pAcGFP1-Lam

pAcGFP1-Lam载体基本信息

pAcGFP1-Lam载体质粒图谱和多克隆位点信息

pAcGFP1-Lam载体描述

pAcGFP1-Lamin encodes a green fluorescent protein (GFP) from Aequorea coerulescens (excitation maximum = 475 nm; emission maximum = 505 nm) and the gene encoding the human nuclear lamin (1). SV40 polyadenylation signals downstream of the AcGFP1-Lamin fusion direct proper processing of the 3’end of the AcGFP1-Lamin mRNA. AcGFP1 contains silent mutations that create an open reading frame comprised almost entirely of optimized human codons. These changes increase the translational efficiency of the AcGFP1 mRNA and consequently the expression of AcGFP1 in mammalian and plant cells. The vector backbone also contains an SV40 origin for replication in any mammalian cell line that expresses 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 this cassette drives expression of the gene encoding kanamycin resistance in E. coli. The pAcGFP1-Lamin backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

The pAcGFP1-Lamin is provided in the Matchmaker Chemiluminescent Co-IP Vector Set (Cat. No. 630458) as a negative bait control construct. In mammalian cells, this vector expresses the AcGFP1-Lamin fusion protein—the AcGFP1 serves as a tag, through which a polyclonal antibody against AcGFP1 can be used for immunoprecipitation, while lamin serves as the bait for the capture of interacting proteins via coimmunoprecipitation. Because lamin does not interact with SV40 large T antigen (2), in lysates of cells cotransfected with pAcGFP1-Lamin and pProLabel-T, there is no ProLabel activity detected in the Co-IP of this control for noninteracting pairs of proteins. The fluorescence from AcGFP1-Lamin fusion protein also allows for easy detection of fusion expression and localization in vivo, as well as offering a mode for determining transfection efficiency.

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: ~500

 Plasmid incompatibility group: pMB1/ColE1

References

1. Sturman, N. et al. (1998) J. Stuct. Biol. 122(1–2) 42–66.

2. Matchmaker Co-IP Kit (April 1999) Clontechniques XIV(2): 14–15.

3. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II. Ed. D.M. Glover. (IRL Press, Oxford, UK) pp. 143–190.

pAcGFP1-Lam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 CCGGACTTAA GGCCTCTGTC GACGCCGCCG AGGAGCAGAA GCTGATCTCA

     1381 GAGGAGGACC TGCATATGGC CATGGAGGCC GAATTCCCGG GGATCTCTGA AGAGGTGGTC

     1441 AGCCGCGAGG TGTCCGGCAT CAAGGCCGCC TACGAGGCCG AGCTCGGGGA TGCCCGCAAG

     1501 ACCCTTGACT CAGTAGCCAA GGAGCGCGCC CGCCTGCAGC TGGAGCTGAG CAAAGTGCGT

     1561 GAGGAGTTTA AGGAGCTGAA AGCGCGCAAT ACCAAGAAGG AGGGTGACCT GATAGCTGCT

     1621 CAGGCTCGGC TGAAGGACCT GGAGGCTCTG CTGAACTCCA AGGAGGCCGC ACTGAGCACT

     1681 GCTCTCAGTG AGAAGCGCAC GCTGGAGGGC GAGCTGCATG ATCTGCGGGG CCAGGTGGCC

     1741 AAGCTTGAGG CAGCCCTAGG TGAGGCCAAG AAGCAACTTC AGGATGAGAT GCTGCGGCGG

     1801 GTGGATGCTG AGAACAGGCT GCAGACCATG AAGGAGGAAC TGGACTTCCA GAAGAACATC

     1861 TACAGTGAGG AGCTGCGTGA GACCAAGCGC CGTCATGAGA CCCGACTGGT GGAGATTGAC

     1921 AGGATCCGTC GACCTGCAGC GGCCGCATAA CTAGCATAAC CCCTTGGGGC CTCTAAACGG

     1981 GTCTTGAGGG GTTTTTTGCG CGCTTGCAGC CAAGCTAATT CCGGGCGAAT TTCTTATGAT

     2041 TTATGATTTT TATTATTAAA TAAGTTATAA AAAAAATAAG TGTATACAAA TTTTAAAGTG

     2101 ACAAGCTTGC GAATTCTGGC GGATCCACCG GATCTAGATA ACTGATCATA ATCAGCCATA

     2161 CCACATTTGT AGAGGTTTTA CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA

     2221 AACATAAAAT GAATGCAATT GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA

     2281 AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT

     2341 GTGGTTTGTC CAAACTCATC AATGTATCTT AACGCGTAAA TTGTAAGCGT TAATATTTTG

     2401 TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT TTAACCAATA GGCCGAAATC

     2461 GGCAAAATCC CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT TGTTCCAGTT

     2521 TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG TCAAAGGGCG AAAAACCGTC

     2581 TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTTTTTT GGGGTCGAGG

     2641 TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC GATTTAGAGC TTGACGGGGA

     2701 AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG

     2761 CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT TAATGCGCCG

     2821 CTACAGGGCG CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA

     2881 TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT

     2941 CAATAATATT GAAAAAGGAA GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG AATGTGTGTC

     3001 AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC

     3061 TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC

     3121 AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC

     3181 CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT

     3241 ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT

     3301 TTGGAGGCCT AGGCTTTTGC AAAGATCGAT CAAGAGACAG GATGAGGATC GTTTCGCATG

     3361 ATTGAACAAG ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC

     3421 TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG

     3481 CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAA

     3541 GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC

     3601 GACGTTGTCA CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT

     3661 CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG

     3721 CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC

     3781 GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG

     3841 CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGAGCAT GCCCGACGGC

     3901 GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC

     3961 CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA

     4021 GCGTTGGCTA CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC

     4081 GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC

     4141 GAGTTCTTCT GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC

     4201 CATCACGAGA TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT

     4261 TCCGGGACGC CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC

     4321 ACCCTAGGGG GAGGCTAACT GAAACACGGA AGGAGACAAT ACCGGAAGGA ACCCGCGCTA

     4381 TGACGGCAAT AAAAAGACAG AATAAAACGC ACGGTGTTGG GTCGTTTGTT CATAAACGCG

     4441 GGGTTCGGTC CCAGGGCTGG CACTCTGTCG ATACCCCACC GAGACCCCAT TGGGGCCAAT

     4501 ACGCCCGCGT TTCTTCCTTT TCCCCACCCC ACCCCCCAAG TTCGGGTGAA GGCCCAGGGC

     4561 TCGCAGCCAA CGTCGGGGCG GCAGGCCCTG CCATAGCCTC AGGTTACTCA TATATACTTT

     4621 AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA

     4681 ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG

     4741 AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA

     4801 CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT

     4861 TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTTCTT CTAGTGTAGC

     4921 CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA

     4981 TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA

     5041 GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC

     5101 CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA

     5161 GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA

     5221 CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG

     5281 GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC

     5341 TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG

     5401 CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCATGC

     5461 AT

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