pAcGFP1-C In-Fusion Ready

pAcGFP1-C In-Fusion Ready载体基本信息

pAcGFP1-C In-Fusion Ready载体质粒图谱和多克隆位点信息

pAcGFP1-C In-Fusion Ready载体简介

pAcGFP1-C In-Fusion Ready vector is a linearized mammalian expression vector that encodes a Green Fluorescent Protein (GFP) from Aequorea coerulescens. This 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 the translation efficiency in eukaryotic cells.

The primers that will be used to amplify In-Fusion Ready PCR products require the following 15 nucleotides on their 5’ends:

Sense primer: 5’ AAGGCCTCTGTCGAC followed by sequence of amplification target 3’

Antisense primer: 5’ AGAATTCGCAAGCTT followed by sequence of amplification target 3’

If the gene of interest is added in-frame immediately after the 15 nucleotides mentioned above, this sequence will automatically be in-frame with the AcGFP1 sequence upstream and therefore be 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, andpolyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allow selection of stable transformants in eukaryotic cells using G418. A bacterial promoter upstream of the gene expresses kanamycin resistance in E. coli. The pAcGFP1-C In-Fusion Ready Vector 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 and allow monitoring of the fusion protein localization in vivo. The target gene can be fused into pAcGFP1-C In-Fusion Ready Vector so that it is in-frame with the AcGFP1 coding sequence with no intervening in-frame stop codons. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. Stable transformants can be selected using G418 (2).

Propagation in E. coli

 Suitable host strains: Stellar Competent Cells.

 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-C In-Fusion Ready载体序列

ORIGIN

    1 AGCTTGCGAA TTCTGGCGGA TCCACCGGAT CTAGATAACT GATCATAATC AGCCATACCA

   61 CATTTGTAGA GGTTTTACTT GCTTTAAAAA ACCTCCCACA CCTCCCCCTG AACCTGAAAC

  121 ATAAAATGAA TGCAATTGTT GTTGTTAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT

  181 AAAGCAATAG CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG

  241 GTTTGTCCAA ACTCATCAAT GTATCTTAAC GCGTAAATTG TAAGCGTTAA TATTTTGTTA

  301 AAATTCGCGT TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGC CGAAATCGGC

  361 AAAATCCCTT ATAAATCAAA AGAATAGACC GAGATAGGGT TGAGTGTTGT TCCAGTTTGG

  421 AACAAGAGTC CACTATTAAA GAACGTGGAC TCCAACGTCA AAGGGCGAAA AACCGTCTAT

  481 CAGGGCGATG GCCCACTACG TGAACCATCA CCCTAATCAA GTTTTTTGGG GTCGAGGTGC

  541 CGTAAAGCAC TAAATCGGAA CCCTAAAGGG AGCCCCCGAT TTAGAGCTTG ACGGGGAAAG

  601 CCGGCGAACG TGGCGAGAAA GGAAGGGAAG AAAGCGAAAG GAGCGGGCGC TAGGGCGCTG

  661 GCAAGTGTAG CGGTCACGCT GCGCGTAACC ACCACACCCG CCGCGCTTAA TGCGCCGCTA

  721 CAGGGCGCGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG GAACCCCTAT TTGTTTATTT

  781 TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA

  841 TAATATTGAA AAAGGAAGAG TCCTGAGGCG GAAAGAACCA GCTGTGGAAT GTGTGTCAGT

  901 TAGGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA

  961 ATTAGTCAGC AACCAGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA

 1021 GCATGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC

 1081 TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG

 1141 CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG

 1201 GAGGCCTAGG CTTTTGCAAA GATCGATCAA GAGACAGGAT GAGGATCGTT TCGCATGATT

 1261 GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG TGGAGAGGCT ATTCGGCTAT

 1321 GACTGGGCAC AACAGACAAT CGGCTGCTCT GATGCCGCCG TGTTCCGGCT GTCAGCGCAG

 1381 GGGCGCCCGG TTCTTTTTGT CAAGACCGAC CTGTCCGGTG CCCTGAATGA ACTGCAAGAC

 1441 GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC CTTGCGCAGC TGTGCTCGAC

 1501 GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG AAGTGCCGGG GCAGGATCTC

 1561 CTGTCATCTC ACCTTGCTCC TGCCGAGAAA GTATCCATCA TGGCTGATGC AATGCGGCGG

 1621 CTGCATACGC TTGATCCGGC TACCTGCCCA TTCGACCACC AAGCGAAACA TCGCATCGAG

 1681 CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG ATGATCTGGA CGAAGAGCAT

 1741 CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG CGAGCATGCC CGACGGCGAG

 1801 GATCTCGTCG TGACCCATGG CGATGCCTGC TTGCCGAATA TCATGGTGGA AAATGGCCGC

 1861 TTTTCTGGAT TCATCGACTG TGGCCGGCTG GGTGTGGCGG ACCGCTATCA GGACATAGCG

 1921 TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT GGGCTGACCG CTTCCTCGTG

 1981 CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT TCTATCGCCT TCTTGACGAG

 2041 TTCTTCTGAG CGGGACTCTG GGGTTCGAAA TGACCGACCA AGCGACGCCC AACCTGCCAT

 2101 CACGAGATTT CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC

 2161 GGGACGCCGG CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC TTCGCCCACC

 2221 CTAGGGGGAG GCTAACTGAA ACACGGAAGG AGACAATACC GGAAGGAACC CGCGCTATGA

 2281 CGGCAATAAA AAGACAGAAT AAAACGCACG GTGTTGGGTC GTTTGTTCAT AAACGCGGGG

 2341 TTCGGTCCCA GGGCTGGCAC TCTGTCGATA CCCCACCGAG ACCCCATTGG GGCCAATACG

 2401 CCCGCGTTTC TTCCTTTTCC CCACCCCACC CCCCAAGTTC GGGTGAAGGC CCAGGGCTCG

 2461 CAGCCAACGT CGGGGCGGCA GGCCCTGCCA TAGCCTCAGG TTACTCATAT ATACTTTAGA

 2521 TTGATTTAAA ACTTCATTTT TAATTTAAAA GGATCTAGGT GAAGATCCTT TTTGATAATC

 2581 TCATGACCAA AATCCCTTAA CGTGAGTTTT CGTTCCACTG AGCGTCAGAC CCCGTAGAAA

 2641 AGATCAAAGG ATCTTCTTGA GATCCTTTTT TTCTGCGCGT AATCTGCTGC TTGCAAACAA

 2701 AAAAACCACC GCTACCAGCG GTGGTTTGTT TGCCGGATCA AGAGCTACCA ACTCTTTTTC

 2761 CGAAGGTAAC TGGCTTCAGC AGAGCGCAGA TACCAAATAC TGTTCTTCTA GTGTAGCCGT

 2821 AGTTAGGCCA CCACTTCAAG AACTCTGTAG CACCGCCTAC ATACCTCGCT CTGCTAATCC

 2881 TGTTACCAGT GGCTGCTGCC AGTGGCGATA AGTCGTGTCT TACCGGGTTG GACTCAAGAC

 2941 GATAGTTACC GGATAAGGCG CAGCGGTCGG GCTGAACGGG GGGTTCGTGC ACACAGCCCA

 3001 GCTTGGAGCG AACGACCTAC ACCGAACTGA GATACCTACA GCGTGAGCTA TGAGAAAGCG

 3061 CCACGCTTCC CGAAGGGAGA AAGGCGGACA GGTATCCGGT AAGCGGCAGG GTCGGAACAG

 3121 GAGAGCGCAC GAGGGAGCTT CCAGGGGGAA ACGCCTGGTA TCTTTATAGT CCTGTCGGGT

 3181 TTCGCCACCT CTGACTTGAG CGTCGATTTT TGTGATGCTC GTCAGGGGGG CGGAGCCTAT

 3241 GGAAAAACGC CAGCAACGCG GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC

 3301 ACATGTTCTT TCCTGCGTTA TCCCCTGATT CTGTGGATAA CCGTATTACC GCCATGCATT

 3361 AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC

 3421 GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG

 3481 ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA

 3541 TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA

 3601 AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC

 3661 ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC

 3721 ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA

 3781 TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG

 3841 GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA

 3901 CGGTGGGAGG TCTATATAAG CAGAGCTGGT TTAGTGAACC GTCAGATCCG CTAGCGCTAC

 3961 CGGTCGCCAC CATGGTGAGC AAGGGCGCCG AGCTGTTCAC CGGCATCGTG CCCATCCTGA

 4021 TCGAGCTGAA TGGCGATGTG AATGGCCACA AGTTCAGCGT GAGCGGCGAG GGCGAGGGCG

 4081 ATGCCACCTA CGGCAAGCTG ACCCTGAAGT TCATCTGCAC CACCGGCAAG CTGCCTGTGC

 4141 CCTGGCCCAC CCTGGTGACC ACCCTGAGCT ACGGCGTGCA GTGCTTCTCA CGCTACCCCG

 4201 ATCACATGAA GCAGCACGAC TTCTTCAAGA GCGCCATGCC TGAGGGCTAC ATCCAGGAGC

 4261 GCACCATCTT CTTCGAGGAT GACGGCAACT ACAAGTCGCG CGCCGAGGTG AAGTTCGAGG

 4321 GCGATACCCT GGTGAATCGC ATCGAGCTGA CCGGCACCGA TTTCAAGGAG GATGGCAACA

 4381 TCCTGGGCAA TAAGATGGAG TACAACTACA ACGCCCACAA TGTGTACATC ATGACCGACA

 4441 AGGCCAAGAA TGGCATCAAG GTGAACTTCA AGATCCGCCA CAACATCGAG GATGGCAGCG

 4501 TGCAGCTGGC CGACCACTAC CAGCAGAATA CCCCCATCGG CGATGGCCCT GTGCTGCTGC

 4561 CCGATAACCA CTACCTGTCC ACCCAGAGCG CCCTGTCCAA GGACCCCAAC GAGAAGCGCG

 4621 ATCACATGAT CTACTTCGGC TTCGTGACCG CCGCCGCCAT CACCCACGGC ATGGATGAGC

 4681 TGTACAAGTC CGGACTTAAG GCCTCTGTCG A

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