pAcGFP1-p53

pAcGFP1-p53

pAcGFP1-p53载体基本信息

pAcGFP1-p53载体描述

pAcGFP1-p53 Vector encodes a green fluorescent protein (GFP) from Aequorea coerulescens (excitation maximum = 475 nm; emission maximum = 505 nm) and the gene encoding the human tumor suppressor p53 (ref.). SV40 polyadenylation signals downstream of the AcGFP1- p53 fusion direct proper processing of the 3’end of the AcGFP1-p53 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-p53 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

The pAcGFP1-p53 is provided in the Matchmaker Chemiluminescent Co-IP Vector Set as a positive bait control construct. In mammalian cells, this vector expresses the AcGFP1-p53 fusion protein—AcGFP1 serves as a tag, through which a polyclonal antibody against AcGFP1 can be used for immunoprecipitation, while p53 serves as the bait for the capture of interacting proteins via coimmunoprecipitation. Because p53 interacts with SV40 large T (1), in lysates of cells cotransfected with pAcGFP1-p53 and ProLabel-T, there is a high level of ProLabel activity detected in the Co-IP of this positive control for interacting pairs of proteins. The fluorescence from AcGFP1-p53 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

pAcGFP1-p53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 GAATTCCCTG TCACCGAGAC CCCTGGGCCA

     1441 GTGGCCCCTG CCCCAGCCAC TCCATGGCCC CTGTCATCTT TTGTCCCTTC TCAAAAAACT

     1501 TACCAGGGCA ACTATGGCTT CCACCTGGGC TTCCTGCAGT CTGGGACAGC CAAGTCTGTT

     1561 ATGTGCACGT ACTCTCCTCC CCTCAATAAG CTATTCTGCC AGCTGGCGAA GACGTGCCCT

     1621 GTGCAGTTGT GGGTCAGCGC CACACCTCCA GCTGGGAGCC GTGTCCGCGC CATGGCCATC

     1681 TACAAGAAGT CACAGCACAT GACGGAGGTC GTGAGACGCT GCCCCCACCA TGAGCGCTGC

     1741 TCCGATGGTG ATGGCCTGGC TCCTCCCCAG CATCTTATCC GGGTGGAAGG AAATTTGTAT

     1801 CCCGAGTATC TGGAAGACAG GCAGACTTTT CGCCACAGCG TGGTGGTACC TTATGAGCCA

     1861 CCCGAGGCCG GCTCTGAGTA TACCACCATC CACTACAAGT ACATGTGTAA TAGCTCCTGC

     1921 ATGGGGGGCA TGAACCGCCG ACCTATCCTT ACCATCATCA CACTGGAAGA CTCCAGTGGG

     1981 AACCTTCTGG GACGGGACAG CTTTGAGGTT CGTGTTTGTG CCTGCCCTGG GAGAGACCGC

     2041 CGTACAGAAG AAGAAAATTT CCGCAAAAAG GAAGTCCTTT GCCCTGAACT GCCCCCAGGG

     2101 AGCGCAAAGA GAGCGCTGCC CACCTGCACA AGCGCCTCTC CCCCGCAAAA GAAAAAACCA

     2161 CTTGATGGAG AGTATTTCAC CCTCAAGATC CGCGGGCGTA AACGCTTCGA GATGTTCCGG

     2221 GAGCTGAATG AGGCCTTAGA GTTAAAGGAT GCCCATGCTA CAGAGGAGTC TGGAGACAGC

     2281 AGGGCTCACT CCAGCTACCT GAAGACCAAG AAGGGCCAGT CTACTTCCCG CCATAAAAAA

     2341 ACAATGGTCA AGAAAGTGGG GCCTGACTCA GACTGACTGC CTCTGCATCC CGGATCCGTC

     2401 GACCTGCAGC GGCCGCATAA CTAGCATAAC CCCTTGGGGC CTCTAAACGG GTCTTGAGGG

     2461 GTTTTTTGCG CGCTTGCAGC CAAGCTAATT CCGGGCGAAT TTCTTATGAT TTATGATTTT

     2521 TATTATTAAA TAAGTTATAA AAAAAATAAG TGTATACAAA TTTTAAAGTG ACAAGCTTGC

     2581 GAATTCTGGC GGATCCACCG GATCTAGATA ACTGATCATA ATCAGCCATA CCACATTTGT

     2641 AGAGGTTTTA CTTGCTTTAA AAAACCTCCC ACACCTCCCC CTGAACCTGA AACATAAAAT

     2701 GAATGCAATT GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA

     2761 TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC

     2821 CAAACTCATC AATGTATCTT AACGCGTAAA TTGTAAGCGT TAATATTTTG TTAAAATTCG

     2881 CGTTAAATTT TTGTTAAATC AGCTCATTTT TTAACCAATA GGCCGAAATC GGCAAAATCC

     2941 CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT TGTTCCAGTT TGGAACAAGA

     3001 GTCCACTATT AAAGAACGTG GACTCCAACG TCAAAGGGCG AAAAACCGTC TATCAGGGCG

     3061 ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTTTTTT GGGGTCGAGG TGCCGTAAAG

     3121 CACTAAATCG GAACCCTAAA GGGAGCCCCC GATTTAGAGC TTGACGGGGA AAGCCGGCGA

     3181 ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG CGCTAGGGCG CTGGCAAGTG

     3241 TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT TAATGCGCCG CTACAGGGCG

     3301 CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA

     3361 TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT

     3421 GAAAAAGGAA GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG AATGTGTGTC AGTTAGGGTG

     3481 TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC TCAATTAGTC

     3541 AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA

     3601 TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC CCCTAACTCC

     3661 GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT ATGCAGAGGC

     3721 CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT TTGGAGGCCT

     3781 AGGCTTTTGC AAAGATCGAT CAAGAGACAG GATGAGGATC GTTTCGCATG ATTGAACAAG

     3841 ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG

     3901 CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC

     3961 CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAA GACGAGGCAG

     4021 CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA

     4081 CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT

     4141 CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA

     4201 CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC

     4261 GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC

     4321 TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGAGCAT GCCCGACGGC GAGGATCTCG

     4381 TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG

     4441 GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA

     4501 CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG

     4561 GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT

     4621 GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGAGA

     4681 TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC GGAATCGTTT TCCGGGACGC

     4741 CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG TTCTTCGCCC ACCCTAGGGG

     4801 GAGGCTAACT GAAACACGGA AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT

     4861 AAAAAGACAG AATAAAACGC ACGGTGTTGG GTCGTTTGTT CATAAACGCG GGGTTCGGTC

     4921 CCAGGGCTGG CACTCTGTCG ATACCCCACC GAGACCCCAT TGGGGCCAAT ACGCCCGCGT

     4981 TTCTTCCTTT TCCCCACCCC ACCCCCCAAG TTCGGGTGAA GGCCCAGGGC TCGCAGCCAA

     5041 CGTCGGGGCG GCAGGCCCTG CCATAGCCTC AGGTTACTCA TATATACTTT AGATTGATTT

     5101 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC

     5161 CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA

     5221 AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA CAAAAAAACC

     5281 ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT

     5341 AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTTCTT CTAGTGTAGC CGTAGTTAGG

     5401 CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC

     5461 AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT

     5521 ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC CCAGCTTGGA

     5581 GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT

     5641 TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG

     5701 CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA

     5761 CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA

     5821 CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG CTCACATGTT

     5881 CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCATGC AT

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