pAcGFP1-N In-Fusion Ready

pAcGFP1-N In-Fusion Ready

pAcGFP1-N In-Fusion Ready载体基本信息

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

pAcGFP1-N In-Fusion Ready载体描述 

pAcGFP1-N 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. The linearized vector allows direct cloning of PCR products without any need for restriction digestion when using Clontech's In-Fusion HD Cloning Plus (638909). This is accomplished by the use of a specific 15 nucleotide long sequence within the sense and antisense primers that overlap with the cut ends created by initial digestion of the vector with Sal I and Hind III.

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 sequence of 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 N-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 allows the plasmid to express kanamycin resistance in E. coli. The pAcGFP1-N 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 N-terminus of AcGFP1 retain the fluorescent properties of the native protein and allow monitoring of fusion protein localization in vivo. The PCR-amplified gene of interest is directly cloned into pAcGFP1-N In-Fusion Ready vector so that it is in-frame with the AcGFP1 coding sequence with no intervening in-frame stop codons. This can be accomplished by using the suggested primers. The inserted gene must include the initiating ATG codon. 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-N In-Fusion Ready载体序列

ORIGIN

    1 AGCTTGCGAA TTCTGGCCTA CCGGTCGCGA GCAAGGGCGC CGAGCTGTTC ACCGGCATCG

   61 TGCCCATCCT GATCGAGCTG AATGGCGATG TGAATGGCCA CAAGTTCAGC GTGAGCGGCG

  121 AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC ACCACCGGCA

  181 AGCTGCCTGT GCCCTGGCCC ACCCTGGTGA CCACCCTGAG CTACGGCGTG CAGTGCTTCT

  241 CACGCTACCC CGATCACATG AAGCAGCACG ACTTCTTCAA GAGCGCCATG CCTGAGGGCT

  301 ACATCCAGGA GCGCACCATC TTCTTCGAGG ATGACGGCAA CTACAAGTCG CGCGCCGAGG

  361 TGAAGTTCGA GGGCGATACC CTGGTGAATC GCATCGAGCT GACCGGCACC GATTTCAAGG

  421 AGGATGGCAA CATCCTGGGC AATAAGATGG AGTACAACTA CAACGCCCAC AATGTGTACA

  481 TCATGACCGA CAAGGCCAAG AATGGCATCA AGGTGAACTT CAAGATCCGC CACAACATCG

  541 AGGATGGCAG CGTGCAGCTG GCCGACCACT ACCAGCAGAA TACCCCCATC GGCGATGGCC

  601 CTGTGCTGCT GCCCGATAAC CACTACCTGT CCACCCAGAG CGCCCTGTCC AAGGACCCCA

  661 ACGAGAAGCG CGATCACATG ATCTACTTCG GCTTCGTGAC CGCCGCCGCC ATCACCCACG

  721 GCATGGATGA GCTGTACAAG TGAGCGGCCG CGACTCTAGA TCATAATCAG CCATACCACA

  781 TTTGTAGAGG TTTTACTTGC TTTAAAAAAC CTCCCACACC TCCCCCTGAA CCTGAAACAT

  841 AAAATGAATG CAATTGTTGT TGTTAACTTG TTTATTGCAG CTTATAATGG TTACAAATAA

  901 AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC TAGTTGTGGT

  961 TTGTCCAAAC TCATCAATGT ATCTTAAGGC GTAAATTGTA AGCGTTAATA TTTTGTTAAA

 1021 ATTCGCGTTA AATTTTTGTT AAATCAGCTC ATTTTTTAAC CAATAGGCCG AAATCGGCAA

 1081 AATCCCTTAT AAATCAAAAG AATAGACCGA GATAGGGTTG AGTGTTGTTC CAGTTTGGAA

 1141 CAAGAGTCCA CTATTAAAGA ACGTGGACTC CAACGTCAAA GGGCGAAAAA CCGTCTATCA

 1201 GGGCGATGGC CCACTACGTG AACCATCACC CTAATCAAGT TTTTTGGGGT CGAGGTGCCG

 1261 TAAAGCACTA AATCGGAACC CTAAAGGGAG CCCCCGATTT AGAGCTTGAC GGGGAAAGCC

 1321 GGCGAACGTG GCGAGAAAGG AAGGGAAGAA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC

 1381 AAGTGTAGCG GTCACGCTGC GCGTAACCAC CACACCCGCC GCGCTTAATG CGCCGCTACA

 1441 GGGCGCGTCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT

 1501 CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA

 1561 ATATTGAAAA AGGAAGAGTC CTGAGGCGGA AAGAACCAGC TGTGGAATGT GTGTCAGTTA

 1621 GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT

 1681 TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC

 1741 ATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA CTCCGCCCAT CCCGCCCCTA

 1801 ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC TAATTTTTTT TATTTATGCA

 1861 GAGGCCGAGG CCGCCTCGGC CTCTGAGCTA TTCCAGAAGT AGTGAGGAGG CTTTTTTGGA

 1921 GGCCTAGGCT TTTGCAAAGA TCGATCAAGA GACAGGATGA GGATCGTTTC GCATGATTGA

 1981 ACAAGATGGA TTGCACGCAG GTTCTCCGGC CGCTTGGGTG GAGAGGCTAT TCGGCTATGA

 2041 CTGGGCACAA CAGACAATCG GCTGCTCTGA TGCCGCCGTG TTCCGGCTGT CAGCGCAGGG

 2101 GCGCCCGGTT CTTTTTGTCA AGACCGACCT GTCCGGTGCC CTGAATGAAC TGCAAGACGA

 2161 GGCAGCGCGG CTATCGTGGC TGGCCACGAC GGGCGTTCCT TGCGCAGCTG TGCTCGACGT

 2221 TGTCACTGAA GCGGGAAGGG ACTGGCTGCT ATTGGGCGAA GTGCCGGGGC AGGATCTCCT

 2281 GTCATCTCAC CTTGCTCCTG CCGAGAAAGT ATCCATCATG GCTGATGCAA TGCGGCGGCT

 2341 GCATACGCTT GATCCGGCTA CCTGCCCATT CGACCACCAA GCGAAACATC GCATCGAGCG

 2401 AGCACGTACT CGGATGGAAG CCGGTCTTGT CGATCAGGAT GATCTGGACG AAGAGCATCA

 2461 GGGGCTCGCG CCAGCCGAAC TGTTCGCCAG GCTCAAGGCG AGCATGCCCG ACGGCGAGGA

 2521 TCTCGTCGTG ACCCATGGCG ATGCCTGCTT GCCGAATATC ATGGTGGAAA ATGGCCGCTT

 2581 TTCTGGATTC ATCGACTGTG GCCGGCTGGG TGTGGCGGAC CGCTATCAGG ACATAGCGTT

 2641 GGCTACCCGT GATATTGCTG AAGAGCTTGG CGGCGAATGG GCTGACCGCT TCCTCGTGCT

 2701 TTACGGTATC GCCGCTCCCG ATTCGCAGCG CATCGCCTTC TATCGCCTTC TTGACGAGTT

 2761 CTTCTGAGCG GGACTCTGGG GTTCGAAATG ACCGACCAAG CGACGCCCAA CCTGCCATCA

 2821 CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG GCTTCGGAAT CGTTTTCCGG

 2881 GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC TGGAGTTCTT CGCCCACCCT

 2941 AGGGGGAGGC TAACTGAAAC ACGGAAGGAG ACAATACCGG AAGGAACCCG CGCTATGACG

 3001 GCAATAAAAA GACAGAATAA AACGCACGGT GTTGGGTCGT TTGTTCATAA ACGCGGGGTT

 3061 CGGTCCCAGG GCTGGCACTC TGTCGATACC CCACCGAGAC CCCATTGGGG CCAATACGCC

 3121 CGCGTTTCTT CCTTTTCCCC ACCCCACCCC CCAAGTTCGG GTGAAGGCCC AGGGCTCGCA

 3181 GCCAACGTCG GGGCGGCAGG CCCTGCCATA GCCTCAGGTT ACTCATATAT ACTTTAGATT

 3241 GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC

 3301 ATGACCAAAA TCCCTTAACG TGAGTTTTCG TTCCACTGAG CGTCAGACCC CGTAGAAAAG

 3361 ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT CTGCGCGTAA TCTGCTGCTT GCAAACAAAA

 3421 AAACCACCGC TACCAGCGGT GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG

 3481 AAGGTAACTG GCTTCAGCAG AGCGCAGATA CCAAATACTG TTCTTCTAGT GTAGCCGTAG

 3541 TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT GCTAATCCTG

 3601 TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA CCGGGTTGGA CTCAAGACGA

 3661 TAGTTACCGG ATAAGGCGCA GCGGTCGGGC TGAACGGGGG GTTCGTGCAC ACAGCCCAGC

 3721 TTGGAGCGAA CGACCTACAC CGAACTGAGA TACCTACAGC GTGAGCTATG AGAAAGCGCC

 3781 ACGCTTCCCG AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA

 3841 GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC TGTCGGGTTT

 3901 CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT CAGGGGGGCG GAGCCTATGG

 3961 AAAAACGCCA GCAACGCGGC CTTTTTACGG TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC

 4021 ATGTTCTTTC CTGCGTTATC CCCTGATTCT GTGGATAACC GTATTACCGC CATGCATTAG

 4081 TTATTAATAG TAATCAATTA CGGGGTCATT AGTTCATAGC CCATATATGG AGTTCCGCGT

 4141 TACATAACTT ACGGTAAATG GCCCGCCTGG CTGACCGCCC AACGACCCCC GCCCATTGAC

 4201 GTCAATAATG ACGTATGTTC CCATAGTAAC GCCAATAGGG ACTTTCCATT GACGTCAATG

 4261 GGTGGAGTAT TTACGGTAAA CTGCCCACTT GGCAGTACAT CAAGTGTATC ATATGCCAAG

 4321 TACGCCCCCT ATTGACGTCA ATGACGGTAA ATGGCCCGCC TGGCATTATG CCCAGTACAT

 4381 GACCTTATGG GACTTTCCTA CTTGGCAGTA CATCTACGTA TTAGTCATCG CTATTACCAT

 4441 GGTGATGCGG TTTTGGCAGT ACATCAATGG GCGTGGATAG CGGTTTGACT CACGGGGATT

 4501 TCCAAGTCTC CACCCCATTG ACGTCAATGG GAGTTTGTTT TGGCACCAAA ATCAACGGGA

 4561 CTTTCCAAAA TGTCGTAACA ACTCCGCCCC ATTGACGCAA ATGGGCGGTA GGCGTGTACG

 4621 GTGGGAGGTC TATATAAGCA GAGCTGGTTT AGTGAACCGT CAGATCCGCT AGCTAAGGCC

 4681 TCTGTCGA

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