pAcGFP1-N2

pAcGFP1-N2

pAcGFP1-N2载体基本信息

pAcGFP1-N2载体质粒图谱和多克隆位点信息

pAcGFP1-N2载体描述

pAcGFP1-N2 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-N2 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-N2 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-N2 such that it is in frame with the AcGFP1 coding sequences and contains 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-N2 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 JM109 or XL1-Blue.

 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ≈500

 Plasmid incompatibility group: pMB1/ColE1

pAcGFP1-N2载体序列

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 CCGGTCATGG TGAGCAAGGG CGCCGAGCTG TTCACCGGCA TCGTGCCCAT

  721 CCTGATCGAG CTGAATGGCG ATGTGAATGG CCACAAGTTC AGCGTGAGCG GCGAGGGCGA

  781 GGGCGATGCC ACCTACGGCA AGCTGACCCT GAAGTTCATC TGCACCACCG GCAAGCTGCC

  841 TGTGCCCTGG CCCACCCTGG TGACCACCCT GAGCTACGGC GTGCAGTGCT TCTCACGCTA

  901 CCCCGATCAC ATGAAGCAGC ACGACTTCTT CAAGAGCGCC ATGCCTGAGG GCTACATCCA

  961 GGAGCGCACC ATCTTCTTCG AGGATGACGG CAACTACAAG TCGCGCGCCG AGGTGAAGTT

 1021 CGAGGGCGAT ACCCTGGTGA ATCGCATCGA GCTGACCGGC ACCGATTTCA AGGAGGATGG

 1081 CAACATCCTG GGCAATAAGA TGGAGTACAA CTACAACGCC CACAATGTGT ACATCATGAC

 1141 CGACAAGGCC AAGAATGGCA TCAAGGTGAA CTTCAAGATC CGCCACAACA TCGAGGATGG

 1201 CAGCGTGCAG CTGGCCGACC ACTACCAGCA GAATACCCCC ATCGGCGATG GCCCTGTGCT

 1261 GCTGCCCGAT AACCACTACC TGTCCACCCA GAGCGCCCTG TCCAAGGACC CCAACGAGAA

 1321 GCGCGATCAC ATGATCTACT TCGGCTTCGT GACCGCCGCC GCCATCACCC ACGGCATGGA

 1381 TGAGCTGTAC AAGTGAGCGG CCGCGACTCT AGATCATAAT CAGCCATACC ACATTTGTAG

 1441 AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA

 1501 ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA

 1561 GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA

 1621 AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA ATATTTTGTT AAAATTCGCG

 1681 TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG CCGAAATCGG CAAAATCCCT

 1741 TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT

 1801 CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT

 1861 GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA

 1921 CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC

 1981 GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA

 2041 GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG

 2101 TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT TTTCTAAATA

 2161 CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA ATAATATTGA

 2221 AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA TGTGTGTCAG TTAGGGTGTG

 2281 GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG

 2341 CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC

 2401 TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC CATCCCGCCC CTAACTCCGC

 2461 CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT TTTTATTTAT GCAGAGGCCG

 2521 AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG AGGCTTTTTT GGAGGCCTAG

 2581 GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT TTCGCATGAT TGAACAAGAT

 2641 GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC TATTCGGCTA TGACTGGGCA

 2701 CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC TGTCAGCGCA GGGGCGCCCG

 2761 GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG AACTGCAAGA CGAGGCAGCG

 2821 CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG CTGTGCTCGA CGTTGTCACT

 2881 GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG GGCAGGATCT CCTGTCATCT

 2941 CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG CAATGCGGCG GCTGCATACG

 3001 CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC ATCGCATCGA GCGAGCACGT

 3061 ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG ACGAAGAGCA TCAGGGGCTC

 3121 GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC CCGACGGCGA GGATCTCGTC

 3181 GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG AAAATGGCCG CTTTTCTGGA

 3241 TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC AGGACATAGC GTTGGCTACC

 3301 CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC GCTTCCTCGT GCTTTACGGT

 3361 ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC TTCTTGACGA GTTCTTCTGA

 3421 GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CAACCTGCCA TCACGAGATT

 3481 TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG

 3541 GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCTAGGGGGA

 3601 GGCTAACTGA AACACGGAAG GAGACAATAC CGGAAGGAAC CCGCGCTATG ACGGCAATAA

 3661 AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA TAAACGCGGG GTTCGGTCCC

 3721 AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG GGGCCAATAC GCCCGCGTTT

 3781 CTTCCTTTTC CCCACCCCAC CCCCCAAGTT CGGGTGAAGG CCCAGGGCTC GCAGCCAACG

 3841 TCGGGGCGGC AGGCCCTGCC ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA

 3901 AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA

 3961 AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG

 4021 GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC

 4081 CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA

 4141 CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTTCTTCT AGTGTAGCCG TAGTTAGGCC

 4201 ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG

 4261 TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC

 4321 CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC

 4381 GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC

 4441 CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA

 4501 CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC

 4561 TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG

 4621 CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT

 4681 TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCATGCAT

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