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基本信息

質(zhì)粒圖譜和多克隆位點信息 

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載體描述

pAcGFP1-N1 encodes a green fluorescent protein (GFP) from Aequorea coerulescens (excitation maximum = 475 nm; emission maximum = 505 nm). The coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (1). The MCS in pAcGFP1-N1 is between the immediate early promoter of CMV (PCMV IE) and the AcGFP1 coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of AcGFP1 if they are in the same reading frame as AcGFP1 and there are no intervening stop codons. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of the gene expresses kanamycin resistance in E. coli. The pAcGFP1-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

Fusions to the N terminus of AcGFP1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pAcGFP1-N1 so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (2). pAcGFP1-N1 can also be used simply to express AcGFP1 in a cell line of interest (e.g., as a transfection marker).

Propagation in E. coli

 Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires

a host containing an F plasmid such as JM101 or XL1-Blue.

 Selectable marker: plasmid confers resistance to kanamycin (30 μg/ml) to E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ≈500

 Plasmid incompatibility group: pMB1/ColE1

載體序列

ORIGIN

        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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg

      661 gatccaccgg tcatggtgag caagggcgcc gagctgttca ccggcatcgt gcccatcctg

      721 atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga gggcgagggc

      781 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcctgtg

      841 ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc acgctacccc

      901 gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta catccaggag

      961 cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt gaagttcgag

     1021 ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga ggatggcaac

     1081 atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat catgaccgac

     1141 aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga ggatggcagc

     1201 gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc tgtgctgctg

     1261 cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa cgagaagcgc

     1321 gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg catggatgag

     1381 ctgtacaagt gagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt

     1441 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc

     1501 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat

     1561 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact

     1621 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa

     1681 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata

     1741 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac

     1801 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc

     1861 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa

     1921 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg

     1981 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg

     2041 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag

     2101 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt

     2161 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa

     2221 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa

     2281 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac

     2341 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa

     2401 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag

     2461 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc

     2521 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt

     2581 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat

     2641 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac

     2701 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc

     2761 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc

     2821 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag

     2881 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc

     2941 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg

     3001 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc

     3061 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc

     3121 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga

     3181 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca

     3241 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg

     3301 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg

     3361 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg

     3421 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga

     3481 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg

     3541 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct

     3601 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag

     3661 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg

     3721 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc

     3781 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg

     3841 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact

     3901 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat

     3961 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc

     4021 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct

     4081 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg

     4141 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca

     4201 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc

     4261 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga

     4321 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac

     4381 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga

     4441 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag

     4501 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg

     4561 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag

     4621 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc

     4681 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat

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