pAcGFP1-N3

pAcGFP1-N3

pAcGFP1-N3载体基本信息

pAcGFP1-N3载体质粒图谱和多克隆位点信息

pAcGFP1-N3载体描述

pAcGFP1-N3 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-N3 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-N3 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-N3 such that it is in frame with the AcGFP1 coding sequences, with no intervening inframe 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-N3 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-N3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 GATCCATCAT GGTGAGCAAG GGCGCCGAGC TGTTCACCGG CATCGTGCCC ATCCTGATCG

  721 AGCTGAATGG CGATGTGAAT GGCCACAAGT TCAGCGTGAG CGGCGAGGGC GAGGGCGATG

  781 CCACCTACGG CAAGCTGACC CTGAAGTTCA TCTGCACCAC CGGCAAGCTG CCTGTGCCCT

  841 GGCCCACCCT GGTGACCACC CTGAGCTACG GCGTGCAGTG CTTCTCACGC TACCCCGATC

  901 ACATGAAGCA GCACGACTTC TTCAAGAGCG CCATGCCTGA GGGCTACATC CAGGAGCGCA

  961 CCATCTTCTT CGAGGATGAC GGCAACTACA AGTCGCGCGC CGAGGTGAAG TTCGAGGGCG

 1021 ATACCCTGGT GAATCGCATC GAGCTGACCG GCACCGATTT CAAGGAGGAT GGCAACATCC

 1081 TGGGCAATAA GATGGAGTAC AACTACAACG CCCACAATGT GTACATCATG ACCGACAAGG

 1141 CCAAGAATGG CATCAAGGTG AACTTCAAGA TCCGCCACAA CATCGAGGAT GGCAGCGTGC

 1201 AGCTGGCCGA CCACTACCAG CAGAATACCC CCATCGGCGA TGGCCCTGTG CTGCTGCCCG

 1261 ATAACCACTA CCTGTCCACC CAGAGCGCCC TGTCCAAGGA CCCCAACGAG AAGCGCGATC

 1321 ACATGATCTA CTTCGGCTTC GTGACCGCCG CCGCCATCAC CCACGGCATG GATGAGCTGT

 1381 ACAAGTGAGC GGCCGCGACT CTAGATCATA ATCAGCCATA CCACATTTGT AGAGGTTTTA

 1441 CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT GAATGCAATT

 1501 GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA

 1561 AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC

 1621 AATGTATCTT AAGGCGTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG CGTTAAATTT

 1681 TTGTTAAATC AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC CTTATAAATC

 1741 AAAAGAATAG ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA GTCCACTATT

 1801 AAAGAACGTG GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG ATGGCCCACT

 1861 ACGTGAACCA TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG CACTAAATCG

 1921 GAACCCTAAA GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA ACGTGGCGAG

 1981 AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC

 2041 GCTGCGCGTA ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG CGTCAGGTGG

 2101 CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA TACATTCAAA

 2161 TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA

 2221 GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG AATGTGTGTC AGTTAGGGTG TGGAAAGTCC

 2281 CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCAGG

 2341 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG

 2401 TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC GCCCAGTTCC

 2461 GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC CGAGGCCGCC

 2521 TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC

 2581 AAAGATCGAT CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG ATGGATTGCA

 2641 CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC

 2701 AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT

 2761 TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAA GACGAGGCAG CGCGGCTATC

 2821 GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG

 2881 AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC

 2941 TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA CGCTTGATCC

 3001 GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC GTACTCGGAT

 3061 GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC

 3121 CGAACTGTTC GCCAGGCTCA AGGCGAGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA

 3181 TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA

 3241 CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA CCCGTGATAT

 3301 TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC

 3361 TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GAGCGGGACT

 3421 CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGAGA TTTCGATTCC

 3481 ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC CGGCTGGATG

 3541 ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCTAGGGG GAGGCTAACT

 3601 GAAACACGGA AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT AAAAAGACAG

 3661 AATAAAACGC ACGGTGTTGG GTCGTTTGTT CATAAACGCG GGGTTCGGTC CCAGGGCTGG

 3721 CACTCTGTCG ATACCCCACC GAGACCCCAT TGGGGCCAAT ACGCCCGCGT TTCTTCCTTT

 3781 TCCCCACCCC ACCCCCCAAG TTCGGGTGAA GGCCCAGGGC TCGCAGCCAA CGTCGGGGCG

 3841 GCAGGCCCTG CCATAGCCTC AGGTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT

 3901 TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT

 3961 TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA AGGATCTTCT

 4021 TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA

 4081 GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC

 4141 AGCAGAGCGC AGATACCAAA TACTGTTCTT CTAGTGTAGC CGTAGTTAGG CCACCACTTC

 4201 AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT

 4261 GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG

 4321 GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC

 4381 TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG

 4441 AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG

 4501 CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA CCTCTGACTT

 4561 GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA CGCCAGCAAC

 4621 GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG

 4681 TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCATGC AT

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