[发明专利]用于T－A克隆的pG7X－T载体及其制备方法

摘要

本发明涉及用于T－A克隆的pG7X－T载体及其制备方法，属于重组DNA技术的载体构建领域，其技术方案是利用自行设计的引物对质粒pGEM7Zf(+)进行扩增，扩增产物经HindIII消化、自连、转化、筛选等过程，获得新质粒pG7X。相比pGEM7Zf(+)而言，pG7X增加了2个XcmI识别位点，可用于制备T－A克隆载体。用XcmI酶切后得到的两端带有3′dT粘性末端的DNA即为T载体，命名为pG7X－T。该T载体与带有dA末端DNA片段或PCR产物进行连接，阳性重组率达95％以上。利用本发明制备T载体的成本在60元/微克以下，仅为市场价格的十分之一，而且操作简便、性能稳定、重组率高。

主权项

1.用于T-A克隆的pG7X-T载体及其制备方法，其特征在于，该载体DNA序列如下：0001 GACTGGGGA TCCGGAGAGC TCCCAACGCG TTGGATGCAT AGCTTGAGTA TTCTATAGTG TCACCTAAAT AGCTTGGCGT AATCATGGTC TCTGACCCCT AGGCCTCTCG AGGGTTGCGC AACCTACGTA TCGAACTCAT AAGATATCAC AGTGGATTTA TCGAACCGCA TTAGTACCAG0091 ATAGCTGTTT CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGCCT GGGGTGCCTA TATCGACAAA GGACACACTT TAACAATAGG CGAGTGTTAA GGTGTGTTGT ATGCTCGGCC TTCGTATTTC ACATTTCGGA CCCCACGGAT0181 ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC CAGCTGCATT AATGAATCGG TACTCACTCG ATTGAGTGTA ATTAACGCAA CGCGAGTGAC GGGCGAAAGG TCAGCCCTTT GGACAGCACG GTCGACGTAA TTACTTAGCC0271 CCAACGCGCG GGGAGAGGCG GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG GGTTGCGCGC CCCTCTCCGC CAAACGCATA ACCCGCGAGA AGGCGAAGGA GCGAGTGACT GAGCGACGCG AGCCAGCAAG CCGACGCCGC0361 AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA TCGCCATAGT CGAGTGAGTT TCCGCCATTA TGCCAATAGG TGTCTTAGTC CCCTATTGCG TCCTTTCTTG TACACTCGTT TTCCGGTCGT0451 AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG TTTCCGGTCC TTGGCATTTT TCCGGCGCAA CGACCGCAAA AAGGTATCCG AGGCGGGGGG ACTGCTCGTA GTGTTTTTAG CTGCGAGTTC0541 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC AGTCTCCACC GCTTTGGGCT GTCCTGATAT TTCTATGGTC CGCAAAGGGG GACCTTCGAG GGAGCACGCG AGAGGACAAG GCTGGGACGG0631 GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGAATGGCCT ATGGACAGGC GGAAAGAGGG AAGCCCTTCG CACCGCGAAA GAGTATCGAG TGCGACATCC ATAGAGTCAA GCCACATCCA0721 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GCAAGCGAGG TTCGACCCGA CACACGTGCT TGGGGGGCAA GTCGGGCTGG CGACGCGGAA TAGGCCATTG ATAGCAGAAC TCAGGTTGGG0811 GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA CCATTCTGTG CTGAATAGCG GTGACCGTCG TCGGTGACCA TTGTCCTAAT CGTCTCGCTC CATACATCCG CCACGATGTC TCAAGAACTT0901 GTGGTGGCCT AACTACGGCT ACACTAGAAG AACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CACCACCGGA TTGATGCCGA TGTGATCTTC TTGTCATAAA CCATAGACGC GAGACGACTT CGGTCAATGG AAGCCTTTTT CTCAACCATC0991 CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA GAGAACTAGG CCGTTTGTTT GGTGGCGACC ATCGCCACCA AAAAAACAAA CGTTCGTCGT CTAATGCGCG TCTTTTTTTC CTAGAGTTCT1081 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT TCTAGGAAAC TAGAAAAGAT GCCCCAGACT GCGAGTCACC TTGCTTTTGA GTGCAATTCC CTAAAACCAG TACTCTAATA GTTTTTCCTA1171 CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT GAAGTGGATC TAGGAAAATT TAATTTTTAC TTCAAAATTT AGTTAGATTT CATATATACT CATTTGAACC AGACTGTCAA TGGTTACGAA1261 AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA TTAGTCACTC CGTGGATAGA GTCGCTAGAC AGATAAAGCA AGTAGGTATC AACGGACTGA GGGGCAGCAC ATCTATTGAT GCTATGCCCT1351 GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG CCCGAATGGT AGACCGGGGT CACGACGTTA CTATGGCGCT CTGGGTGCGA GTGGCCGAGG TCTAAATAGT CGTTATTTGG TCGGTCGGCC1441 AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC TTCCCGGCTC GCGTCTTCAC CAGGACGTTG AAATAGGCGG AGGTAGGTCA GATAATTAAC AACGGCCCTT CGATCTCATT CATCAAGCGG1531 AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC TCAATTATCA AACGCGTTGC AACAACGGTA ACGATGTCCG TAGCACCACA GTGCGAGCAG CAAACCATAC CGAAGTAAGT CGAGGCCAAG1621 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGTTGCTAGT TCCGCTCAAT GTACTAGGGG GTACAACACG TTTTTTCGCC AATCGAGGAA GCCAGGAGGC TAGCAACAGT CTTCATTCAA1711 GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA CCGGCGTCAC AATAGTGAGT ACCAATACCG TCGTGACGTA TTAAGAGAAT GACAGTACGG TAGGCATTCT ACGAAAAGAC ACTGACCACT1801 GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CATGAGTTGG TTCAGTAAGA CTCTTATCAC ATACGCCGCT GGCTCAACGA GAACGGGCCG CAGTTATGCC CTATTATGGC GCGGTGTATC1891 CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA GTCTTGAAAT TTTCACGAGT AGTAACCTTT TGCAAGAAGC CCCGCTTTTG AGAGTTCCTA GAATGGCGAC AACTCTAGGT CAAGCTACAT1981 ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA TGGGTGAGCA CGTGGGTTGA CTAGAAGTCG TAGAAAATGA AAGTGGTCGC AAAGACCCAC TCGTTTTTGT CCTTCCGTTT TACGGCGTTT2071 AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT TTTCCCTTAT TCCCGCTGTG CCTTTACAAC TTATGAGTAT GAGAAGGAAA AAGTTATAAT AACTTCGTAA ATAGTCCCAA TAACAGAGTA2161 GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ATGCGGTGTG CTCGCCTATG TATAAACTTA CATAAATCTT TTTATTTGTT TATCCCCAAG GCGCGTGTAA AGGGGCTTTT CACGGTGGAC TACGCCACAC2251 AAATACCGCA CAGATGCGTA AGGAGAAAAT ACCGCATCAG GAAATTGTAA GCGTTAATAT TTTGTTAAAA TTCGCGTTAA ATTTTTGTTA TTTATGGCGT GTCTACGCAT TCCTCTTTTA TGGCGTAGTC CTTTAACATT CGCAATTATA AAACAATTTT AAGCGCAATT TAAAAACAAT2341 AATCAGCTCA TTTTTTAACC AATAGGCCGA AATCGGCAAA ATCCCTTATA AATCAAAAGA ATAGACCGAG ATAGGGTTGA GTGTTGTTCC TTAGTCGAGT AAAAAATTGG TTATCCGGCT TTAGCCGTTT TAGGGAATAT TTAGTTTTCT TATCTGGCTC TATCCCAACT CACAACAAGG2431 AGTTTGGAAC AAGAGTCCAC TATTAAAGAA CGTGGACTCC AACGTCAAAG GGCGAAAAAC CGTCTATCAG GGCGATGGCC CACTACGTGA TCAAACCTTG TTCTCAGGTG ATAATTTCTT GCACCTGAGG TTGCAGTTTC CCGCTTTTTG GCAGATAGTC CCGCTACCGG GTGATGCACT2521 ACCATCACCC TAATCAAGTT TTTTGGGGTC GAGGTGCCGT AAAGCACTAA ATCGGAACCC TAAAGGGAGC CCCCGATTTA GAGCTTGACG TGGTAGTGGG ATTAGTTCAA AAAACCCCAG CTCCACGGCA TTTCGTGATT TAGCCTTGGG ATTTCCCTCG GGGGCTAAAT CTCGAACTGC2611 GGGAAAGCCG GCGAACGTGG CGAGAAAGGA AGGGAAGAAA GCGAAAGGAG CGGGCGCTAG GGCGCTGGCA AGTGTAGCGG TCACGCTGCG CCCTTTCGGC CGCTTGCACC GCTCTTTCCT TCCCTTCTTT CGCTTTCCTC GCCCGCGATC CCGCGACCGT TCACATCGCC AGTGCGACGC2701 CGTAACCACC ACACCCGCCG CGCTTAATGC GCCGCTACAG GGCGCGTCCA TTCGCCATTC AGGCTGCGCA ACTGTTGGGA AGGGCGATCG GCATTGGTGG TGTGGGCGGC GCGAATTACG CGGCGATGTC CCGCGCAGGT AAGCGGTAAG TCCGACGCGT TGACAACCCT TCCCGCTAGC2791 GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA CACGCCCGGA GAAGCGATAA TGCGGTCGAC CGCTTTCCCC CTACACGACG TTCCGCTAAT TCAACCCATT GCGGTCCCAA AAGGGTCAGT2881 CGACGTTGTA AAACGACGGC CAGTGAATTG TAATACGACT CACTATAGGG CGAATTGGGC CCGACGTCGC ATGCTCCTCT AGACTCGAGG GCTGCAACAT TTTGCTGCCG GTCACTTAAC ATTATGCTGA GTGATATCCC GCTTAACCCG GGCTGCAGCG TACGAGGAGA TCTGAGCTCC2971 AATTCGGTAC CCCAGGTCT TTAAGCCATG GGGTCCAG