat 780

ccgcttatta tcacttattc aggcgtagca ccaggcgttt aagggcacca ataactgcct 840

taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt 900

ctgccgacat ggaagccatc acagacggca tgatgaacct gaatcgccag cggcatcagc 960

accttgtcgc cttgcgtata atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc 1020

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 1080

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 1140

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 1200

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 1260

accagctcac cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca 1320

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 1380

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 1440

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 1500

ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt 1560

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 1620

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 1680

tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct 1740

gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc 1800

tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc 1860

cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc 1920

agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt 1980

gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc 2040

ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac 2100

ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga 2160

caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag 2220

ataccaggcg tttcccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt 2280

accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc 2340

cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg 2400

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc 2460

actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt 2520

aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt 2580

tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt 2640

tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc 2700

agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca 2760

gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga 2820

gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag 2880

ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 2940

ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca 3000

gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc 3060

cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt 3120

tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta 3180

ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg 3240

ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca 3300

tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa 3360

tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac 3420

ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc 3480

ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga 3540

catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt 3600

catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg 3660

ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca 3720

gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac 3780

tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt 3840

tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa 3900

cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg 3960

cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc 4020

gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc 4080

tctcccttat gcgactcctg cattaggaaa ttaatacgac tcactata 4128

<210> 24

<211> 4128

<212> DNA

<213> 人工序列

<220>

<223> GFP-crRNA/pACYCDuet-1

<220>

<221> 前体_RNA

<222> (81)..(262)

<223> pre-crRNA

<400> 24

ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60

gagatatacc atggcatatg tggatgtgtt gtttgtgtga tactataaag ttggtagatt 120

gtgactggct taaaaaatca ttaattaata ataggttatg tttagagtgt tccccgcgcc 180

agcggggata aaccgatccg ccacaacatc gaggacggca gcgtgcagtg ttccccgcgc 240

cagcggggat aaaccgtttt ttctcgaggc ggccgcataa tgcttaagtc gaacagaaag 300

taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt 360

gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata 420

tggcagatct caattggata tcggccggcc acgcgatcgc tgacgtcggt accctcgagt 480

ctggtaaaga aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg 540

cagcttaatt aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg 600

cctctaaacg ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg 660

tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt 720

aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc tgccattcat 780

ccgcttatta tcacttattc aggcgtagca ccaggcgttt aagggcacca ataactgcct 840

taaaaaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt 900

ctgccgacat ggaagccatc acagacggca tgatgaacct gaatcgccag cggcatcagc 960

accttgtcgc cttgcgtata atatttgccc atagtgaaaa cgggggcgaa gaagttgtcc 1020

atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa 1080

aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca 1140

tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat 1200

gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc 1260

accagctcac cgtctttcat tgccatacgg aactccggat gagcattcat caggcgggca 1320

agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag 1380

gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc 1440

tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt 1500

ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt 1560

agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat 1620

tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat 1680

tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct 1740

gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg cttctgtttc 1800

tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc 1860

cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc 1920

agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt 1980

gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc 2040

ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac 2100

ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga 2160

caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag 2220

ataccaggcg tttcccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt 2280

accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc 2340

cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg 2400

ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc 2460

actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt 2520

aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt 2580

tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt 2640

tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc 2700

agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca 2760

gccccatacg atataagttg taattctcat gttagtcatg ccccgcgccc accggaagga 2820

gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag 2880

ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 2940

ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca 3000

gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc 3060

cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt 3120

tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta 3180

ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg 3240

ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca 3300

tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa 3360

tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac 3420

ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc 3480

ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga 3540

catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt 3600

catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg 3660

ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca 3720

gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac 3780

tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt 3840

tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa 3900

cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg 3960

cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc 4020

gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc 4080

tctcccttat gcgactcctg cattaggaaa ttaatacgac tcactata 4128

Claims (8)

1.Cas3蛋白质的制造方法，该方法包括：

(a)将导入有Cas3基因的昆虫细胞在20～28℃培养，在该昆虫细胞内使Cas3蛋白质表达的工序，和

(b)回收所表达的Cas3蛋白质的工序。

2.根据权利要求1所述的方法，Cas3蛋白质是大肠杆菌来源的。

3.根据权利要求1或2所述的方法，昆虫细胞是Sf9细胞。

4.根据权利要求1～3的任一项所述的方法，所表达的Cas3蛋白质的回收包括Cas3蛋白质的纯化。

5.根据权利要求4所述的方法，Cas3蛋白质添加有标签，Cas3蛋白质的纯化包括针对该标签的亲和纯化。

6.根据权利要求5所述的方法，标签包含HN标签。

7.根据权利要求4～6的任一项所述的方法，Cas3蛋白质的纯化包括利用凝胶过滤层析的纯化。

8.根据权利要求4～7的任一项所述的方法，纯化中使用的缓冲液是磷酸缓冲液。