pAcGFP1-C

pAcGFP1-C载体基本信息

pAcGFP1-C载体质粒图谱和多克隆位点信息

pAcGFP1-C载体描述

The fluorescent protein coding sequence in this construct has been human-codon-optimized for efficient expression and enhanced brightness. AcGFP1 protein has an excitation maximum at 475 nm and an emission maximum at 505 nm. Sequences flanking AcGFP1 have been converted to a Kozak consensus translation initiation site (1) to further increase translation efficiency in eukaryotic cells. A gene of interest can be added in-frame downstream of the AcGFP1 coding sequence and expressed as a fusion protein to the C-terminus of AcGFP1. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3’end of the mRNA transcript. In addition, the vector also contains a SV40 origin of replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor) containing an SV40 early promoter, the neomycin/ kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allows selection of stable transformants in eukaryotic cells using G418. A bacterial promoter upstream of the gene expresses kanamycin resistance in E. coli. pAcGFP-C Vector also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

Provided with the Matchmaker Chemiluminescent CoIP Vector Set, the pAcGFP1-C Vector allows the generation of a N-terminal AcGFP1-bait fusion. Any bait sequence in our current Matchmaker Yeast Two Hybrid System 3 pGBKT7 bait vector can be PCR-amplified using the AcGFP BD FWD/REV universal In-Fusion primer set, and directionally and efficiently cloned into the SalI/HindIII sites of the pAcGFP1-C using our In-Fusion Dry-Down PCR Cloning Kit. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. The resulting expressed AcGFP1- bait fusion protein will automatically be in-frame and can be used to monitor transfection efficiency as well as AcGFP1-bait fusion expression and localization in vivo since the AcGFP1-bait fusion retains the fluorescent properties of the native AcGFP1 protein.

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. Kozak, M. (1987) Nucleic Acids Res. 15(20):8125–8148.

2. Gorman, C. (1985) In DNA cloning: A practical approach, Vol. II. Ed. D.M. Glover. (IRL Press, Oxford, UK) pp. 143–190.

pAcGFP1-C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 GACCAGATCT CTAAGCTTGC GAATTCTGGC

     1381 GGATCCACCG GATCTAGATA ACTGATCATA 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 AACGCGTAAA 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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