CN113278651A - 一种可实现靶基因两种剂量表达的基因修饰模型构建方法 - Google Patents
一种可实现靶基因两种剂量表达的基因修饰模型构建方法 Download PDFInfo
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Abstract
本发明提供了控制同一靶基因在宿主中以不同剂量进行表达的方法及产品，在插入序列中，外源强启动子两端设置了两个终止元件，每个终止元件两端具有不同的重组酶识别位点序列，当插入到宿主基因组编码外显子之前的内含子之中时，两个终止元件将阻止启动子的功能从而抑制靶基因表达，而通过不同的重组酶控制相应的终止元件及启动子的剔除实现内源启动子或外源强启动子调控的基因低剂量或高剂量表达。
Description
技术领域
本发明属于生物技术领域，具体涉及一种可实现靶基因两种剂量表达的基因修饰模型的构建方法。
背景技术
利用基因过表达制备细胞或者动物模型，是研究基因功能的常用技术，广泛应用于生命科学和医学基础研究以及临床前转化研究。常用的转基因动物模型为研究早期发育、疾病的发病过程和临床前药物评价提供了强有力的工具。研究发现，在这些模型中基因的表达剂量和表型密切相关，如：FUS基因发现与肌萎缩性侧索硬化症（ALS）和额颞叶变性（FTLD）有关，在过表达野生型人FUS基因的转基因纯合子小鼠中（FUS蛋白表达量是对照组的1.9倍），早期出现震颤，随后后肢逐渐瘫痪，12周的时候小鼠死亡；然而在过表达野生型人FUS基因的转基因杂合子小鼠中（FUS蛋白表达量是对照组的1.4倍），没有出现运动障碍的表型或者病理病变。这说明人FUS基因过表达导致进行性神经元病变是剂量依赖的(Jacqueline C. Mitchell et al., Overexpression of human wild-type FUS causesprogressive motor neuron degeneration in an age- and dose-dependent fashion. Acta Neuropathol (2013) 125:273–288, DOI 10.1007/s00401-012-1043-z）。同样的情况也发生在人源TLR8转基因小鼠模型中(Cristiana Guiducci, et.al, RNA recognitionby human TLR8 can lead to autoimmune inflammation, J. Exp. Med. 2013 Vol. 210No. 13 2903-2919),人源TLR8的表达会导致小鼠自身免疫性疾病的发生，小鼠的存活率及表型和人TLR8的表达量呈明显的负相关。
由此可见，不同剂量的基因表达水平，可以使基因展现出不同的生物学功能；不同剂量的表达模型可以为我们理解基因功能提供更全面的线索。而我们目前常用的不同表达剂量模型，通常有：1）杂合子和纯合子动物模型间的基因表达剂量差异，但两种基因型间的基因表达水平差异有限；2）通过传统的随机转基因方式，因为插入位点和基因拷贝数不同，可以获得不同表达水平的动物模型，但因为常规的转基因模型需要复杂的建系过程，从多个品系中筛选出可传代，目的基因表达的动物模型，增加了时间和成本。
鉴于此，通过一种小鼠模型，高效获得两种或者多种不同表达剂量的模型，对于基因生命科学、医学研究具有重要意义，亦有需求，特提出本发明。
发明内容
本发明的目的在于提供一种可实现靶基因高、低两种剂量表达的基因修饰模型构建方法以及用于实施该方法的核酸序列、载体和宿主，利用该方法制备的细胞和动物模型中，可以实现敲入基因分别由内源启动子驱动表达，实现较低水平的转基因表达或由强启动子（如CAG启动子）来驱动目的基因高表达。
根据本发明的一个方面，提供一种用于基因重组表达的核酸序列，其包含：外源强启动子序列和至少一个控制启动子序列功能的调控元件。优选地，所述在所述外源强自动子上游和下游各有一个控制启动子序列功能的调控元件，其能够调控外源强启动子和/或所述核酸序列插入位置的内源启动子的功能。优选地，所述调控序列为终止元件，其两端具有重组酶识别元件。
本发明还提供了包含上述核酸序列的载体、宿主，以及构建所述载体和宿主的方法，所述方法包括讲所述核酸序列引入所述载体以及将所述核酸序列或载体引入所述宿主。
进一步地，本发明还提供了利用所述核酸序列、载体或宿主表达外源基因的方法。
敲入基因可以根据需求实现不同强度的过表达。具体的方法为：如图1所示，通过定点敲入的方式，将RNA剪接受体序列（SA）-重组酶1识别元件-终止元件1-CAG强启动子-重组酶1识别元件-重组酶2识别元件-终止元件2-重组酶2识别元件-靶基因-polyA片段插入基因编码外显子之前的内含子中。正常情况下，因为转录终止元件1和2的存在，所插入位置基因内源启动子和CAG强启动子的转录都被终止，目的基因不表达；当通过重组酶2作用后，STOP2元件将被剔除掉，目的基因为CAG强启动子所驱动，实现目的基因高表达；在此基础上，可通过重组酶1进一步作用，将STOP1元件和CAG启动子剔除掉，目的基因受插入位置基因内源启动子驱动表达，实现一个较低剂量表达。
上述名词中，CAG启动子为巨细胞病毒（CMV）增强子元件、鸡β-肌动蛋白基因的启动子和兔β-珠蛋白基因的剪接受体组合而成的一个人工启动子，具有较高的转录活性，强启动子也可以选择其他启动子，优选CAG启动子；重组酶识别元件为各种重组酶所识别的DNA序列，常用的有loxp，FRT, Rox序列等，重组酶1识别元件可以为loxp，FRT, Rox序列的任意一种，优选非loxp序列；重组酶2识别元件可以为loxp，FRT, Rox序列的任意一种，优选loxp序列；重组酶1识别元件和重组酶2识别元件需为不同种类的识别元件，对应的重组酶应为两种不同的重组酶；终止元件1为转录终止元件，发挥终止内源基因转录的作用，避免对外源强启动子表达有影响；终止元件2为转录或者翻译终止元件，用于终止强启动子的转录或者终止强启动子转录出mRNA的翻译，避免外源基因、蛋白表达。PolyA为Polyadenylation缩写，用于终止转录。
本发明可以通过构建一个模型，实现靶基因的两种剂量表达，为观察不同靶基因表达剂量对基因功能的影响研究提供了简便的方法。
附图说明
为了更清楚的说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需使用的附图做简单的介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1是本发明内容的示意图。
图2 tdTomato-Luciferase条件性高低剂量过表达小鼠胚胎干细胞模型构建。图A为模型构建验证示意图；图B为tdTomato-Luciferase酶切验证结果。载体经EcoRI限制性内切酶，酶切后理论条带大小为9831bp、5617bp、3969bp、3640bp、932bp，结果符合预期（M为1kb DNA marker）；图C为同源重组ES细胞鉴定电泳结果，上图为5’同源臂鉴定结果，下图为3’同源臂鉴定结果（M为1kb DNA marker，图上方编号为ES细胞克隆编号）。
图3tdTomato-Luciferase阳性ES细胞克隆目的基因高低剂量表达验证。图A为Cre作用后阳性ES细胞克隆PCR鉴定电泳结果；图B为Cre/Flp两种重组酶作用后阳性ES细胞克隆PCR鉴定电泳结果；图C为野生型ES细胞克隆、tdTomato-Luciferase阳性ES细胞克隆、Cre作用后阳性ES细胞克隆、Cre/Flp两种重组酶作用后阳性ES细胞克隆EGFP和tdTomato荧光表达检测结果；图D为野生型ES细胞克隆、tdTomato-Luciferase阳性ES细胞克隆、Cre作用后阳性ES细胞克隆、Cre/Flp两种重组酶作用后阳性ES细胞克隆Luciferase荧光素酶活性表达检测结果。
图4EGFP高、低剂量过表达小鼠模型构建及验证。图A为模型构建验证示意图；图B为同源重组F1代小鼠鉴定电泳结果，左图为5’同源臂鉴定结果，右图为3’同源臂鉴定结果（M为1kb DNA marker，图上方数字为F1代小鼠编号）；图C-F图为分别检测野生型小鼠（WT）、Cre重组酶去STOP2小鼠（CAG-EGFP）、Cre/Flp重组酶去STOP1和STOP2小鼠（R26-EGFP）的脑（C）、肾脏（D）、肝脏（E）、肺（F）四个组织EGFP荧光表达结果，左图为荧光显微镜拍照结果，右图为左图荧光结果的定量分析结果。
具体实施方式
实施例一
tdTomato-Luciferase条件性高低剂量过表达小鼠胚胎干细胞模型
为验证本发明在细胞水平实现高低剂量过表达调控的可行性，发明人通过同源重组方式将SA-FRT-EGFP-STOP1-CAG-FRT-Loxp-STOP2-Loxp-tdTomato-IRES-luciferase-WPRE-polyA-PGK-Neo-polyA元件插入在小鼠Rosa26基因的内含子1中，策略示意图2所示。利用该策略，发明人可通过检测tdTomato红色荧光和luciferase荧光素酶催化底物发光的活性来反映是否可以实现目的基因tdTomato和luciferase高低剂量表达。同时该策略引入了一个EGFP绿色荧光蛋白，便于验证片段插入后Rosa26启动子的活性。该模型的构建和验证过程如下：
1）同源重组载体构建
通过全基因合成获得同源重组左臂(SEQ ID NO:1 )，同源重组右臂(SEQ ID NO:2 )，SA-FRT-EGFP-STOP1(SA、FRT、EGFP、STOP1序列依次为SEQ ID NO:3-6)，FRT-Loxp-STOP2-Loxp(Loxp、STOP2序列依次为SEQ ID NO:7-8)，tdTomato-IRES-luciferase-WPRE-polyA(tdTomato、IRES、Luciferase、WPRE-polyA序列依次为SEQ ID NO:9-12), PGK-Neo-polyA(SEQ ID NO:13)片段，通过PCR扩增的方式以plvct-tTR-KRAB质粒为模板扩增获得CAG启动子(SEQ ID NO:14)，然后通过In-Fusion的方法，将同源重组左臂、SA-FRT-EGFP-STOP1、CAG启动子、FRT-Loxp-STOP2-Loxp、tdTomato-IRES-luciferase-WPRE-polyA、 PGK-Neo-polyA和同源重组右臂片段按序插入到PBR322骨架载体（SEQ ID NO:15）中，构建获得tdTomato-Luciferase同源重组载体(SEQ ID NO:16)，该载体经酶切（EcoRI酶切结果如图2B所示）、测序确认正确后进行后续的ES细胞转染。
2)tdTomato-Luciferase阳性ES细胞克隆获得
验证正确的重组载体经大肠杆菌转化扩增，抽提纯化后，将重组打靶载体利用电转的方式转染小鼠胚胎干细胞（ES细胞）。利用Neomycine抗生素进行筛选，共计获得96个抗性克隆，通过PCR技术对抗性克隆进行鉴定，共计获得10个同源重组阳性克隆，阳性克隆两个同源臂重组的PCR鉴定结果如图2C所示（上图为5’臂同源重组阳性克隆鉴定结果，下图为3’臂同源重组阳性克隆鉴定结果，编号为克隆编号，M为1kb DNA marker），5’臂同源重组阳性克隆可扩增出3.4kb大小的阳性条带，阴性克隆只能扩增出6.8kb的阴性条带；3’臂同源重组阳性克隆应扩增出7.7kb片段，阴性克隆应无产物。PCR信息和反应条件如下：
引物序列信息：
| 引物名称 | 序列信息（5’->3'） |
| 5'臂鉴定正向引物 | GCCGGGCCTCGTCGTCT(SEQ ID NO: 17 ) |
| 5'臂鉴定反向引物 | TTTTTGGGGGTGATGGTGGTC (SEQ ID NO: 18 ) |
| 3’臂鉴定正向引物 | TCGCAGCCTACCGTGGTGTTCGTT (SEQ ID NO: 19 ) |
| 3’臂鉴定反向引物 | GGTGTTAATTGGCAGTCTATGAGC (SEQ ID NO: 20 ) |
PCR反应体系：
| PCR反应组成 | 体积 (µl) |
| ddH2O | 31 |
| PCR Buffer | 10 |
| 2.5 mM dNTP | 4 |
| 正向引物 | 1 |
| 反向引物 | 1 |
| DNA Polymerase | 2 |
| genomic DNA | 1 |
| 总计 | 50 |
PCR反应程序：
| 步骤 | 温度（℃） | 时间 | 备注 |
| 1 | 98 | 2 min | |
| 2 | 98 | 15 sec | |
| 3 | 68 | 15 sec | |
| 4 | 68 | 4 min | 重复步骤2-4共34个循环 |
| 5 | 68 | 1 min | |
| 6 | 12 | 10 min |
3)tdTomato-Luciferase ES细胞克隆Cre重组酶、Cre/Flp重组酶处理
tdTomato-Luciferase 阳性ES细胞克隆分别转染Cre重组酶表达质粒、Cre/Flp重组酶表达质粒,转染72小时后，进行单克隆培养，分别获得剔除STOP2元件、剔除STOP1和STOP2元件的单克隆。
tdTomato-Luciferase阳性ES细胞克隆，转染Cre质粒，获得剔除STOP2元件，由CAG启动子驱动tdTomato-Luciferase表达的ES细胞克隆，PCR鉴定方法如下所示：
引物序列信息如下表，PCR反应体系和反应程序除退火温度为63℃外，其他同上
| 引物名称 | 序列信息（5’->3'） |
| 鉴定正向引物 | CGCCGCCGCACCCCCTCTA(SEQ ID NO: 21 ) |
| 鉴定反向引物 | GGCACCAGCAGCGCACTTTGAATC (SEQ ID NO: 22 ) |
PCR鉴定阳性克隆应可扩增出3.4kb大小的条带，电泳结果如图3A所示，经PCR产物测序确认D7和D11为去除STOP2的阳性克隆。
tdTomato-Luciferase阳性ES细胞克隆，转染Cre/Flp质粒，获得剔除STOP1和STOP2元件，由Rosa26基因启动子驱动tdTomato-Luciferase表达的ES细胞克隆，PCR鉴定方法如下所示：
引物序列信息如下表，PCR反应体系和反应程序同上
| 引物名称 | 序列信息（5’->3'） |
| 鉴定正向引物 | TCCGCCCACCGCCCCACACT(SEQ ID NO: 23 ) |
| 鉴定反向引物 | GGCACCAGCAGCGCACTTTGAATC (SEQ ID NO: 24 ) |
PCR鉴定阳性克隆应可扩增出7.3kb大小的条带，电泳结果如图3A所示，经PCR产物测序确认G4和G6为去除STOP1和STOP2的阳性克隆。
4）重组酶作用前后目的基因tdTomato和Luciferase表达量检测。
①tdTomato荧光表达检测：分别对C57BL/6野生型细胞、tdTomato-Luciferase打靶阳性ES细胞、转染Cre质粒去STOP2的ES细胞和转染Cre+Flp质粒去STOP1及STOP2的ES细胞，观察每组细胞的荧光表达情况。如图3C所示，C57BL/6野生型细胞作为对照组没有检测到任何荧光表达；tdTomato-Luciferase打靶阳性ES细胞只能检测到Rosa26内源启动子驱动的绿色荧光表达，无tdTomato荧光表达；tdTomato-Luciferase打靶阳性转染Cre质粒ES细胞可以检测到Rosa26内源启动子驱动的绿色荧光和CAG强启动子驱动的红色荧光表达；tdTomato-Luciferase打靶阳性转染Cre+Flp质粒的ES细胞只能检测到Rosa26基因启动子驱动的tdTomato红色荧光的表达，而且红色荧光的表达强度明显弱于转染Cre质粒ES细胞组，这是因为在转染Cre质粒ES细胞组中红色荧光是受CAG启动子驱动表达，在转染Cre+Flp质粒ES细胞中红色荧光是受Rosa26基因内源性启动子驱动表达。这说明本发明的基因双表达系统可以通过重组酶作用实现诱导不同强度的靶基因过表达。
②体外Luciferase荧光素酶活性检测:
在上述tdTomato荧光检测部分，发明人通过荧光对目的基因表达进行了比较分析，为进一步说明本应用的可行性，发明人通过检测另一个目的基因Luciferase催化底物产生荧光的能力，从功能层面上也对目的基因表达进行了检测。分别对C57BL/6野生型细胞、tdTomato-Luciferase打靶阳性ES细胞、转染Cre质粒去STOP2的ES细胞和转染Cre+Flp质粒去STOP1及STOP2的ES细胞进行培养，培养至80%密度时，去培养基，PBS洗两次后，分别加入500ml裂解液（Promega），4℃，12000rpm，离心15分钟，取上清即为蛋白样品。使用Luciferase Assay System试剂盒（Promega），用化学发光检测仪Luminometer TD-20/20(Lumat LB9507)检测荧光强度。细胞蛋白浓度采用BCA法测定（Pierce）,定量后将测得的荧光数值用蛋白浓度来标准化，得到每毫克蛋白的荧光值。如图3D所示，C57BL/6野生型ES细胞和tdTomato-Luciferase打靶阳性ES细胞不能检测到Luciferase表达，而转染Cre质粒后Luciferase受CAG启动子驱动表达强度是转染Cre/Flp质粒后Luciferase受Rosa26基因内源性启动子驱动表达强度的3倍左右。
实施例二
EGFP高、低剂量过表达小鼠模型
为验证本发明在动物体内实现高低剂量目的基因过表达调控的可行性，发明人通过同源重组方式将SA-FRT-STOP1-CAG-FRT-Loxp-STOP2-Loxp-EGFP-WPRE-polyA-PGK-Neo-polyA元件插入在小鼠Rosa26基因的内含子1中，制备策略如示意图4A，具体构建及检测过程描述如下：
1）同源重组载体构建
利用实施例一中构建获得的tdTomato-Luciferase同源重组载体(SEQ ID NO:16)为模板，PCR扩增获得同源重组左臂(SEQ ID NO:1 )，同源重组右臂(SEQ ID NO:2 )、SA-FRT-STOP1、CAG、FRT-Loxp-STOP2-Loxp、EGFP-WPRE-polyA等载体元件，然后通过In-Fusion的方法，将各元件插入到PBR322骨架载体（SEQ ID NO:15）中，构建获得EGFP同源重组载体，载体经酶切、测序确认后进行后续的受精卵注射。
2）EGFP基因敲入小鼠获得
EGFP同源重组载体质粒与Cas9、gRNA混合后进行小鼠受精卵显微注射，注射后的受精卵经培养箱短暂培养后，移植至受体母鼠的输卵管，获得基因修饰小鼠F0代小鼠，同源重组阳性的F0代小鼠通过与野生型小鼠交配，繁育获得F1代小鼠。通过PCR方法对F1代小鼠是否为正确发生同源重组的阳性小鼠进行鉴定，发生5’臂同源重组阳性的小鼠应可扩增出3.4kb的PCR产物，阴性小鼠只能扩增出6.8kb的PCR产物；发生3’臂同源重组阳性的小鼠应可扩增出4.1kb的PCR产物，阴性小鼠无PCR产物扩增出。部分小鼠的鉴定结果如图4B所示，1-4号小鼠为5’和3’臂同源重组阳性的小鼠（该小鼠简称：EGFP小鼠）；该小鼠通过和Dppa3-Cre小鼠（一种在胚胎早期及生殖细胞系中高表达Cre的小鼠品系，可用于小鼠整体全身性的去除loxp之间的元件）交配，获得去掉STOP2元件的EGFP小鼠,在该小鼠中EGFP表达受CAG启动子驱动（简称:CAG-EGFP）；将CAG-EGFP小鼠与CAG-Flp转基因小鼠交配，获得同时去掉STOP1和STOP2元件的EGFP小鼠,在该小鼠中EGFP表达受Rosa26内源启动子驱动（简称:R26-EGFP）。
3）EGFP小鼠、R26-EGFP小鼠、CAG-EGFP小鼠体内各组织EGFP荧光表达检测
分别取EGFP小鼠、R26-EGFP小鼠、CAG-EGFP小鼠的脑（图4C）、肾脏(图4D)、肝脏(图4E)和肺(图4F)组织进行EGFP荧光检测,并对荧光结果进行定量比较分析（图4C-F,左图为荧光照片结果，右图为荧光定量分析）。结果显示：EGFP小鼠各组织中无EGFP荧光检测到，R26-EGFP小鼠和CAG-EGFP小鼠4个组织均可以检测到EGFP荧光的表达，CAG-EGFP小鼠各组织中荧光显著高于R26-EGFP小鼠，4个组织中CAG-EGFP小鼠较R26-EGFP小鼠荧光强度的比值略有不同，CAG-EGFP小鼠组织荧光约是R26-EGFP小鼠的3-4倍。该结果说明本发明的两种剂量表达系统可以在体内通过重组酶作用实现诱导不同强度的靶基因过表达。
综上所述，给予两个实施例的结果，发明人通过在Rosa26基因位点分别插入不同的目的基因，借助重组酶系统，分别在细胞水平和小鼠体内水平证明本发明“一种可实现靶基因两种剂量表达的基因修饰模型构建方法”，可以通过构建一个模型，实现靶基因的两种剂量表达，为观察不同靶基因表达剂量对基因功能的影响研究提供了简便的方法。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
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<110> 上海南方模式生物科技股份有限公司
上海砥石生物科技有限公司
广东南模生物科技有限公司
<120> 一种可实现靶基因两种剂量表达的基因修饰模型构建方法
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3300
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gccagcgggg gcggcgagga ggcgctccca ggttccggcc ctcccctcgg ccccgcgccg 60
cagagtctgg ccgcgcgccc ctgcgcaacg tggcaggaag cgcgcgctgg gggcggggac 120
gggcagtagg gctgagcggc tgcggggcgg gtgcaagcac gtttccgact tgagttgcct 180
caagaggggc gtgctgagcc agacctccat cgcgcactcc ggggagtgga gggaaggagc 240
gagggctcag ttgggctgtt ttggaggcag gaagcacttg ctctcccaaa gtcgctctga 300
gttgttatca gtaagggagc tgcagtggag taggcgggga gaaggccgca cccttctccg 360
gaggggggag gggagtgttg caataccttt ctgggagttc tctgctgcct cctggcttct 420
gaggaccgcc ctgggcctgg gagaatccct tccccctctt ccctcgtgat ctgcaactcc 480
agtctttcta gaagatgggc gggagtcttc tgggcaggct taaaggctaa cctggtgtgt 540
gggcgttgtc ctgcagggga attgaacagg tgtaaaattg gagggacaag acttcccaca 600
gattttcggt tttgtcggga agttttttaa taggggcaaa taaggaaaat gggaggatag 660
gtagtcatct ggggttttat gcagcaaaac tacaggttat tattgcttgt gatccgcctc 720
ggagtatttt ccatcgaggt agattaaaga catgctcacc cgagttttat actctcctgc 780
ttgagatcct tactacagta tgaaattaca gtgtcgcgag ttagactatg taagcagaat 840
tttaatcatt tttaaagagc ccagtacttc atatccattt ctcccgctcc ttctgcagcc 900
ttatcaaaag gtattttaga acactcattt tagccccatt ttcatttatt atactggctt 960
atccaacccc tagacagagc attggcattt tccctttcct gatcttagaa gtctgatgac 1020
tcatgaaacc agacagatta gttacataca ccacaaatcg aggctgtagc tggggcctca 1080
acactgcagt tcttttataa ctccttagta cactttttgt tgatcctttg ccttgatcct 1140
taattttcag tgtctatcac ctctcccgtc aggtggtgtt ccacatttgg gcctattctc 1200
agtccaggga gttttacaac aatagatgta ttgagaatcc aacctaaagc ttaactttcc 1260
actcccatga atgcctctct cctttttctc catttataaa ctgagctatt aaccattaat 1320
ggtttccagg tggatgtctc ctcccccaat attacctgat gtatcttaca tattgccagg 1380
ctgatatttt aagacattaa aaggtatatt tcattattga gccacatggt attgattact 1440
gcttactaaa attttgtcat tgtacacatc tgtaaaaggt ggttcctttt ggaatgcaaa 1500
gttcaggtgt ttgttgtctt tcctgaccta aggtcttgtg agcttgtatt ttttctattt 1560
aagcagtgct ttctcttgga ctggcttgac tcatggcatt ctacacgtta ttgctggtct 1620
aaatgtgatt ttgccaagct tcttcaggac ctataatttt gcttgacttg tagccaaaca 1680
caagtaaaat gattaagcaa caaatgtatt tgtgaagctt ggtttttagg ttgttgtgtt 1740
gtgtgtgctt gtgctctata ataatactat ccaggggctg gagaggtggc tcggagttca 1800
agagcacaga ctgctcttcc agaagtcctg agttcaattc ccagcaacca catggtggct 1860
cacaaccatc tgtaatggga tctgatgccc tcttctggtg tgtctgaaga ccacaagtgt 1920
attcacatta aataaataaa tcctccttct tcttcttttt ttttttttta aagagaatac 1980
tgtctccagt agaatttact gaagtaatga aatactttgt gtttgttcca atatggtagc 2040
caataatcaa attactcttt aagcactgga aatgttacca aggaactaat ttttatttga 2100
agtgtaactg tggacagagg agccataact gcagacttgt gggatacaga agaccaatgc 2160
agactttaat gtcttttctc ttacactaag caataaagaa ataaaaattg aacttctagt 2220
atcctatttg tttaaactgc tagctttact taacttttgt gcttcatcta tacaaagctg 2280
aaagctaagt ctgcagccat tactaaacat gaaagcaagt aatgataatt ttggatttca 2340
aaaatgtagg gccagagttt agccagccag tggtggtgct tgcctttatg cctttaatcc 2400
cagcactctg gaggcagaga caggcagatc tctgagtttg agcccagcct ggtctacaca 2460
tcaagttcta tctaggatag ccaggaatac acacagaaac cctgttgggg aggggggctc 2520
tgagatttca taaaattata attgaagcat tccctaatga gccactatgg atgtggctaa 2580
atccgtctac ctttctgatg agatttgggt attatttttt ctgtctctgc tgttggttgg 2640
gtcttttgac actgtgggct ttctttaaag cctccttcct gccatgtggt ctcttgtttg 2700
ctactaactt cccatggctt aaatggcatg gctttttgcc ttctaagggc agctgctgag 2760
atttgcagcc tgatttccag ggtggggttg ggaaatcttt caaacactaa aattgtcctt 2820
taattttttt tttaaaaaat gggttatata ataaacctca taaaatagtt atgaggagtg 2880
aggtggacta atattaaatg agtccctccc ctataaaaga gctattaagg ctttttgtct 2940
tatacttaac ttttttttta aatgtggtat ctttagaacc aagggtctta gagttttagt 3000
atacagaaac tgttgcatcg cttaatcaga ttttctagtt tcaaatccag agaatccaaa 3060
ttcttcacag ccaaagtcaa attaagaatt tctgactttt aatgttaatt tgcttactgt 3120
gaatataaaa atgatagctt ttcctgaggc agggtctcac tatgtatctc tgcctgatct 3180
gcaacaagat atgtagacta aagttctgcc tgcttttgtc tcctgaatac taaggttaaa 3240
atgtagtaat acttttggaa cttgcaggtc agattctttt ataggggaca cactaaggga 3300
<210> 2
<211> 3300
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gcttgggtga tagttggtaa aatgtgtttc aagtgatgaa aacttgaatt attatcaccg 60
caacctactt tttaaaaaaa aaagccaggc ctgttagagc atgcttaagg gatccctagg 120
acttgctgag cacacaagag tagttacttg gcaggctcct ggtgagagca tatttcaaaa 180
aacaaggcag acaaccaaga aactacagtt aaggttacct gtctttaaac catctgcata 240
tacacaggga tattaaaata ttccaaataa tatttcattc aagttttccc ccatcaaatt 300
gggacatgga tttctccggt gaataggcag agttggaaac taaacaaatg ttggttttgt 360
gatttgtgaa attgttttca agtgatagtt aaagcccatg agatacagaa caaagctgct 420
atttcgaggt ctcttggttt atactcagaa gcacttcttt gggtttccct gcactatcct 480
gatcatgtgc taggcctacc ttaggctgat tgttgttcaa ataaacttaa gtttcctgtc 540
aggtgatgtc atatgatttc atatatcaag gcaaaacatg ttatatatgt taaacatttg 600
tacttaatgt gaaagttagg tctttgtggg tttgattttt aattttcaaa acctgagcta 660
aataagtcat ttttacatgt cttacatttg gtggaattgt ataattgtgg tttgcaggca 720
agactctctg acctagtaac cctacctata gagcactttg ctgggtcaca agtctaggag 780
tcaagcattt caccttgaag ttgagacgtt ttgttagtgt atactagttt atatgttgga 840
ggacatgttt atccagaaga tattcaggac tatttttgac tgggctaagg aattgattct 900
gattagcact gttagtgagc attgagtggc ctttaggctt gaattggagt cacttgtata 960
tctcaaataa tgctggcctt ttttaaaaag cccttgttct ttatcaccct gttttctaca 1020
taatttttgt tcaaagaaat acttgtttgg atctcctttt gacaacaata gcatgttttc 1080
aagccatatt ttttttcctt tttttttttt tttttggttt ttcgagacag ggtttctctg 1140
tatagccctg gctgtcctgg aactcacttt gtagaccagg ctggcctcga actcagaaat 1200
ccgcctgcct ctgcctcctg agtgccggga ttaaaggcgt gcaccaccac gcctggctaa 1260
gttggatatt ttgttatata actataacca atactaactc cactgggtgg atttttaatt 1320
cagtcagtag tcttaagtgg tctttattgg cccttcatta aaatctactg ttcactctaa 1380
cagaggctgt tggtactagt ggcacttaag caacttccta cggatatact agcagattaa 1440
gggtcaggga tagaaactag tctagcgttt tgtataccta ccagctttat actaccttgt 1500
tctgatagaa atatttcagg acatctagag tgtactataa ggttgatggt aagcttataa 1560
ggaacttgaa agtggagtaa ctactccatt tctctgaggg gagaattaaa atttttgacc 1620
aagtgttgtt gagccactga gaatggtctc agaacataac ttcttaagga accttcccag 1680
attgccctca acactgcacc acatttggtc ctgcttgaac attgccatgg ctcttaaagt 1740
cttaattaag aatattaatt gtgtaattat tgtttttcct cctttagatc attccttgag 1800
gacaggacag tgcttgttta aggctatatt tctgctgtct gagcagcaac aggtcttcga 1860
gatcaacatg atgttcataa tcccaagatg ttgccattta tgttctcaga agcaagcaga 1920
ggcatgatgg tcagtgacag taatgtcact gtgttaaatg ttgctatgca gtttggattt 1980
ttctaatgta gtgtaggtag aacatatgtg ttctgtatga attaaactct taagttacac 2040
cttgtataat ccatgcaatg tgttatgcaa ttaccatttt aagtattgta gctttctttg 2100
tatgtgagga taaaggtgtt tgtcataaaa tgttttgaac atttccccaa agttccaaat 2160
tataaaacca caacgttaga acttatttat gaacaatggt tgtagtttca tgcttttaaa 2220
atgcttaatt attcaattaa caccgtttgt gttataatat atataaaact gacatgtaga 2280
agtgtttgtc cagaacattt cttaaatgta tactgtcttt agagagttta atatagcatg 2340
tcttttgcaa catactaact tttgtgttgg tgcgagcaat attgtgtagt cattttgaaa 2400
ggagtcattt caatgagtgt cagattgttt tgaatgttat tgaacatttt aaatgcagac 2460
ttgttcgtgt tttagaaagc aaaactgtca gaagctttga actagaaatt aaaaagctga 2520
agtatttcag aagggaaata agctacttgc tgtattagtt gaaggaaagt gtaatagctt 2580
agaaaattta aaaccatata gttgtcattg ctgaatatct ggcagatgaa aagaaatact 2640
cagtggttct tttgagcaat ataacagctt gttatattaa aaattttccc cacagatata 2700
aactctaatc tataactcat aaatgttaca aatggatgaa gcttacaaat gtggcttgac 2760
ttgtcactgt gcttgtttta gttatgtgaa agtttggcaa taaacctatg tcctaaatag 2820
tcaaactgtg gaatgacttt ttaatctatt ggtttgtcta gaacagttat gttgccattt 2880
gccctaatgg tgaaagaaaa agtggggagt gccttggcac tgttcatttg tggtgtgaac 2940
caaagagggg ggcatgcact tacacttcaa acatcctttt gaaagactga caagtttggg 3000
tcttcacagt tggaattggg catccctttt gtcagggagg gagggaggga gggaggctgg 3060
cttgttatgc tgacaagtgt gattaaattc aaactttgag gtaagttgga ggaacttgta 3120
cattgttagg agtgtgacaa tttggactct taatgatttg gtcatacaaa atgaacctag 3180
accaacttct ggaagatgta tataataact ccatgttaca ttgatttcac ctgactaata 3240
cttatccctt atcaattaaa tacagaagat gccagccatc tgggcctttt aacccagaaa 3300
<210> 3
<211> 182
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttaattaagg gatctgtagg gcgcagtagt ccagggtttc cttgatgatg tcatacttat 60
cctgtccctt ttttttccac agctcgcggt tgaggacaaa ctcttcgcgg tctttccagt 120
ggggatcgac ggtatcgtag agtcgaggcc gctctagaac tagtggatcc ggaaccctta 180
at 182
<210> 4
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gaagttccta ttctctagaa agtataggaa cttc 34
<210> 5
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 6
<211> 555
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tcgcgatgaa taaatgaaag cttgcagatc tgcgactcta gaggatctgc gactctagag 60
gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca 120
cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 180
agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 240
ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat 300
ctgcgactct agaggatcat aatcagccat accacatttg tagaggtttt acttgcttta 360
aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 420
aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 480
aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 540
tatcatgtct ggatc 555
<210> 7
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ataacttcgt atagcataca ttatacgaag ttat 34
<210> 8
<211> 1731
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cctcagcacc atggctagcg gcagcctcgg agtttgaata gatagaataa aatatcttta 60
ttttcattcc atctgtgtgt tggttttttg tgtgagatct acgggtggca tccctgtgac 120
ccctccccag tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc 180
ctaataaaat taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg 240
tggagggggg tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg 300
gtctattggg aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc 360
tcctgggttc aagcgattct cctgcctcag gctcccgagt tgttgggatt ccaggcatgc 420
atgaccaggc tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc 480
aggctggtct ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg 540
ggattacagg cgtgaaccac tgctcccttc cctgtccttc tgcctcagct aattgagtag 600
gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg 660
caataaaaag acagaataaa acgcacgggt gttgggtcgt ttgttcataa acgcggggtt 720
cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc 780
cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca 840
gccaacgtcg gggcggcagg ccgtggaatt cgtaaatgaa ttttctgtat gaggtcgcga 900
tgaataaatg aaagcttgca gatctgcgac tctagaggat ctgcgactct agaggatcat 960
aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc 1020
cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta 1080
taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 1140
gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgcga 1200
ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac 1260
ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg 1320
tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 1380
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat 1440
gtctggatct gcgactctag aggatcataa tcagccatac cacatttgta gaggttttac 1500
ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg 1560
ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 1620
atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 1680
atgtatctta tcatgtctgg atccccatca agctgatccg gaacccttaa t 1731
<210> 9
<211> 1431
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60
ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120
ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180
atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240
cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300
gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360
tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag 420
accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480
gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540
atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600
gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660
cgccaccacc tgttcctggg gcatggcacc ggcagcaccg gcagcggcag ctccggcacc 720
gcctcctccg aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc 780
atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 840
tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc 900
tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc 960
gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 1020
aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg 1080
ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag 1140
aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 1200
aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 1260
aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc 1320
aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc 1380
gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaagta a 1431
<210> 10
<211> 585
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60
gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120
ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180
gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240
aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300
tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360
acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420
aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480
gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540
gggacgtggt tttcctttga aaaacacgat gataatatgg ccaca 585
<210> 11
<211> 1658
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga 60
accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120
gcttttacag atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc 180
gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240
tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300
gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt 360
tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420
aaaaagctcc caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga 480
tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540
tttgtgccag agtccttcga tagggacaag acaattgcac tgatcatgaa ctcctctgga 600
tctactggtc tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg 660
catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720
gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780
cgagtcgtct taatgtatag atttgaagaa gagctgtttc tgaggagcct tcaggattac 840
aagattcaaa gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg 900
attgacaaat acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct 960
aaggaagtcg gggaagcggt tgccaagagg ttccatctgc caggtatcag gcaaggatat 1020
gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080
gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140
acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt 1200
tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260
ggagacatag cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct 1320
ctgattaagt acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa 1380
caccccaaca tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt 1440
cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500
tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560
gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620
aaggccaaga agggcggaaa gatcgccgtg taactcga 1658
<210> 12
<211> 828
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 60
ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 120
ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 180
gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 240
ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 300
tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 360
gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 420
ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 480
tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 540
ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg 600
atcctgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 660
ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 720
gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 780
attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg 828
<210> 13
<211> 1638
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggtctgaaga ggagtttacg tccagccaag ctagcttggc tgcaggtcgt cgaaattcta 60
ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct 120
gggcacttgg cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag 180
gcgccaaccg gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt 240
caggaagttc ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc 300
acgtctcact agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct 360
ttggggcagc ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag 420
gggtgggtcc gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc 480
ctccggaggc ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc 540
ttcctcatct ccgggccttt cgacctgcag cctgttgaca attaatcatc ggcatagtat 600
atcggcatag tataatacga caaggtgagg aactaaacca tgggatcggc cattgaacaa 660
gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg 720
gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc 780
ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca 840
gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc 900
actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca 960
tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat 1020
acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca 1080
cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg 1140
ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgatgatctc 1200
gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct 1260
ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct 1320
acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac 1380
ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc 1440
tgaggggatc aattctctag agctcgctga tcagcctcga ctgtgccttc tagttgccag 1500
ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 1560
gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 1620
ctggggggtg gggtgggg 1638
<210> 14
<211> 1598
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 60
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 120
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 180
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 240
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 300
gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca 360
attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg 420
gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt 480
gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 540
cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg ggagtcgctg cgacgctgcc 600
ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg 660
cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct 720
tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg 780
agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga 840
gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc 900
tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg 960
gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag 1020
ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg 1080
agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc 1140
cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg 1200
agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga 1260
gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc 1320
aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg 1380
cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc 1440
gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac ggctgccttc 1500
gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct 1560
ctgctaacca tgttcatgcc ttcttctttt tcctacag 1598
<210> 15
<211> 5869
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gatcctctag agtcgagcag tgtggttttc aagaggaagc aaaaagcctc tccacccagg 60
cctggaatgt ttccacccaa tgtcgagcag tgtggttttg caagaggaag caaaaagcct 120
ctccacccag gcctggaatg tttccaccca atgtcgagca aaccccgccc agcgtcttgt 180
cattggcgaa ttcgaacacg cagatgcagt cggggcggcg cggtcccagg tccacttcgc 240
atattaaggt gacgcgtgtg gcctcgaaca ccgagcgacc ctgcagcgac ccgcttaaca 300
gcgtcaacag cgtgccgcag atcttggtgg cgtgaaactc ccgcacctct tcggccagcg 360
ccttgtagaa gcgcgtatgg cttcgtaccc cggccatcag cacgcgtctg cgttcgacca 420
ggctgcgcgt tctcgcggcc atagcaaccg acgtacggcg ttgcgccctc gccggcagca 480
agaagccacg gaagtccgcc cggagcagaa aatgcccacg ctactgcggg tttatataga 540
cggtccccac gggatgggga aaaccaccac cacgcaactg ctggtggccc tgggttcgcg 600
cgacgatatc gtctacgtac ccgagccgat gacttactgg cgggtgctgg gggcttccga 660
gacaatcgcg aacatctaca ccacacaaca ccgccttgac cagggtgaga tatcggccgg 720
ggacgcggcg gtggtaatga caagcgccca gataacaatg ggcatgcctt atgccgtgac 780
cgacgccgtt ctggctcctc atatcggggg ggaggctggg agctcacatg ccccgccccc 840
ggccctcacc ctcatcttcg accgccatcc catcgccgcc ctcctgtgct acccggccgc 900
gcgatacctt atgggcagca tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc 960
gccgaccttg cccggcacaa acatcgtgtt gggggccctt ccggaggaca gacacatcga 1020
ccgcctggcc aaacgccagc gccccggcga gcggcttgac ctggctatgc tggccgcgat 1080
tcgccgcgtt tacgggctgc ttgccaatac ggtgcggtat ctgcagggcg gcgggtcgtg 1140
gcgggaggat tggggacagc tttcggggac ggccgtgccg ccccagggtg ccgagcccca 1200
gagcaacgcg ggcccacgac cccatatcgg ggacacgtta tttaccctgt ttcgggcccc 1260
cgagttgctg gcccccaacg gcgacctgta caacgtgttt gcctgggcct tggacgtctt 1320
ggccaaacgc ctccgtccca tgcacgtctt tatcctggat tacgaccaat cgcccgccgg 1380
ctgccgggac gccctgctgc aacttacctc cgggatgatc cagacccacg tcaccacccc 1440
aggctccata ccgacgatct gcgacctggc gcgcacgttt gcccgggaga tgggggaggc 1500
taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa 1560
gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag 1620
ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct 1680
tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc 1740
ggggcggcag gccctgccat agccacgggc cccgtgggtt agggacgggg tcccccatgg 1800
ggaatggttt atggttcgtg ggggttatta ttttgggcgt tgcgtggggt cagtccacga 1860
ctggactgag cagacagacc catggttttt ggatggcctg ggcatggacc gcatgtactg 1920
gcgcgacacg aacaccgggc gtctgtggct gccaaacacc cccgaccccc aaaaaccacc 1980
gcgcggattt ctggcgccgc cggacgaact aaacctgact acggcatctc tgccccttct 2040
tcgctggtac gaggagcgct tttgttttgt attggtcacc acggccgagt ttcctcgacc 2100
gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat 2160
cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc 2220
gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc 2280
gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac 2340
caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta 2400
cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc 2460
ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga 2520
ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg 2580
accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg 2640
gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag 2700
ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca 2760
agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag 2820
aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggcagcgtt 2880
gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg 2940
cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac 3000
tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt 3060
tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc 3120
gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat 3180
tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca 3240
caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct 3300
cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg 3360
accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg 3420
cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt 3480
cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa 3540
aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 3600
agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 3660
acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 3720
ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 3780
accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3840
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3900
ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3960
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 4020
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 4080
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 4140
ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 4200
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 4260
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 4320
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 4380
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 4440
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 4500
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 4560
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4620
gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4680
aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4740
aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4800
cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4860
ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4920
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4980
ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 5040
ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 5100
ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 5160
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 5220
ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 5280
ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 5340
gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 5400
ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 5460
cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 5520
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 5580
aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 5640
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5700
gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5760
cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaagaattc tcatgtttga 5820
cagcttatca tcgataagct gcggccgcaa aggccgcggt cgacaagct 5869
<210> 16
<211> 23989
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gaattctcat gtttgacagc ttatcatcga taagctgcgg ccgcaaaggc cgcggtcgac 60
aagctgccag cgggggcggc gaggaggcgc tcccaggttc cggccctccc ctcggccccg 120
cgccgcagag tctggccgcg cgcccctgcg caacgtggca ggaagcgcgc gctgggggcg 180
gggacgggca gtagggctga gcggctgcgg ggcgggtgca agcacgtttc cgacttgagt 240
tgcctcaaga ggggcgtgct gagccagacc tccatcgcgc actccgggga gtggagggaa 300
ggagcgaggg ctcagttggg ctgttttgga ggcaggaagc acttgctctc ccaaagtcgc 360
tctgagttgt tatcagtaag ggagctgcag tggagtaggc ggggagaagg ccgcaccctt 420
ctccggaggg gggaggggag tgttgcaata cctttctggg agttctctgc tgcctcctgg 480
cttctgagga ccgccctggg cctgggagaa tcccttcccc ctcttccctc gtgatctgca 540
actccagtct ttctagaaga tgggcgggag tcttctgggc aggcttaaag gctaacctgg 600
tgtgtgggcg ttgtcctgca ggggaattga acaggtgtaa aattggaggg acaagacttc 660
ccacagattt tcggttttgt cgggaagttt tttaataggg gcaaataagg aaaatgggag 720
gataggtagt catctggggt tttatgcagc aaaactacag gttattattg cttgtgatcc 780
gcctcggagt attttccatc gaggtagatt aaagacatgc tcacccgagt tttatactct 840
cctgcttgag atccttacta cagtatgaaa ttacagtgtc gcgagttaga ctatgtaagc 900
agaattttaa tcatttttaa agagcccagt acttcatatc catttctccc gctccttctg 960
cagccttatc aaaaggtatt ttagaacact cattttagcc ccattttcat ttattatact 1020
ggcttatcca acccctagac agagcattgg cattttccct ttcctgatct tagaagtctg 1080
atgactcatg aaaccagaca gattagttac atacaccaca aatcgaggct gtagctgggg 1140
cctcaacact gcagttcttt tataactcct tagtacactt tttgttgatc ctttgccttg 1200
atccttaatt ttcagtgtct atcacctctc ccgtcaggtg gtgttccaca tttgggccta 1260
ttctcagtcc agggagtttt acaacaatag atgtattgag aatccaacct aaagcttaac 1320
tttccactcc catgaatgcc tctctccttt ttctccattt ataaactgag ctattaacca 1380
ttaatggttt ccaggtggat gtctcctccc ccaatattac ctgatgtatc ttacatattg 1440
ccaggctgat attttaagac attaaaaggt atatttcatt attgagccac atggtattga 1500
ttactgctta ctaaaatttt gtcattgtac acatctgtaa aaggtggttc cttttggaat 1560
gcaaagttca ggtgtttgtt gtctttcctg acctaaggtc ttgtgagctt gtattttttc 1620
tatttaagca gtgctttctc ttggactggc ttgactcatg gcattctaca cgttattgct 1680
ggtctaaatg tgattttgcc aagcttcttc aggacctata attttgcttg acttgtagcc 1740
aaacacaagt aaaatgatta agcaacaaat gtatttgtga agcttggttt ttaggttgtt 1800
gtgttgtgtg tgcttgtgct ctataataat actatccagg ggctggagag gtggctcgga 1860
gttcaagagc acagactgct cttccagaag tcctgagttc aattcccagc aaccacatgg 1920
tggctcacaa ccatctgtaa tgggatctga tgccctcttc tggtgtgtct gaagaccaca 1980
agtgtattca cattaaataa ataaatcctc cttcttcttc tttttttttt ttttaaagag 2040
aatactgtct ccagtagaat ttactgaagt aatgaaatac tttgtgtttg ttccaatatg 2100
gtagccaata atcaaattac tctttaagca ctggaaatgt taccaaggaa ctaattttta 2160
tttgaagtgt aactgtggac agaggagcca taactgcaga cttgtgggat acagaagacc 2220
aatgcagact ttaatgtctt ttctcttaca ctaagcaata aagaaataaa aattgaactt 2280
ctagtatcct atttgtttaa actgctagct ttacttaact tttgtgcttc atctatacaa 2340
agctgaaagc taagtctgca gccattacta aacatgaaag caagtaatga taattttgga 2400
tttcaaaaat gtagggccag agtttagcca gccagtggtg gtgcttgcct ttatgccttt 2460
aatcccagca ctctggaggc agagacaggc agatctctga gtttgagccc agcctggtct 2520
acacatcaag ttctatctag gatagccagg aatacacaca gaaaccctgt tggggagggg 2580
ggctctgaga tttcataaaa ttataattga agcattccct aatgagccac tatggatgtg 2640
gctaaatccg tctacctttc tgatgagatt tgggtattat tttttctgtc tctgctgttg 2700
gttgggtctt ttgacactgt gggctttctt taaagcctcc ttcctgccat gtggtctctt 2760
gtttgctact aacttcccat ggcttaaatg gcatggcttt ttgccttcta agggcagctg 2820
ctgagatttg cagcctgatt tccagggtgg ggttgggaaa tctttcaaac actaaaattg 2880
tcctttaatt ttttttttaa aaaatgggtt atataataaa cctcataaaa tagttatgag 2940
gagtgaggtg gactaatatt aaatgagtcc ctcccctata aaagagctat taaggctttt 3000
tgtcttatac ttaacttttt ttttaaatgt ggtatcttta gaaccaaggg tcttagagtt 3060
ttagtataca gaaactgttg catcgcttaa tcagattttc tagtttcaaa tccagagaat 3120
ccaaattctt cacagccaaa gtcaaattaa gaatttctga cttttaatgt taatttgctt 3180
actgtgaata taaaaatgat agcttttcct gaggcagggt ctcactatgt atctctgcct 3240
gatctgcaac aagatatgta gactaaagtt ctgcctgctt ttgtctcctg aatactaagg 3300
ttaaaatgta gtaatacttt tggaacttgc aggtcagatt cttttatagg ggacacacta 3360
agggagacca ccatcacccc caaaaactcg agttaattaa gggatctgta gggcgcagta 3420
gtccagggtt tccttgatga tgtcatactt atcctgtccc ttttttttcc acagctcgcg 3480
gttgaggaca aactcttcgc ggtctttcca gtggggatcg acggtatcgt agagtcgagg 3540
ccgctctaga actagtggat ccggaaccct taatgaagtt cctattctct agaaagtata 3600
ggaacttcta ggtccctcga cctgcagccc aagctagatc gaattcggcc ggccttcacg 3660
agccgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt 3720
cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga 3780
tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc 3840
ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga 3900
ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg 3960
caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg 4020
cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat 4080
cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa 4140
gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt 4200
gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc 4260
cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga 4320
tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct 4380
gtacaagtaa tcgcgatgaa taaatgaaag cttgcagatc tgcgactcta gaggatctgc 4440
gactctagag gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa 4500
acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 4560
tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 4620
aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 4680
atgtctggat ctgcgactct agaggatcat aatcagccat accacatttg tagaggtttt 4740
acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat 4800
tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 4860
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 4920
caatgtatct tatcatgtct ggatcctcga acataactta cggtaaatgg cccgcctggc 4980
tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 5040
ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 5100
gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 5160
tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 5220
atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact 5280
ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt 5340
tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc 5400
gaggggcggg gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc 5460
cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg 5520
cggcgggcgg ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct 5580
cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg 5640
gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt 5700
ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg 5760
ggggtgcgtg cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg 5820
gctgtgagcg ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga 5880
gcgcggccgg gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg 5940
tgcggggtgt gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac 6000
cccccctgca cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc 6060
gtacggggcg tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc 6120
cgggcggggc ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc 6180
ggagcgccgg cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt 6240
gcgagagggc gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc 6300
gccgccgcac cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa 6360
tgggcgggga gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc 6420
ggggctgtcc gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct 6480
tctggcgtgt gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt 6540
tcctacagct cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat 6600
tgatttgata ccgcgggccc taaacgcgta ccaacgtgaa aaaattatta ttcgcgaagt 6660
tcctattctc tagaaagtat aggaacttcc gcggactcgt ggcagcgagc ataacttcgt 6720
atagcataca ttatacgaag ttatcctcag caccatggct agcggcagcc tcggagtttg 6780
aatagataga ataaaatatc tttattttca ttccatctgt gtgttggttt tttgtgtgag 6840
atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc 6900
actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt gtctgactag 6960
gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg gcaagttggg 7020
aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc agtggcacaa 7080
tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc tcaggctccc 7140
gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt tttttggtag 7200
agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca ggtgatctac 7260
ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc cttccctgtc 7320
cttctgcctc agctaattga gtagggggga ggctaactga aacacggaag gagacaatac 7380
cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac gggtgttggg 7440
tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg 7500
agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt 7560
tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccgtgg aattcgtaaa 7620
tgaattttct gtatgaggtc gcgatgaata aatgaaagct tgcagatctg cgactctaga 7680
ggatctgcga ctctagagga tcataatcag ccataccaca tttgtagagg ttttacttgc 7740
tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt 7800
tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 7860
cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 7920
atcttatcat gtctggatct gcgactctag aggatcataa tcagccatac cacatttgta 7980
gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg 8040
aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat 8100
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 8160
aaactcatca atgtatctta tcatgtctgg atctgcgact ctagaggatc ataatcagcc 8220
ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 8280
tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt 8340
acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 8400
gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcccc atcaagctga 8460
tccggaaccc ttaatataac ttcgtatagc atacattata cgaagttatt aggtccctcg 8520
acctgcagcc caagctagat cgaattcggc cggccttcac gagccgccac cctcgagcca 8580
ccatggtgag caagggcgag gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg 8640
agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg 8700
agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg 8760
acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca 8820
tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact 8880
tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga 8940
tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga 9000
agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg 9060
gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga 9120
ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc 9180
tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg 9240
gccgccacca cctgttcctg gggcatggca ccggcagcac cggcagcggc agctccggca 9300
ccgcctcctc cgaggacaac aacatggccg tcatcaaaga gttcatgcgc ttcaaggtgc 9360
gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc 9420
cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg 9480
cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg 9540
ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga 9600
tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca 9660
cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc 9720
agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc 9780
tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt 9840
tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca 9900
ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct 9960
ccgagggccg ccaccacctg ttcctgtacg gcatggacga gctgtacaag taagcccctc 10020
tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt 10080
tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc 10140
tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca 10200
aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 10260
gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg 10320
ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt 10380
gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct 10440
gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg 10500
ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt 10560
ggttttcctt tgaaaaacac gatgataata tggccacaac catggaagac gccaaaaaca 10620
taaagaaagg cccggcgcca ttctatccgc tggaagatgg aaccgctgga gagcaactgc 10680
ataaggctat gaagagatac gccctggttc ctggaacaat tgcttttaca gatgcacata 10740
tcgaggtgga catcacttac gctgagtact tcgaaatgtc cgttcggttg gcagaagcta 10800
tgaaacgata tgggctgaat acaaatcaca gaatcgtcgt atgcagtgaa aactctcttc 10860
aattctttat gccggtgttg ggcgcgttat ttatcggagt tgcagttgcg cccgcgaacg 10920
acatttataa tgaacgtgaa ttgctcaaca gtatgggcat ttcgcagcct accgtggtgt 10980
tcgtttccaa aaaggggttg caaaaaattt tgaacgtgca aaaaaagctc ccaatcatcc 11040
aaaaaattat tatcatggat tctaaaacgg attaccaggg atttcagtcg atgtacacgt 11100
tcgtcacatc tcatctacct cccggtttta atgaatacga ttttgtgcca gagtccttcg 11160
atagggacaa gacaattgca ctgatcatga actcctctgg atctactggt ctgcctaaag 11220
gtgtcgctct gcctcataga actgcctgcg tgagattctc gcatgccaga gatcctattt 11280
ttggcaatca aatcattccg gatactgcga ttttaagtgt tgttccattc catcacggtt 11340
ttggaatgtt tactacactc ggatatttga tatgtggatt tcgagtcgtc ttaatgtata 11400
gatttgaaga agagctgttt ctgaggagcc ttcaggatta caagattcaa agtgcgctgc 11460
tggtgccaac cctattctcc ttcttcgcca aaagcactct gattgacaaa tacgatttat 11520
ctaatttaca cgaaattgct tctggtggcg ctcccctctc taaggaagtc ggggaagcgg 11580
ttgccaagag gttccatctg ccaggtatca ggcaaggata tgggctcact gagactacat 11640
cagctattct gattacaccc gagggggatg ataaaccggg cgcggtcggt aaagttgttc 11700
cattttttga agcgaaggtt gtggatctgg ataccgggaa aacgctgggc gttaatcaaa 11760
gaggcgaact gtgtgtgaga ggtcctatga ttatgtccgg ttatgtaaac aatccggaag 11820
cgaccaacgc cttgattgac aaggatggat ggctacattc tggagacata gcttactggg 11880
acgaagacga acacttcttc atcgttgacc gcctgaagtc tctgattaag tacaaaggct 11940
atcaggtggc tcccgctgaa ttggaatcca tcttgctcca acaccccaac atcttcgacg 12000
caggtgtcgc aggtcttccc gacgatgacg ccggtgaact tcccgccgcc gttgttgttt 12060
tggagcacgg aaagacgatg acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa 12120
caaccgcgaa aaagttgcgc ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta 12180
ccggaaaact cgacgcaaga aaaatcagag agatcctcat aaaggccaag aagggcggaa 12240
agatcgccgt gtaactcgag gggccacggt acccgtatca agcttatcga taatcaacct 12300
ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 12360
ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc 12420
attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt 12480
gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc 12540
attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg 12600
gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact 12660
gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt 12720
gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg 12780
gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc 12840
cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgatcctgt 12900
gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 12960
aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 13020
taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 13080
agacaatagc aggcatgctg gggatgcggt gggctctatg ggaggtgcca gggcgtgccc 13140
ttgggctccc cgggcgcggc ggccgcatcg aatggtctga agaggagttt acgtccagcc 13200
aagctagctt ggctgcaggt cgtcgaaatt ctaccgggta ggggaggcgc ttttcccaag 13260
gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca caagtggcct 13320
ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt tctttggtgg 13380
ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct 13440
cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga 13500
cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg 13560
ctccttcgct ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg 13620
cgggctcagg ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct 13680
tcaaaagcgc acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgacctg 13740
cagcctgttg acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg 13800
aggaactaaa ccatgggatc ggccattgaa caagatggat tgcacgcagg ttctccggcc 13860
gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 13920
gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 13980
tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg 14040
ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 14100
ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 14160
tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 14220
gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 14280
gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 14340
ctcaaggcgc gcatgcccga cggcgatgat ctcgtcgtga cccatggcga tgcctgcttg 14400
ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 14460
gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 14520
ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 14580
atcgccttct atcgccttct tgacgagttc ttctgagggg atcaattctc tagagctcgc 14640
tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 14700
ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 14760
gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gtgggttttt 14820
cgtcagcttg cagaagatct ccccaactgg ggtaaccttt gagttctctc agttgggggc 14880
tcgaggcttg ggtgatagtt ggtaaaatgt gtttcaagtg atgaaaactt gaattattat 14940
caccgcaacc tactttttaa aaaaaaaagc caggcctgtt agagcatgct taagggatcc 15000
ctaggacttg ctgagcacac aagagtagtt acttggcagg ctcctggtga gagcatattt 15060
caaaaaacaa ggcagacaac caagaaacta cagttaaggt tacctgtctt taaaccatct 15120
gcatatacac agggatatta aaatattcca aataatattt cattcaagtt ttcccccatc 15180
aaattgggac atggatttct ccggtgaata ggcagagttg gaaactaaac aaatgttggt 15240
tttgtgattt gtgaaattgt tttcaagtga tagttaaagc ccatgagata cagaacaaag 15300
ctgctatttc gaggtctctt ggtttatact cagaagcact tctttgggtt tccctgcact 15360
atcctgatca tgtgctaggc ctaccttagg ctgattgttg ttcaaataaa cttaagtttc 15420
ctgtcaggtg atgtcatatg atttcatata tcaaggcaaa acatgttata tatgttaaac 15480
atttgtactt aatgtgaaag ttaggtcttt gtgggtttga tttttaattt tcaaaacctg 15540
agctaaataa gtcattttta catgtcttac atttggtgga attgtataat tgtggtttgc 15600
aggcaagact ctctgaccta gtaaccctac ctatagagca ctttgctggg tcacaagtct 15660
aggagtcaag catttcacct tgaagttgag acgttttgtt agtgtatact agtttatatg 15720
ttggaggaca tgtttatcca gaagatattc aggactattt ttgactgggc taaggaattg 15780
attctgatta gcactgttag tgagcattga gtggccttta ggcttgaatt ggagtcactt 15840
gtatatctca aataatgctg gcctttttta aaaagccctt gttctttatc accctgtttt 15900
ctacataatt tttgttcaaa gaaatacttg tttggatctc cttttgacaa caatagcatg 15960
ttttcaagcc atattttttt tccttttttt tttttttttt ggtttttcga gacagggttt 16020
ctctgtatag ccctggctgt cctggaactc actttgtaga ccaggctggc ctcgaactca 16080
gaaatccgcc tgcctctgcc tcctgagtgc cgggattaaa ggcgtgcacc accacgcctg 16140
gctaagttgg atattttgtt atataactat aaccaatact aactccactg ggtggatttt 16200
taattcagtc agtagtctta agtggtcttt attggccctt cattaaaatc tactgttcac 16260
tctaacagag gctgttggta ctagtggcac ttaagcaact tcctacggat atactagcag 16320
attaagggtc agggatagaa actagtctag cgttttgtat acctaccagc tttatactac 16380
cttgttctga tagaaatatt tcaggacatc tagagtgtac tataaggttg atggtaagct 16440
tataaggaac ttgaaagtgg agtaactact ccatttctct gaggggagaa ttaaaatttt 16500
tgaccaagtg ttgttgagcc actgagaatg gtctcagaac ataacttctt aaggaacctt 16560
cccagattgc cctcaacact gcaccacatt tggtcctgct tgaacattgc catggctctt 16620
aaagtcttaa ttaagaatat taattgtgta attattgttt ttcctccttt agatcattcc 16680
ttgaggacag gacagtgctt gtttaaggct atatttctgc tgtctgagca gcaacaggtc 16740
ttcgagatca acatgatgtt cataatccca agatgttgcc atttatgttc tcagaagcaa 16800
gcagaggcat gatggtcagt gacagtaatg tcactgtgtt aaatgttgct atgcagtttg 16860
gatttttcta atgtagtgta ggtagaacat atgtgttctg tatgaattaa actcttaagt 16920
tacaccttgt ataatccatg caatgtgtta tgcaattacc attttaagta ttgtagcttt 16980
ctttgtatgt gaggataaag gtgtttgtca taaaatgttt tgaacatttc cccaaagttc 17040
caaattataa aaccacaacg ttagaactta tttatgaaca atggttgtag tttcatgctt 17100
ttaaaatgct taattattca attaacaccg tttgtgttat aatatatata aaactgacat 17160
gtagaagtgt ttgtccagaa catttcttaa atgtatactg tctttagaga gtttaatata 17220
gcatgtcttt tgcaacatac taacttttgt gttggtgcga gcaatattgt gtagtcattt 17280
tgaaaggagt catttcaatg agtgtcagat tgttttgaat gttattgaac attttaaatg 17340
cagacttgtt cgtgttttag aaagcaaaac tgtcagaagc tttgaactag aaattaaaaa 17400
gctgaagtat ttcagaaggg aaataagcta cttgctgtat tagttgaagg aaagtgtaat 17460
agcttagaaa atttaaaacc atatagttgt cattgctgaa tatctggcag atgaaaagaa 17520
atactcagtg gttcttttga gcaatataac agcttgttat attaaaaatt ttccccacag 17580
atataaactc taatctataa ctcataaatg ttacaaatgg atgaagctta caaatgtggc 17640
ttgacttgtc actgtgcttg ttttagttat gtgaaagttt ggcaataaac ctatgtccta 17700
aatagtcaaa ctgtggaatg actttttaat ctattggttt gtctagaaca gttatgttgc 17760
catttgccct aatggtgaaa gaaaaagtgg ggagtgcctt ggcactgttc atttgtggtg 17820
tgaaccaaag aggggggcat gcacttacac ttcaaacatc cttttgaaag actgacaagt 17880
ttgggtcttc acagttggaa ttgggcatcc cttttgtcag ggagggaggg agggagggag 17940
gctggcttgt tatgctgaca agtgtgatta aattcaaact ttgaggtaag ttggaggaac 18000
ttgtacattg ttaggagtgt gacaatttgg actcttaatg atttggtcat acaaaatgaa 18060
cctagaccaa cttctggaag atgtatataa taactccatg ttacattgat ttcacctgac 18120
taatacttat cccttatcaa ttaaatacag aagatgccag ccatctgggc cttttaaccc 18180
agaaagatcc tctagagtcg agcagtgtgg ttttcaagag gaagcaaaaa gcctctccac 18240
ccaggcctgg aatgtttcca cccaatgtcg agcagtgtgg ttttgcaaga ggaagcaaaa 18300
agcctctcca cccaggcctg gaatgtttcc acccaatgtc gagcaaaccc cgcccagcgt 18360
cttgtcattg gcgaattcga acacgcagat gcagtcgggg cggcgcggtc ccaggtccac 18420
ttcgcatatt aaggtgacgc gtgtggcctc gaacaccgag cgaccctgca gcgacccgct 18480
taacagcgtc aacagcgtgc cgcagatctt ggtggcgtga aactcccgca cctcttcggc 18540
cagcgccttg tagaagcgcg tatggcttcg taccccggcc atcagcacgc gtctgcgttc 18600
gaccaggctg cgcgttctcg cggccatagc aaccgacgta cggcgttgcg ccctcgccgg 18660
cagcaagaag ccacggaagt ccgcccggag cagaaaatgc ccacgctact gcgggtttat 18720
atagacggtc cccacgggat ggggaaaacc accaccacgc aactgctggt ggccctgggt 18780
tcgcgcgacg atatcgtcta cgtacccgag ccgatgactt actggcgggt gctgggggct 18840
tccgagacaa tcgcgaacat ctacaccaca caacaccgcc ttgaccaggg tgagatatcg 18900
gccggggacg cggcggtggt aatgacaagc gcccagataa caatgggcat gccttatgcc 18960
gtgaccgacg ccgttctggc tcctcatatc gggggggagg ctgggagctc acatgccccg 19020
cccccggccc tcaccctcat cttcgaccgc catcccatcg ccgccctcct gtgctacccg 19080
gccgcgcgat accttatggg cagcatgacc ccccaggccg tgctggcgtt cgtggccctc 19140
atcccgccga ccttgcccgg cacaaacatc gtgttggggg cccttccgga ggacagacac 19200
atcgaccgcc tggccaaacg ccagcgcccc ggcgagcggc ttgacctggc tatgctggcc 19260
gcgattcgcc gcgtttacgg gctgcttgcc aatacggtgc ggtatctgca gggcggcggg 19320
tcgtggcggg aggattgggg acagctttcg gggacggccg tgccgcccca gggtgccgag 19380
ccccagagca acgcgggccc acgaccccat atcggggaca cgttatttac cctgtttcgg 19440
gcccccgagt tgctggcccc caacggcgac ctgtacaacg tgtttgcctg ggccttggac 19500
gtcttggcca aacgcctccg tcccatgcac gtctttatcc tggattacga ccaatcgccc 19560
gccggctgcc gggacgccct gctgcaactt acctccggga tgatccagac ccacgtcacc 19620
accccaggct ccataccgac gatctgcgac ctggcgcgca cgtttgcccg ggagatgggg 19680
gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat 19740
aaaaagacag aataaaacgc acgggtgttg ggtcgtttgt tcataaacgc ggggttcggt 19800
cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg 19860
tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca 19920
acgtcggggc ggcaggccct gccatagcca cgggccccgt gggttaggga cggggtcccc 19980
catggggaat ggtttatggt tcgtgggggt tattattttg ggcgttgcgt ggggtcagtc 20040
cacgactgga ctgagcagac agacccatgg tttttggatg gcctgggcat ggaccgcatg 20100
tactggcgcg acacgaacac cgggcgtctg tggctgccaa acacccccga cccccaaaaa 20160
ccaccgcgcg gatttctggc gccgccggac gaactaaacc tgactacggc atctctgccc 20220
cttcttcgct ggtacgagga gcgcttttgt tttgtattgg tcaccacggc cgagtttcct 20280
cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg 20340
actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg 20400
gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc 20460
ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc 20520
gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgacgcgctg 20580
ggctacgtct tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt 20640
ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat 20700
gacgaccatc agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc 20760
actggaccgc tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg 20820
gcatggattg taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca 20880
tggagccggg ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca 20940
ctccaagaat tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt 21000
ggcagaacat atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca 21060
gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag 21120
gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa 21180
gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc 21240
gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct 21300
gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct 21360
ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac 21420
cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc 21480
tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat 21540
cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat 21600
taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat 21660
cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg 21720
gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg 21780
ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag 21840
ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga 21900
gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag 21960
aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 22020
tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 22080
aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 22140
aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 22200
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 22260
ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 22320
cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 22380
ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 22440
ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 22500
gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 22560
agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg 22620
cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 22680
aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 22740
aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 22800
ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 22860
aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 22920
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 22980
agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 23040
cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 23100
ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 23160
gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 23220
cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 23280
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 23340
ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 23400
catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 23460
tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 23520
ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct 23580
catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 23640
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 23700
cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 23760
acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 23820
ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 23880
tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac 23940
attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaa 23989
<210> 17
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gccgggcctc gtcgtct 17
<210> 18
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tttttggggg tgatggtggt c 21
<210> 19
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tcgcagccta ccgtggtgtt cgtt 24
<210> 20
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ggtgttaatt ggcagtctat gagc 24
<210> 21
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cgccgccgca ccccctcta 19
<210> 22
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ggcaccagca gcgcactttg aatc 24
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tccgcccacc gccccacact 20
<210> 24
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
ggcaccagca gcgcactttg aatc 24
Claims (19)
1.一种用于基因重组表达的核酸序列，其包含：外源强启动子序列和至少一个控制启动子序列功能的调控元件。
2.如权利要求1所述的核酸序列，在所述外源强启动子上游和下游各有一个控制启动子序列功能的调控元件。
3.如权利要求1-2的核酸序列，所述调控元件可调控外源强启动子和/或所述核酸序列插入位置的内源启动子的功能。
4.如权利要求1-3核酸序列，所述调控元件为终止元件。
5.如权利要求1-4核酸序列，所述调控元件两端具有重组酶识别元件。
6.如权利要求1-5所述的核酸序列，其在5’端至3’端依次具有RNA剪接受体序列（SA）-重组酶1识别元件-终止元件1-外源强启动子-重组酶1识别元件-重组酶2识别元件-终止元件2-重组酶2识别元件-靶基因。
7.如权利要求1-6所述的核酸序列，所述外源强启动子为CAG启动子。
8.如权利要求6-7所述的核酸序列，所述重组酶1识别元件和重组酶2识别元件需为不同种类的识别元件，对应的重组酶应为两种不同的重组酶。
9.如权利要求6-8所述的核酸序列，所述终止元件1为转录终止元件，终止元件2为转录或者翻译终止元件。
10.一种转基因载体，包含权利要求1-9任一项所述的核酸序列。
11.一种宿主细胞，包含权利要求1-9任一项所述核酸序列或权利要求10所述转基因载体。
12.如权利要求11所述宿主细胞，其包含小鼠胚胎干细胞或小鼠受精卵细胞。
13.如权利要求11或12所述的宿主细胞，其还包括具有编码重组酶序列的载体。
14.构建转基因载体的方法，包括将权利要求1-9任一项所述核酸序列引入载体中。
15.构建表达外源基因的宿主的方法，其包括：将权利要求1-9任一项所述外源核酸序列或权利要求10所述载体导入到宿主中，所述外源核酸序列包含外源高强度启动子序列和控制启动子序列功能的调控元件。
16.如权利要求14所述的方法，所述宿主为动物或植物细胞、组织或个体。
17.如权利要求15所述的方法，所述动物个体、细胞来源于小鼠，所述动物个体为小鼠。
18.权利要求1-9任一项所述核酸序列、权利要求10所述的载体、权利要求11-13任一项所述的宿主或权利要求15-17任一项所述的方法构建的宿主在表达外源基因中的应用。
19.如权利要求18所述的应用，其包括将具有重组酶序列的载体导入宿主细胞。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103409448A (zh) * | 2013-08-30 | 2013-11-27 | 北京百奥赛图基因生物技术有限公司 | 一种模型小鼠制备方法及条件性细胞剔除重组载体 |
| CN111705072A (zh) * | 2020-08-18 | 2020-09-25 | 江苏集萃药康生物科技有限公司 | 利用重组酶调控基因表达的方法 |
| CN112029797A (zh) * | 2020-06-22 | 2020-12-04 | 北京工业大学 | 一种哺乳动物启动子活性评价质粒载体及应用 |
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| CN103409448A (zh) * | 2013-08-30 | 2013-11-27 | 北京百奥赛图基因生物技术有限公司 | 一种模型小鼠制备方法及条件性细胞剔除重组载体 |
| CN112029797A (zh) * | 2020-06-22 | 2020-12-04 | 北京工业大学 | 一种哺乳动物启动子活性评价质粒载体及应用 |
| CN111705072A (zh) * | 2020-08-18 | 2020-09-25 | 江苏集萃药康生物科技有限公司 | 利用重组酶调控基因表达的方法 |
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