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基本信息

質(zhì)粒圖譜和多克隆位點(diǎn)信息

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載體描述

    pacAd5 CMVK-NpA質(zhì)粒是一個(gè)腺病毒質(zhì)粒，CMV啟動(dòng)子驅(qū)動(dòng)目的基因的表達(dá)。

Recombinant adenoviruses have tremendous potential in both research and therapeutic applications. There are numerous advantages they provide when introducing genetic material into host cells. The permissive host cell range is very wide. The virus has been used to infect many mammalian cell types (both replicative and non-replicative) for high expression of the recombinant protein. Recombinant adenoviruses are especially useful for gene transfer and protein expression in cell lines that have low transfection efficiency with liposome. After entering cells, the virus remains epichromosomal (i.e. does not integrate into the host chromosome so does not activate or inactivate host genes). Recently, recombinant adenoviruses have been used to deliver RNAi into cells.
    Two methods have traditionally been used to generate recombinant adenoviruses. The first involves homologous recombination of a shuttle vector containing gene of interest and an adenoviral backbone plasmid vector (restricted in E1/E3) in an adenovirus packaging cell line. The isolation of recombinant adenovirus by this method involves performing multiple plaque isolations to avoid wild-type virus and is extremely laborious and time consuming. The second method, pAdEasy system, employs the homologous recombination machinery in E. coli, a recombinant adenovirus is produced by a doublerecombination event between cotransformed adenoviral backbone plasmid vector and a shuttle vector carrying the gene of interest. For the pAdEasy method, the system is high fidelity, but inefficient and requires the screening of many bacterial colonies. This results in a significant time commitment even before transfection of recombinant DNA into E1-expressing cells such as HEK293 cells. 

載體序列

ORIGIN

        1 aattaattaa gctagcatca tcaataatat accttatttt ggattgaagc caatatgata

       61 atgagggggt ggagtttgtg acgtggcgcg gggcgtggga acggggcggg tgacgtagta

      121 gtgtggcgga agtgtgatgt tgcaagtgtg gcggaacaca tgtaagcgac ggatgtggca

      181 aaagtgacgt ttttggtgtg cgccggtgta cacaggaagt gacaattttc gcgcggtttt

      241 aggcggatgt tgtagtaaat ttgggcgtaa ccgagtaaga tttggccatt ttcgcgggaa

      301 aactgaataa gaggaagtga aatctgaata attttgtgtt actcatagcg cgtaatattt

      361 gtctagggag atcagcctgc aggtcgttac ataacttacg gtaaatggcc cgcctggctg

      421 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc

      481 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc

      541 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg

      601 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat

      661 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg

      721 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag

      781 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt

      841 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt

      901 gaaccgtcag ttggtaccgt ttaaactcga ggtcgacggt atcgataagc ttgatatcga

      961 attcctgcag cccgggggat ccactagttc tagagcggcc gccaccgcgg ggagatccag

     1021 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat

     1081 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata

     1141 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcasggg gaggtgtggg

     1201 aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat gatcccggct

     1261 gcctcgcgcg tttcggtgat gacggtgaaa acctcttgac acatgcagct cccggagacg

     1321 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg

     1381 ggtgttggcg ggtgtcgggg cgcagccatg aggtcgactc tagtccccgc ggtggcagat

     1441 ctggaaggtg ctgaggtacg atgagacccg caccaggtgc agaccctgcg agtgtggcgg

     1501 taaacatatt aggaaccagc ctgtgatgct ggatgtgacc gaggagctga ggcccgatca

     1561 cttggtgctg gcctgcaccc gcgctgagtt tggctctagc gatgaagata cagattgagg

     1621 tactgaaatg tgtgggcgtg gcttaagggt gggaaagaat atataaggtg ggggtcttat

     1681 gtagttttgt atctgttttg cagcagccgc cgccgccatg agcaccaact cgtttgatgg

     1741 aagcattgtg agctcatatt tgacaacgcg catgccccca tgggccgggg tgcgtcagaa

     1801 tgtgatgggc tccagcattg atggtcgccc cgtcctgccc gcaaactcta ctaccttgac

     1861 ctacgagacc gtgtctggaa cgccgttgga gactgcagcc tccgccgccg cttcagccgc

     1921 tgcagccacc gcccgcggga ttgtgactga ctttgctttc ctgagcccgc ttgcaagcag

     1981 tgcagcttcc cgttcatccg cccgcgatga caagttgacg gctcttttgg cacaattgga

     2041 ttctttgacc cgggaactta atgtcgtttc tcagcagctg ttggatctgc gccagcaggt

     2101 ttctgccctg aaggcttcct cccctcccaa tgcggtttaa aacataaata aaaaaccaga

     2161 ctctgtttgg atttggatca agcaagtgtc ttgctgtctt tatttagggg ttttgcgcgc

     2221 gcggtaggcc cgggaccagc ggtctcggtc gttgagggtc ctgtgtattt tttccaggac

     2281 gtggtaaagg tgactctgga tgttcagata catgggcata agcccgtctc tggggtggag

     2341 gtagcaccac tgcagagctt catgctgcgg ggtggtgttg tagatgatcc agtcgtagca

     2401 ggagcgctgg gcgtggtgcc taaaaatgtc tttcagtagc aagctgattg ccaggggcag

     2461 gcccttggtg taagtgttta caaagcggtt aagctgggat gggtgcatac gtggggatat

     2521 gagatgcatc ttggactgta tttttaggtt ggctatgttc ccagccatat ccctccgggg

     2581 attcatgttg tgcagaacca ccagcacagt gtatccggtg cacttgggaa atttgtcatg

     2641 tagcttagaa ggaaatgcgt ggaagaactt ggagacgccc ttgtgacctc caagattttc

     2701 catgcattcg tccataatga tggcaatggg cccacgggcg gcggcctggg cgaagatatt

     2761 tctgggatca ctaacgtcat agttgtgttc caggatgaga tcgtcatagg ccatttttac

     2821 aaagcgcggg cggagggtgc cagactgcgg tataatggtt ccatccggcc caggggcgta

     2881 gttaccctca cagatttgca tttcccacgc tttgagttca gatgggggga tcatgtctac

     2941 ctgcggggcg atgaagaaaa cggtttccgg ggtaggggag atcagctggg aagaaagcag

     3001 gttcctgagc agctgcgact taccgcagcc ggtgggcccg taaatcacac ctattaccgg

     3061 gtgcaactgg tagttaagag agctgcagct gccgtcatcc ctgagcaggg gggccacttc

     3121 gttaagcatg tccctgactc gcatgttttc cctgaccaaa tccgccagaa ggcgctcgcc

     3181 gcccagcgat agcagttctt gcaaggaagc aaagtttttc aacggtttga gaccgtccgc

     3241 cgtaggcatg cttttgagcg tttgaccaag cagttccagg cggtcccaca gctcggtcac

     3301 ctgctctacg gcatctcgat ccagcatatc tcctcgtttc gcgggttggg gcggctttcg

     3361 ctgtacggca gtagtcggtg ctcgtccaga cgggccaggg tcatgtcttt ccacgggcgc

     3421 agggtcctcg tcagcgtagt ctgggtcacg gtgaaggggt gcgctccggg ctgcgcgctg

     3481 gccagggtgc gcttgaggct ggtcctgctg gtgctgaagc gctgccggtc ttcgccctgc

     3541 gcgtcggcca ggtagcattt gaccatggtg tcatagtcca gcccctccgc ggcgtggccc

     3601 ttggcgcgca gcttgccctt ggaggaggcg ccgcacgagg ggcagtgcag acttttgagg

     3661 gcgtagagct tgggcgcgag aaataccgat tccggggagt aggcatccgc gccgcaggcc

     3721 ccgcagacgg tctcgcattc cacgagccag gtgagctctg gccgttcggg gtcaaaaacc

     3781 aggtttcccc catgcttttt gatgcgtttc ttacctctgg tttccatgag ccggtgtcca

     3841 cgctcggtga cgaaaaggct gtccgtgtcc ccgtatacag acttgagagg cctgtcctcg

     3901 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac

     3961 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc

     4021 agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct

     4081 gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc

     4141 caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg acgcgctggg

     4201 ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta tgattcttct

     4261 cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc aggtagatga

     4321 cgaccatcag ggacagcttc aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt

     4381 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag

     4441 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc

     4501 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc

     4561 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt

     4621 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt

     4681 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc

     4741 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa

     4801 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa

     4861 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg

     4921 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga

     4981 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg

     5041 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg

     5101 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt

     5161 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact

     5221 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat

     5281 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg

     5341 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg

     5401 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat

     5461 tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc

     5521 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt

     5581 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc

     5641 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga

     5701 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc

     5761 gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa

     5821 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta

     5881 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg

     5941 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg

     6001 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat

     6061 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt

     6121 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa

     6181 aaataggcgt atcacgaggc cctttcgtct tcaagaa

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