pAcGFP1-Hyg-C1

pAcGFP1-Hyg-C1

pAcGFP1-Hyg-C1载体基本信息

pAcGFP1-Hyg-C1载体质粒图谱和多克隆位点信息

pAcGFP1-Hyg-C1载体描述

pAcGFP1-Hyg-C1 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-Hyg-C1 is located downstream of the AcGFP1 coding region, allowing the construction of a C-terminal fusion protein with AcGFP1 when genes are cloned 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 backbonealso contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A hygromycin resistance cassette (Hygr), consisting of the SV40 early promoter, the hygromycin resistance gene, and SV40 polyadenylation signals, allows stably transfected eukaryotic cells to be selected using hygromycin. A bacterial promoter upstream of the ampicillin gene expresses ampicillin resistance in E. coli. The pAcGFP1-Hyg-C1 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 C 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-Hyg-C1 so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using hygromycin. pAcGFP1-Hyg-C1 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 and other general purpose strains.

 Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

Reference

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

pAcGFP1-Hyg-C1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 CCGGTCGCCA CCATGGTGAG CAAGGGCGCC GAGCTGTTCA CCGGCATCGT GCCCATCCTG

      661 ATCGAGCTGA ATGGCGATGT GAATGGCCAC AAGTTCAGCG TGAGCGGCGA GGGCGAGGGC

      721 GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCTGTG

      781 CCCTGGCCCA CCCTGGTGAC CACCCTGAGC TACGGCGTGC AGTGCTTCTC ACGCTACCCC

      841 GATCACATGA AGCAGCACGA CTTCTTCAAG AGCGCCATGC CTGAGGGCTA CATCCAGGAG

      901 CGCACCATCT TCTTCGAGGA TGACGGCAAC TACAAGTCGC GCGCCGAGGT GAAGTTCGAG

      961 GGCGATACCC TGGTGAATCG CATCGAGCTG ACCGGCACCG ATTTCAAGGA GGATGGCAAC

     1021 ATCCTGGGCA ATAAGATGGA GTACAACTAC AACGCCCACA ATGTGTACAT CATGACCGAC

     1081 AAGGCCAAGA ATGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGATGGCAGC

     1141 GTGCAGCTGG CCGACCACTA CCAGCAGAAT ACCCCCATCG GCGATGGCCC TGTGCTGCTG

     1201 CCCGATAACC ACTACCTGTC CACCCAGAGC GCCCTGTCCA AGGACCCCAA CGAGAAGCGC

     1261 GATCACATGA TCTACTTCGG CTTCGTGACC GCCGCCGCCA TCACCCACGG CATGGATGAG

     1321 CTGTACAAGT CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC

     1381 GCGGGCCCGG GATCCACCGG ATCTAGATAA CTGATCATAA TCAGCCATAC CACATTTGTA

     1441 GAGGTTTTAC TTGCTTTAAA AAACCTCCCA CACCTCCCCC TGAACCTGAA ACATAAAATG

     1501 AATGCAATTG TTGTTGTTAA CTTGTTTATT GCAGCTTATA ATGGTTACAA ATAAAGCAAT

     1561 AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG TGGTTTGTCC

     1621 AAACTCATCA ATGTATCTTA ACGCGTAAAT TGTAAGCGTT AATATTTTGT TAAAATTCGC

     1681 GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC

     1741 TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG

     1801 TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA

     1861 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC

     1921 ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGAA

     1981 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT

     2041 AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC

     2101 GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT

     2161 ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG

     2221 AAAAAGGAAG AGTCCTGAGG CGGAAAGAAC CAGCTGTGGA ATGTGTGTCA GTTAGGGTGT

     2281 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA

     2341 GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT

     2401 CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG

     2461 CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC

     2521 GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA

     2581 GGCTTTTGCA AAGATCGATA TGAAAAAGCC TGAACTCACC GCGACGTCTG TCGAGAAGTT

     2641 TCTGATCGAA AAGTTCGACA GCGTCTCCGA CCTGATGCAG CTCTCGGAGG GCGAAGAATC

     2701 TCGTGCTTTC AGCTTCGATG TAGGAGGGCG TGGATATGTC CTGCGGGTAA ATAGCTGCGC

     2761 CGATGGTTTC TACAAAGATC GTTATGTTTA TCGGCACTTT GCATCGGCCG CGCTCCCGAT

     2821 TCCGGAAGTG CTTGACATTG GGGAATTCAG CGAGAGCCTG ACCTATTGCA TCTCCCGCCG

     2881 TGCACAGGGT GTCACGTTGC AAGACCTGCC TGAAACCGAA CTGCCCGCTG TTCTGCAGCC

     2941 GGTCGCGGAG GCCATGGATG CGATCGCTGC GGCCGATCTT AGCCAGACGA GCGGGTTCGG

     3001 CCCATTCGGA CCGCAAGGAA TCGGTCAATA CACTACATGG CGTGATTTCA TATGCGCGAT

     3061 TGCTGATCCC CATGTGTATC ACTGGCAAAC TGTGATGGAC GACACCGTCA GTGCGTCCGT

     3121 CGCGCAGGCT CTCGATGAGC TGATGCTTTG GGCCGAGGAC TGCCCCGAAG TCCGGCACCT

     3181 CGTGCACGCG GATTTCGGCT CCAACAATGT CCTGACGGAC AATGGCCGCA TAACAGCGGT

     3241 CATTGACTGG AGCGAGGCGA TGTTCGGGGA TTCCCAATAC GAGGTCGCCA ACATCTTCTT

     3301 CTGGAGGCCG TGGTTGGCTT GTATGGAGCA GCAGACGCGC TACTTCGAGC GGAGGCATCC

     3361 GGAGCTTGCA GGATCGCCGC GGCTCCGGGC GTATATGCTC CGCATTGGTC TTGACCAACT

     3421 CTATCAGAGC TTGGTTGACG GCAATTTCGA TGATGCAGCT TGGGCGCAGG GTCGATGCGA

     3481 CGCAATCGTC CGATCCGGAG CCGGGACTGT CGGGCGTACA CAAATCGCCC GCAGAAGCGC

     3541 GGCCGTCTGG ACCGATGGCT GTGTAGAAGT ACTCGCCGAT AGTGGAAACC GACGCCCCAG

     3601 CACTCGTCCG AGGGCAAAGG AATAGAGTTC TAGAGGATCA TAATCAGCCA TACCACATTT

     3661 GTAGAGGTTT TACTTGCTTT AAAAAACCTC CCACACCTCC CCCTGAACCT GAAACATAAA

     3721 ATGAATGCAA TTGTTGTTGT TAACTTGTTT ATTGCAGCTT ATAATGGTTA CAAATAAAGC

     3781 AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCCTCGCGA TCCACAACCA

     3841 TGGCGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA ATGTCATGAT AATAATGGTT

     3901 TCTTAGACGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG GAACCCCTAT TTGTTTATTT

     3961 TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA

     4021 TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC GTGTCGCCCT TATTCCCTTT

     4081 TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA CGCTGGTGAA AGTAAAAGAT

     4141 GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC TGGATCTCAA CAGCGGTAAG

     4201 ATCCTTGAGA GTTTTCGCCC CGAAGAACGT TTTCCAATGA TGAGCACTTT TAAAGTTCTG

     4261 CTATGTGGCG CGGTATTATC CCGTGTTGAC GCCGGGCAAG AGCAACTCGG TCGCCGCATA

     4321 CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA CAGAAAAGCA TCTTACGGAT

     4381 GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA TGAGTGATAA CACTGCGGCC

     4441 AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA CCGCTTTTTT GCACAACATG

     4501 GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC TGAATGAAGC CATACCAAAC

     4561 GACGAGCGTG ACACCACGAT GCCTGTAGCA ATGGCAACAA CGTTGCGCAA ACTATTAACT

     4621 GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG ACTGGATGGA GGCGGATAAA

     4681 GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT GGTTTATTGC TGATAAATCT

     4741 GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC TGGGGCCAGA TGGTAAGCCC

     4801 TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA CTATGGATGA ACGAAATAGA

     4861 CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT AAGGCGCGCC AGGCCCTGCC

     4921 ATAGCCTCAG GTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA

     4981 AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT

     5041 TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT

     5101 TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT

     5161 TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG

     5221 ATACCAAATA CTGTTCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA

     5281 GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT

     5341 AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG

     5401 GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG

     5461 AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC

     5521 AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA

     5581 AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT

     5641 TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA

     5701 CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT

     5761 TCTGTGGATA ACCGTATTAC CGCCATGCAT

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