CN110564770B - 一种适用地中海贫血和镰刀型贫血基因治疗的慢病毒载体 - Google Patents

一种适用地中海贫血和镰刀型贫血基因治疗的慢病毒载体 Download PDF

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CN110564770B
CN110564770B CN201910824134.8A CN201910824134A CN110564770B CN 110564770 B CN110564770 B CN 110564770B CN 201910824134 A CN201910824134 A CN 201910824134A CN 110564770 B CN110564770 B CN 110564770B
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汪啸渊
徐婷
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Shanghai Bendao Gene Technology Co ltd
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Abstract

本发明公开了一种适用地中海贫血和镰刀型贫血基因治疗的慢病毒载体；该慢病毒载体包含beta珠蛋白表达盒，所述表达盒包含：微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；beta珠蛋白的基因序列；beta珠蛋白的基因序列上游侧翼的启动子序列；beta珠蛋白的基因序列下游的侧翼序列。与现有技术相比，本发明筛选出beta珠蛋白表达盒兼具效率与特异性，并且不会导致慢病毒滴度降低；筛选的一段来自泡沫病毒的仅36bp的绝缘子序列，不含有隐秘的RNA剪接信号，具有维持基因表达和防止所在区域基因激活的功能；本发明可以用于地中海贫血和镰刀型贫血的基因治疗。

Description

一种适用地中海贫血和镰刀型贫血基因治疗的慢病毒载体
技术领域
本发明属于医疗技术领域，具体涉及一种适用地中海贫血和镰刀型贫血基因治疗的慢病毒载体。
背景技术
迄今为止，人类共发现有7000余种罕见病，但仅5％的罕见病有药可用。目前，几乎所有的罕见病药物都需要终身使用。基因治疗则为罕见病提供了一次性治愈的可能。地中海贫血(地贫)是一种全球分布最广、累积患病人群最多的单基因遗传性血红蛋白病，是罕见病中最常见的人类遗传性疾病。
基因治疗的安全性是其能够成为常规疗法，使更多患者受益的前提。慢病毒载体以不可控的方式插入到基因组(约75％整合至转录活跃的基因编码区域)，伴随有较高的激活癌基因和失活抑癌基因的风险。事实上，慢病毒载体曾在基因治疗地中海贫血的过程中激活HMGA2基因，导致转导的造血干细胞克隆大量扩增。该克隆的异常扩增是由于慢病毒基因治疗载体中使用的绝缘子元件，也即鸡beta珠蛋白脱氧核糖核酸酶I超敏感部位4核心部位(core chicken hypersensitivity site 4，cHS4)造成的(Cavazzana-Calvo,M.etal.Transfusion independence and HMGA2 activation after gene therapy of humanbeta-thalassaemia.Nature 467,318-22(2010).)。
绝缘子元件是具有阻止基因沉默或者防止所在区域基因激活功能的基因元件。cHS4绝缘子含有隐秘的RNA剪接信号，造成克隆扩增，影响基因治疗的安全性，并且使得慢病毒的滴度大幅下降(Cavazzana-Calvo,M.et al.Transfusion independence and HMGA2activation after gene therapy of human beta-thalassaemia.Nature 467,318-22(2010).Negre,O.et al.Preclinical evaluation of efficacy and safety of animproved lentiviral vector for the treatment of beta-thalassemia and sicklecell disease.Curr Gene Ther 15,64-81(2015).)。本发明所包含的绝缘子元件来自泡沫病毒(Foamy virus)，仅36bp的绝缘子序列，不含有隐秘的RNA剪接信号，并具有维持基因表达和防止所在区域的癌基因激活的功能。
Beta珠蛋白基因(HBB)在发育过程中的特异性表达由一段位于其上游的基因座控制区(LCR)调控(Kim,A.&Dean,A.Chromatin loop formation in the beta-globin locusand its role in globin gene transcription.Mol Cells 34,1-5(2012).)。该段序列长达16kb，远远超出了慢病毒载体的运载能力。为了构建用于基因治疗的beta珠蛋白基因表达盒，必须从LCR区域中挑选出具有调控beta珠蛋白基因表达功能的、大小合适的核心元件。LCR由HS2、HS3、HS4等脱氧核糖核酸酶I(DNase I)超敏感位点(hypersensitive sites)组成。这些超敏感位点长度依然太大，必须对其进一步缩减才能包装进慢病毒载体。由于这些超敏感位点功能不同，必须相互配合才能发挥精准的调控功能，因此，必须找到有利beta珠蛋白基因高效和特异表达的组合。例如，由HS4、HS3、HS2脱氧核糖核酸酶I超敏感位点的核心元件组成的微基因座(miniLCR)导致病毒效价显著降低(Weber,L.et al.AnOptimized Lentiviral Vector Efficiently Corrects the Human Sickle CellDisease Phenotype.Mol Ther Methods Clin Dev 10,268-280(2018)。本发明的筛选的微基因座不会导致病毒效价的显著降低。
发明内容
本发明的目的在于解决现有技术中慢病毒载体用于地中海贫血与镰刀型贫血基因治疗的安全性与有效性问题，提供一种适用地中海贫血和镰刀型贫血基因治疗的慢病毒载体。
本发明的目的通过以下技术方案来实现的：
本发明涉及一种包含beta珠蛋白表达盒的慢病毒载体，所述表达盒包含：
a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；
b、beta珠蛋白的基因序列；
c、beta珠蛋白的基因序列上游侧翼的启动子序列；
d、beta珠蛋白的基因序列下游的侧翼序列。
作为本发明的一个实施方案，所述微基因座控制区是从beta珠蛋白16kb的基因座控制区LCR的HS4、HS3、HS2区域分别截取长度约380bp、1900bp、720bp的脱氧核糖核苷酸序列组成总长3kb的微型基因座调控元件。本发明所使用的HS3区序列更好地保留了原完整的HS3序列的功能，对球蛋白基因的表达有潜在的促进作用。
作为本发明的一个实施方案，所述微型基因座调控元件的序列如SEQ ID NO.1所示。
作为本发明的一个实施方案，所述微基因座控制区是从beta珠蛋白16kb的基因座控制区的HS3、HS2区域分别截取长度约1900bp、720bp的脱氧核糖核苷酸序列组成总长2.6kb的微型基因座调控元件。
作为本发明的一个实施方案，所述微型基因座调控元件的序列如SEQ ID NO.2所示。
作为本发明的一个实施方案，所述beta珠蛋白的基因序列含外显子序列和内含子序列；且在密码子87处编码包含突变T87Q。
所述beta珠蛋白的基因序列如如SEQ ID NO.3所示。
作为本发明的一个实施方案，所述启动子序列为265bp，具体序列如SEQ ID NO.4所示。
作为本发明的一个实施方案，所述侧翼序列为300bp，具体序列如SEQ ID NO.5所示。
作为本发明的一个实施方案，在慢病毒载体的LTR U3区域添加一段来自泡沫病毒(Foamy virus)的仅36bp的绝缘子序列。
作为本发明的一个实施方案，所述绝缘子序列如SEQ ID NO.6所示。
与现有技术相比，本发明具有如下有益效果：
1)筛选出beta珠蛋白表达盒兼具效率与特异性，并且不会导致慢病毒滴度降低；
2)一段来自泡沫病毒(Foamy virus)的仅36bp的绝缘子序列，不含有隐秘的RNA剪接信号，具有维持基因表达和防止所在区域基因激活的功能；
3)本发明可以用于地中海贫血和镰刀型贫血的基因治疗。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1是本发明的用于基因治疗的慢病毒载体设计示意图；
图2是实施例1的两种表达beta珠蛋白的慢病毒载体的滴度；其中，实施例1中载体1的微基因座控制区大小约为2.6kb；实施例1中载体2的微基因座控制区大小约为3kb。常规慢病毒载体是不含微基因座控制区的普通慢病毒载体。载体1和载体2均不含绝缘子序列。病毒的滴度由ELISA法测得；ns＝差异不显著；
图3是实施例1的表达人源beta珠蛋白的慢病毒载体1的电子显微镜照片；
图4是RT-qPCR分析实施例1的两种表达人源beta珠蛋白的慢病毒载体在MEL细胞中beta珠蛋白基因(Huβ)的相对表达情况；其中，实施例1中载体1的微基因座控制区大小约为2.6kb，实施例1中载体2的微基因座控制区大小约为3kb；载体1和载体2均不含绝缘子序列。MEL细胞经HMBA诱导分化成红细胞。人源beta珠蛋白的相对表达量经鼠源beta珠蛋白标准化；
图5是ELISA分析实施例2的两种表达beta珠蛋白的慢病毒载体的滴度；其中，实施例2中载体1的微基因座控制区大小约为2.6kb；实施21中载体2的微基因座控制区大小约为3kb。常规慢病毒载体是不含微基因座控制区的普通慢病毒载体。载体1和载体2均含绝缘子序列；ns＝差异不显著；
图6是RT-qPCR分析实施例2的两种表达人源beta珠蛋白的慢病毒载体在MEL细胞中beta珠蛋白基因(Huβ)的相对表达量；其中，实施例2中载体1的微基因座控制区大小约为2.6kb，实施例2中载体2的微基因座控制区大小约为3kb；实施例2中的载体1和载体2均不含36bp的绝缘子序列。MEL细胞经HMBA诱导分化成红细胞。人源beta珠蛋白的相对表达量经mGAPDH(鼠源甘油醛-3-磷酸脱氢酶)标准化；
图7是表达绿色荧光蛋白GFP的慢病毒载体的LTR U3区域添加源自泡沫病毒的36bp的绝缘子FVin序列维持基因长期的表达。GFP的荧光强度通过流式细胞术记录。ns＝差异不显著；*＝显著差异；
图8是在慢病毒载体的LTR U3区域添加源自泡沫病毒的36bp的绝缘子FVin序列可以防止激活癌基因。表达绿色荧光蛋白GFP的慢病毒载体通过基因编辑技术插入到原癌基因LMO2附近，36bp的绝缘子FVin序列对LMO2表达强度的影响(RT-qPCR分析)。*＝显著差异。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1
本发明筛选了一个具有红细胞特异性、能高效表达的人源beta珠蛋白的表达盒，进而获得含该表达盒的用于基因治疗的慢病毒载体。载体设计如图1所示，该表达盒包含以下几种元件：(1)微基因座控制区(miniLCR)，即一段从beta珠蛋白16-kb的基因座控制区(LCR)中筛选出来的长度3kb(序列如SEQ ID NO.1所示)或2.6kb(序列如SEQ ID NO.2所示)的调控元件；(2)beta珠蛋白的基因序列(序列如SEQ ID NO.3所示)；其中外显子2含T87Q突变。(3)265-bp的beta珠蛋白的基因序列上游启动子序列(序列如SEQ ID NO.4所示)；(4)下游300-bp的beta珠蛋白的基因序列polyA序列(序列如SEQ ID NO.5所示)。将该表达盒置于慢病毒载体。含3kb微基因座控制区的慢病毒载体的序列如SEQ ID NO.7所示。含2.6kb微基因座控制区的慢病毒载体的序列如SEQ ID NO.8所示。本实施例慢病毒载体不含绝缘子序列。将本发明(13μg)与表达膜蛋白的质粒，如3.75μg pMD2G(序列如SEQ ID NO.9所示)、表达HIV-1来源的GagPol长链蛋白的质粒如13μg pMDlg/pRRE(序列如SEQ ID NO.10所示)、表达REV蛋白的质粒如3μg pRSV-REV(序列如SEQ ID NO.11所示)共转染病毒4X106生产细胞(如293T)，可以生产出表达beta珠蛋白的慢病毒。慢病毒通过超速离心或者柱层析的方法的富集或者纯化。制备好的病毒通过p24 ELISA的方法，测定其滴度(图2)；由图2可知，本发明的微基因座控制区不会造成慢病毒包装滴度下降。此外，也可以通过电子显微镜，观察病毒载体的形态(图3)。该慢病毒可以用于地中海贫血或者镰刀型贫血的基因治疗。
图4为验证两种慢病毒载体表达beta珠蛋白在MEL细胞表达情况。载体1的微基因座控制区大小约为2.6kb，载体2的微基因座控制区大小约为3kb。MEL细胞经4-羟甲基苯甲酸(HMBA)诱导分化成红细胞。由图4可知，载体1和载体2在MEL细胞经HMBA诱导分化成红细胞后，均高效表达人源的beta珠蛋白。载体1和载体2表达人源beta珠蛋白(Huβ)的水平分别达到鼠源beta珠蛋白(Muβ)的60％和90％左右。
实施例2
本发明筛选了一个具有红细胞特异性、能高效表达的人源beta珠蛋白的表达盒，进而获得含该表达盒的用于基因治疗的慢病毒载体。载体设计如图1所示，该表达盒包含以下几种元件：(1)微基因座控制区(miniLCR)，即一段从beta珠蛋白16-kb的基因座控制区(LCR)中筛选出来的长度3kb(序列如SEQ ID NO.1所示)或2.6kb(序列如SEQ ID NO.2所示)的调控元件；(2)完整的beta珠蛋白的基因序列。其中外显子2含T87Q突变。(3)265-bp的beta珠蛋白的基因序列上游启动子序列(序列如SEQ ID NO.4所示)；(4)下游300-bp的beta珠蛋白的基因序列polyA序列(序列如SEQ ID NO.5所示)。将该表达盒置于慢病毒载体，慢病毒载体选择添加绝缘子序列。在添加绝缘子序列的情况下，在慢病毒载体的LTR U3区域添加一段来自泡沫病毒(Foamy virus)的仅36bp的绝缘子序列(序列如SEQ ID NO.6所示：aagggagacatctagtgatataagtgtgaactacac)。该绝缘子序列具有维持基因长期表达和阻止整合位点附近基因激活的功能，且不会降低慢病毒载体的滴度。将该表达盒置于慢病毒载体，同时慢病毒载体添加来自泡沫病毒的36bp的绝缘子序列(FVin)。将本发明(13μg)与表达膜蛋白的质粒(如3.75μg pMD2G(序列如SEQ ID NO.9所示))、表达HIV-1来源的GagPol长链蛋白的质粒(通常为13μg pMDlg/pRRE(序列如SEQ ID NO.10所示))、表达REV蛋白(通常为3μg pRSV-REV(序列如SEQ ID NO.11所示))的质粒共转染病毒4X106生产细胞(如293T)，可以生产出表达beta珠蛋白的慢病毒。慢病毒通过超速离心或者柱层析的方法的富集或者纯化。制备好的病毒通过p24 ELISA的方法，测定其滴度(图5)；由图5可知，该绝缘子不会对病毒滴度造成负面影响。该慢病毒可以用于地中海贫血或者镰刀型贫血的基因治疗。
图6为验证两种慢病毒载体表达beta珠蛋白在MEL细胞表达情况。载体1的微基因座控制区大小约为2.6kb，载体2的微基因座控制区大小约为3kb。MEL细胞经HMBA诱导分化成红细胞。由图6可知，载体1和载体2在未经分化诱导的小鼠红白血病细胞系MEL不表达人源的beta珠蛋白(Huβ)，而在MEL细胞经HMBA诱导分化成红细胞后，载体1和载体2均高效表达人源的beta珠蛋白。载体1和载体2表达人源beta珠蛋白(Huβ)的水平分别达到鼠源GAPDH(mGAPDH)的3倍和5倍左右。
图7为在慢病毒载体的LTR U3区域添加源自泡沫病毒的36bp的绝缘子FVin序列以增加基因长期的表达的稳定性。图7为表达绿色荧光蛋白GFP的慢病毒载体感染293T细胞5天和15天后，绿色荧光蛋白的表达强度变化情况。由图7可知，含36bp的绝缘子FVin序列的慢病毒载体(FVin+)，其绿色荧光蛋白的表达强度在感染15天以后得以维持，具有维持基因长久表达的功能；缺少36bp的绝缘子FVin序列的慢病毒载体(FVin-)，其绿色荧光蛋白的表达强度在感染15天以后显著降低。ns＝差异不显著；*＝显著差异。并且，
图8为在慢病毒载体的LTR U3区域添加源自泡沫病毒的36bp的绝缘子FVin序列以防止激活癌基因。图8为表达绿色荧光蛋白GFP的慢病毒载体通过基因编辑技术插入到癌基因LMO2附近，对LMO2表达强度的影响。由图8可知，含36bp的绝缘子FVin序列的慢病毒载体(FVin+)在整合到癌基因LMO2附近时，其对LMO2的表达水平的提升显著低于缺少36bp的绝缘子FVin序列的慢病毒载体(FVin-)；可见具有防止癌基因激活的功能，安全隐患小。*＝显著差异。
综上所述，本发明的核心特点是(1)包含红细胞特异的珠蛋白表达盒的慢病毒载体。(2)在慢病毒载体的LTR U3区域添加一段来自泡沫病毒(Foamy virus)的仅36bp的绝缘子序列。在添加绝缘子序列的情况下，在慢病毒载体的LTR U3区域添加一段来自泡沫病毒(Foamy virus)的仅36bp的绝缘子序列。该绝缘子序列具有维持珠蛋白基因长期表达和阻止癌基因激活的功能，且不会降低慢病毒载体的滴度。将本发明与表达膜蛋白的质粒(如VSV-G)、表达HIV-1来源的GagPol长链蛋白的质粒、表达REV蛋白的质粒共转染病毒生产细胞(如293T)，可以生产出表达beta珠蛋白的慢病毒载体。该慢病毒载体可以用于地中海贫血或者镰刀型贫血的基因治疗。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。
序列表
<110> 上海本导基因技术有限公司
<120> 一种适用地中海贫血和镰刀型贫血基因治疗的慢病毒载体
<130> DD05562A
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3013
<212> DNA
<213> beta珠蛋白的微型基因座调控元件( beta-globin)
<400> 1
gatctgaccc cgtatgtgag catgtgtcct ctaacagcac aggccttttg ccacctagct 60
gtccaggggt gccttaaaat ggcaaacaag gtttgttttc ttttcctgtt ttcatgcctt 120
cctcttccat atccttgttt catattaata catgtgtata gatcctaaaa atctatacac 180
atgtattaat aaagcctgat tctgccgctt ctaggtatag aggccacctg caagataaat 240
atttgattca caataactaa tcattctatg gcaattgata acaacaaata tatatatata 300
tatatatata cgtatatgtg tatatatata tatatattca ggaaataata tattctagaa 360
tatgtcacat tctgtctcag gcatccattt tctttatgat gccgtttgag gtggagtttt 420
agtcaggtgg tcagcttctc cttttttttg ccatctgccc tgtaagcatc ctgctgggga 480
cccagatagg agtcatcact ctaggctgag aacatctggg cacacaccct aagcctcagc 540
atgactcatc atgactcagc attgctgtgc ttgagccaga aggtttgctt agaaggttac 600
acagaaccag aaggcggggg tggggcactg accccgacag gggcctggcc agaactgctc 660
atgcttggac tatgggaggt cactaatgga gacacacaga aatgtaacag gaactaagga 720
aaaactgttc attaaaaaaa gtctaaccag ctgcattcga ctttgactgc agcagctggt 780
tagaaggttc tactggagga gggtcccagc ccattgctaa attaacatca ggctctgaga 840
ctggcagtat atctctaaca gtggttgatg ctatcttctg gaacttgcct gctacattga 900
gaccactgac ccatacatag gaagcccata gctctgtcct gaactgttag gccactggtc 960
cagagagtgt gcatctcctt tgatcctcat aataacccta tgagatagac acaattatta 1020
ctcttacttt atagatgatg atcctgaaaa cataggagtc aaggcacttg cccctagctg 1080
ggggtatagg ggagcagtcc catgtagtag tagaatgaaa aatgctgcta tgctgtgcct 1140
cccccacctt tcccatgtct gccctctact catggtctat ctctcctggc tcctgggagt 1200
catggactcc acccagcacc accaacctga cctaaccacc tatctgagcc tgccagccta 1260
taacccatct gggccctgat agctggtggc cagccctgac cccaccccac cctccctgga 1320
acctctgata gacacatctg gcacaccagc tcgcaaagtc accgtgaggg tcttgtgttt 1380
gctgagtcaa aattccttga aatccaagtc cttagagact cctgctccca aatttacagt 1440
catagacttc ttcatggctg tctcctttat ccacagaatg attcctttgc ttcattgccc 1500
catccatctg atcctcctca tcagtgcagc acagggccca tgagcagtag ctgcagagtc 1560
tcacataggt ctggcactgc ctctgacatg tccgacctta ggcaaatgct tgactcttct 1620
gagctcagtc ttgtcatggc aaaataaaga taataatagt gtttttttat ggagttagcg 1680
tgaggatgga aaacaatagc aaaattgatt agactataaa aggtctcaac aaatagtagt 1740
agattttatc gtccattaat ccttccctct cctctcttac tcatcccatc acgtatgcct 1800
cttaattttc ccttacctat aataagagtt attcctctta ttatattctt cttatagtga 1860
ttctggatat taaagtggga atgaggggca ggccactaac gaagaagatg tttctcaaag 1920
aagccattct ccccacatag atcatctcag cagggttcag gaagataaag gaggatcaag 1980
gtcgaaggta ggaactaagg aagaacactg ggcaagtgga tccaggttgt ccaacgctga 2040
aagtaggaat ctaagcacta gtctctggat gctaggaggg cctctgcatg ggtaactctt 2100
tcaactagcc aggggctgga ctgtggagaa accatttcca gatagaagtg aggagattcc 2160
agcaggaaac acttaagaga ggatccctgg aagttcgggg caggaggctc cctgtcacat 2220
gaaggaaacc tgctcagcgt aggctctagg ttcttcccta ctcttatcca atggggcttc 2280
tgattttaag ccagtcttca caaagcccca gatttctaca tgctggagcc ctgaatgggc 2340
aggaagcatt gcggtttcca tttcattcag agctctttca taccctgctt ccccatagtt 2400
tgtctcaaca tttctgttga taatctgatt catgcaacca agaattatga gagagccttc 2460
agtcatgcct aggcctgcat ttattgttgt gcatatgatg gggtagcaga caagaaaacg 2520
ttaacagcat agactttctc agagctatat gagtttggga attaatcaag accagccttt 2580
tgctcagggt agaaatcccc tatagggatt tattcaatga tgggttaaaa gcttctacat 2640
agtttccatg tcacagccag ggctgggcag tctcctgtta tttcttttaa aataaatata 2700
tcatttaaat gcataaataa gcaaaccctg ctcgggaatg ggagggagag tctctggagt 2760
ccaccccttc tcggccctgg ctctgcagat agtgctatca aagccctgac agagccctgc 2820
ccattgctgg gccttggagt gagtcagcct agtagagagg cagggcaagc catctcatag 2880
ctgctgagtg ggagagagaa aagggctcat tgtctataaa ctcaggtcat ggctattctt 2940
attctcacac taagaaaaag aatgagatgt ctacatatac cctgcgtccc ctcttgtgta 3000
ctggggtccc caa 3013
<210> 2
<211> 2634
<212> DNA
<213> beta珠蛋白的微型基因座调控元件( beta-globin)
<400> 2
gatctgaccc cgtatgtgag catgtgtcct ctaacagcac aggccttttg ccacctagct 60
gtccaggggt gccttaaaat ggcaaacaag gtttgttttc ttttcctgtt ttcatgcctt 120
cctcttccat atccttgttt catattaata catgtgtata gatcctaaaa atctatacac 180
atgtattaat aaagcctgat tctgccgctt ctaggtatag aggccacctg caagataaat 240
atttgattca caataactaa tcattctatg gcaattgata acaacaaata tatatatata 300
tatatatata cgtatatgtg tatatatata tatatattca ggaaataata tattctagaa 360
tatgtcacat tctgtctcag gcatccattt tctttatgat gccgtttgag gtggagtttt 420
agtcaggtgg tcagcttctc cttttttttg ccatctgccc tgtaagcatc ctgctgggga 480
cccagatagg agtcatcact ctaggctgag aacatctggg cacacaccct aagcctcagc 540
atgactcatc atgactcagc attgctgtgc ttgagccaga aggtttgctt agaaggttac 600
acagaaccag aaggcggggg tggggcactg accccgacag gggcctggcc agaactgctc 660
atgcttggac tatgggaggt cactaatgga gacacacaga aatgtaacag gaactaagga 720
aaaactgttc attaaaaaaa gtctaaccag ctgcattcga ctttgactgc agcagctggt 780
tagaaggttc tactggagga gggtcccagc ccattgctaa attaacatca ggctctgaga 840
ctggcagtat atctctaaca gtggttgatg ctatcttctg gaacttgcct gctacattga 900
gaccactgac ccatacatag gaagcccata gctctgtcct gaactgttag gccactggtc 960
cagagagtgt gcatctcctt tgatcctcat aataacccta tgagatagac acaattatta 1020
ctcttacttt atagatgatg atcctgaaaa cataggagtc aaggcacttg cccctagctg 1080
ggggtatagg ggagcagtcc catgtagtag tagaatgaaa aatgctgcta tgctgtgcct 1140
cccccacctt tcccatgtct gccctctact catggtctat ctctcctggc tcctgggagt 1200
catggactcc acccagcacc accaacctga cctaaccacc tatctgagcc tgccagccta 1260
taacccatct gggccctgat agctggtggc cagccctgac cccaccccac cctccctgga 1320
acctctgata gacacatctg gcacaccagc tcgcaaagtc accgtgaggg tcttgtgttt 1380
gctgagtcaa aattccttga aatccaagtc cttagagact cctgctccca aatttacagt 1440
catagacttc ttcatggctg tctcctttat ccacagaatg attcctttgc ttcattgccc 1500
catccatctg atcctcctca tcagtgcagc acagggccca tgagcagtag ctgcagagtc 1560
tcacataggt ctggcactgc ctctgacatg tccgacctta ggcaaatgct tgactcttct 1620
gagctcagtc ttgtcatggc aaaataaaga taataatagt gtttttttat ggagttagcg 1680
tgaggatgga aaacaatagc aaaattgatt agactataaa aggtctcaac aaatagtagt 1740
agattttatc gtccattaat ccttccctct cctctcttac tcatcccatc acgtatgcct 1800
cttaattttc ccttacctat aataagagtt attcctctta ttatattctt cttatagtga 1860
ttctggatat taaagtggga atgaggggca ggccactaac gaagaagatg tttctcaaag 1920
aagccattct ccccacatag atcatctcag cagggttcag gaagataaag gaggatcaag 1980
gtcgaaggta ggaactaagg aagaacactg ggcaagtgga tccaggttgt ccaacgctga 2040
aagtaggaat ctaagcacta gtctctggat gctaggaggg cctctgcatg ggtaactctt 2100
tcaactagcc aggggctgga ctgtggagaa accatttcca gatagaagtg aggagattcc 2160
agcaggaaac acttaagaga ggatccctgg aagttcgggg caggaggctc cctgtcacat 2220
gaaggaaacc tgctcagcgt aggctctagg ttcttcccta ctcttatcca atggggcttc 2280
tgattttaag ccagtcttca caaagcccca gatttctaca tgctggagcc ctgaatgggc 2340
aggaagcatt gcggtttcca tttcattcag agctctttca taccctgctt ccccatagtt 2400
tgtctcaaca tttctgttga taatctgatt catgcaacca agaattatga gagagccttc 2460
agtcatgcct aggcctgcat ttattgttgt gcatatgatg gggtagcaga caagaaaacg 2520
ttaacagcat agactttctc agagctatat gagtttggga attaatcaag accagccttt 2580
tgctcagggt agaaatcccc tatagggatt tattcaatga tgggttaaaa gctt 2634
<210> 3
<211> 1424
<212> DNA
<213> beta珠蛋白的基因序列( beta-globin)
<400> 3
atggtgcatc tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac 60
gtggatgaag ttggtggtga ggccctgggc aggttggtat caaggttaca agacaggttt 120
aaggagacca atagaaactg ggcatgtgga gacagagaag actcttgggt ttctgatagg 180
cactgactct ctctgcctat tggtctattt tcccaccctt aggctgctgg tggtctaccc 240
ttggacccag aggttctttg agtcctttgg ggatctgtcc actcctgatg ctgttatggg 300
caaccctaag gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc 360
tcacctggac aacctcaagg gcacctttgc ccagctgagt gagctgcact gtgacaagct 420
gcacgtggat cctgagaact tcagggtgag tctatgggac gcttgatgtt ttctttcccc 480
ttcttttcta tggttaagtt catgtcatag gaaggggata agtaacaggg tacagtttag 540
aatgggaaac agacgaatga ttgcatcagt gtggaagtct caggatcgtt ttagtttctt 600
ttatttgctg ttcataacaa ttgttttctt ttgtttaatt cttgctttct ttttttttct 660
tctccgcaat ttttactatt atacttaatg ccttaacatt gtgtataaca aaaggaaata 720
tctctgagat acattaagta acttaaaaaa aaactttaca cagtctgcct agtacattac 780
tatttggaat atatgtgtgc ttatttgcat attcataatc tccctacttt attttctttt 840
atttttaatt gatacataat cattatacat atttatgggt taaagtgtaa tgttttaata 900
tgtgtacaca tattgaccaa atcagggtaa ttttgcattt gtaattttaa aaaatgcttt 960
cttcttttaa tatacttttt tgtttatctt atttctaata ctttccctaa tctctttctt 1020
tcagggcaat aatgatacaa tgtatcatgc ctctttgcac cattctaaag aataacagtg 1080
ataatttctg ggttaaggca atagcaatat ctctgcatat aaatatttct gcatataaat 1140
tgtaactgat gtaagaggtt tcatattgct aatagcagct acaatccagc taccattctg 1200
cttttatttt atggttggga taaggctgga ttattctgag tccaagctag gcccttttgc 1260
taatcatgtt catacctctt atcttcctcc cacagctcct gggcaacgtg ctggtctgtg 1320
tgctggccca tcactttggc aaagaattca ccccaccagt gcaggctgcc tatcagaaag 1380
tggtggctgg tgtggctaat gccctggccc acaagtatca ctaa 1424
<210> 4
<211> 264
<212> DNA
<213> beta珠蛋白的启动子序列( beta-globin)
<400> 4
gtgtctgttt gaggttgcta gtgaacacag ttgtgtcaga agcaaatgta agcaatagat 60
ggctctgccc tgacttttat gcccagccct ggctcctgcc ctccctgctc ctgggagtag 120
attggccaac cctagggtgt ggctccacag ggtgaggtct aagtgatgac agccgtacct 180
gtccttggct cttctggcac tggcttagga gttggacttc aaaccctcag ccctccctct 240
aagatatatc tcttggcccc atac 264
<210> 5
<211> 300
<212> DNA
<213> beta珠蛋白的侧翼序列( beta-globin)
<400> 5
gccaatgtgc attagctgtt tgcagcctca ccttctttca tggagtttaa gatatagtgt 60
attttcccaa ggtttgaact agctcttcat ttctttatgt tttaaatgca ctgacctccc 120
acattccctt tttagtaaaa tattcagaaa taatttaaat acatcattgc aatgaaaata 180
aatgtttttt attaggcaga atccagatgc tcaaggccct tcataatatc ccccagttta 240
gtagttggac ttagggaaca aaggaacctt taatagaaat tggacagcaa gaaagcgagc 300
<210> 6
<211> 36
<212> DNA
<213> 来自泡沫病毒的绝缘子序列(Foamy virus)
<400> 6
aagggagaca tctagtgata taagtgtgaa ctacac 36
<210> 7
<211> 11497
<212> DNA
<213> 含微基因座控制区的慢病毒载体(Artificial Sequence)
<400> 7
aagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat 60
gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 120
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 180
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 240
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 300
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 360
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 420
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 480
acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 540
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 600
actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 660
gagctcgttt agtgaaccgg ggtctctctg gttagaccag atctgagcct gggagctctc 720
tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 780
agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 840
agtgtggaaa atctctagca gtggcgcccg aacagggacc tgaaagcgaa agggaaacca 900
gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 960
cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 1020
gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 1080
ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 1140
cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 1200
cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 1260
gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 1320
aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 1380
cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 1440
agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 1500
agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 1560
cactatgggc gcagcctcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 1620
agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 1680
cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 1740
ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 1800
gccttggaat gctagttgga gtaataaatc tctggaacag attggaatca cacgacctgg 1860
atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa 1920
tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt 1980
ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 2040
gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt 2100
aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac 2160
aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta 2220
gtgaacggat ctcgacggta tcggttaact tttaaaagaa aaggggggat tggggggtac 2280
agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa 2340
aaacaaatta caaaaattca aaattttatc gatcacgaga ctagcctcga cgatggtcgc 2400
caatgtgcat tagctgtttg cagcctcacc ttctttcatg gagtttaaga tatagtgtat 2460
tttcccaagg tttgaactag ctcttcattt ctttatgttt taaatgcact gacctcccac 2520
attccctttt tagtaaaata ttcagaaata atttaaatac atcattgcaa tgaaaataaa 2580
tgttttttat taggcagaat ccagatgctc aaggcccttc ataatatccc ccagtttagt 2640
agttggactt agggaacaaa ggaaccttta atagaaattg gacagcaaga aagcgagctt 2700
agtgatactt gtgggccagg gcattagcca caccagccac cactttctga taggcagcct 2760
gcactggtgg ggtgaattct ttgccaaagt gatgggccag cacacagacc agcacgttgc 2820
ccaggagctg tgggaggaag ataagaggta tgaacatgat tagcaaaagg gcctagcttg 2880
gactcagaat aatccagcct tatcccaacc ataaaataaa agcagaatgg tagctggatt 2940
gtagctgcta ttagcaatat gaaacctctt acatcagtta caatttatat gcagaaatat 3000
ttatatgcag agatattgct attgccttaa cccagaaatt atcactgtta ttctttagaa 3060
tggtgcaaag aggcatgata cattgtatca ttattgccct gaaagaaaga gattagggaa 3120
agtattagaa ataagataaa caaaaaagta tattaaaaga agaaagcatt ttttaaaatt 3180
acaaatgcaa aattaccctg atttggtcaa tatgtgtaca catattaaaa cattacactt 3240
taacccataa atatgtataa tgattatgta tcaattaaaa ataaaagaaa ataaagtagg 3300
gagattatga atatgcaaat aagcacacat atattccaaa tagtaatgta ctaggcagac 3360
tgtgtaaagt ttttttttaa gttacttaat gtatctcaga gatatttcct tttgttatac 3420
acaatgttaa ggcattaagt ataatagtaa aaattgcgga gaagaaaaaa aaagaaagca 3480
agaattaaac aaaagaaaac aattgttatg aacagcaaat aaaagaaact aaaacgatcc 3540
tgagacttcc acactgatgc aatcattcgt ctgtttccca ttctaaactg taccctgtta 3600
cttatcccct tcctatgaca tgaacttaac catagaaaag aaggggaaag aaaacatcaa 3660
gcgtcccata gactcaccct gaagttctca ggatccacgt gcagcttgtc acagtgcagc 3720
tcactcagct gggcaaaggt gcccttgagg ttgtccaggt gagccaggcc atcactaaag 3780
gcaccgagca ctttcttgcc atgagccttc accttagggt tgcccataac agcatcagga 3840
gtggacagat ccccaaagga ctcaaagaac ctctgggtcc aagggtagac caccagcagc 3900
ctaagggtgg gaaaatagac caataggcag agagagtcag tgcctatcag aaacccaaga 3960
gtcttctctg tctccacatg cccagtttct attggtctcc ttaaacctgt cttgtaacct 4020
tgataccaac ctgcccaggg cctcaccacc aacttcatcc acgttcacct tgccccacag 4080
ggcagtaacg gcagacttct cctcaggagt cagatgcacc atggtgtctg tttgaggttg 4140
ctagtgaaca cagttgtgtc agaagcaaat gtaagcaata gatggctctg ccctgacttt 4200
tatgcccagc cctggctcct gccctccctg ctcctgggag tagattggcc aaccctaggg 4260
tgtggctcca cagggtgagg tctaagtgat gacagccgta cctgtccttg gctcttctgg 4320
cactggctta ggagttggac ttcaaaccct cagccctccc tctaagatat atctcttggc 4380
cccatacctc gaggatctga ccccgtatgt gagcatgtgt cctctaacag cacaggcctt 4440
ttgccaccta gctgtccagg ggtgccttaa aatggcaaac aaggtttgtt ttcttttcct 4500
gttttcatgc cttcctcttc catatccttg tttcatatta atacatgtgt atagatccta 4560
aaaatctata cacatgtatt aataaagcct gattctgccg cttctaggta tagaggccac 4620
ctgcaagata aatatttgat tcacaataac taatcattct atggcaattg ataacaacaa 4680
atatatatat atatatatat atacgtatat gtgtatatat atatatatat tcaggaaata 4740
atatattcta gaatatgtca cattctgtct caggcatcca ttttctttat gatgccgttt 4800
gaggtggagt tttagtcagg tggtcagctt ctcctttttt ttgccatctg ccctgtaagc 4860
atcctgctgg ggacccagat aggagtcatc actctaggct gagaacatct gggcacacac 4920
cctaagcctc agcatgactc atcatgactc agcattgctg tgcttgagcc agaaggtttg 4980
cttagaaggt tacacagaac cagaaggcgg gggtggggca ctgaccccga caggggcctg 5040
gccagaactg ctcatgcttg gactatggga ggtcactaat ggagacacac agaaatgtaa 5100
caggaactaa ggaaaaactg ttcattaaaa aaagtctaac cagctgcatt cgactttgac 5160
tgcagcagct ggttagaagg ttctactgga ggagggtccc agcccattgc taaattaaca 5220
tcaggctctg agactggcag tatatctcta acagtggttg atgctatctt ctggaacttg 5280
cctgctacat tgagaccact gacccataca taggaagccc atagctctgt cctgaactgt 5340
taggccactg gtccagagag tgtgcatctc ctttgatcct cataataacc ctatgagata 5400
gacacaatta ttactcttac tttatagatg atgatcctga aaacatagga gtcaaggcac 5460
ttgcccctag ctgggggtat aggggagcag tcccatgtag tagtagaatg aaaaatgctg 5520
ctatgctgtg cctcccccac ctttcccatg tctgccctct actcatggtc tatctctcct 5580
ggctcctggg agtcatggac tccacccagc accaccaacc tgacctaacc acctatctga 5640
gcctgccagc ctataaccca tctgggccct gatagctggt ggccagccct gaccccaccc 5700
caccctccct ggaacctctg atagacacat ctggcacacc agctcgcaaa gtcaccgtga 5760
gggtcttgtg tttgctgagt caaaattcct tgaaatccaa gtccttagag actcctgctc 5820
ccaaatttac agtcatagac ttcttcatgg ctgtctcctt tatccacaga atgattcctt 5880
tgcttcattg ccccatccat ctgatcctcc tcatcagtgc agcacagggc ccatgagcag 5940
tagctgcaga gtctcacata ggtctggcac tgcctctgac atgtccgacc ttaggcaaat 6000
gcttgactct tctgagctca gtcttgtcat ggcaaaataa agataataat agtgtttttt 6060
tatggagtta gcgtgaggat ggaaaacaat agcaaaattg attagactat aaaaggtctc 6120
aacaaatagt agtagatttt atcgtccatt aatccttccc tctcctctct tactcatccc 6180
atcacgtatg cctcttaatt ttcccttacc tataataaga gttattcctc ttattatatt 6240
cttcttatag tgattctgga tattaaagtg ggaatgaggg gcaggccact aacgaagaag 6300
atgtttctca aagaagccat tctccccaca tagatcatct cagcagggtt caggaagata 6360
aaggaggatc aaggtcgaag gtaggaacta aggaagaaca ctgggcaagt ggatccaggt 6420
tgtccaacgc tgaaagtagg aatctaagca ctagtctctg gatgctagga gggcctctgc 6480
atgggtaact ctttcaacta gccaggggct ggactgtgga gaaaccattt ccagatagaa 6540
gtgaggagat tccagcagga aacacttaag agaggatccc tggaagttcg gggcaggagg 6600
ctccctgtca catgaaggaa acctgctcag cgtaggctct aggttcttcc ctactcttat 6660
ccaatggggc ttctgatttt aagccagtct tcacaaagcc ccagatttct acatgctgga 6720
gccctgaatg ggcaggaagc attgcggttt ccatttcatt cagagctctt tcataccctg 6780
cttccccata gtttgtctca acatttctgt tgataatctg attcatgcaa ccaagaatta 6840
tgagagagcc ttcagtcatg cctaggcctg catttattgt tgtgcatatg atggggtagc 6900
agacaagaaa acgttaacag catagacttt ctcagagcta tatgagtttg ggaattaatc 6960
aagaccagcc ttttgctcag ggtagaaatc ccctataggg atttattcaa tgatgggtta 7020
aaagcttcta catagtttcc atgtcacagc cagggctggg cagtctcctg ttatttcttt 7080
taaaataaat atatcattta aatgcataaa taagcaaacc ctgctcggga atgggaggga 7140
gagtctctgg agtccacccc ttctcggccc tggctctgca gatagtgcta tcaaagccct 7200
gacagagccc tgcccattgc tgggccttgg agtgagtcag cctagtagag aggcagggca 7260
agccatctca tagctgctga gtgggagaga gaaaagggct cattgtctat aaactcaggt 7320
catggctatt cttattctca cactaagaaa aagaatgaga tgtctacata taccctgcgt 7380
cccctcttgt gtactggggt ccccaactcg agggaattcc gataatcaac ctctggatta 7440
caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg 7500
atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc 7560
ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca 7620
acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac 7680
cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact 7740
catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc 7800
cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg 7860
gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc 7920
ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac 7980
gagtcggatc tccctttggg ccgcctcccc gcatcgggaa ttcgagctcg gtacctttaa 8040
gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac 8100
tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc 8160
tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 8220
agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 8280
ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatct 8340
agctagaatt aattccgtgt attctatagt gtcacctaaa tcgtatgtgt atgatacata 8400
aggttatgta ttaattgtag ccgcgttcta acgacaatat gtacaagcct aattgtgtag 8460
catctggctt actgaagcag accctatcat ctctctcgta aactgccgtc agagtcggtt 8520
tggttggacg aaccttctga gtttctggta acgccgtccc gcacccggaa atggtcagcg 8580
aaccaatcag cagggtcatc gctagcctag gcttttgcgt cgagacgtac ccaattcgcc 8640
ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga 8700
aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 8760
taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 8820
atggcgcgac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 8880
cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 8940
tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 9000
ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 9060
tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 9120
taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 9180
tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 9240
aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttcccagg tggcactttt 9300
cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 9360
ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 9420
agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 9480
tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 9540
gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 9600
gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 9660
attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 9720
gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 9780
agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 9840
ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 9900
cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 9960
gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 10020
cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 10080
gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 10140
ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 10200
acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 10260
ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 10320
aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 10380
aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 10440
ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 10500
ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 10560
actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 10620
caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 10680
gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 10740
ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 10800
cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 10860
cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 10920
acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 10980
ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 11040
gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 11100
tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 11160
accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 11220
cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac 11280
gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc 11340
actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 11400
gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca 11460
attaaccctc actaaaggga acaaaagctg gagctgc 11497
<210> 8
<211> 11118
<212> DNA
<213> 含微基因座控制区的慢病毒载体(Artificial Sequence)
<400> 8
aagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat 60
gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 120
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 180
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 240
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 300
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 360
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 420
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 480
acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 540
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 600
actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 660
gagctcgttt agtgaaccgg ggtctctctg gttagaccag atctgagcct gggagctctc 720
tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 780
agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 840
agtgtggaaa atctctagca gtggcgcccg aacagggacc tgaaagcgaa agggaaacca 900
gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 960
cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 1020
gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 1080
ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 1140
cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 1200
cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 1260
gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 1320
aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 1380
cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 1440
agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 1500
agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 1560
cactatgggc gcagcctcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 1620
agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 1680
cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 1740
ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 1800
gccttggaat gctagttgga gtaataaatc tctggaacag attggaatca cacgacctgg 1860
atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa 1920
tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt 1980
ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 2040
gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt 2100
aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac 2160
aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta 2220
gtgaacggat ctcgacggta tcggttaact tttaaaagaa aaggggggat tggggggtac 2280
agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa 2340
aaacaaatta caaaaattca aaattttatc gatcacgaga ctagcctcga cgatggtcgc 2400
caatgtgcat tagctgtttg cagcctcacc ttctttcatg gagtttaaga tatagtgtat 2460
tttcccaagg tttgaactag ctcttcattt ctttatgttt taaatgcact gacctcccac 2520
attccctttt tagtaaaata ttcagaaata atttaaatac atcattgcaa tgaaaataaa 2580
tgttttttat taggcagaat ccagatgctc aaggcccttc ataatatccc ccagtttagt 2640
agttggactt agggaacaaa ggaaccttta atagaaattg gacagcaaga aagcgagctt 2700
agtgatactt gtgggccagg gcattagcca caccagccac cactttctga taggcagcct 2760
gcactggtgg ggtgaattct ttgccaaagt gatgggccag cacacagacc agcacgttgc 2820
ccaggagctg tgggaggaag ataagaggta tgaacatgat tagcaaaagg gcctagcttg 2880
gactcagaat aatccagcct tatcccaacc ataaaataaa agcagaatgg tagctggatt 2940
gtagctgcta ttagcaatat gaaacctctt acatcagtta caatttatat gcagaaatat 3000
ttatatgcag agatattgct attgccttaa cccagaaatt atcactgtta ttctttagaa 3060
tggtgcaaag aggcatgata cattgtatca ttattgccct gaaagaaaga gattagggaa 3120
agtattagaa ataagataaa caaaaaagta tattaaaaga agaaagcatt ttttaaaatt 3180
acaaatgcaa aattaccctg atttggtcaa tatgtgtaca catattaaaa cattacactt 3240
taacccataa atatgtataa tgattatgta tcaattaaaa ataaaagaaa ataaagtagg 3300
gagattatga atatgcaaat aagcacacat atattccaaa tagtaatgta ctaggcagac 3360
tgtgtaaagt ttttttttaa gttacttaat gtatctcaga gatatttcct tttgttatac 3420
acaatgttaa ggcattaagt ataatagtaa aaattgcgga gaagaaaaaa aaagaaagca 3480
agaattaaac aaaagaaaac aattgttatg aacagcaaat aaaagaaact aaaacgatcc 3540
tgagacttcc acactgatgc aatcattcgt ctgtttccca ttctaaactg taccctgtta 3600
cttatcccct tcctatgaca tgaacttaac catagaaaag aaggggaaag aaaacatcaa 3660
gcgtcccata gactcaccct gaagttctca ggatccacgt gcagcttgtc acagtgcagc 3720
tcactcagct gggcaaaggt gcccttgagg ttgtccaggt gagccaggcc atcactaaag 3780
gcaccgagca ctttcttgcc atgagccttc accttagggt tgcccataac agcatcagga 3840
gtggacagat ccccaaagga ctcaaagaac ctctgggtcc aagggtagac caccagcagc 3900
ctaagggtgg gaaaatagac caataggcag agagagtcag tgcctatcag aaacccaaga 3960
gtcttctctg tctccacatg cccagtttct attggtctcc ttaaacctgt cttgtaacct 4020
tgataccaac ctgcccaggg cctcaccacc aacttcatcc acgttcacct tgccccacag 4080
ggcagtaacg gcagacttct cctcaggagt cagatgcacc atggtgtctg tttgaggttg 4140
ctagtgaaca cagttgtgtc agaagcaaat gtaagcaata gatggctctg ccctgacttt 4200
tatgcccagc cctggctcct gccctccctg ctcctgggag tagattggcc aaccctaggg 4260
tgtggctcca cagggtgagg tctaagtgat gacagccgta cctgtccttg gctcttctgg 4320
cactggctta ggagttggac ttcaaaccct cagccctccc tctaagatat atctcttggc 4380
cccatacctc gaggatctga ccccgtatgt gagcatgtgt cctctaacag cacaggcctt 4440
ttgccaccta gctgtccagg ggtgccttaa aatggcaaac aaggtttgtt ttcttttcct 4500
gttttcatgc cttcctcttc catatccttg tttcatatta atacatgtgt atagatccta 4560
aaaatctata cacatgtatt aataaagcct gattctgccg cttctaggta tagaggccac 4620
ctgcaagata aatatttgat tcacaataac taatcattct atggcaattg ataacaacaa 4680
atatatatat atatatatat atacgtatat gtgtatatat atatatatat tcaggaaata 4740
atatattcta gaatatgtca cattctgtct caggcatcca ttttctttat gatgccgttt 4800
gaggtggagt tttagtcagg tggtcagctt ctcctttttt ttgccatctg ccctgtaagc 4860
atcctgctgg ggacccagat aggagtcatc actctaggct gagaacatct gggcacacac 4920
cctaagcctc agcatgactc atcatgactc agcattgctg tgcttgagcc agaaggtttg 4980
cttagaaggt tacacagaac cagaaggcgg gggtggggca ctgaccccga caggggcctg 5040
gccagaactg ctcatgcttg gactatggga ggtcactaat ggagacacac agaaatgtaa 5100
caggaactaa ggaaaaactg ttcattaaaa aaagtctaac cagctgcatt cgactttgac 5160
tgcagcagct ggttagaagg ttctactgga ggagggtccc agcccattgc taaattaaca 5220
tcaggctctg agactggcag tatatctcta acagtggttg atgctatctt ctggaacttg 5280
cctgctacat tgagaccact gacccataca taggaagccc atagctctgt cctgaactgt 5340
taggccactg gtccagagag tgtgcatctc ctttgatcct cataataacc ctatgagata 5400
gacacaatta ttactcttac tttatagatg atgatcctga aaacatagga gtcaaggcac 5460
ttgcccctag ctgggggtat aggggagcag tcccatgtag tagtagaatg aaaaatgctg 5520
ctatgctgtg cctcccccac ctttcccatg tctgccctct actcatggtc tatctctcct 5580
ggctcctggg agtcatggac tccacccagc accaccaacc tgacctaacc acctatctga 5640
gcctgccagc ctataaccca tctgggccct gatagctggt ggccagccct gaccccaccc 5700
caccctccct ggaacctctg atagacacat ctggcacacc agctcgcaaa gtcaccgtga 5760
gggtcttgtg tttgctgagt caaaattcct tgaaatccaa gtccttagag actcctgctc 5820
ccaaatttac agtcatagac ttcttcatgg ctgtctcctt tatccacaga atgattcctt 5880
tgcttcattg ccccatccat ctgatcctcc tcatcagtgc agcacagggc ccatgagcag 5940
tagctgcaga gtctcacata ggtctggcac tgcctctgac atgtccgacc ttaggcaaat 6000
gcttgactct tctgagctca gtcttgtcat ggcaaaataa agataataat agtgtttttt 6060
tatggagtta gcgtgaggat ggaaaacaat agcaaaattg attagactat aaaaggtctc 6120
aacaaatagt agtagatttt atcgtccatt aatccttccc tctcctctct tactcatccc 6180
atcacgtatg cctcttaatt ttcccttacc tataataaga gttattcctc ttattatatt 6240
cttcttatag tgattctgga tattaaagtg ggaatgaggg gcaggccact aacgaagaag 6300
atgtttctca aagaagccat tctccccaca tagatcatct cagcagggtt caggaagata 6360
aaggaggatc aaggtcgaag gtaggaacta aggaagaaca ctgggcaagt ggatccaggt 6420
tgtccaacgc tgaaagtagg aatctaagca ctagtctctg gatgctagga gggcctctgc 6480
atgggtaact ctttcaacta gccaggggct ggactgtgga gaaaccattt ccagatagaa 6540
gtgaggagat tccagcagga aacacttaag agaggatccc tggaagttcg gggcaggagg 6600
ctccctgtca catgaaggaa acctgctcag cgtaggctct aggttcttcc ctactcttat 6660
ccaatggggc ttctgatttt aagccagtct tcacaaagcc ccagatttct acatgctgga 6720
gccctgaatg ggcaggaagc attgcggttt ccatttcatt cagagctctt tcataccctg 6780
cttccccata gtttgtctca acatttctgt tgataatctg attcatgcaa ccaagaatta 6840
tgagagagcc ttcagtcatg cctaggcctg catttattgt tgtgcatatg atggggtagc 6900
agacaagaaa acgttaacag catagacttt ctcagagcta tatgagtttg ggaattaatc 6960
aagaccagcc ttttgctcag ggtagaaatc ccctataggg atttattcaa tgatgggtta 7020
aaagcttctc gagggaattc cgataatcaa cctctggatt acaaaatttg tgaaagattg 7080
actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct 7140
ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg 7200
ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact 7260
gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc 7320
gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc 7380
cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag 7440
ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc 7500
ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg 7560
gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg 7620
gccgcctccc cgcatcggga attcgagctc ggtaccttta agaccaatga cttacaaggc 7680
agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc 7740
ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg 7800
agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc 7860
ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 7920
cagacccttt tagtcagtgt ggaaaatctc tagcagcatc tagctagaat taattccgtg 7980
tattctatag tgtcacctaa atcgtatgtg tatgatacat aaggttatgt attaattgta 8040
gccgcgttct aacgacaata tgtacaagcc taattgtgta gcatctggct tactgaagca 8100
gaccctatca tctctctcgt aaactgccgt cagagtcggt ttggttggac gaaccttctg 8160
agtttctggt aacgccgtcc cgcacccgga aatggtcagc gaaccaatca gcagggtcat 8220
cgctagccta ggcttttgcg tcgagacgta cccaattcgc cctatagtga gtcgtattac 8280
gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 8340
cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 8400
accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcga cgcgccctgt 8460
agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 8520
agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 8580
tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 8640
cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 8700
tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 8760
caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 8820
ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 8880
aacaaaatat taacgtttac aatttcccag gtggcacttt tcggggaaat gtgcgcggaa 8940
cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 9000
cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 9060
tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 9120
tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 9180
atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 9240
gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 9300
aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 9360
aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 9420
gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 9480
cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 9540
atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 9600
tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 9660
ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 9720
ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 9780
ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 9840
tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 9900
tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 9960
aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 10020
tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 10080
tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 10140
gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 10200
agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 10260
tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 10320
ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 10380
cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 10440
tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 10500
acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 10560
gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 10620
ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 10680
tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 10740
attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 10800
cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 10860
ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 10920
aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 10980
ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 11040
acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg 11100
aacaaaagct ggagctgc 11118
<210> 9
<211> 5824
<212> DNA
<213> pMD2G质粒(Artificial Sequence)
<400> 9
ggatcccctg agggggcccc catgggctag aggatccggc ctcggcctct gcataaataa 60
aaaaaattag tcagccatga gcttggccca ttgcatacgt tgtatccata tcataatatg 120
tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt 180
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 240
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 300
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 360
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 420
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 480
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 540
gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 600
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 660
tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 720
tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 780
ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctt 840
acatgtggta ccgagctcgg atcctgagaa cttcagggtg agtctatggg acccttgatg 900
ttttctttcc ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag 960
ggtacacata ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct 1020
tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc 1080
agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat 1140
aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg 1200
taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct 1260
tttattttat ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta 1320
atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg 1380
ctggcccatc actttggcaa agcacgtgag atctgaattc aacagagatc gatctgtttc 1440
cttgacacta tgaagtgcct tttgtactta gcctttttat tcattggggt gaattgcaag 1500
ttcaccatag tttttccaca caaccaaaaa ggaaactgga aaaatgttcc ttctaattac 1560
cattattgcc cgtcaagctc agatttaaat tggcataatg acttaatagg cacagccata 1620
caagtcaaaa tgcccaagag tcacaaggct attcaagcag acggttggat gtgtcatgct 1680
tccaaatggg tcactacttg tgatttccgc tggtatggac cgaagtatat aacacagtcc 1740
atccgatcct tcactccatc tgtagaacaa tgcaaggaaa gcattgaaca aacgaaacaa 1800
ggaacttggc tgaatccagg cttccctcct caaagttgtg gatatgcaac tgtgacggat 1860
gccgaagcag tgattgtcca ggtgactcct caccatgtgc tggttgatga atacacagga 1920
gaatgggttg attcacagtt catcaacgga aaatgcagca attacatatg ccccactgtc 1980
cataactcta caacctggca ttctgactat aaggtcaaag ggctatgtga ttctaacctc 2040
atttccatgg acatcacctt cttctcagag gacggagagc tatcatccct gggaaaggag 2100
ggcacagggt tcagaagtaa ctactttgct tatgaaactg gaggcaaggc ctgcaaaatg 2160
caatactgca agcattgggg agtcagactc ccatcaggtg tctggttcga gatggctgat 2220
aaggatctct ttgctgcagc cagattccct gaatgcccag aagggtcaag tatctctgct 2280
ccatctcaga cctcagtgga tgtaagtcta attcaggacg ttgagaggat cttggattat 2340
tccctctgcc aagaaacctg gagcaaaatc agagcgggtc ttccaatctc tccagtggat 2400
ctcagctatc ttgctcctaa aaacccagga accggtcctg ctttcaccat aatcaatggt 2460
accctaaaat actttgagac cagatacatc agagtcgata ttgctgctcc aatcctctca 2520
agaatggtcg gaatgatcag tggaactacc acagaaaggg aactgtggga tgactgggca 2580
ccatatgaag acgtggaaat tggacccaat ggagttctga ggaccagttc aggatataag 2640
tttcctttat acatgattgg acatggtatg ttggactccg atcttcatct tagctcaaag 2700
gctcaggtgt tcgaacatcc tcacattcaa gacgctgctt cgcaacttcc tgatgatgag 2760
agtttatttt ttggtgatac tgggctatcc aaaaatccaa tcgagcttgt agaaggttgg 2820
ttcagtagtt ggaaaagctc tattgcctct tttttcttta tcatagggtt aatcattgga 2880
ctattcttgg ttctccgagt tggtatccat ctttgcatta aattaaagca caccaagaaa 2940
agacagattt atacagacat agagatgaac cgacttggaa agtaactcaa atcctgcaca 3000
acagattctt catgtttgga ccaaatcaac ttgtgatacc atgctcaaag aggcctcaat 3060
tatatttgag tttttaattt ttatggaatt caccccacca gtgcaggctg cctatcagaa 3120
agtggtggct ggtgtggcta atgccctggc ccacaagttt cactaagctc gcttccttgc 3180
tgtccaattt ctattaaagg ttccttggtt ccctaagtcc aactactaaa ctgggggata 3240
ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 3300
tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 3360
tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 3420
actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gccctgatgc 3480
ctatgcctta ttcatccctc agaaaaggat tcaagtagag gcttgatttg gaggttaaag 3540
tttggctatg ctgtatttta cattacttat tgttttagct gtcctcatga atgtcttttc 3600
actacccatt tgcttatcct gcatctctca gccttgactc cactcagttc tcttgcttag 3660
agataccacc tttcccctga agtgttcctt ccatgtttta cggcgagatg gtttctcctc 3720
gcctggccac tcagccttag ttgtctctgt tgtcttatag aggtctactt gaagaaggaa 3780
aaacaggggg catggtttga ctgtcctgtg agcccttctt ccctgcctcc cccactcaca 3840
gtgacccgga atccctcgac atggcagtct agcactagtg cggccgcaga tctgcttcct 3900
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3960
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 4020
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 4080
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 4140
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 4200
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 4260
ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4320
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4380
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4440
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4500
acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4560
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4620
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4680
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 4740
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4800
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4860
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4920
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4980
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 5040
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 5100
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 5160
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 5220
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 5280
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 5340
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 5400
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 5460
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 5520
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 5580
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 5640
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 5700
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 5760
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5820
acgt 5824
<210> 10
<211> 8895
<212> DNA
<213> pMDlg/pRRE 质粒(Artificial Sequence)
<400> 10
ggatcccctg agggggcccc catgggctag aggatccggc ctcggcctct gcataaataa 60
aaaaaattag tcagccatga gcttggccca ttgcatacgt tgtatccata tcataatatg 120
tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt 180
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 240
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 300
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 360
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 420
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 480
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 540
gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 600
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 660
tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 720
tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 780
ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctt 840
acatgtggta ccgagctcgg atcctgagaa cttcagggtg agtctatggg acccttgatg 900
ttttctttcc ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag 960
ggtacacata ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct 1020
tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc 1080
agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat 1140
aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg 1200
taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct 1260
tttattttat ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta 1320
atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg 1380
ctggcccatc actttggcaa agcacgtgag atctgaattc gagatctgcc gccgccatgg 1440
gtgcgagagc gtcagtatta agcgggggag aattagatcg atgggaaaaa attcggttaa 1500
ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag 1560
aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg 1620
gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag 1680
tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag 1740
acaagataga ggaagagcaa aacaaaagta agaaaaaagc acagcaagca gcagctgaca 1800
caggacacag caatcaggtc agccaaaatt accctatagt gcagaacatc caggggcaaa 1860
tggtacatca ggccatatca cctagaactt taaatgcatg ggtaaaagta gtagaagaga 1920
aggctttcag cccagaagtg atacccatgt tttcagcatt atcagaagga gccaccccac 1980
aagatttaaa caccatgcta aacacagtgg ggggacatca agcagccatg caaatgttaa 2040
aagagaccat caatgaggaa gctgcagaat gggatagagt gcatccagtg catgcagggc 2100
ctattgcacc aggccagatg agagaaccaa ggggaagtga catagcagga actactagta 2160
ctagtaccct tcaggaacaa ataggatgga tgacacataa tccacctatc ccagtaggag 2220
aaatctataa aagatggata atcctgggat taaataaaat agtaagaatg tatagcccta 2280
ccagcattct ggacataaga caaggaccaa aggaaccctt tagagactat gtagaccgat 2340
tctataaaac tctaagagcc gagcaagctt cacaagaggt aaaaaattgg atgacagaaa 2400
ccttgttggt ccaaaatgcg aacccagatt gtaagactat tttaaaagca ttgggaccag 2460
gagcgacact agaagaaatg atgacagcat gtcagggagt ggggggaccc ggccataaag 2520
caagagtttt ggctgaagca atgagccaag taacaaatcc agctaccata atgatacaga 2580
aaggcaattt taggaaccaa agaaagactg ttaagtgttt caattgtggc aaagaagggc 2640
acatagccaa aaattgcagg gcccctagga aaaagggctg ttggaaatgt ggaaaggaag 2700
gacaccaaat gaaagattgt actgagagac aggctaattt tttagggaag atctggcctt 2760
cccacaaggg aaggccaggg aattttcttc agagcagacc agagccaaca gccccaccag 2820
aagagagctt caggtttggg gaagagacaa caactccctc tcagaagcag gagccgatag 2880
acaaggaact gtatccttta gcttccctca gatcactctt tggcagcgac ccctcgtcac 2940
aataaagata ggggggcaat taaaggaagc tctattagat acaggagcag atgatacagt 3000
attagaagaa atgaatttgc caggaagatg gaaaccaaaa atgatagggg gaattggagg 3060
ttttatcaaa gtaggacagt atgatcagat actcatagaa atctgcggac ataaagctat 3120
aggtacagta ttagtaggac ctacacctgt caacataatt ggaagaaatc tgttgactca 3180
gattggctgc actttaaatt ttcccattag tcctattgag actgtaccag taaaattaaa 3240
gccaggaatg gatggcccaa aagttaaaca atggccattg acagaagaaa aaataaaagc 3300
attagtagaa atttgtacag aaatggaaaa ggaaggaaaa atttcaaaaa ttgggcctga 3360
aaatccatac aatactccag tatttgccat aaagaaaaaa gacagtacta aatggagaaa 3420
attagtagat ttcagagaac ttaataagag aactcaagat ttctgggaag ttcaattagg 3480
aataccacat cctgcagggt taaaacagaa aaaatcagta acagtactgg atgtgggcga 3540
tgcatatttt tcagttccct tagataaaga cttcaggaag tatactgcat ttaccatacc 3600
tagtataaac aatgagacac cagggattag atatcagtac aatgtgcttc cacagggatg 3660
gaaaggatca ccagcaatat tccagtgtag catgacaaaa atcttagagc cttttagaaa 3720
acaaaatcca gacatagtca tctatcaata catggatgat ttgtatgtag gatctgactt 3780
agaaataggg cagcatagaa caaaaataga ggaactgaga caacatctgt tgaggtgggg 3840
atttaccaca ccagacaaaa aacatcagaa agaacctcca ttcctttgga tgggttatga 3900
actccatcct gataaatgga cagtacagcc tatagtgctg ccagaaaagg acagctggac 3960
tgtcaatgac atacagaaat tagtgggaaa attgaattgg gcaagtcaga tttatgcagg 4020
gattaaagta aggcaattat gtaaacttct taggggaacc aaagcactaa cagaagtagt 4080
accactaaca gaagaagcag agctagaact ggcagaaaac agggagattc taaaagaacc 4140
ggtacatgga gtgtattatg acccatcaaa agacttaata gcagaaatac agaagcaggg 4200
gcaaggccaa tggacatatc aaatttatca agagccattt aaaaatctga aaacaggaaa 4260
atatgcaaga atgaagggtg cccacactaa tgatgtgaaa caattaacag aggcagtaca 4320
aaaaatagcc acagaaagca tagtaatatg gggaaagact cctaaattta aattacccat 4380
acaaaaggaa acatgggaag catggtggac agagtattgg caagccacct ggattcctga 4440
gtgggagttt gtcaataccc ctcccttagt gaagttatgg taccagttag agaaagaacc 4500
cataatagga gcagaaactt tctatgtaga tggggcagcc aatagggaaa ctaaattagg 4560
aaaagcagga tatgtaactg acagaggaag acaaaaagtt gtccccctaa cggacacaac 4620
aaatcagaag actgagttac aagcaattca tctagctttg caggattcgg gattagaagt 4680
aaacatagtg acagactcac aatatgcatt gggaatcatt caagcacaac cagataagag 4740
tgaatcagag ttagtcagtc aaataataga gcagttaata aaaaaggaaa aagtctacct 4800
ggcatgggta ccagcacaca aaggaattgg aggaaatgaa caagtagatg ggttggtcag 4860
tgctggaatc aggaaagtac tatttttaga tggaatagat aaggcccaag aagaacatga 4920
gaaatatcac agtaattgga gagcaatggc tagtgatttt aacctaccac ctgtagtagc 4980
aaaagaaata gtagccagct gtgataaatg tcagctaaaa ggggaagcca tgcatggaca 5040
agtagactgt agcccaggaa tatggcagct agattgtaca catttagaag gaaaagttat 5100
cttggtagca gttcatgtag ccagtggata tatagaagca gaagtaattc cagcagagac 5160
agggcaagaa acagcatact tcctcttaaa attagcagga agatggccag taaaaacagt 5220
acatacagac aatggcagca atttcaccag tactacagtt aaggccgcct gttggtgggc 5280
ggggatcaag caggaatttg gcattcccta caatccccaa agtcaaggag taatagaatc 5340
tatgaataaa gaattaaaga aaattatagg acaggtaaga gatcaggctg aacatcttaa 5400
gacagcagta caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg 5460
gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt 5520
acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc 5580
agtttggaaa ggaccagcaa agctcctctg gaaaggtgaa ggggcagtag taatacaaga 5640
taatagtgac ataaaagtag tgccaagaag aaaagcaaag atcatcaggg attatggaaa 5700
acagatggca ggtgatgatt gtgtggcaag tagacaggat gaggattaac acatggaatt 5760
ccggagcggc cgcaggagct ttgttccttg ggttcttggg agcagcagga agcactatgg 5820
gcgcagcctc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc 5880
agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct 5940
ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac 6000
agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga 6060
atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt 6120
gggacagaga aattaacaat tacacaagct tccgcggaat tcaccccacc agtgcaggct 6180
gcctatcaga aagtggtggc tggtgtggct aatgccctgg cccacaagtt tcactaagct 6240
cgcttccttg ctgtccaatt tctattaaag gttccttggt tccctaagtc caactactaa 6300
actgggggat attatgaagg gccttgagca tctggattct gcctaataaa aaacatttat 6360
tttcattgca atgatgtatt taaattattt ctgaatattt tactaaaaag ggaatgtggg 6420
aggtcagtgc atttaaaaca taaagaaatg aagagctagt tcaaaccttg ggaaaataca 6480
ctatatctta aactccatga aagaaggtga ggctgcaaac agctaatgca cattggcaac 6540
agccctgatg cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt 6600
ggaggttaaa gtttggctat gctgtatttt acattactta ttgttttagc tgtcctcatg 6660
aatgtctttt cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt 6720
ctcttgctta gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat 6780
ggtttctcct cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact 6840
tgaagaagga aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc 6900
ccccactcac agtgacccgg aatccctcga catggcagtc tagcactagt gcggccgcag 6960
atctgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 7020
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 7080
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 7140
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 7200
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 7260
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 7320
cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 7380
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 7440
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 7500
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 7560
taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 7620
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 7680
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 7740
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 7800
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 7860
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 7920
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 7980
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 8040
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 8100
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 8160
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 8220
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 8280
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 8340
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 8400
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 8460
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 8520
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 8580
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 8640
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 8700
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 8760
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 8820
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 8880
agtgccacct gacgt 8895
<210> 11
<211> 4174
<212> DNA
<213> pRSV-REV质粒(Artificial Sequence)
<400> 11
agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60
acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120
tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180
ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gaattcgatg 240
tacgggccag atatacgcgt atctgagggg actaggtgtg tttaggcgaa aagcggggct 300
tcggttgtac gcggttagga gtcccctcag gattagtagt ttcgcttttg catagggagg 360
gggaaatgta gtcttatgca atacacttgt agtcttgcaa catggtaacg atgagttagc 420
aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 480
acgatcgtgc cttattagga aggcaacaga caggtctgac atggattgga cgaaccactg 540
aattccgcat tgcagagata attgtattta agtgcctagc tcgatacaat aaacgccatt 600
tgaccattca ccacattggt gtgcacctcc aagctcgagc tcgtttagtg aaccgtcaga 660
tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca 720
gcctcccctc gaagctagtc gattaggcat ctcctatggc aggaagaagc ggagacagcg 780
acgaagacct cctcaaggca gtcagactca tcaagtttct ctatcaaagc aacccacctc 840
ccaatcccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 900
agagacagat ccattcgatt agtgaacgga tccttagcac ttatctggga cgatctgcgg 960
agcctgtgcc tcttcagcta ccaccgcttg agagacttac tcttgattgt aacgaggatt 1020
gtggaacttc tgggacgcag ggggtgggaa gccctcaaat attggtggaa tctcctacaa 1080
tattggagtc aggagctaaa gaatagtgct gttagcttgc tcaatgccac agctatagca 1140
gtagctgagg ggacagatag ggttatagaa gtagtacaag aagcttggca ctggccgtcg 1200
ttttacatga tctgagcctg ggagatctct ggctaactag ggaacccact gcttaagcct 1260
caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 1320
aactagagat caggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 1380
tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 1440
cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 1500
ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 1560
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 1620
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 1680
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 1740
cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 1800
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 1860
aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 1920
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 1980
acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 2040
cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 2100
tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 2160
ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 2220
ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 2280
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2340
taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 2400
cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 2460
aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 2520
aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 2580
tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 2640
ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 2700
catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 2760
aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 2820
ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 2880
gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 2940
aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 3000
gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 3060
gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 3120
gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 3180
gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3240
gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3300
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3360
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3420
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 3480
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 3540
ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3600
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 3660
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3720
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3780
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3840
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 3900
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 3960
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4020
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 4080
gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 4140
gagcgcagcg agtcagtgag cgaggaagcg gaag 4174

Claims (4)

1.一种包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述表达盒包含：
a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；所述微型基因座调控元件的序列如SEQ ID NO.1、或如SEQ ID NO.2所示；
b、beta珠蛋白的基因序列；
c、beta珠蛋白的基因序列上游侧翼的启动子序列；
d、beta珠蛋白的基因序列下游的侧翼序列；所述侧翼序列为300bp，具体序列如SEQ IDNO.5所示。
2.根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述beta珠蛋白的基因序列含外显子序列和内含子序列；且在密码子87处编码包含突变T87Q。
3.根据权利要求2所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述beta珠蛋白的基因序列如SEQ ID NO.3所示。
4.根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述启动子序列为265bp，具体序列如SEQ ID NO.4所示。
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