[发明专利]siRNA筛选系统的通用性绿色荧光蛋白融合靶基因表达载体

摘要

本发明公开了siRNA筛选系统的通用性绿色荧光蛋白融合靶基因表达载体，该载体以pEGFP－C1载体为基础，包括人巨细胞病毒CMV启动子，增强的绿色荧光蛋白基因EGFP，单纯疱疹病毒HSV胸苷激酶TK多腺苷酸化信号，SV40早期启动子组成元件，在1336－1363增加SSe8387I和NotI两个内切酶位点，其载体大小为4695bp，载体命名为pEGFP－RNAi。本发明通过检测荧光强度可以非常方便快速地鉴定相应siRNA序列的沉默效果，从而筛选出最有效的siRNA序列。

主权项

1.siRNA筛选系统的通用性绿色荧光蛋白融合靶基因表达载体，其特征在于，该载体以pEGFP-C1载体为基础，在1336-1363改造SSe8387I和NotI两个内切酶位点，其载体大小为4695bp，载体命名为pEGFP-RNAi，序列如下： 1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG 61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT 121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC 241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC 361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG 421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA 601 CCGGTCGCCA CCATGGTGAG CAAGGGCGAG GAGCTGTTCA CCGGGGTGGT GCCCATCCTG 661 GTCGAGCTGG ACGGCGACGT AAACGGCCAC AAGTTCAGCG TGTCCGGCGA GGGCGAGGGC 721 GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCCGTG 781 CCCTGGCCCA CCCTCGTGAC CACCCTGACC TACGGCGTGC AGTGCTTCAG CCGCTACCCC 841 GACCACATGA AGCAGCACGA CTTCTTCAAG TCCGCCATGC CCGAAGGCTA CGTCCAGGAG 901 CGCACCATCT TCTTCAAGGA CGACGGCAAC TACAAGACCC GCGCCGAGGT GAAGTTCGAG 961 GGCGACACCC TGGTGAACCG CATCGAGCTG AAGGGCATCG ACTTCAAGGA GGACGGCAAC1021 ATCCTGGGGC ACAAGCTGGA GTACAACTAC AACAGCCACA ACGTCTATAT CATGGCCGAC1081 AAGCAGAAGA ACGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGACGGCAGC1141 GTGCAGCTCG CCGACCACTA CCAGCAGAAC ACCCCCATCG GCGACGGCCC CGTGCTGCTG1201 CCCGACAACC ACTACCTGAG CACCCAGTCC GCCCTGAGCA AAGACCCCAA CGAGAAGCGC1261 GATCACATGG TCCTGCTGGA GTTCGTGACC GCCGCCGGGA TCACTCTCGG CATGGACGAG1321 CTGTACAAGT CCGGACCTGC AGGGAATTCC TCGAGGCGGC CGCATTCTAG ATAACTGATC1381 ATAATCAGCC ATACCACATT TGTAGAGGTT TTACTTGCTT TAAAAAACCT CCCACACCTC1441 CCCCTGAACC TGAAACATAA AATGAATGCA ATTGTTGTTG TTAACTTGTT TATTGCAGCT1501 TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA1561 CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTAACGCGT AAATTGTAAG1621 CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA ATCAGCTCAT TTTTTAACCA1681 ATAGGCCGAA ATCGGCAAAA TCCCTTATAA ATCAAAAGAA TAGACCGAGA TAGGGTTGAG1741 TGTTGTTCCA GTTTGGAACA AGAGTCCACT ATTAAAGAAC GTGGACTCCA ACGTCAAAGG1801 GCGAAAAACC GTCTATCAGG GCGATGGCCC ACTACGTGAA CCATCACCCT AATCAAGTTT1861 TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT AAAGGGAGCC CCCGATTTAG1921 AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA GGGAAGAAAG CGAAAGGAGC1981 GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT CACGCTGCGC GTAACCACCA CACCCGCCGC2041 GCTTAATGCG CCGCTACAGG GCGCGTCAGG TGGCACTTTT CGGGGAAATG TGCGCGGAAC2101 CCCTATTTGT TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA GACAATAACC2701 CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT2761 GAATGAACTG CAAGACGAGG CAGCGCGGCT ATCGTGGCTG GCCACGACGG GCGTTCCTTG2821 CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT2881 GCCGGGGCAG GATCTCCTGT CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC2941 TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC3001 GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC GGTCTTGTCG ATCAGGATGA3061 TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAACTG TTCGCCAGGC TCAAGGCGAG3121 CATGCCCGAC GGCGAGGATC TCGTCGTGAC CCATGGCGAT GCCTGCTTGC CGAATATCAT3181 GGTGGAAAAT GGCCGCTTTT CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG3241 CTATCAGGAC ATAGCGTTGG CTACCCGTGA TATTGCTGAA GAGCTTGGCG GCGAATGGGC3301 TGACCGCTTC CTCGTGCTTT ACGGTATCGC CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA3361 TCGCCTTCTT GACGAGTTCT TCTGAGCGGG ACTCTGGGGT TCGAAATGAC CGACCAAGCG3421 ACGCCCAACC TGCCATCACG AGATTTCGAT TCCACCGCCG CCTTCTATGA AAGGTTGGGC3481 TTCGGAATCG TTTTCCGGGA CGCCGGCTGG ATGATCCTCC AGCGCGGGGA TCTCATGCTG3541 GAGTTCTTCG CCCACCCTAG GGGGAGGCTA ACTGAAACAC GGAAGGAGAC AATACCGGAA3601 GGAACCCGCG CTATGACGGC AATAAAAAGA CAGAATAAAA CGCACGGTGT TGGGTCGTTT3661 GTTCATAAAC GCGGGGTTCG GTCCCAGGGC TGGCACTCTG TCGATACCCC ACCGAGACCC3721 CATTGGGGCC AATACGCCCG CGTTTCTTCC TTTTCCCCAC CCCACCCCCC AAGTTCGGGT3781 GAAGGCCCAG GGCTCGCAGC CAACGTCGGG GCGGCAGGCC CTGCCATAGC CTCAGGTTAC3841 TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT TTAAAAGGAT CTAGGTGAAG3901 ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG AGTTTTCGTT CCACTGAGCG3961 TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC CTTTTTTTCT GCGCGTAATC4021 TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG TTTGTTTGCC GGATCAAGAG4081 CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG CGCAGATACC AAATACTGTC4141 CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT CTGTAGCACC GCCTACATAC4201 CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG GCGATAAGTC GTGTCTTACC4261 GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC GGTCGGGCTG AACGGGGGGT4681 ATTACCGCCA TGCAT。