pIRES2-ZsGreen1

pIRES2-ZsGreen1

pIRES2-ZsGreen1载体基本信息

pIRES2-ZsGreen1载体质粒图谱和多克隆位点信息

pIRES2-ZsGreen1载体描述

pIRES2-ZsGreen1 contains the internal ribosome entry site (IRES; 1, 2) of the encephalomyocarditis virus (ECMV) between an MCS and the Zoanthus sp. green fluorescent protein (ZsGreen; 3, 4)coding region. This design permits both the gene of interest (cloned into the MCS) and the ZsGreen1 gene to be translated from a single bicistronic mRNA. pIRES2-ZsGreen1 is designed for the efficient selection (by flow cytometry or other methods) of transiently transfected mammalian cells expressing ZsGreen1 and the protein of interest. This vector can be used to obtain stably transfected cell lines without time-consuming drug and clonal selection.

ZsGreen1 is a human codon-optimized (5) ZsGreen variant that encodes the brightest commercially available green fluorescent protein. The MCS in pIRES2-ZsGreen1 is located between the immediate early promoter of cytomegalovirus (PCMV IE) and the IRES sequence. SV40 polyadenylation signals downstream of the ZsGreen1 gene direct proper processing of the 3' end of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing 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 expresses kanamycin resistance in E.coli. The pIRES2-ZsGreen1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

pIRES2-ZsGreen1 can be used to quickly identify cells expressing a gene of interest by screening for ZsGreen1 fluorescence. Genes inserted into the MCS should include an initiating ATG codon. Selection of ZsGreen1-positive cells is possible 24–36 hours after transfection by flow cytometry or fluorescence microscopy. pIRES2-ZsGreen1 and its derivatives can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).

Selection of Stable Transfectants

 Selectable marker: plasmid confers resistance to G418 (500 μg/ml)

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: high

pIRES2-ZsGreen1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 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG

  661 GATCCGCCCC TCTCCCTCCC CCCCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG

  721 CCGGTGTGCG TTTGTCTATA TGTTATTTTC CACCATATTG CCGTCTTTTG GCAATGTGAG

  781 GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT CCCCTCTCGC

  841 CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG

  901 AAGACAAACA ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG

  961 GTGCCTCTGC GGCCAAAAGC CACGTGTATA AGATACACCT GCAAAGGCGG CACAACCCCA

 1021 GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT CAAGCGTATT

 1081 CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC

 1141 TCGGTACACA TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA

 1201 CCACGGGGAC GTGGTTTTCC TTTGAAAAAC ACGATGATAA TATGGCCACA ACCATGGCCC

 1261 AGTCCAAGCA CGGCCTGACC AAGGAGATGA CCATGAAGTA CCGCATGGAG GGCTGCGTGG

 1321 ACGGCCACAA GTTCGTGATC ACCGGCGAGG GCATCGGCTA CCCCTTCAAG GGCAAGCAGG

 1381 CCATCAACCT GTGCGTGGTG GAGGGCGGCC CCTTGCCCTT CGCCGAGGAC ATCTTGTCCG

 1441 CCGCCTTCAT GTACGGCAAC CGCGTGTTCA CCGAGTACCC CCAGGACATC GTCGACTACT

 1501 TCAAGAACTC CTGCCCCGCC GGCTACACCT GGGACCGCTC CTTCCTGTTC GAGGACGGCG

 1561 CCGTGTGCAT CTGCAACGCC GACATCACCG TGAGCGTGGA GGAGAACTGC ATGTACCACG

 1621 AGTCCAAGTT CTACGGCGTG AACTTCCCCG CCGACGGCCC CGTGATGAAG AAGATGACCG

 1681 ACAACTGGGA GCCCTCCTGC GAGAAGATCA TCCCCGTGCC CAAGCAGGGC ATCTTGAAGG

 1741 GCGACGTGAG CATGTACCTG CTGCTGAAGG ACGGTGGCCG CTTGCGCTGC CAGTTCGACA

 1801 CCGTGTACAA GGCCAAGTCC GTGCCCCGCA AGATGCCCGA CTGGCACTTC ATCCAGCACA

 1861 AGCTGACCCG CGAGGACCGC AGCGACGCCA AGAACCAGAA GTGGCACCTG ACCGAGCACG

 1921 CCATCGCCTC CGGCTCCGCC TTGCCCTGAG CGGCCGCGAC TCTAGATCAT AATCAGCCAT

 1981 ACCACATTTG TAGAGGTTTT ACTTGCTTTA AAAAACCTCC CACACCTCCC CCTGAACCTG

 2041 AAACATAAAA TGAATGCAAT TGTTGTTGTT AACTTGTTTA TTGCAGCTTA TAATGGTTAC

 2101 AAATAAAGCA ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT

 2161 TGTGGTTTGT CCAAACTCAT CAATGTATCT TAAGGCGTAA ATTGTAAGCG TTAATATTTT

 2221 GTTAAAATTC GCGTTAAATT TTTGTTAAAT CAGCTCATTT TTTAACCAAT AGGCCGAAAT

 2281 CGGCAAAATC CCTTATAAAT CAAAAGAATA GACCGAGATA GGGTTGAGTG TTGTTCCAGT

 2341 TTGGAACAAG AGTCCACTAT TAAAGAACGT GGACTCCAAC GTCAAAGGGC GAAAAACCGT

 2401 CTATCAGGGC GATGGCCCAC TACGTGAACC ATCACCCTAA TCAAGTTTTT TGGGGTCGAG

 2461 GTGCCGTAAA GCACTAAATC GGAACCCTAA AGGGAGCCCC CGATTTAGAG CTTGACGGGG

 2521 AAAGCCGGCG AACGTGGCGA GAAAGGAAGG GAAGAAAGCG AAAGGAGCGG GCGCTAGGGC

 2581 GCTGGCAAGT GTAGCGGTCA CGCTGCGCGT AACCACCACA CCCGCCGCGC TTAATGCGCC

 2641 GCTACAGGGC GCGTCAGGTG GCACTTTTCG GGGAAATGTG CGCGGAACCC CTATTTGTTT

 2701 ATTTTTCTAA ATACATTCAA ATATGTATCC GCTCATGAGA CAATAACCCT GATAAATGCT

 2761 TCAATAATAT TGAAAAAGGA AGAGTCCTGA GGCGGAAAGA ACCAGCTGTG GAATGTGTGT

 2821 CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT

 2881 CTCAATTAGT CAGCAACCAG GTGTGGAAAG TCCCCAGGCT CCCCAGCAGG CAGAAGTATG

 2941 CAAAGCATGC ATCTCAATTA GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG

 3001 CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT

 3061 TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT

 3121 TTTGGAGGCC TAGGCTTTTG CAAAGATCGA TCAAGAGACA GGATGAGGAT CGTTTCGCAT

 3181 GATTGAACAA GATGGATTGC ACGCAGGTTC TCCGGCCGCT TGGGTGGAGA GGCTATTCGG

 3241 CTATGACTGG GCACAACAGA CAATCGGCTG CTCTGATGCC GCCGTGTTCC GGCTGTCAGC

 3301 GCAGGGGCGC CCGGTTCTTT TTGTCAAGAC CGACCTGTCC GGTGCCCTGA ATGAACTGCA

 3361 AGACGAGGCA GCGCGGCTAT CGTGGCTGGC CACGACGGGC GTTCCTTGCG CAGCTGTGCT

 3421 CGACGTTGTC ACTGAAGCGG GAAGGGACTG GCTGCTATTG GGCGAAGTGC CGGGGCAGGA

 3481 TCTCCTGTCA TCTCACCTTG CTCCTGCCGA GAAAGTATCC ATCATGGCTG ATGCAATGCG

 3541 GCGGCTGCAT ACGCTTGATC CGGCTACCTG CCCATTCGAC CACCAAGCGA AACATCGCAT

 3601 CGAGCGAGCA CGTACTCGGA TGGAAGCCGG TCTTGTCGAT CAGGATGATC TGGACGAAGA

 3661 GCATCAGGGG CTCGCGCCAG CCGAACTGTT CGCCAGGCTC AAGGCGAGCA TGCCCGACGG

 3721 CGAGGATCTC GTCGTGACCC ATGGCGATGC CTGCTTGCCG AATATCATGG TGGAAAATGG

 3781 CCGCTTTTCT GGATTCATCG ACTGTGGCCG GCTGGGTGTG GCGGACCGCT ATCAGGACAT

 3841 AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC GAATGGGCTG ACCGCTTCCT

 3901 CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC GCCTTCTATC GCCTTCTTGA

 3961 CGAGTTCTTC TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCAACCTG

 4021 CCATCACGAG ATTTCGATTC CACCGCCGCC TTCTATGAAA GGTTGGGCTT CGGAATCGTT

 4081 TTCCGGGACG CCGGCTGGAT GATCCTCCAG CGCGGGGATC TCATGCTGGA GTTCTTCGCC

 4141 CACCCTAGGG GGAGGCTAAC TGAAACACGG AAGGAGACAA TACCGGAAGG AACCCGCGCT

 4201 ATGACGGCAA TAAAAAGACA GAATAAAACG CACGGTGTTG GGTCGTTTGT TCATAAACGC

 4261 GGGGTTCGGT CCCAGGGCTG GCACTCTGTC GATACCCCAC CGAGACCCCA TTGGGGCCAA

 4321 TACGCCCGCG TTTCTTCCTT TTCCCCACCC CACCCCCCAA GTTCGGGTGA AGGCCCAGGG

 4381 CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT GCCATAGCCT CAGGTTACTC ATATATACTT

 4441 TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT

 4501 AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA

 4561 GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC GCGTAATCTG CTGCTTGCAA

 4621 ACAAAAAAAC CACCGCTACC AGCGGTGGTT TGTTTGCCGG ATCAAGAGCT ACCAACTCTT

 4681 TTTCCGAAGG TAACTGGCTT CAGCAGAGCG CAGATACCAA ATACTGTTCT TCTAGTGTAG

 4741 CCGTAGTTAG GCCACCACTT CAAGAACTCT GTAGCACCGC CTACATACCT CGCTCTGCTA

 4801 ATCCTGTTAC CAGTGGCTGC TGCCAGTGGC GATAAGTCGT GTCTTACCGG GTTGGACTCA

 4861 AGACGATAGT TACCGGATAA GGCGCAGCGG TCGGGCTGAA CGGGGGGTTC GTGCACACAG

 4921 CCCAGCTTGG AGCGAACGAC CTACACCGAA CTGAGATACC TACAGCGTGA GCTATGAGAA

 4981 AGCGCCACGC TTCCCGAAGG GAGAAAGGCG GACAGGTATC CGGTAAGCGG CAGGGTCGGA

 5041 ACAGGAGAGC GCACGAGGGA GCTTCCAGGG GGAAACGCCT GGTATCTTTA TAGTCCTGTC

 5101 GGGTTTCGCC ACCTCTGACT TGAGCGTCGA TTTTTGTGAT GCTCGTCAGG GGGGCGGAGC

 5161 CTATGGAAAA ACGCCAGCAA CGCGGCCTTT TTACGGTTCC TGGCCTTTTG CTGGCCTTTT

 5221 GCTCACATGT TCTTTCCTGC GTTATCCCCT GATTCTGTGG ATAACCGTAT TACCGCCATG

 5281 CAT

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