CN108424931A - CRISPR/Cas9技术介导山羊VEGF基因定点整合的方法 - Google Patents

CRISPR/Cas9技术介导山羊VEGF基因定点整合的方法 Download PDF

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CN108424931A
CN108424931A CN201810266144.XA CN201810266144A CN108424931A CN 108424931 A CN108424931 A CN 108424931A CN 201810266144 A CN201810266144 A CN 201810266144A CN 108424931 A CN108424931 A CN 108424931A
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呼啸
刘东军
郝斐
高源�
梁浩
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Abstract

本发明利用CRISPER/Cas9系统介导完成山羊VEGF基因定点整合，其是依据山羊的FGF5基因序列，构建基于CRISPER/Cas9系统的Cas9‑gRNA表达载体及VEGF打靶载体；然后将优化后的Cas9‑gRNA表达载体及VEGF打靶载体共同转染至山羊胎儿成纤维细胞中，获得VEGF基因在FGF5位点定点整合的细胞株。本发明所构建的基于CRISPER/Cas9系统的打靶载体为山羊VEGF基因定点整合及FGF5基因靶向敲除提供了一种简单快捷安全的途径。该方法在细胞株筛选过程中没有涉及任何筛选标记基因，从而大大提高了转基因动物的安全性，对山羊的遗传育种以及基因功能的研究具有重要价值。

Description

CRISPR/Cas9技术介导山羊VEGF基因定点整合的方法
技术领域
本发明涉及分子生物学及动物遗传育种领域，具体地说，涉及CRISPR/Cas9技术介导的山羊VEGF基因定点整合及FGF5基因敲除的方法。
背景技术
成簇短间隔回文重复系统（Clustered Regulatory Interspaced ShortPalindromic Repeat，CRISPR）作为第三代基因编辑技术，因其史无前例的特异性，有效性和多功能性而闻名。其来源于原核生物的获得性免疫系统，提供获得性免疫能力用来抵抗外来基因原件如噬菌体等的入侵，广泛存在于古生菌和原核生物中。CRISPR/Cas9系统近来发展成为可以对真核生物基因组进行特异性编辑的有效工具。与传统的锌指核酸酶（znicfinger nuclease，ZFNs）、转录激活因子样效应物核酸酶（transcriptionactivator-likeeffector nuclease，TALENs）相比，CRISPR/Cas9系统有着更高的基因编辑效率及更低的制作成本，可以灵活的应用于不同的核苷酸序列，是更为强力的基因编辑工具。CRISPR/Cas9系统应用广泛，包括用于研究的转基因动物制备，大规模筛选基因功能，潜在的临床基因治疗等。
山羊绒是一种稀有的特种动物纤维，具有极高的经济价值。内蒙古白绒山羊是经过长期自然选育形成的优良地方性绒肉兼用型品种，但相对于其它绒山羊而言，产绒量偏低，故培育高产绒的绒山羊新品系显得十分重要。相对于传统育种，现代生物技术极大程度地缩短了育种所需时间，农业生物新品种培育提供了新的技术途径。
血管内皮细胞生长因子(Vascular Endothelial Cell Growth Factor，VEGF)作用于血管内皮细胞，促使血管内皮细胞分裂增殖、转移、并改变血管通透性和促进新血管生成，具有诱导毛囊形态发生及调控毛发生长周期的作用。成纤维细胞生长因子5(Fibroblast growth factor 5， FGF5) 对毛发生长有负调控作用，敲除FGF5后能增加次级毛乳头的数量，增加产绒量，延长毛发的生长期，延缓进入退行期，从而延长毛发生长时间，获得长毛的新性状。
本实验在FGF5基因上，用KAP6.1作为目的基因VEGF的毛囊特异表达的启动子，在抑制FGF5基因表达的同时，使得VEGF基因只在毛囊细胞中表达，既可以加速山羊毛囊的形成进而提高羊绒产量，又可以防止外源基因在山羊的其他组织中表达，影响其他生理活动。
利用CRISPR/Cas9系统介导山羊VEGF基因在FGF5位点定点整合的方法未见报道。
发明内容
发明的目的是提供CRISPR/Cas9系统介导山羊VEGF基因定点整合的方法。
为了实现本发明目的，本发明提供的CRISPR/Cas9系统介导山羊VEGF基因定点整合的方法，其是依据山羊FGF5基因序列（NC_030813.1），在FGF5基因的第一外显子上设计靶序列，构建基于CRISPR/Cas9系统的Cas9-gRNA表达载体及VEGF打靶载体。然后将优化后的Cas9-gRNA表达载体及VEGF打靶载体转入山羊胎儿成纤维细胞中，获得山羊VEGF基因在FGF5位点定点整合的细胞株。
本发明中述及的山羊包括但不限于阿尔巴斯绒山羊
前述的方法，gRNA作用靶位点位于山羊FGF5基因的1号外显子上。sgRNA作用位点的DNA序列为5 ′- agcccctcggggcgccggac-3 ′。
前述的方法，所述的Cas9-gRNA表达载体的核苷酸序列如SEQ ID NO:1所示 （Cas9-gRNA表达载体图谱见图1）；所述VEGF打靶载体的核苷酸序列如SEQ ID NO:16所示（VEGF打靶载体质粒图谱见图4）。
本发明还提供根据上述方法获得山羊VEGF基因定点整合的细胞株。
本发明进一步提供上述方法在制备VEGF基因定点整合的体细胞克隆山羊的应用。该应用是指将所述山羊VEGF基因定点整合的细胞为核移植供体细胞，离体的山羊卵母细胞为核移植受体细胞，通过体细胞核移植技术获得山羊克隆胚胎，然后将克隆胚胎通过胚胎移植技术移入受体山羊子宫内进行妊娠，获得VEGF基因定点整合的山羊。
本发明的目的还可以采用以下的技术措施来进一步实现。
1）Cas9-gRNA表达载体的优化；2）根据山羊的FGF5基因序列，设计基于CRISPER/Cas9系统的sgRNA作用位点；3）构建VEGF打靶载体；4）将上述优化的Cas9-gRNA表达载体及VEGF打靶载体共同转染到山羊胎儿成纤维细胞中，通过流式细胞仪筛选单克隆细胞系；5）通过PCR技术鉴定筛选单克隆细胞株，获得VEGF基因定点整合的单克隆细胞株。
步骤4）中利用流式细胞仪挑取单克隆细胞系是指转染48h后的细胞，用胰蛋白酶消化，用含10%胎牛血清的DMEM/F12培养液重悬终止消化，离心，弃上清，用PBS清洗细胞，此过程重复三次，最后用1mL PBS重悬细胞，利用流式细胞仪将单个细胞接种于预先处理好的96孔板中。
步骤5）中鉴定VEGF基因定点整合单克隆细胞株主要采用设计跨靶序列的检测引物，通过PCR技术扩增并对扩增片段进行电泳及测序分析，目的是确定VEGF基因定点整合的情况。
本发明利用流式细胞仪法筛选得到121株单克隆细胞系，通过跨靶序列的检测引物进行扩增并进行葡聚糖凝胶电泳及测序分析发现有5株在FGF5位点实现定点整合，定点整合效率为4.1%。跨靶序列检测结果见图6。测序结果如SEQ ID NO:17。
本发明具有以下优点。
（一）CRISPR/Cas9基因编辑技术与常规的同源重组技术、ZFNs技术、TALEN技术相比，基因编辑效率显著提高。
(二)在FGF5基因上设计sgRNA，敲除FGF5后能增加次级毛乳头的数量，增加产绒量，延长毛发的生长期，延缓进入退行期，从而延长毛发生长时间，获得长毛的新性状。
(三) 用KAP6.1作为目的基因VEGF的毛囊特异表达的启动子,使得VEGF基因只在毛囊细胞中表达,既可以加速山羊毛囊的形成进而提高羊绒产量，又可以防止外源基因在山羊的其他组织中表达，影响其他生理活动。
(四) 通过CRISPR/Cas9系统的介导，实现了不添加任何筛选标记即可筛选出VEGF基因定点整合的细胞株，这是传统同源重组技术、ZFNs技术和TALEN技术无法实现的，很大程度提高了转基因动物的安全性。
(五) 通过体细胞核移植技术制备VEGF基因定点整合山羊，为构建成熟基因修饰动物的研究和生产奠定基础。
附图说明
图1为本发明实施例1中Cas9-gRNA表达载体图谱。
图2为本发明实施例2中CRISPR/Cas9系统介导山羊VEGF基因定点整合模式图。
图3为本发明实施例4中利用Surveyor突变检测试剂盒检测靶位点的电泳结果；其中，M:DL2000 Ladder marker;1:DNA突变情况。
图4为本发明实施例5中VEGF基因打靶载体图谱。
图5为本发明实施例6中单克隆生长到10d时细胞图。
图6为本发明实施例7中单克隆细胞的跨靶序列检测结果图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册（ Sambrook J & Russell DW ,Molecular cloning: a laboratory manual, 2001），或按照制造厂商说明书建议的条件。
以下实施例中测序工作由华大基因完成。
实施例1 Cas9-gRNA表达载体的优化
对购自北京唯尚立德生物科技有限公司的Cas9-gRNA表达载体进行优化，优化后的Cas9-gRNA表达载体的核苷酸序列如SEQ ID NO:1所示，质粒图谱见图1。
实施例2 Cas9-gRNA表达载体的构建
根据山羊的FGF5基因序列（NC_030813.1），基因序列如SEQ ID NO:2所示。在FGF5基因的第1号外显子序列上设计gRNA序列，并构建基于CRISPER/Cas9系统的gRNA表达载体。gRNA表达载体包括4部分：U6启动子、靶序列、gRNA骨架和终止信号。基于CRISPR/Cas9系统介导山羊VEGF基因定点敲入模式图见图2 。利用生物学软件根据sgRNA作用位点设计sgRNA序列，序列如SEQ ID NO:3所示。合成Target-Sense和Target-Anti，序列如SEQ ID NO:6、7所示，使其退火形成部分互补的oligo二聚体，后将oligo二聚体插入到Cas9/gRNA表达载体中，得到完整的Cas9/gRNA表达载体，转化大肠杆菌Trans1-T1，涂板，9h后，挑取单菌落，摇菌，对菌液进行PCR鉴定，确定阳性后，保菌，送华大基因公司测序，将测序正确的单菌落接种于含Amp的LB培养基中，37℃、220rpm过夜揺菌，提取质粒备用。
实施例3 电穿孔法转染山羊胎儿成纤维细胞
将原代山羊胎儿成纤维细胞解冻在100mm培养皿中培养，隔天换液。电穿孔法转染前应对细胞进行一次传代，改善其生长状态，以便提高转染后的细胞存活率及转染效率。传代后当细胞生长汇合度达到80%至90%时，首先用PBS缓冲液洗去残余代谢产物及培养液中残留的血清，加入0.25%胰蛋白酶2 mL消化2 min，用含10% FBS的DMEM/F12培养液终止消化，使用移液枪轻轻吹吸混匀后收集细胞于15 mL离心管中，800rpm离心5min，加入Opti-MEM清洗两遍并弃去上清液。为保证转染效率，可根据细胞沉淀量加入适量Opti-MEM重悬计数，调整浓度至100μL约含有1×106个细胞即可。将90 μL细胞悬液与5 μL的 Cas9/gRNA表达载体混合均匀后加入严格灭菌后的电转杯，应尽量避免产生气泡而影响电穿孔。将装好的电击杯置于电转杯腔中，测定电阻值，电阻值在一定程度上能够反映细胞溶液的浓度。利用最佳转染条件电压225V、脉冲时间2.5ms进行电穿孔。电转后将培养液加入电击杯中，因此时细胞极脆弱，应避免反复吹打。将细胞悬液分别转移到60 mm培养皿中置于37℃、5% CO2 、饱和湿度条件下的培养箱中培养，24h后换液。
实施例4 CRISPER/Cas9系统中sgRNA的活性检测
将Cas9/gRNA表达载体通过电穿孔法转染山羊胎儿成纤维细胞，提取细胞的基因组DNA，以提取的基因组为模板跨靶序列设计的上、下游引物进行PCR扩增，扩增引物为TS1-F、TS1-R，序列如SEQ ID NO:4、5所示。扩增体系（50µL）为： La TaqTMII 25µL，基因组模板1.5µL（浓度应小于500ng），上游引物2µL，下游引物2µL，无酶水 19.5µL。反应条件为：95℃预变性5min；95℃变性30s、58℃退火30s，72℃延伸50s，35个循环；72℃额外延伸10min，4℃保存。扩增得到大小为684bp的DNA片段，配制1％的琼脂糖凝胶，进行电泳检测，胶回收目的条带，胶回收产物逐步退火使其进行DNA杂交，杂交体系为：胶回收产物30µL，10×La PCRBuffer 3µL。杂交程序见表1。
表1 DNA杂交程序
反应温度 反应时间
95℃ 10 min
95℃ to 85℃ -2.0℃/s
85℃ 1 min
95℃ to 85℃ -0.3℃/s
95℃ 1 min
85℃ to 75℃ -0.3℃/s
75℃ 1 min
75℃ to 65℃ -0.3℃/s
65℃ 1 min
65℃ to 55℃ -0.3℃/s
55℃ 1 min
55℃ to 45℃ -0.3℃/s
45℃ 1 min
45℃ to 35℃ -0.3℃/s
35℃ 1 min
35℃ to 25℃ -0.3℃/s
25℃ 1 min
4℃ Hold
得到了杂交DNA后，通过用Surveyor突变检测试剂盒来检测突变活性。根据Surveyor突变检测试剂盒的使用说明书来完成实验，具体的反应体系见表2
表2 Surveyor突变检测体系
名称 用量
杂交DNA 20µL
0.15M MgCl2 Solution 3µL
Surveyor Enhancer S 1µL
Surveyor Nuclease S 1µL
Total 25µL
将反应体系配置完毕后，置于42℃反应1小时，最后将入2.5µL的Stop Solution后，将上述反应液通过2%琼脂糖凝胶电泳检测DNA片段的大小。检测结果见图3，两种sgRNA均具有活性，均可以介导Cas9对靶位序列进行切割。
实施例5 VEGF基因打靶载体的构建
将靶位点上下游1000bp左右的序列作为同源臂序列，设计含有BamHⅠ单酶切位点的上游同源臂引物和含有KpnⅠ、MfeⅠ酶切位点的下游同源臂引物，序列如SEQ ID NO:8-11所示。以山羊胎儿成纤维细胞的基因组为模板，扩增长度分别为1099bp的上游同源臂和926bp的下游同源臂，上游同源臂核苷酸序列如SEQ ID NO:14所示，下游同源臂的核苷酸序列如SEQID NO:15所示。反应体系（50µL）为：La TaqTMII 25µL，上、下游引物（引物序列如SEQ IDNO:8-11所示）各2µL，基因模板1.5µL，灭菌水19.5µL。PCR扩增程序为：95℃预变性5min；95℃变性30s、70℃退火30s，72℃延伸50s，35个循环；72℃额外延伸10min，4℃保存。PCR产物进行电泳检测，条带正确，PCR产物胶回收后连入pMD19-T，转化Trans1-T1，涂板，9h后，挑取单菌落，摇菌，保菌并送华大基因公司测序，测序正确的菌液留用。
在pCCR5-P1-Kap6.1-VEGF-polyA-P2载体为骨架，通过酶切连接得到VEGF基因打靶载体，构建顺序及过程如下：使用GeneJET Plasmid Miniprep Kit提取测序正确的pMD19-T-5hr和骨架载体pCCR5-P1-Kap6.1-VEGF-polyA-P2的质粒，具体步骤参照产品说明书； 使用BamHⅠ对pMD19-T-5hr单酶切，可适量加入碱性磷酸酶防止自连，胶回收长度为1099bp的上游同源臂； BamHⅠ对pCCR5-P1-Kap6.1-VEGF-polyA-P2单酶切，胶回收长度为6321bp的质粒骨架；随后用T4连接酶将上游同源臂与质粒骨架连接，转化Trans1-T1感受态大肠杆菌，挑菌、摇菌、酶切检验后送测序，将测序正确的质粒命名为pCCR5-Kap6.1-VEGF-polyA-P2。提取测序正确的pMD19-T-3hr和pCCR5-Kap6.1-VEGF-polyA-P2质粒；使用KpnI、MfeⅠ对pMD19-T-3hr进行双酶切，酶切体系见表1.10胶回收长度为926bp的下游同源臂；使用Kpn I、Mfe Ⅰ对pCCR5-Kap6.1-VEGF-polyA-P2进行双酶切，回收5931bp的质粒骨架；随后用T4连接酶将下游同源臂与质粒骨架连接，转化Trans1-T1感受态大肠杆菌，挑菌、摇菌、酶切检验后阳性菌株送测序，将测序正确的质粒命名为pFGF5-Kap6.1-VEGF-polyA，即VEGF基因打靶载体构建成功。VEGF基因打靶载体构建见图4，VEGF基因打靶载体构建的核苷酸序列如SEQ ID NO:16所示。
实施例6 单克隆细胞株的筛选
本实施例旨在筛选VEGF基因定点整合单克隆细胞株。
提取CRISPR/Cas9表达载体及VEGF打靶载体pFGF5-Kap6.1-VEGF-polyA；为提高VEGF基因的整合效率，也为避免卡那霉素抗性基因整合到绒山羊基因组中，使用限制性内切酶SfiI、PciⅠ将pFGF5-Kap6.1-VEGF-polyA载体线性化，胶回收含有上游同源臂、Kap6.1启动子、VEGF基因、PolyA和下游同源臂的大片段，-20℃保存。
将从雄性胎儿分离得到的原代GFFs解冻在100mm培养皿中培养，隔天换液。电穿孔法转染前应对细胞进行一次传代，改善其生长状态，以便提高转染后的细胞存活率及转染效率。传代后当细胞生长汇合度达到80%至90%时，首先用PBS缓冲液洗去残余代谢产物及培养液中残留的血清，加入0.25%胰蛋白酶2 mL消化2 min，用含10% FBS的DMEM/F12培养液终止消化，使用移液枪轻轻吹吸混匀后收集细胞于15 mL离心管中，800rpm离心5min，加入Opti-MEM清洗两遍并弃去上清液。为保证转染效率，可根据细胞沉淀量加入适量Opti-MEM重悬计数，调整浓度至100μL约含有1×106个细胞即可。将90 μL细胞悬液与2μg Cas9/gRNA表达载体及4μg线性化后的VEGF打靶载体混合均匀后加入电转杯，应尽量避免产生气泡而影响电穿孔。将装好的电击杯置于电转杯腔中，测定电阻值。利用最佳转染条件电压225V、脉冲时间2.5ms进行电穿孔，电转后将培养液加入电击杯中，应避免反复吹打。将细胞悬液分别转移到60 mm培养皿中置于37℃、5% CO2 、饱和湿度条件下的培养箱中培养，24h后换液洗去没有贴壁的细胞。
转染48h后，用胰蛋白酶消化细胞，用含血清的培养液终止消化，离心，弃掉上清，用PBS清洗细胞三次，务必洗净培养液。最后用少量PBS重悬细胞，通过流式细胞仪将单个细胞接种到预先加入含15%FBS DMEM/F12培养液的96孔板中。培养至10d左右时，观察细胞（见图5），将生长汇合度到达80%至90%的单细胞克隆传代至24孔板内继续培养，隔天换液，洗掉残留胰蛋白酶。当细胞在24孔板内再次生长至80%时，蛋白酶消化，吸取一部分细胞悬液提取基因组鉴定，剩余细胞悬液在孔内继续培养。
实施例7 单克隆细胞系的鉴定
以单克隆细胞株的基因组DNA作为模板，以跨靶位点的引物进行PCR扩增。具体的PCR反应引物为VEGF-IDF、VEGF-IDR，序列如SEQ ID NO:12、13所示，反应体系（25µL）为：LaTaqTMII 12.5µL，上、下游引物各1µL，模板DNA 1µL，灭菌水9.5µL。PCR扩增程序为：95℃预变性5min；95℃变性30s、59℃退火30s，72℃延伸50s，35个循环；72℃额外延伸10min，4℃保存。PCR产物进行琼脂糖凝胶电泳检测，结果如图6。将PCR产物送往华大基因公司测序。经测序分析共有5株单克隆细胞株在FGF5位点实现了VEGF基因定点整合。测序结果如SEQ IDNO:17所示。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
SEQUENCE LISTING
<110> 内蒙古大学
<120> CRISPR/Cas9技术介导山羊VEGF基因定点整合的方法
<130> CRISPR/Cas9技术介导山羊VEGF基因定点整合的方法
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 8360
<212> DNA
<213> 人工序列
<400> 1
aagtttcaat ggcgttccca aagggagtta agtcacgtat agaagcgaat atatattaaa 60
tacatatata tatataatat atatcaacct acatgctttc aataagatca caggccgggc 120
agtaaccttt gggctacagt cgctagcaag aaaatgattc aaggcctata aaaaatgcct 180
ttgatgaaag acacaatact agacaaaaag tttattcttt acaaatatag taaaatatgg 240
agtttcttaa accccctaaa ccgcagccat aaaaaaccta gcatacgccc ttgccttttg 300
tatacttcat cgctctggtc atacaacacc acatatctgt ggacggacgg acactccctt 360
gagcggacaa gaaaactgtc taatttggta gcaatttcga cagataaaag tcttcatcgc 420
tcactagtaa tgttcgtcga caaatatgcc atcgggaaga gtcagaaaac ctagaacctc 480
gctcactttg gaagagaaaa tggaggtaat ccagtcccag gagcgaaaca agctatcggt 540
tcgggatctg gccaagaggt aagatcaatg cctgatacat acatatgaat gggtaaaaat 600
aacaagtttc cgaaggttta acattggaaa gacacaagca gcagacattt tgaagcacaa 660
acagtcgatt aaagagggtc tgttgagtgg agaacttaag ttgaatcaga tgcggaggaa 720
tcccctctct cagcggggcg cccaaatcga tgagatgtgc tttgattggt tctcccgtgt 780
ccgtaccgag aatataccca tatcggggga aatggtgcgg aagaaggcca agcagttagc 840
tgtggagctg ggccactcca atttctctgc ctcctccgga tggctggaga aatggcgaaa 900
gcgacacaac gtccgataca atgacaccgg tgacagcctg gatctgcagg agttcgaggc 960
gattctggtg aaaagcgaac ccatctcgaa taaggacgat tgtgatgagc cataccccgt 1020
gactctgata gagcctatct attccaccga ggaggccatg atgcagctgg cccggctgaa 1080
ggagttcgca aaggatgact atgcttccta ccagcagttg atcagtctgg aaaatcaatg 1140
gagctggaaa tggaacatct ttaagaagga gcttccctga cccaagcatg caatttaggt 1200
gcaatataaa tacatattta tctcgaatct agtagtaaaa gttatttaat gtacgtttct 1260
tttataagta acatcgttga agataactta tcccttctac gcgttgaatt taaacctaaa 1320
actattgggt tattctattc atgacgatgc tcgtaaatat acgaaataca tgaccgtttg 1380
ctaatacgac tcactatagg caccttcctc ttcttcttgg ggtcagccct gcttacctgt 1440
tcttcatgag gtaacccgag ctatagccgt gtttgtcgca gccgacccgg cagtaatgcc 1500
tgctcatgtt ccacggctcg ttttttaagt ttcaagaccc gttatggcta gcggtgtcgt 1560
atttcttctt ggagtaaccg cgggaggaca agctgaggcc cctctgccgg cttcggtgcg 1620
ccgagttttc ttgtcgtgcc gcgtctatat gggcgtcttt cttagcctag acgatggacg 1680
tcctctagaa atcattactc taccgattcc acctactgag aaagaaggta tccgacctcc 1740
tcaggaaaaa ccacctcctc ctatttttcg tgctcgcggt gggttagaaa ccgttatagc 1800
acctgctcca ccgcatggta cttttcatgg gttggtatat agtagactcc ttcttcgaac 1860
atctgtcatg actattccga ctgaacgcca actagataga gcgcgaccgc gtatactagt 1920
ttaaagcccc tgtgaaggag tagctccccc tggacttggg tctgttgtcg ctacagctgt 1980
ttgagaaata ggttgaccaa gtctgaatgt tagtcgaaaa gcttctcttg ggctagttgc 2040
gtaggcctca actgcggttt cgttaggact cgcgatccga caggtttagg gccgccgagc 2100
ttttggagta gcgtgtcgag ggacccctct tcttcttgcc ggacaaacca ttagaatagc 2160
gggacagtga gcccgactgg gggttgaaat ttagattgaa gctggaccgg cttctacggt 2220
tcgaagttga ctcgtttctg tggatgctac tactagagct gttagacgac cgggtctagc 2280
cgctggtcat gcgtctggaa aaaaaccgcc gtttcttgga cagtctgcgg taagacgact 2340
cactataaga cgctcacttg tgcctctagt ggtttcgagg cgactcgcga tcatactagt 2400
tcgcgatact actcgtggtg gttctgaact gaaacgactt ccgggaacag tctgtcgttg 2460
acggactctt catgttcctt taaaagaagc tagtcagatt tttaccgatg cggcctatgt 2520
aactgccgcc tcgttcggtc ctccttaaaa tgtttaaata attcgggtag aacctttttt 2580
acctgccgtg gctcctcgac gaccatttcg aattgtctct tctagacaac gcgtttgtcg 2640
cgtgaaagct gttaccttcg tagggggtgg tctaagtgga cccgcttgac gtgcgatagg 2700
agtccgccgt tctcctaaag atggggaaaa actttctatt gtcccttttc taactctttt 2760
aggagtgtaa agcctatggg atgatacatc cgggggagcg ggccccttta aggtctaagc 2820
gcacctactg agcgtttagt cttctctggt agtgagggac cttgaagctc cttcagcacc 2880
tattcccccg gagacgggtc aggaagtagc tttcctactg attgaaacta tttttagacg 2940
gattgctttt ccacgaagga tttgtgagag acgacatgct catgaagtgt caaatattgc 3000
tcgagtggtt ccagtttatg cagtgtcttc cctactcttt cggtcgtaag gacagacctc 3060
tcgtcttctt tcgatagcac ctggaggaga agttctgctt ggcctttcaa tggcactttg 3120
tcgagtttct tctgataaag tttttctaac ttacaaagct gagacaactt tagtcgcctc 3180
acctcctagc gaagttgcgt agggaccctt gcatagtgct agaggacttt tagtaatttc 3240
tgttcctgaa ggacctgtta ctcctcttgc tcctgtaaga actcctgtaa caggagtggg 3300
aatgcaacaa acttctatcc ctctactaac ttcttgcgaa cttttgaatg cgagtagaga 3360
agctgctgtt tcagtacttt gtcgagttct ccgcggctat atgtcctacc cccgccgaca 3420
gttcttttga ctagttaccc taggctctgt tcgtctcacc tttctgttag gacctaaaag 3480
aattcaggct acctaaacgg ttggccttga agtacgtcaa ctaggtacta ctgagagagt 3540
ggaaattcct cctgtaggtc tttcgtgttc aaagaccggt ccccctgtca gaagtgctcg 3600
tgtagcgatt agaacgtcca tcgggtcgat agtttttccc ttatgacgtc tggcaattcc 3660
agcacctact tgagcagttt cattaccctt ccgtattcgg gctcttatag caatagctct 3720
accgggctct cttggtttga tgggtcttcc ctgtcttctt gtcatccctt tcctacttct 3780
cctaacttct cccatatttt cttgacccca gggtttagga attccttgtg ggtcaacttt 3840
tgtgggtcga agtcttactc ttcgagatgg acatgatgga cgtcttgccg tccctgtaca 3900
tgcacctagt ccttgacctg tagttagccg agaggctgat gctgcaccta gtatagcacg 3960
gggtcagaaa agagtttcta ctaagataac tattatttca caactgttct aggctatttt 4020
tatctccctt ctcactattg caggggagtc ttcttcaaca gttcttttac tttttaataa 4080
ccgccgtcga cgacttgcgg tttgactagt gtgttgcctt caagctatta gactgattcc 4140
gacttgctcc accggacaga ctcaacctat ttcggccgaa gtagttttcc gtcgaacaac 4200
tctgtgcggt ctagtggttc gtgcaccggg tttaagagct aagtgcgtac ttgtggttca 4260
tgctactttt actgtttgac taagctctcc actttcaata atgagacttc agattcgacc 4320
agagtctaaa gtctttcctg aaagtcaaaa tattccactc tctctagttg ttaatggtgg 4380
tacgcgtact acggatggac ttacgtcacc atccgtgacg tgaatagttt tttatagggt 4440
tcgaacttag acttaaacaa atgcctctga tatttcacat gctacaatcc ttttactagc 4500
gtttcagact cgtcctttat ccgttccggt ggcgattcat gaagaaaatg tcgttataat 4560
acttaaaaaa gttctggctc taatgtgacc ggttacctct ctaagccttc gctggtgaat 4620
agctttgttt gcctctttgt cctctttagc acaccctgtt cccatcccta aagcgctgtc 4680
aggccttcca ggacaggtac ggcgtccact tgtagcaatt tttctggctt catgtctggc 4740
ctccgaagag gttcctttca taggagggct tttccttgtc gctgttcgac tagcgtgcgt 4800
tttttctaac cctggggttc tttatgccgc ctaagctaag aggatgtcag cgaatgtcac 4860
atgaccaaca ccggtttcac ctctttccct tcagattttt tgagttttcg cagttccttg 4920
acgacccgta gtgttagtac ctcgctagtt cgaagctttt tttggggtag ctgaaagagc 4980
tccgctttcc tatatttctc cagttttttc tggagtagta attcgaaggg ttcatgagag 5040
agaaactcga acttttgccg gcctttgctt acgagcgatc acgcccgctc gacgtctttc 5100
cattgctcga ccgtgacggg agatttatgc aattaaagaa catagaccgg tcggtgatac 5160
ttttcgagtt tcccagaggg cttctattac tcgtcttcgt cgacaagcac cttgttgtgt 5220
ttgtgatgga actactctag tagctcgttt attcgcttaa gaggttttct cactaggagc 5280
ggctgcgatt ggagctattc cacgaaagac gaatgttatt cgtgtcccta ttcgggtagt 5340
ccctcgtccg tcttttgtaa taggtgaaca aatgagactg gttgaacccg cgcggacgtc 5400
ggaagttcat gaagctgtgg tggtatctgt ctttcgccat gtggagatgt ttcctccagg 5460
acctgcggtg tgactaagta gtcagttaat gccccgagat actttgttct tagctggaga 5520
gagtcgagcc acctctgtaa gatacattga tgagtttgga caaaccacaa ctagaatgca 5580
gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 5640
aagctgcaat aaacaagttt gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac 5700
tggatccggt accaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt 5760
gcatatacga tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa 5820
gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 5880
aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 5940
cttggcttta tatatcttgt ggaaaggacg aaacaccgtc gtgggggaga agttccgagt 6000
tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 6060
caccgagtcg gtgctttttt tctagaccca gctttcttgt acaaagttgg cattagctcc 6120
cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 6180
cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg 6240
tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 6300
gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 6360
cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 6420
taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg 6480
gtacccgggg atcctctaga gattatcgtc gacctgcagg catgcaagct tggcgtaatc 6540
atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 6600
agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 6660
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 6720
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 6780
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 6840
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 6900
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6960
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 7020
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 7080
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 7140
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 7200
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 7260
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 7320
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 7380
tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 7440
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 7500
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 7560
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 7620
aagatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 7680
cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 7740
gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 7800
cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 7860
tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 7920
ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 7980
ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 8040
atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 8100
cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 8160
atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 8220
gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 8280
cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 8340
tctcgcggta tcattgcagc 8360
<210> 2
<211> 21133
<212> DNA
<213> goat
<400> 2
gtgaggggaa gcttcgcagg cgtgcacgga gcagtgagat cactggcgtt ataaatatcc 60
cagtgccagc gccgagatcc gttcgggtgg cctccctctc tctctctccc cctctctccc 120
tctctctctt ccccgaggct atgtccaccc ggtgcggcga ggggggcagc gccagaggca 180
cgcagccgcg cgggggctac ggagcccaga accaaccctg caagatgcac ttaggacccc 240
cgcggctgga agcatgagct tgtccttcct cctcctcctc ttccttagcc acctgatcct 300
cagcgcctgg gctcaagggg agaagcgcct cgcacccaaa gggcagcccg gaccggctgc 360
cacggagagg aacccgggag gcgccagcag ccgccggagc agcagtagca ccgcgtcttc 420
ctcttcttcc cctgcctcct cctcctccgc ggcttctcgg ggcggcccag gaagcggctt 480
ggagcagagc agcttccagt ggagcccctc ggggcgccgg accggtagcc tctactgcag 540
agtgggcatc ggtttccatc tgcagatcta cccggatggc aaagtcaatg gctcccacga 600
agccaatatg ttaagtaagt tgcttgctct cctccagaac ctgtcctgag cgggtggcgg 660
gagcaccgag aggggccgcc ggttattccc cgggctgtaa gcgcaccttc cggagcttgg 720
gccactggga tccagccggc tctcgaaaca gcccggaggg ttgggttgga ggtgcgtctg 780
ggggccacgc acacacttac cggtctcatg gggatgggga atggcgattc ctgaggtaca 840
ggctactccg aggctggtgc agacaggcac cttgtttcca gcttgccctg gtcccaaaga 900
ggagctccaa cctgtgcccg gagcaaaacc aaaaatcttt ctccttgttt agtttctgtt 960
taccttctgc ttctgtttac cagactagca tttttcactg ggcagcaaag tgtacccctt 1020
tacctgccca taactgaaca gttcttctaa aaataaccaa atctcacata ctcccctctc 1080
acaaccgcta ccacaaatcc gaaattgcta cataatgtgt gtgcgtatat gtgtgtgtgt 1140
gtatctgtat atgtctcttg taggtacgta catatttgca catcatatac atatgggata 1200
tataggtata tcagacggga gaccagacgt tctctcattg gaagaagaca gtggtctaga 1260
aaactggaca cgtttccaga gggagacgtg atggacattc gaattattaa ttccggagtt 1320
gggtttagca acgtttagta tcttaagacc atgaccagac cttgtgaggg agagtggccg 1380
gccttttgtc cccaaggaca attaaaggaa ggttctgtat tttagagagc atttagaggc 1440
agttcagttc ggtacaggtg aaatttttgt gggtttttta atttaagcca agttttaaaa 1500
tttccttctt agtttagctc tctttctttc cctccttcgt tgtctcctcc cttccactct 1560
gagtttccaa accatcactg cggttcctga ttcttctcac tgtctctccg aggcaacaga 1620
ggggaaagat gtgtgggact ggggacatct ctgcggacgt tttcctcggt agggccagat 1680
ggctcttcca gccggggctg gtccaacaga tagacgcaca gccccagcct cagggaccca 1740
gggagaacgg aaacgaggcc aggagacagc gcacagtcat ccggcccgga gtgtcagcct 1800
ggtggttccc aggtgtactt aactttctct gcctcaagcc aatgacccta cagatttctt 1860
cttaaccaag ctccagagga gcgggggcgc cactgcgagg ctggcagctc caacccccac 1920
gggggggttg gagatgcgca catccgtggg gtggccggct ggcaagctct ccctcgggag 1980
ttcaggaggc gggaagtgga acgcgagcgg gtccacgcct gccccttgcc cctgactgct 2040
cctaccgcct ctgccttcag cttgctgtgc tgcccgtggt gaggtggctt ggagcaccga 2100
accccagaga gggccttccg cgtcagcccg agcgaatgcc agggcctccc acacgctctt 2160
cagaggggac tgttttgtgt gtgtgtgtgt gtgtgttttt aagaatgtat gtatttttga 2220
ctgggtcttc cttgcagtgg cagtctttct gtactttcag gagcccgggc taccctttct 2280
tgtggtgcat gggcttctga ttgcagcggt ttctcttgtt gcagagtgca gacttcacta 2340
gtcccagtgt gtgggcgcgg tagttgaggc accgggctta gttgctcagg gggatatgca 2400
gtttccctgg gaccagggat tgatcctctg tcccctgctt tgggaggtgg acacttatcc 2460
actgcatcac cgggaaagtg cgaggggcct acttctgtgt tttaaggttc attgcactcg 2520
ttaaacttct cgccatattt ggtgataaaa ggcaaggtca attagggtga aggggaattt 2580
ccggagattt cagggtaagc aaatgttatg tctaagcaag tgttatttcc atctgtcctc 2640
atttggtccc acccagctct ccttccacca gacatgagag ctcatttccc cacttgtggt 2700
aatcttttcc cctttctctt acaggagcac tctttctatt ctgttcttcc tgttgttcag 2760
tagctaagtt gtgtctgact cttttgagac gccatggact gtagcccacc aggctcctct 2820
gtccatgaga tttcccaagc aagaatactg gagtgggttg ccatttcctt ctccaaggga 2880
tcttcccaac ccaggggtcg aacccacatc tcctgcattg gcagatggat tctttaccac 2940
tcagccacca gggaaaccca ttcttcctgt agtttatccc caaactcagc ctcaacagag 3000
aatgacttct gtgttgccaa gggaagattc aaagctaacc tgcaaggagc cgcaggttgg 3060
atccaaggat gggtaaaagg tggtgttgga gtgggttttg gtgtgctagg gatctctgaa 3120
aaccagactc attcccacgg tatttacctt cttctcttgg aagaaaccgc aagagacaaa 3180
gattttttgc agcttaggcc ttttggaggg gaagttatag aaaagcatag aaaggaaaat 3240
gagaacagat caacagagaa tacattcagc taaagaagac attggctggc attgatttcc 3300
aaggtacatt ttattttaaa atccactttg aaagatatct cacaaagaac agacacaaaa 3360
tgctgctttg tttcttaagc aaaactaaat aaaagtgcct aaataaaact ttcttaaatt 3420
aactaacgct ttcctaattc attccagttt ataacagcct ttcagagttt agtctgtatc 3480
ttttgcttgg cttttgatca gttcacttat tcactcatgc atttgttcat gtacccaaca 3540
caggatgcct agctcagcaa gcttgatatt ctggttactt gatattgggt tagttgggat 3600
ttttttttcc tttgccttct ataggttttt ctcacagagg aaagtataat ttgtttaaat 3660
catggtgatt ttcagctttg tgatgtctgc aatgtcatac actgtatcat aagattttgc 3720
taccattgat ccatgaagaa tttatgaaat atgtcatgtt taaaaggata gcatgtaatt 3780
tgaaagttta aaatgaatct ctgtctttaa gtagctgtga aagagaaaca agatctgcta 3840
attgcagttg gtcatagacc tctttgaaaa tagcacatga cggtttcctg gtacatatgt 3900
aatcacagga gttgtttatg gactattggt tggagtagat gaggaaacat ttgcccagat 3960
ctcttaagat atgctgagag agcttggacc atgagaacca tggaggatac atcagtttta 4020
cctcatcatc taatttgact gtctgtgagt tagcagtaat ggaaaacata cattattgtg 4080
acacaacttg ttacttctgt aaagagtcaa aactctgcca tgaaactgca ttctaacagg 4140
ttcttctaaa ctccataatt ttctgtaatg tttgtcagaa gagagaaata tatatgcatg 4200
tatgtttcat gactatcatt tgacattaaa atttttgtaa accctgaata tttttttggt 4260
ttgttatcaa aagtacattt cccatagatt ttgaaacata atttatttag ttaaggtata 4320
ccccttaatg tagtcttagc ttctcataca agagttatcc caagttttaa agacaagaat 4380
caagctcatt gcacataaac tcttatcata aggaaacgct aaggaaggat gaagatgggc 4440
aagacaagat agatgataga attccactac tgaataaaaa caaaactttc ctttaatctc 4500
tacattttca agtttagttt caatacaatg cttctgttgg cttatcttgg gccactgaag 4560
aagaggcaat caagtaactg tcagttcagg gagcaaaagc ttaaggctat agctacacaa 4620
agactgactg ttcacataaa aatatggaga ttacagtcat gtattttggc tagcttttaa 4680
aaatctgaat ctactgtcaa ccttgatgtt aatgcagagt caacctttat atagcccccc 4740
gaagcccaaa taaaccagaa tctatacgga aaaactcaag tggcaaattt gctaacttaa 4800
tgaagactgc taaactcaga tttcaagact cggatttcat aatgacttgt catatttgtt 4860
gctgtgtgaa gccaagtaga gaaacatcag ttttaaatga ttaatctttg ggagttttca 4920
gatgggctgt tgaaatcttc tgaatacatg gaatatttgt gtatcttatt cacagataaa 4980
acagaaaaaa gggagacgtc tacataactg tatcaaacat cattgataag agtcttattg 5040
tattaaaaat tctagagggg actaagtgaa caaatctaga tagagatttt cttatgggaa 5100
taagtttatt ttgtaatgaa cacctaatgc ttgtaaatgg gttaagcagt gcgagactct 5160
gaacccctgc atcctttaac ctagatgtca atcccagaat tccaccaaga gagaataatt 5220
ccttcagttt aacactgctt tttacttttg atcctcttaa atatgagcac cttaggaaag 5280
ataacacatt gccatttata ctttaatgtg aattaattta tactgatctc tctccctttc 5340
ccattccccc agcaggacca aaatagcagg agattactaa gacatatatt catacatgtg 5400
tacacatgga gtatcctgaa aaggaagaga ggtagtgaga tggtcaaaga aacaatccaa 5460
tttacaaaca ttcactaact gtaatctgag acacttggcc ttccaggttt tctttgggct 5520
accatttggg cttccgtggg aattgtaaat tcctatcaaa tttattattg ctgactctta 5580
aacagaaatt actaaagttg aaaaaaaatc aattttagag aaatgctttc tgaaatttat 5640
atacatgtga tttagtgcac ataggtttaa aatacttaga attctttaat gagttgagat 5700
ttggcatgaa aatcaataag cctgtgatct tttcaggcag gtgaggggaa agaggttgaa 5760
ttgtggggct tgaagaatct actccaatat tctatgaaaa aagacattgt gaaattactg 5820
ttgaaatgac tttaaaacta taatcttaac aatgttccca atagtacact gtaattacat 5880
taaaatttag aacatcatac caaagttaac ttaatgtttc tttccttcat cttcataact 5940
tgaaatagaa tgacttttgt agaaaaatca ttttcaaggt ttcaattaga tataattttc 6000
ttaaaattat aaaatccttt tattctcagg caatatatca tttttttgta ctggaactta 6060
caagaaaaga agccatgtag tagtatgtaa taaaataaca ctttcacaca tatcatttca 6120
tcttccccat atttaacatt tactataatg caaattttat ataacatata catgcagttg 6180
ggaaatgagt tgagtcaaaa tgggtctttc tttaaaatag aatatgttgt cagaagccag 6240
ttatataaac aaaacagttt cccattaaac aaagatttac agagcccttt tgtcttgacc 6300
tcttaaatgt taacctgcaa acacatgaga attaatgaga aattttttcc ccaactaata 6360
cgcaatagat ttgcatttac aaaaccaggt gattttccag acaataatac agaaaacctt 6420
taccaactca gatccaggag ttaattaagg gaaacataca atcaagtctt ttctaccaac 6480
tccacattgt ctgttttgta tttgtcattc tacacataga cgatttcttg tgttctccct 6540
gtctcatttt ttaacagatg tctacatagt attctcaatt gtattcacca tctccgaaat 6600
atccaaaatc attaatgcta ttttcaatat tacccggtaa ttttgataga aatttaactt 6660
tgcatcagat attgtaatca tgctttatta ataatattac tatcatacta cattgtgtag 6720
taaatttatc tttacaaagc atttttcatg ctttgttact atttgataaa ttgataatgc 6780
ttctaagtct ttggatatgc atcacaatta gtctgtatca taaaatctac tataaattga 6840
attttgaaaa cattttgaag aaaagaattc ctaagacaca gcggttttct tttcctgaaa 6900
gcaaaagtag ttcctttcta atagttttta gatttggggg agttcgtttt aaagaaacac 6960
ccactaataa agtggtagcg gtagttttct tgagatgtgt tttatttact ccaggctaag 7020
aacacttatt cacaagaaca gtagtttccc tatgaacttt ctgttcaagt attttttaaa 7080
aatgaagttg aataaataag tggcaggatt tgctacaatt ataggtatta gatcacatta 7140
tgcaacattt aagtgttcta taaagatact cataaatgtg ttttaggtat tttatacatg 7200
tgtatccaca atacactgct tcttgtatat tgaataattt tatatgtact tcaaatactg 7260
caggaggaga ttattatgtt aagaaaatct ggtgaacttc cctattcaac agagtcctat 7320
tgaaatattc taaccagaat gtatgcttaa aatactacag attgaaatat tgatattttt 7380
ttcgttttcc cttccactcc cacttgcttc cttcctttct tccagggcag gtccctttat 7440
agctttaata aataatatta ttttatcttt tttttttttg caatttgatg taggcagtta 7500
acaaaaagcc ccatgctttt ctcttgttca tttagcatta catatgagtt tatggtaaac 7560
aggcaggaat tccaattata tacagactat gagtttcagc ttcttaaaaa tactctatga 7620
actgtggtgt gcaactcttt tgacccagtg gatgtgaact ttcaaattac tatagcagac 7680
agagggacat agacttggta attctcacta tgaaatacca aatataaatg ataacataat 7740
aaaatacctc atgaaatttt ttccaggaga gtcattcaaa acgcaagatg tctcaaacca 7800
taatccatca aacacttcat tgagctaaca accccaaatt atctatggtg ctttcagtgt 7860
cttgttatgt gtaatcttga aataagcaac cactaaatga tagggtatga tatatattga 7920
tttgtacttt tataattatg gtcatttgag aagagggatt tttttattta attgaatatt 7980
attttcaaag cttccaatga tgtatattaa tcttgagtgc agttgtatgt gctttaatat 8040
tcaatgtact gtgttatctt tgacagagca acaatgcatg agaattattt tgtaaaacat 8100
tttaaaaagt cttttccatt tccttagaat ttgaaaagat tcttctgcca aacactgatt 8160
actaacagtg acatcatttt caggaaaaaa tgaatactaa tgaagcacaa ctaaatttta 8220
aaccttgaaa tttatctttt taaatgaata cttgagcccc aaaccttttt ttccctttta 8280
cataataaat gtcacaataa acaaggatgt caaatactta aaaaaaaatc actgtaataa 8340
agaatggaaa gagtatctgt agaaatttaa atcttcacaa tgatatatag aggagtctgt 8400
gttttatttt gggatttctg tcatcctagg tattttggaa atatttgctg tgtctcaggg 8460
gattgtagga atacgaggag ttttcagcaa caaattttta gcgatgtcaa aaaaaggaaa 8520
actccatgca agtgtaagta gaaccacttt atatttgtta aaggtgttac agagatttta 8580
catgtataaa atggaatgag tgattttcca aattgataga taagacaatt tgcccagaga 8640
actttatgtt tttcattttt ataagattat gtatccatta aaccaaaaga attgtatgtg 8700
tttttataat aatttaaata ttttatttat ttgaaaagat aatgtagaat ccacaaaaag 8760
gtttaaggat aagtttattt gaaaaaagta ttctctaagt gattgtcatt atttatgtct 8820
taatagtggt ccaagtaagg agaaatatta tggaaatact atattataaa ttaaatttac 8880
atactgatgg tgatggtgta atcagttgat catgttctta atttttaaag ctacttatta 8940
tgtcaaaaat ggagtctaag taataaaaca catatttatt ttaataagta tggtgggaag 9000
aaaccaataa tgagatgatt ttgaccttta atagagaaat ttcatttgag acattttcat 9060
cttgtagttt ttgttgtatc aaaaacaatt acttcttaaa gctacgaaga ctgattctgc 9120
actcttaaac actttttttg gactagttac acagtttgta atcaatttat aaattataga 9180
tttctatgct ttgttctgaa aatgctcatt acttgggaaa cactgaagtg tttttaaaga 9240
tagtaaaata catttagctt aactttcctg ttgagagatt ctttcattag tattagtgtg 9300
tgcgcacagc ccttaaaaag ttatttatgc tttgtctgtg ctgtactaag ttttatcaca 9360
ctaaatcatt ttaaaaagca gttcttcttt aaagctactc ctttaatcct tattcacttt 9420
aatttttatt aaattggcga aatatgactt ttccagtttt caaagtatat aggaagtttt 9480
aatcgagtgc agtcagttga aacttactaa ttatgatttc tgctttacag acttttaaaa 9540
aattgaaaga gaatttcact tggtttaatg tttaagactt tatgttttcc tttctctagc 9600
atctaaaggt ggcttgttaa tatcttttgg aggagatcat aaaactgaag ctatttctac 9660
agttatttgg ggattaaatg agataatata tacaaagatt ttagaacaat agaaggtata 9720
atgtaagtgc tcaaaaaata tctattatta gataattcaa atttaaatac aataaactac 9780
tataatctca gtttattaaa tctttgttaa cctcttcgat catccattgc aagagttata 9840
actagattta cttatctcat tttctgagct gattatttta ttctagcaaa ttataatatt 9900
cataagataa ttgctctggt ttataaccta ttttaaggga caaaattttg ctatggaaag 9960
caggacatgc cattttattt ttaaaaagat gtttgacttc taaaaatcca catacaggat 10020
ggaaaacaat catcaattgc ttctgcactg aataactttt cctctatcta ctttggtcat 10080
taaagattga aacaccttca gactctaagc attacaataa tgtgcaattc tgaaaaaaat 10140
ttctgttatt ggtttgctta ttttgggtga aaatcttaat ttttaaaata ttctgattca 10200
agccttagca catgcaaaaa atagactacc ttggaccagc ttaaggataa ttgtagcagc 10260
aagcaaagta ttacaaatgg atgccaatta gaaaggtcaa actgagaggt taggatacat 10320
gattcctctg tcaaatgcaa gtaacaaatt tccagaaact tgacccaaat gtctaaggaa 10380
ggctggccga aataatgcag ataatgcaaa aacagcatct gttattcttt ctcatcttgg 10440
caggctaaaa gttatttcat ggagtaatga cttttaaaca gtacaaagca agccttgctc 10500
ttgtcactca ctgaagctct ttgatataaa agtcattctg atgactttga gctgaattgt 10560
agataaaaaa ttaagcccta tgattatgtg aatttttttt aaaccaattg tccaagccca 10620
ctgtctctca tttgagcttt tattagcact ttttgtctcg atagaaacct aattaaatta 10680
taaagtgact tatttttgag gaagttgaat ttagatatag tattagccct aatgtttatt 10740
agttgtatgc cagaggattt ccttcttctt tttagttact aatgaaacat gttgaaaaat 10800
tgttcttata actttaaata taattatttg atttagttcg acttacaaag tttcatcacc 10860
actaaaaact ctaaaatgcc taacactttc aggattaaat gtataataag tgaattgctt 10920
taaattaata attgtgcaga ggcatttaaa gagtaacttt ataatcattt caaaaaatag 10980
ctatataaaa tcatcatgtt gtatatctta aacttacaca gtgttgtatg tcaattgtat 11040
ctcaataaag ctggaaaaat tttaaattaa tgaagagtaa ctttggggtt ctctttacaa 11100
agtaagtgtg aactataaat gtaaagtatt catgtaattc tggggaaagt aaacccatag 11160
tatgttgaga aatattcatg gattatagta gatttttctt cttttatata gtaacaatca 11220
gaccattaaa agttagttac taggtagaga taaaatatct gtatatttct ttcaaagttc 11280
tttcactcac attctctcat tacctggtta ggtgaggcaa cagtctcaga gatgttaaat 11340
attttcctaa gttaccacag ttaacaaaaa cttgaaccag aataacatac tttaaaaatt 11400
ctctctccat ttaaatgcta taatgcttgg tagggaaaag ttagcttgaa agaagatatt 11460
ttgtttcatt tgaaactatt tacactattt tcagctatta ttttatggaa attaatagtt 11520
gcatgtttca ataatatttt tatgctacat tttaaagagt gaatactgtg gttcatcctt 11580
agactatttt tgctttcttc tctctctttc tctctctctc attccaagct ctttgggagg 11640
atctttttcc cccatctgct tctgtgtctc ctgtacctga ttttaaagtc tagaaaggaa 11700
gagacgtgtg caccatgaaa cttatcacac tgttttgtaa atgttggtta ttcagtgcga 11760
tgaggagctg catcaggaac ctcttttagg agatacttcc ggaagagaaa ggagatctgt 11820
ttcattcaca gaaaatcttc ttttagaaaa tgcttcatgg tcagtggaca ggaagattgt 11880
gaacagtatt atcagcctgg agagctcaga tttaggcaac atttatgctg acctcctttg 11940
tttgctaaat ttgttaattc cgtaaaagga gttttatccg cccttcagcc atctcagtgt 12000
gctctgctct gtgcactcag tcaaccaaca cgctcatatt aatctccacg cagctctgcc 12060
tgagaccttt aactggaaga ccctaaatag acaatgcagt cagatcccta gtgtgcaggt 12120
ttttttcagc atccattaca aggatgaagg tgtctcgtct tcttctcaag atgtcttgag 12180
aatcactgag agaagcacga ggtgattatt tccccaagag cctcgagggt tagtcagaac 12240
cttcactgtg gcctctgtcc tttctccact tattcacagg tactaggtac taggtgaggc 12300
tgagcaccct tctctttcgg tccctctctg cctgcctatc tgttcatgct ccctcctcac 12360
tctgttcttg ctctcttctc aggctggttg tctttatcca gagtctcata tacagtgcct 12420
ttctggattt atctgagatg ggtctgaata taagagaggt agttaaattg tgtcattctt 12480
aaactgtgtt gttttactta atctttctat tccttggctt tatcagctgt aaaaggaaga 12540
aaattaaatt aacaatacag ggtgatagtg gagcatttta aatgacataa tacatataaa 12600
acatttagaa tagttcctgg aaagtattca gcatttaatg ttagaacaga tcttcctgaa 12660
tttgctctca tgcagctgtt tcatgatcct atatctcctc tctcctcttc ctgattttgg 12720
ggtcaggcag ctgtgacccc ctcggatatt tagaccagcc tcaaatcaca ttcattattg 12780
gcctgttttt tttttttttt tcctttaggt cccaactcgt cttgcgtaca gtagcctctt 12840
atgtcctggg ttttcctttt cccaaggaac ttaatttgaa agcaaagatt tccaatggtc 12900
ataatctcat aattttttaa gatccttctc gtgcatcatg gttttttttc cccttaaaaa 12960
tctaaatctt ggaaaaagtg gttatcatga atttgtcagt cttttcataa actatacgtg 13020
atcacccatt ggttgttggg ggaaaacatt ttagcttatt ttaaacatac agaaagaagc 13080
atgtggtgga attatttttg tttctcaggg ttttagggga tcatgcttgg aaggaaagct 13140
aattgcagag tttacgaaac aggcatggaa tgtatgcttg atcagccctg agcaaggact 13200
agaaaagcct cctttcttgg tttcagaggg tgccagctgg cttgcccagg atgagagtct 13260
ggactggact tcctgagctg ggggcagagt gaggaggagt ccgcagcccc agagctgggc 13320
atgtttattg aaaggagaaa tgaaggtttg agtgatggtt atcatcatca ttaaaaaaat 13380
aatttattgg cctctgtaaa ggaattgaat agaaacttcc cttatcctct gtgttgtgct 13440
gtgctaagtc actttagtca tgtgccactc tttgcgaccg tatggactgt agcccacccc 13500
ctcttctgtc cgtgggattc tccaggtggc aatactggag tgggttgcca tgcctttctc 13560
cagggtatct tcccaaccca gggatcaaac ccttgtctgt tatgtttcct acattggcag 13620
gcaggttctt taccagtagc gccacctggg aagcaccccc attttctaag aacttttcaa 13680
tttactcagt aattgagtac ctagtaactc aggctaaaaa aaaaaaaatg ggttcaggat 13740
tctacctttg cagtaaatgt cagttgtccc ttaatttcac actatgttta tctggctgtg 13800
ctgtgctcaa ttgtgtccga ctctttggga tcccatgaac tgtagcctgc caggctcctc 13860
tgtccatggg gattctccag gcaagaatac tggagtagat tgccatgcct tcctctaggg 13920
gatcttccca acactgaagc tctattttat tgagaaaaat ttcatgtaat gaattgcctt 13980
gtagcttcag agatccattt cagcctaaag tgagagatgg tgttctatgt ctccggtggt 14040
ttggggaatg gcacttattt tgaccagtat tttaacatgg agcaacttta gggcaaggta 14100
gtaatgtaca gaagcctagg agatgccctt ggatttgtta ccgaaagagc ttgaatgtag 14160
gattcagtag atgagattta agtcttaggc ttaccttggc atttggtaaa tgtggggatt 14220
tagaggaact attttcttta tttttctggc tggaagatca gataatagca tccaaatagt 14280
tgtggaatta tgtgacaatg aaataatata tgtaaaaccc ctggcacatg gtagacctca 14340
atagcccagc tcttttgagc aaggtgactc agatggtaaa gaatctgatt caatgcagga 14400
gacctgggtt cgatccttag gtcaggaaga ttcccctgga gaaggggatg gcaacccact 14460
ccagtattct cgcctgggaa atcccgtgga cagagaagcc tggctgtcca ttaggttaca 14520
aagagtcaga catgactaag gggctgagaa ctcatgcctg agcactcaca tatattccct 14580
tggttgctca gcctgaagtt ttgagacata gaatggccag atagcaattg ctaagcagga 14640
aatatggcca agctctgggg atagcatggc ttgcctacaa ctgacccata caatgcttgt 14700
aaagaataga aggcaaccct cagggtgcgt gctgccctgt tctggagttc tctgcttggg 14760
agcagttcga gcagggtctg gctttcggct ttcatccact accaaaggtt ggtacttttg 14820
tgctgggaac atcctgcaca actgtacaaa acagcctggg cttagggcac ctggcctggg 14880
cagtcccatt ggcatgagta gtactgggaa ccaaaacaca ctgaaagaac acagaggaac 14940
cagaagcagg gatgcaggca gcaagaaagt gttgtttaga gcacaatttg gtctataatt 15000
gcgttgatta cagcctataa gagtagttta attatttaat aatattatat agccttcgat 15060
aacattttaa ttatttgtat ttattactta actttataaa gttttgcaag aagcaaatca 15120
aataaaaagt gaaataaaga attacaattg attaaatacc ctgcatactc cagggtttat 15180
actctagggc tttgctatta gcctgaattt aatcctcttt atacatttct ttcatacatc 15240
atgtatttac tttgatagta gcacataatg agaattggga ccctattaac atgccctcag 15300
ctcatttgct ataagattct caatcatctt agagcatgtt gactaagaaa caaaactata 15360
ttaacaagat ttagaaggtt ttttaaaaat attgtttcag aaagaattct ctggtttaat 15420
tgaccttcag atttcaatta agcagacttg ttcttttctg gaatattact ttctttgaac 15480
tgaatttgat ttttctttta aatatccact gtgtgccctc tccctgtgaa gagatgagta 15540
gaatcaaggc aaagtgttcc cctaagccca aaattcttgt tttatttaac agcactgtgt 15600
tgccccacct ctgaattatt tccatagttt caagcaaaag tagtgtaatt tagtttttat 15660
ttgttctgtc aattaagatg attttaaatt atgtagaaat cagacaattg aaacatttca 15720
tttttaattt tgcagttttg gcttgttgac tgtaacttat ttttacactt gcaaaatcaa 15780
ggagaaatta aagagaaaaa cacagataat ttaagtcaac aaagaacaca gacagaacca 15840
tatctaaatt gtctttcttc ataaaaatgg caaccagcag tgtatgggat gtgatacata 15900
gtcaataaac atctattgaa taaattcaga aaataatgaa ttagaaacaa agataggggt 15960
tctttatgaa catgttaatg attggttttg ttgtagtgac tgatggtggg aggatgaagg 16020
tgatgtccat tctgttttca gtagcttatt tgaaatctga ttcacctctc tacttcacac 16080
ctgaactgta gataaagtct ttatggtgac ttcacttttt ctagggaaat tatccagttc 16140
ttttatttat tttttaagtt tttaactgga ggataattgc tttacaatgt tgtgttggtt 16200
tctgctgtaa aataacatga gtcagtgata agtatacatg actctcctcc ctcctgagac 16260
tccctcccac ccccacatcc catcccttga ggacatcaca gaacaccaca gctctattat 16320
tttaaaagta tatgttttgc taaaagagtg tttataattc caaaagaatt tcaaaggtta 16380
aacttagcct ggaagattta ctatctaaaa ttgtatgatg gcaaatgtac atttaaataa 16440
gtctgctgct tgatgactct gctatagaga tgtttaatgg gtaacattaa ggatagccat 16500
gtactgcttc cctggtggct cagaggttaa agcatctgct tgcaatacag gagacctcag 16560
ttcgatccct gggtcgggaa gatcccctgg agaaggaaat ggcaacccac tccagtattc 16620
ttgcctggag aatcccatgg atggaggagc ctggtggact acagtccact ggggtcacaa 16680
agagttggac acgactgagc gacttcactt tcattttcat gcactgcttg gggttaccat 16740
cttgtctgaa aaggaccagg aatattgaca ggaacagtca gtttctccag aagaaactag 16800
atctcctgat gaaaaaaact cactgacatg ttcatttcaa attattctgt tccattgtag 16860
ctagaagaaa ttttcagaca aaacattaag ttagaagtca attaagaaag ccgaaacagt 16920
ctgactgcca ttgcttctgt ttcagtgtga agtttggact ggctaagggc ctacaagtag 16980
tgtgtgggtc ctcagagttc tgtctctgta ccccatgtga caattaagtg gctcttgaaa 17040
aacctggagc ttgaacagtc atcctggcaa aggcttctct taagaaagaa catgaatctt 17100
gagcggagga gggggagagg gagaaaggtt ttagatgcaa taattttaga gggcagtatc 17160
attagatttt cattatagaa attgctgtag gctaatacat tgtgtgggtg tgtgtatcca 17220
tgtgtgtctg ttggccacat taactaaaaa aaaagacagg tttcttcttt attttcgttg 17280
acactgttag gggaaaaagg ttaaattata cagtacactt tttaaaaatt gaattaagta 17340
atcactgcta tctttatttc aggaaaatga tttttgtgtg tgtgtgaaac aggggtagaa 17400
gtcaagtcaa agtataatgt accatacatt agaaatgtta ttcagacata ttttaagaaa 17460
aaaaatctga gcaggatgag tatacgtgct actctttttt aatgaaatct tccacacctt 17520
tctaaaatgc ttgtccattt atgtagcaga aaatattcca gaagactatt gctgttgttc 17580
agttgctaag ctgtatctga ctcttttcta ccccatggac tgcagcatac taggcctccc 17640
tgtctctcac tgtctcccgg cgtttgccca agttcatgtc cattgagttg gtgacactat 17700
ccaaccatct catcctctgc tgccctcttc tcctcttacc acatgactat agtttttatt 17760
ttacaataaa actttgagat tatctaattg aatgagagag cattgatgtc cttttaagag 17820
ctaagttgac aaatctatgt ttggtttgta agaaagcctt tttaattgaa agccaataag 17880
aacaataaat gtgtcctctt ctaaaggaat gtcagcattg cacccttgtc ctaggtatgg 17940
gccagtaggt taggatgtga tgagagaaag ccatagacaa tggttcttta aaacactaga 18000
ttatagcctg ctaaaaaccc agtcaaagga atcgccagta gacagattct gagataacat 18060
agtttcgtag ccctcaaata aagagctttt tgattgaact acactctaca ctcgaatgta 18120
aataacttag gtagactgtc atgtgtaaga taaatgtgct attggaaata ctctgagatt 18180
ggttgtcttc tgatattgtc agtacccttc aaaactcaaa aattctatgt gaccatcaac 18240
agtaggtata tgtagattgt tttgcagtgt tcagacttcc agattacagg gttgagatct 18300
gtactgcaga agaaagcttg atattattag aaaaacaagg aaactagagg actttcagtt 18360
ccagtatgga actgaagcag agcatattaa aaagcagaga cattattttg ccaacaaagg 18420
ttcgtctagt caaagctatg gtttttccag tagtcatgta tggatgtgag agttggacta 18480
taaagaaagc tgagcaccaa agaactgatg cttttgaact gtggtgttgg agaagattct 18540
tgagagtccc ttggcctgca aggagatcca gccagtccat cctaaaggaa atcagtcctg 18600
aatattcatt ggaaggactg atgctgaggc tgaaactcca gtactttggc cacctgatgc 18660
aaagaactga cttatttgaa aagaccctga tgctgggaaa gattgaaggc aggaggaaaa 18720
aggatgacag aggatgagat ggttggatgg catcaccaac tcaatggaca tgagtttgag 18780
caagctccgg gggttggtga tgaacaggga agcctggtgt actgcagtcc acggagttgt 18840
aaagagtcag actgagcgac tgaactgaac tgaactgaag gaaactatga tatataaaga 18900
attatatata taagcatata tatatatata tatagttgtt tagtcactaa ttcatgtcca 18960
actctttttt cagccccatg gactgtagcc tgccggtctc ctctgtccat gggatttccc 19020
aggcaagaat actggagttg gtatttagag agaaggcaat ggcaccccac tccagtactc 19080
ttgcctggaa aatcccatgg acagaggagc ctggtgggct gcagtccatg gggtcactaa 19140
gagtcggaca caactgagtg acttcacttt gacttttcac tttcatgcac tggagaagga 19200
aatggcaacc cactgcagta ttcttgccta gagaatccca gggatggggg agcctggtgg 19260
gctgccgtct acggggtcac acagagtcgg acataactga agcgacttag cagcagtagc 19320
agttcatata tatgggtcaa gatacttggt attccaaagc ccaggacatt tgtacaaatg 19380
gtcctccctg aaattgtgaa acctagtcca ggatagggaa ttctacttct ttgggagtta 19440
tagactcttg catttgataa aagctaaact ctagatcctt ttcctagaaa aatgttttga 19500
ttctcacaat agccttatgg ctgcatgtat tgtgaaaaag gagcctaaag ctaagaactc 19560
aattggcaga actagcattt gattgcaggt ctatttggca ccatcttgcg ctcttcccct 19620
atgctgtgtt cattcaactt cctcagtaca gcacactgag ccttttcaga gcttgtactg 19680
gcctactttg tcatctttat cccttggtcc tttctctatg catcctaagt gaaagtcaca 19740
ttgaacagct tatagtagct tcctgatgac ctgtgaacac accattcctg ttccctgaaa 19800
aacctctcat cacctgatca ctattatctc attgtacttt gagactcagc tcaaaagtca 19860
ccttttctag gaagaccttc ctgccctctt tgctgccacc tctatccttt tggatgtctt 19920
gtaccctctg cataccactg tcataattcc atttatcaaa ttgcaccatg atgaatgtct 19980
acttctttgc ttattccact gtcctgtgag cattttcaag ggcagatgtg tgcgtgcgtg 20040
cgtgcgtgcg tgcgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtatagagg gaaagagagt 20100
gatatctgcc tgaagtgttg ctattaactc taaaaggtaa tttttttctt cttggcatca 20160
aatagactct cctgtatcac tgtgtgagga tgaatttttt tcctaaagaa ctctaacacg 20220
tgcaatatta atagggccat atgctacccc tttctcagca ccaggaagca cactgatgag 20280
actgaaaact tattcttatt tcaaagggta acatgacccc atttctctga atgaattcat 20340
gttttaccag gcaattgttt cttagtaata tagtactatt tactttcttg atggtttagg 20400
agggggaaaa aaatcttacg ttaaaagtca aatttttaac taaaattttc aaagtatgtt 20460
aaaaagagta acagaattta aatatattga attcctgtta ttttaatata cactgatatt 20520
atttcacaat tgcataattc gtagtaaaca acaagcatta caaagatgaa tgaagtagga 20580
atatagaaat gaaaatagta attttaaagc aactttaaaa gaagcaaatg tgtcttcata 20640
aagagaatgg agtcggagcg caggggccag tggaattctt gtttttctta tagtccaaga 20700
cttctttata ttacatgctg aaaatattat cactgcttga atttcctcct cctcttcttt 20760
tttttttttt tttttttccc tgtaggccaa atttacagat gactgcaagt tcagggagcg 20820
atttcaagaa aacagctata atacctatgc ctccgcgata cacagaactg aaaagactgg 20880
gcgggagtgg tacgtggccc tgaacaagag agggaaggct aaacggggct gcagcccccg 20940
ggttaaacct cagcacgtct ctacccactt tctgccaaga ttcaagcaat cggagcagcc 21000
ggaactttct ttcacagtta ctgttcccga aaagaaaaaa ccacctaatc ccgtcaagcc 21060
aaaggttccc ctttccgcac ctcggagaag tcctaacacg gtgaaataca gactcaagtt 21120
tcgctttggt taa 21133
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
agcccctcgg ggcgccggac 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
cgaggggggc agcgccagag 20
<210> 5
<211> 23
<212> DNA
<213> 人工序列
<400> 5
acaaggtgcc tgtctgcacc agc 23
<210> 6
<211> 28
<212> DNA
<213> 人工序列
<400> 6
aaacaccgag cccctcgggg cgccggac 28
<210> 7
<211> 29
<212> DNA
<213> 人工序列
<400> 7
ctctaaaacg tccggcgccc cgaggggct 29
<210> 8
<211> 28
<212> DNA
<213> 人工序列
<400> 8
ggggatcctg ggtaggcgag ggcgcgtt 28
<210> 9
<211> 28
<212> DNA
<213> 人工序列
<400> 9
cgggatccaa gaagaggaag acgcggtg 28
<210> 10
<211> 28
<212> DNA
<213> 人工序列
<400> 10
ggggtaccct actgcagagt gggcatcg 28
<210> 11
<211> 29
<212> DNA
<213> 人工序列
<400> 11
ggcaattgct gtaccgaact gaactgcct 29
<210> 12
<211> 20
<212> DNA
<213> 人工序列
<400> 12
agcagtagca ccgcgtcttc 20
<210> 13
<211> 21
<212> DNA
<213> 人工序列
<400> 13
ctggaggaga gcaagcaact t 21
<210> 14
<211> 1098
<212> DNA
<213> goat
<400> 14
agcgctacca agagtttccc agcaggaaag cagctcgagc cctggagtta atagcttaag 60
gtggctctct aaacactgaa taaatccact ctttagctct tcagtaaatc agtagccaca 120
gccttctacc cagcgcgcca caccaaagcc ttcccaagtt gggctgcgaa tccttgtttg 180
gttttggcca acacttggag cacgggcggg ggttccccca cgccgggatg aatccccctc 240
gggatgcagt tcccgaggca ccccaggggc gggggtggtg acagtcgggt cggctgggca 300
ccggggaagt gaacttggag actggatcct gggtaggcga gggcgcgttg ctgggacgcc 360
agcgttctgc ggaggtgcta gaggatgaga actcactcac tcggcatttc aagcccccat 420
cccaggggct gccccctgga ggagtccggg ggctcgcgct gggctgaact cggcgccgcg 480
ttggctcgag tggaggtggc cgtgcggcgc gtgagaactg ctggggtgcg tggcccgggt 540
gcgcgcgcgc cgggcgtcgg ggtgcagctt ccctccgccc ggcttcccct cccattcgcc 600
ctctcccatc tcctccctcc tccacacccc cgcccctccc cgcaccggcc ggagagtaca 660
caaagcggcg ggtgagggga agcttcgcag gcgtgcacgg agcagtgaga tcactggcgt 720
tataaatatc ccagtgccag cgccgagatc cgttcgggtg gcctccctct ctctctctcc 780
ccctctctcc ctctctctct tccccgaggc tatgtccacc cggtgcggcg aggggggcag 840
cgccagaggc acgcagccgc gcgggggcta cggagcccag aaccaaccct gcaagatgca 900
cttaggaccc ccgcggctgg aagcatgagc ttgtccttcc tcctcctcct cttccttagc 960
cacctgatcc tcagcgcctg ggctcaaggg gagaagcgcc tcgcacccaa agggcagccc 1020
ggaccggctg ccacggagag gaacccggga ggcgccagca gccgccggag cagcagtagc 1080
accgcgtctt cctcttct 1098
<210> 15
<211> 927
<212> DNA
<213> goat
<400> 15
ctactgcaga gtgggcatcg gtttccatct gcagatctac ccggatggca aagtcaatgg 60
ctcccacgaa gccaatatgt taagtaagtt gcttgctctc ctccagaacc tgtcctgagc 120
gggtggcggg agcaccgaga ggggccgccg gttattcccc gggctgtaag cgcaccttcc 180
ggagcttggg ccactgggat ccagccggct ctcgaaacag cccggagggt tgggttggag 240
gtgcgtctgg gggccacgca cacacttacc ggtctcatgg ggatggggaa tggcgattcc 300
tgaggtacag gctactccga ggctggtgca gacaggcacc ttgtttccag cttgccctgg 360
tcccaaagag gagctccaac ctgtgcccgg agcaaaacca aaaatctttc tccttgttta 420
gtttctgttt accttctgct tctgtttacc agactagcat ttttcactgg gcagcaaagt 480
gtaccccttt acctgcccat aactgaacag ttcttctaaa aataaccaaa tctcacatac 540
tcccctctca caaccgctac cacaaatccg aaattgctac ataatgtgtg tgcgtatatg 600
tgtgtgtgtg tatctgtata tgtctcttgt aggtacgtac atatttgcac atcatataca 660
tatgggatat ataggtatat cagacgggag accagacgtt ctctcattgg aagaagacag 720
tggtctagaa aactggacac gtttccagag ggagacgtga tggacattcg aattattaat 780
tccggagttg ggtttagcaa cgtttagtat cttaagacca tgaccagacc ttgtgaggga 840
gagtggccgg ccttttgtcc ccaaggacaa ttaaaggaag gttctgtatt ttagagagca 900
tttagaggca gttcagttcg gtacagg 927
<210> 16
<211> 6857
<212> DNA
<213> 人工序列
<400> 16
ggatcctggg taggcgaggg cgcgttgctg ggacgccagc gttctgcgga ggtgctagag 60
gatgagaact cactcactcg gcatttcaag cccccatccc aggggctgcc ccctggagga 120
gtccgggggc tcgcgctggg ctgaactcgg cgccgcgttg gctcgagtgg aggtggccgt 180
gcggcgcgtg agaactgctg gggtgcgtgg cccgggtgcg cgcgcgccgg gcgtcggggt 240
gcagcttccc tccgcccggc ttcccctccc attcgccctc tcccatctcc tccctcctcc 300
acacccccgc ccctccccgc accggccgga gagtacacaa agcggcgggt gaggggaagc 360
ttcgcaggcg tgcacggagc agtgagatca ctggcgttat aaatatccca gtgccagcgc 420
cgagatccgt tcgggtggcc tccctctctc tctctccccc tctctccctc tctctcttcc 480
ccgaggctat gtccacccgg tgcggcgagg ggggcagcgc cagaggcacg cagccgcgcg 540
ggggctacgg agcccagaac caaccctgca agatgcactt aggacccccg cggctggaag 600
catgagcttg tccttcctcc tcctcctctt ccttagccac ctgatcctca gcgcctgggc 660
tcaaggggag aagcgcctcg cacccaaagg gcagcccgga ccggctgcca cggagaggaa 720
cccgggaggc gccagcagcc gccggagcag cagtagcacc gcgtcttcct cttcttggat 780
cctctagaga ttaatctgca gttcatgggg tcactaagag tcgggcatgg ctgagcgact 840
tcactttcat gtatcacttt catgcattgg agaaggaaat ggcaacgcac tccagtgttc 900
ttgcctggag aatcccaggg ctgggggagc ctggtgcact gccatctctg gggtcgcaca 960
gagtcggaca tgactgaaga gacttagcag cagcagtagc agcatgttga taagggactt 1020
ggtttagcac attaataaac ataaatatgt tagtatattg gatattttct tagaatataa 1080
atctaacact aatgaacaga ctagtttgta taactgtata ttcaatttag aaaaacaagt 1140
ggagaaatca gatttcaaga aataactcct ttttgcagtc cttcaataga aattgagcat 1200
aaatgtgaat tagtcattgg catagacaga aaaatataat gcattttgct cagacttggt 1260
ttactggaaa ctttaactgg ttggattatg atcaacatca tgggaataaa agatacattg 1320
tagtttcaat ataggaaaga aactgaatca ctgaagaaga taatttggat caagaagata 1380
agaatctttg agtaaaaagg agttgttagt cttaagaaaa aaattttaac gtttggtgaa 1440
acaaactgag gtcaagagca aataagatta agaccaacaa atatatttct cactatactg 1500
aaggtgctag gtggttaaaa taaaatgtgt gatctgggac aggactgtgt aggtgtgagt 1560
ctgcatctcc tctcattcaa ttccttaact ggataagagg aatctaaact gagatgtcaa 1620
cacagcaagc ctgctgaatt tctctgaggt ttcatctttg gttgtgaaca acaagctaat 1680
tagtccagtc ataaagttag ccaatggcat gaaggtgtgg tgggtcacac ccacactgag 1740
agcatataaa aggccctctg cagggagaaa tgtccacact caagtgacac ttctactcta 1800
attctctacc cgagaacaac ctcaacaagc aacacctcct agagcaatcg tcgacatgaa 1860
ctttctgctc tcttgggtgc attggagcct tgccttgctg ctctaccttc accatgccaa 1920
gtggtcccag gctgcaccca tggcagaagg agggcagaaa ccccatgaag tgatgaagtt 1980
catggatgtc taccagcgca gcttctgccg tcccattgag accctggtgg acatcttcca 2040
ggagtaccca gatgagattg agttcatttt caagccgtcc tgtgtgcccc tgatgcggtg 2100
cgggggctgc tgtaatgacg aaagtctgga gtgtgtgccc actgaggagt ccaacatcac 2160
catgcagatt atgcggatca aacctcacca aagccagcac ataggagaga tgagtttcct 2220
acagcataac aaatgtgaat gcagaccaaa gaaagataaa gcaaggcaag aaaatccctg 2280
tgggccttgc tcagagcgga gaaagcattt gtttgtacaa gatccgcaga cgtgtaaatg 2340
ttcctgcaaa aacacagact cgcgttgcaa ggcgaggcag cttgagttaa acgaacgtac 2400
ttgcagatgt gacaagccga ggcggtgagc atgcaagctt ctgtgccttc tagttgccag 2460
ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 2520
gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 2580
ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 2640
gctggggatg cggtgggctc tatgggtacc ctactgcaga gtgggcatcg gtttccatct 2700
gcagatctac ccggatggca aagtcaatgg ctcccacgaa gccaatatgt taagtaagtt 2760
gcttgctctc ctccagaacc tgtcctgagc gggtggcggg agcaccgaga ggggccgccg 2820
gttattcccc gggctgtaag cgcaccttcc ggagcttggg ccactgggat ccagccggct 2880
ctcgaaacag cccggagggt tgggttggag gtgcgtctgg gggccacgca cacacttacc 2940
ggtctcatgg ggatggggaa tggcgattcc tgaggtacag gctactccga ggctggtgca 3000
gacaggcacc ttgtttccag cttgccctgg tcccaaagag gagctccaac ctgtgcccgg 3060
agcaaaacca aaaatctttc tccttgttta gtttctgttt accttctgct tctgtttacc 3120
agactagcat ttttcactgg gcagcaaagt gtaccccttt acctgcccat aactgaacag 3180
ttcttctaaa aataaccaaa tctcacatac tcccctctca caaccgctac cacaaatccg 3240
aaattgctac ataatgtgtg tgcgtatatg tgtgtgtgtg tatctgtata tgtctcttgt 3300
aggtacgtac atatttgcac atcatataca tatgggatat ataggtatat cagacgggag 3360
accagacgtt ctctcattgg aagaagacag tggtctagaa aactggacac gtttccagag 3420
ggagacgtga tggacattcg aattattaat tccggagttg ggtttagcaa cgtttagtat 3480
cttaagacca tgaccagacc ttgtgaggga gagtggccgg ccttttgtcc ccaaggacaa 3540
ttaaaggaag gttctgtatt ttagagagca tttagaggca gttcagttcg gtacaggcaa 3600
ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 3660
caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 3720
tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat 3780
ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa 3840
tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta 3900
ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca 3960
ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat 4020
cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg 4080
agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc 4140
acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt 4200
ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 4260
aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 4320
aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt 4380
ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 4440
ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 4500
agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt 4560
ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg 4620
cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt 4680
gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 4740
cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 4800
acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 4860
tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta 4920
tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 4980
ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 5040
gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 5100
ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 5160
atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 5220
gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc 5280
catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 5340
gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 5400
attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 5460
gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga 5520
ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt 5580
ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga 5640
tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg gggaggctaa 5700
ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac 5760
agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg tcccagggct 5820
ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc gtttcttcct 5880
tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg 5940
cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat ttaaaacttc 6000
atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 6060
cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6120
cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6180
cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6240
tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact 6300
tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6360
ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6420
aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6480
cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6540
ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6600
agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6660
ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 6720
acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 6780
cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta ttactagcgc 6840
taccggactc agatcgg 6857
<210> 17
<211> 2015
<212> DNA
<213> 人工序列
<400> 17
agcagtagca ccgcgtcttc ctcttcttgg atcctctaga gattaatctg cagttcatgg 60
ggtcactaag agtcgggcat ggctgagcga cttcactttc atgtatcact ttcatgcatt 120
ggagaaggaa atggcaacgc actccagtgt tcttgcctgg agaatcccag ggctggggga 180
gcctggtgca ctgccatctc tggggtcgca cagagtcgga catgactgaa gagacttagc 240
agcagcagta gcagcatgtt gataagggac ttggtttagc acattaataa acataaatat 300
gttagtatat tggatatttt cttagaatat aaatctaaca ctaatgaaca gactagtttg 360
tataactgta tattcaattt agaaaaacaa gtggagaaat cagatttcaa gaaataactc 420
ctttttgcag tccttcaata gaaattgagc ataaatgtga attagtcatt ggcatagaca 480
gaaaaatata atgcattttg ctcagacttg gtttactgga aactttaact ggttggatta 540
tgatcaacat catgggaata aaagatacat tgtagtttca atataggaaa gaaactgaat 600
cactgaagaa gataatttgg atcaagaaga taagaatctt tgagtaaaaa ggagttgtta 660
gtcttaagaa aaaaatttta acgtttggtg aaacaaactg aggtcaagag caaataagat 720
taagaccaac aaatatattt ctcactatac tgaaggtgct aggtggttaa aataaaatgt 780
gtgatctggg acaggactgt gtaggtgtga gtctgcatct cctctcattc aattccttaa 840
ctggataaga ggaatctaaa ctgagatgtc aacacagcaa gcctgctgaa tttctctgag 900
gtttcatctt tggttgtgaa caacaagcta attagtccag tcataaagtt agccaatggc 960
atgaaggtgt ggtgggtcac acccacactg agagcatata aaaggccctc tgcagggaga 1020
aatgtccaca ctcaagtgac acttctactc taattctcta cccgagaaca acctcaacaa 1080
gcaacacctc ctagagcaat cgtcgacatg aactttctgc tctcttgggt gcattggagc 1140
cttgccttgc tgctctacct tcaccatgcc aagtggtccc aggctgcacc catggcagaa 1200
ggagggcaga aaccccatga agtgatgaag ttcatggatg tctaccagcg cagcttctgc 1260
cgtcccattg agaccctggt ggacatcttc caggagtacc cagatgagat tgagttcatt 1320
ttcaagccgt cctgtgtgcc cctgatgcgg tgcgggggct gctgtaatga cgaaagtctg 1380
gagtgtgtgc ccactgagga gtccaacatc accatgcaga ttatgcggat caaacctcac 1440
caaagccagc acataggaga gatgagtttc ctacagcata acaaatgtga atgcagacca 1500
aagaaagata aagcaaggca agaaaatccc tgtgggcctt gctcagagcg gagaaagcat 1560
ttgtttgtac aagatccgca gacgtgtaaa tgttcctgca aaaacacaga ctcgcgttgc 1620
aaggcgaggc agcttgagtt aaacgaacgt acttgcagat gtgacaagcc gaggcggtga 1680
gcatgcaagc ttctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 1740
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 1800
catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 1860
agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatgggta 1920
ccctactgca gagtgggcat cggtttccat ctgcagatct acccggatgg caaagtcaat 1980
ggctcccacg aagccaatat gttaagtaag ttgct 2015

Claims (8)

1.CRISPR/Cas9技术介导山羊VEGF基因定点整合的方法，其特征在于，其是根据山羊的FGF5基因序列，构建基于CRISPER/Cas9系统的Cas9-gRNA表达载体和VEGF打靶载体，然后将优化后的然后将优化后的Cas9-gRNA表达载体及VEGF打靶载体共同转染至山羊胎儿成纤维细胞中，获得VEGF基因在FGF5位点定点整合的细胞株。
2.根据权利要求1所述的方法，其特征在于，靶位点在FGF5基因的第1外显子上。
3.根 据 权 利 要 求 2 所 述 的 方 法，其 特 征 在 于， s g R N A靶 序 列 为5 ′ - agcccctcggggcgccggac-3′。
4.根据权利要求1-3任一项所述的方法，其特征在于，所述山羊包括阿尔巴斯绒山羊。
5.根据权利要求1-4任一项所述的方法，其特征在于，所述CRISPER/Cas9系统中Cas9-gRNA表达载体的序列如SEQ ID NO:1所示。
6.根据权利要求1-5任一项所述方法获得的山羊VEGF基因定点整合的细胞株。
7.根据权利要求1-6任一项所述方法在生产VEGF基因定点整合的克隆山羊中的应用。
8.根据权利要求7所述的应用，其特征在于，以权利要求6所述山羊VEGF基因定点整合的细胞为核移植供体细胞，离体的山羊卵母细胞为核移植受体细胞，通过核移植技术获得山羊克隆胚胎，然后将克隆胚胎通过胚胎移植技术移入山羊子宫内妊娠，获得VEGF基因在FGF5位点定点整合的山羊。
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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109679923A (zh) * 2019-01-16 2019-04-26 西北农林科技大学 利用CRISPR/Cas9系统生产VEGF164转基因细胞系的方法
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
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US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
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US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5874254A (en) * 1996-03-29 1999-02-23 Director-General Of Agency Of Industrial Science And Technology FGF-5 analogous protein, and pharmaceutical composition containing the same
JP2012505652A (ja) * 2008-10-15 2012-03-08 ダニスコ・ユーエス・インク 修飾変異ボウマンブリックプロテアーゼインヒビター
CN105132427A (zh) * 2015-09-21 2015-12-09 新疆畜牧科学院生物技术研究所 一种以RNA介导的特异性敲除双基因获得基因编辑绵羊的方法及其专用sgRNA
CN106957857A (zh) * 2016-09-23 2017-07-18 西北农林科技大学 一种利用CRISPR/Cas9系统共同敲除山羊MSTN和FGF5基因的方法
CN107072184A (zh) * 2014-09-19 2017-08-18 瑞泽恩制药公司 嵌合抗原受体
CN108570479A (zh) * 2017-12-06 2018-09-25 内蒙古大学 一种基于CRISPR/Cas9技术介导绒山羊VEGF基因定点敲入的方法
CN109868275A (zh) * 2019-03-12 2019-06-11 中国农业大学 CRISPR/Cas9介导的羊FGF5基因敲除和定点整合MTNR1A基因的方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5874254A (en) * 1996-03-29 1999-02-23 Director-General Of Agency Of Industrial Science And Technology FGF-5 analogous protein, and pharmaceutical composition containing the same
JP2012505652A (ja) * 2008-10-15 2012-03-08 ダニスコ・ユーエス・インク 修飾変異ボウマンブリックプロテアーゼインヒビター
CN107072184A (zh) * 2014-09-19 2017-08-18 瑞泽恩制药公司 嵌合抗原受体
CN105132427A (zh) * 2015-09-21 2015-12-09 新疆畜牧科学院生物技术研究所 一种以RNA介导的特异性敲除双基因获得基因编辑绵羊的方法及其专用sgRNA
CN106957857A (zh) * 2016-09-23 2017-07-18 西北农林科技大学 一种利用CRISPR/Cas9系统共同敲除山羊MSTN和FGF5基因的方法
CN108570479A (zh) * 2017-12-06 2018-09-25 内蒙古大学 一种基于CRISPR/Cas9技术介导绒山羊VEGF基因定点敲入的方法
CN109868275A (zh) * 2019-03-12 2019-06-11 中国农业大学 CRISPR/Cas9介导的羊FGF5基因敲除和定点整合MTNR1A基因的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
LI WEN-RONG 等: "CRISPR/Cas9-mediated loss of FGF5 function increases wool staple length in sheep.", 《FEBS JOURNAL》 *
呼啸: "利用CRISPR/Cas9技术制备FGF5位点定点整合VEGF基因绒山羊", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 *
师丙波: "CRISPR/Cas9系统介导的VEGF164基因定点敲入绒山羊转基因细胞的构建", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 *
张婧婧: "VEGF,L-蛋氨酸对绒山羊次级毛囊外根鞘细胞增殖和分化的影响", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 *
阿力玛: "CRISPR/Cas9编辑绒山羊FGF5基因细胞株的建立", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 *

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US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
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US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
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US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
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