pAcGFP1-Mem

pAcGFP1-Mem

pAcGFP1-Mem载体基本信息

pAcGFP1-Mem载体质粒图谱和多克隆位点信息

pAcGFP1-Mem载体描述

pAcGFP1-Mem encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43 (1), and the Aequorea coerulescens green fluorescent protein AcGFP1. (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 (2). The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets AcGFP1 to cellular membranes, and the plasma membrane in particular. Expression of AcGFP1-Mem is driven by the immediate early promoter of CMV (PCMV IE). The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

pAcGFP1-Mem can be transfected into mammalian cells using any standard method. If required, stable transformants can be selected using G418 (3). Expression of AcGFP1-Mem in mammalian cells results in strong labeling of the plasma membrane and allows easy tracking of individual cells in a population. This membrane labeling also permits the study of fine cellular processes such as neuronal axons (4), leading edges of migrating cells, filopodia, or microvilli on cell surfaces. pAc- GFP1-Mem cannot be used as an exclusive plasma membrane marker because it also partially labels intracellular membranes.

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 (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: high

References

1. Skene, J. H. P. & Virag, I. (1989) J. Cell. Biol. 108:613–625.

2. Haas, J., et al. (1996) Curr. Biol. 6:315–324.

3. Gorman, C. (1985) In DNA cloning: a practical approach, vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.

4. Moriyoshi, K., et al. (1996) Neuron 16:255–260.

pAcGFP1-Mem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 TCGCCACCAT GCTGTGCTGT ATGAGAAGAA CCAAACAGGT TGAAAAGAAT

      721 GATGAGGACC AAAAGATCAG CAAGGGCGCC GAGCTGTTCA CCGGCATCGT GCCCATCCTG

      781 ATCGAGCTGA ATGGCGATGT GAATGGCCAC AAGTTCAGCG TGAGCGGCGA GGGCGAGGGC

      841 GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCTGTG

      901 CCCTGGCCCA CCCTGGTGAC CACCCTGAGC TACGGCGTGC AGTGCTTCTC ACGCTACCCC

      961 GATCACATGA AGCAGCACGA CTTCTTCAAG AGCGCCATGC CTGAGGGCTA CATCCAGGAG

     1021 CGCACCATCT TCTTCGAGGA TGACGGCAAC TACAAGTCGC GCGCCGAGGT GAAGTTCGAG

     1081 GGCGATACCC TGGTGAATCG CATCGAGCTG ACCGGCACCG ATTTCAAGGA GGATGGCAAC

     1141 ATCCTGGGCA ATAAGATGGA GTACAACTAC AACGCCCACA ATGTGTACAT CATGACCGAC

     1201 AAGGCCAAGA ATGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGATGGCAGC

     1261 GTGCAGCTGG CCGACCACTA CCAGCAGAAT ACCCCCATCG GCGATGGCCC TGTGCTGCTG

     1321 CCCGATAACC ACTACCTGTC CACCCAGAGC GCCCTGTCCA AGGACCCCAA CGAGAAGCGC

     1381 GATCACATGA TCTACTTCGG CTTCGTGACC GCCGCCGCCA TCACCCACGG CATGGATGAG

     1441 CTGTACAAGT GAAGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACA TTTGTAGAGG

     1501 TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT AAAATGAATG

     1561 CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA AGCAATAGCA

     1621 TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT TTGTCCAAAC

     1681 TCATCAATGT ATCTTAAGGC GTAAATTGTA AGCGTTAATA TTTTGTTAAA ATTCGCGTTA

     1741 AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCCTTAT

     1801 AAATCAAAAG AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA CAAGAGTCCA

     1861 CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA GGGCGATGGC

     1921 CCACTACGTG AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCG TAAAGCACTA

     1981 AATCGGAACC CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC GGCGAACGTG

     2041 GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC AAGTGTAGCG

     2101 GTCACGCTGC GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA GGGCGCGTCA

     2161 GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT

     2221 TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA

     2281 AGGAAGAGTC CTGAGGCGGA AAGAACCAGC TGTGGAATGT GTGTCAGTTA GGGTGTGGAA

     2341 AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA

     2401 CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA

     2461 ATTAGTCAGC AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA

     2521 GTTCCGCCCA TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA GAGGCCGAGG

     2581 CCGCCTCGGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT

     2641 TTTGCAAAGA TCGATATGAA AAAGCCTGAA CTCACCGCGA CGTCTGTCGA GAAGTTTCTG

     2701 ATCGAAAAGT TCGACAGCGT CTCCGACCTG ATGCAGCTCT CGGAGGGCGA AGAATCTCGT

     2761 GCTTTCAGCT TCGATGTAGG AGGGCGTGGA TATGTCCTGC GGGTAAATAG CTGCGCCGAT

     2821 GGTTTCTACA AAGATCGTTA TGTTTATCGG CACTTTGCAT CGGCCGCGCT CCCGATTCCG

     2881 GAAGTGCTTG ACATTGGGGA ATTCAGCGAG AGCCTGACCT ATTGCATCTC CCGCCGTGCA

     2941 CAGGGTGTCA CGTTGCAAGA CCTGCCTGAA ACCGAACTGC CCGCTGTTCT GCAGCCGGTC

     3001 GCGGAGGCCA TGGATGCGAT CGCTGCGGCC GATCTTAGCC AGACGAGCGG GTTCGGCCCA

     3061 TTCGGACCGC AAGGAATCGG TCAATACACT ACATGGCGTG ATTTCATATG CGCGATTGCT

     3121 GATCCCCATG TGTATCACTG GCAAACTGTG ATGGACGACA CCGTCAGTGC GTCCGTCGCG

     3181 CAGGCTCTCG ATGAGCTGAT GCTTTGGGCC GAGGACTGCC CCGAAGTCCG GCACCTCGTG

     3241 CACGCGGATT TCGGCTCCAA CAATGTCCTG ACGGACAATG GCCGCATAAC AGCGGTCATT

     3301 GACTGGAGCG AGGCGATGTT CGGGGATTCC CAATACGAGG TCGCCAACAT CTTCTTCTGG

     3361 AGGCCGTGGT TGGCTTGTAT GGAGCAGCAG ACGCGCTACT TCGAGCGGAG GCATCCGGAG

     3421 CTTGCAGGAT CGCCGCGGCT CCGGGCGTAT ATGCTCCGCA TTGGTCTTGA CCAACTCTAT

     3481 CAGAGCTTGG TTGACGGCAA TTTCGATGAT GCAGCTTGGG CGCAGGGTCG ATGCGACGCA

     3541 ATCGTCCGAT CCGGAGCCGG GACTGTCGGG CGTACACAAA TCGCCCGCAG AAGCGCGGCC

     3601 GTCTGGACCG ATGGCTGTGT AGAAGTACTC GCCGATAGTG GAAACCGACG CCCCAGCACT

     3661 CGTCCGAGGG CAAAGGAATA GAGTTCTAGA GGATCATAAT CAGCCATACC ACATTTGTAG

     3721 AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA

     3781 ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTGTAA TGGTTACAAA TAAAGCAATA

     3841 GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCC TCGCGATCCA CAACCATGGC

     3901 GAAAGGGCCT CGTGATACGC CTATTTTTAT AGGTTAATGT CATGATAATA ATGGTTTCTT

     3961 AGACGTCAGG TGGCACTTTT CGGGGAAATG TGCGCGGAAC CCCTATTTGT TTATTTTTCT

     4021 AAATACATTC AAATATGTAT CCGCTCATGA GACAATAACC CTGATAAATG CTTCAATAAT

     4081 ATTGAAAAAG GAAGAGTATG AGTATTCAAC ATTTCCGTGT CGCCCTTATT CCCTTTTTTG

     4141 CGGCATTTTG CCTTCCTGTT TTTGCTCACC CAGAAACGCT GGTGAAAGTA AAAGATGCTG

     4201 AAGATCAGTT GGGTGCACGA GTGGGTTACA TCGAACTGGA TCTCAACAGC GGTAAGATCC

     4261 TTGAGAGTTT TCGCCCCGAA GAACGTTTTC CAATGATGAG CACTTTTAAA GTTCTGCTAT

     4321 GTGGCGCGGT ATTATCCCGT GTTGACGCCG GGCAAGAGCA ACTCGGTCGC CGCATACACT

     4381 ATTCTCAGAA TGACTTGGTT GAGTACTCAC CAGTCACAGA AAAGCATCTT ACGGATGGCA

     4441 TGACAGTAAG AGAATTATGC AGTGCTGCCA TAACCATGAG TGATAACACT GCGGCCAACT

     4501 TACTTCTGAC AACGATCGGA GGACCGAAGG AGCTAACCGC TTTTTTGCAC AACATGGGGG

     4561 ATCATGTAAC TCGCCTTGAT CGTTGGGAAC CGGAGCTGAA TGAAGCCATA CCAAACGACG

     4621 AGCGTGACAC CACGATGCCT GTAGCAATGG CAACAACGTT GCGCAAACTA TTAACTGGCG

     4681 AACTACTTAC TCTAGCTTCC CGGCAACAAT TAATAGACTG GATGGAGGCG GATAAAGTTG

     4741 CAGGACCACT TCTGCGCTCG GCCCTTCCGG CTGGCTGGTT TATTGCTGAT AAATCTGGAG

     4801 CCGGTGAGCG TGGGTCTCGC GGTATCATTG CAGCACTGGG GCCAGATGGT AAGCCCTCCC

     4861 GTATCGTAGT TATCTACACG ACGGGGAGTC AGGCAACTAT GGATGAACGA AATAGACAGA

     4921 TCGCTGAGAT AGGTGCCTCA CTGATTAAGC ATTGGTAAGG CGCGCCAGGC CCTGCCATAG

     4981 CCTCAGGTTA CTCATATATA CTTTAGATTG ATTTAAAACT TCATTTTTAA TTTAAAAGGA

     5041 TCTAGGTGAA GATCCTTTTT GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT

     5101 TCCACTGAGC GTCAGACCCC GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTC

     5161 TGCGCGTAAT CTGCTGCTTG CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC

     5221 CGGATCAAGA GCTACCAACT CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATAC

     5281 CAAATACTGT TCTTCTAGTG TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC

     5341 CGCCTACATA CCTCGCTCTG CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGT

     5401 CGTGTCTTAC CGGGTTGGAC TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT

     5461 GAACGGGGGG TTCGTGCACA CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGAT

     5521 ACCTACAGCG TGAGCTATGA GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT

     5581 ATCCGGTAAG CGGCAGGGTC GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACG

     5641 CCTGGTATCT TTATAGTCCT GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT

     5701 GATGCTCGTC AGGGGGGCGG AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGT

     5761 TCCTGGCCTT TTGCTGGCCT TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG

     5821 TGGATAACCG TATTACCGCC ATGCAT

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