pIRES

pIRES载体基本信息

pIRES载体质粒图谱和多克隆位点信息

pIRES载体简介

pIRES is a mammalian expression vector that allows high level expression of two genes of interest from the same bicistronic mRNA transcript. The vector contains the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) flanked by two multiple cloning sites (MCS A and B), an arrangement that allows cap-independent translation of the gene cloned into MCS B (1–3). pIRES utilizes a partially disabled IRES sequence (1) that reduces the rate at which the gene cloned into MCS B is translated relative to that of MCS A.

Expression of the bicistronic transcript is driven by the constitutively active cytomegalovirus immediate early promoter (PCMV IE), located upstream of MCS A. An intervening sequence (IVS) known to enhance the stability of mRNA (4) is located between PCMV IE and MCS A, and is efficiently spliced out following transcription. SV40 polyadenylation signals downstream of MCS B direct proper processing of the 3' end of the mRNA. Bacteriophage T7 and T3 promoters are located upstream of MCS A and downstream of MCS B, respectively. pIRES includes a neomycin resistance gene (Neor) to aid in the selection of transfected cells. Neor is expressed from the SV40 enhancer/promoter, and a synthetic polyadenyla¬tion signal directs proper processing of the 3' end of the Neor mRNA. The SV40 origin al¬lows for replication in mammalian cells expressing the SV40 T antigen. The vector also contains an ampicillin resistance gene (Ampr), and a ColE1 origin of replication for se¬lection and propagation in E. coli, and an f1 origin for single-stranded DNA production.

pIRES载体应用

Genes cloned into either MCS must contain ATG start codons. pIRES and derivatives can be introduced into mammalian cells by any standard transfection method. Transformed cells can be selected by growth in medium containing the antibiotic G418. Sense or antisense RNA can be transcribed from the T7 and T3 promoters, respectively.

pIRES载体序列

ORIGIN

        1 TCAATATTGG CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA

       61 TTGGCCATTG CATACGTTGT ATCTATATCA TAATATGTAC ATTTATATTG GCTCATGTCC

      121 AATATGACCG CCATGTTGGC ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG

      181 GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC

      241 GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT

      301 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC

      361 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTCCG CCCCCTATTG ACGTCAATGA

      421 CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTACGGGACT TTCCTACTTG

      481 GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAC

      541 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT

      601 CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTG

      661 CGATCGCCCG CCCCGTTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA

      721 AGCAGAGCTC GTTTAGTGAA CCGTCAGATC ACTAGAAGCT TTATTGCGGT AGTTTATCAC

      781 AGTTAAATTG CTAACGCAGT CAGTGCTTCT GACACAACAG TCTCGAACTT AAGCTGCAGT

      841 GACTCTCTTA AGGTAGCCTT GCAGAAGTTG GTCGTGAGGC ACTGGGCAGG TAAGTATCAA

      901 GGTTACAAGA CAGGTTTAAG GAGACCAATA GAAACTGGGC TTGTCGAGAC AGAGAAGACT

      961 CTTGCGTTTC TGATAGGCAC CTATTGGTCT TACTGACATC CACTTTGCCT TTCTCTCCAC

     1021 AGGTGTCCAC TCCCAGTTCA ATTACAGCTC TTAAGGCTAG AGTACTTAAT ACGACTCACT

     1081 ATAGGCTAGC CTCGAGAATT CACGCGTCGA GCATGCATCT AGGGCGGCCA ATTCCGCCCC

     1141 TCTCCCCCCC CCCCCTCTCC CTCCCCCCCC CCTAACGTTA CTGGCCGAAG CCGCTTGGAA

     1201 TAAGGCCGGT GTGCGTTTGT CTATATGTGA TTTTCCACCA TATTGCCGTC TTTTGGCAAT

     1261 GTGAGGGCCC GGAAACCTGG CCCTGTCTTC TTGACGAGCA TTCCTAGGGG TCTTTCCCCT

     1321 CTCGCCAAAG GAATGCAAGG TCTGTTGAAT GTCGTGAAGG AAGCAGTTCC TCTGGAAGCT

     1381 TCTTGAAGAC AAACAACGTC TGTAGCGACC CTTTGCAGGC AGCGGAACCC CCCACCTGGC

     1441 GACAGGTGCC TCTGCGGCCA AAAGCCACGT GTATAAGATA CACCTGCAAA GGCGGCACAA

     1501 CCCCAGTGCC ACGTTGTGAG TTGGATAGTT GTGGAAAGAG TCAAATGGCT CTCCTCAAGC

     1561 GTATTCAACA AGGGGCTGAA GGATGCCCAG AAGGTACCCC ATTGTATGGG ATCTGATCTG

     1621 GGGCCTCGGT GCACATGCTT TACATGTGTT TAGTCGAGGT TAAAAAAACG TCTAGGCCCC

     1681 CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAAGCTTG CCACAACCCG

     1741 GGATCCTCTA GAGTCGACCC GGGCGGCCGC TTCCCTTTAG TGAGGGTTAA TGCTTCGAGC

     1801 AGACATGATA AGATACATTG ATGAGTTTGG ACAAACCACA ACTAGAATGC AGTGAAAAAA

     1861 ATGCTTTATT TGTGAAATTT GTGATGCTAT TGCTTTATTT GTAACCATTA TAAGCTGCAA

     1921 TAAACAAGTT AACAACAACA ATTGCATTCA TTTTATGTTT CAGGTTCAGG GGGAGATGTG

     1981 GGAGGTTTTT TAAAGCAAGT AAAACCTCTA CAAATGTGGT AAAATCCGAT AAGGATCGAT

     2041 CCGGGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC

     2101 CTGAATGGCG AATGGACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGT GTGGTGGTTA

     2161 CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTC GCTTTCTTCC

     2221 CTTCCTTTCT CGCCACGTTC GCCGGCTTTC CCCGTCAAGC TCTAAATCGG GGGCTCCCTT

     2281 TAGGGTTCCG ATTTAGAGCT TTACGGCACC TCGACCGCAA AAAACTTGAT TTGGGTGATG

     2341 GTTCACGTAG TGGGCCATCG CCCTGATAGA CGGTTTTTCG CCCTTTGACG TTGGAGTCCA

     2401 CGTTCTTTAA TAGTGGACTC TTGTTCCAAA CTGGAACAAC ACTCAACCCT ATCTCGGTCT

     2461 ATTCTTTTGA TTTATAAGGG ATTTTGCCGA TTTCGGCCTA TTGGTTAAAA AATGAGCTGA

     2521 TTTAACAAAT ATTTAACGCG AATTTTAACA AAATATTAAC GTTTACAATT TCGCCTGATG

     2581 CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC GCATACGCGG ATCTGCGCAG

     2641 CACCATGGCC TGAAATAACC TCTGAAAGAG GAACTTGGTT AGGTACCTTC TGAGGCGGAA

     2701 AGAACCAGCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG

     2761 GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CAGGTGTGGA AAGTCCCCAG

     2821 GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCATAGTCC

     2881 CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG TTCCGCCCAT TCTCCGCCCC

     2941 ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC CGCCTCGGCC TCTGAGCTAT

     3001 TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT TTGCAAAAAG CTTGATTCTT

     3061 CTGACACAAC AGTCTCGAAC TTAAGGCTAG AGCCACCATG ATTGAACAAG ATGGATTGCA

     3121 CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG GCTATTCGGC TATGACTGGG CACAACAGAC

     3181 AATCGGCTGC TCTGATGCCG CCGTGTTCCG GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT

     3241 TGTCAAGACC GACCTGTCCG GTGCCCTGAA TGAACTGCAG GACGAGGCAG CGCGGCTATC

     3301 GTGGCTGGCC ACGACGGGCG TTCCTTGCGC AGCTGTGCTC GACGTTGTCA CTGAAGCGGG

     3361 AAGGGACTGG CTGCTATTGG GCGAAGTGCC GGGGCAGGAT CTCCTGTCAT CTCACCTTGC

     3421 TCCTGCCGAG AAAGTATCCA TCATGGCTGA TGCAATGCGG CGGCTGCATA CGCTTGATCC

     3481 GGCTACCTGC CCATTCGACC ACCAAGCGAA ACATCGCATC GAGCGAGCAC GTACTCGGAT

     3541 GGAAGCCGGT CTTGTCGATC AGGATGATCT GGACGAAGAG CATCAGGGGC TCGCGCCAGC

     3601 CGAACTGTTC GCCAGGCTCA AGGCGCGCAT GCCCGACGGC GAGGATCTCG TCGTGACCCA

     3661 TGGCGATGCC TGCTTGCCGA ATATCATGGT GGAAAATGGC CGCTTTTCTG GATTCATCGA

     3721 CTGTGGCCGG CTGGGTGTGG CGGACCGCTA TCAGGACATA GCGTTGGCTA CCCGTGATAT

     3781 TGCTGAAGAG CTTGGCGGCG AATGGGCTGA CCGCTTCCTC GTGCTTTACG GTATCGCCGC

     3841 TCCCGATTCG CAGCGCATCG CCTTCTATCG CCTTCTTGAC GAGTTCTTCT GAGCGGGACT

     3901 CTGGGGTTCG AAATGACCGA CCAAGCGACG CCCAACCTGC CATCACGATG GCCGCAATAA

     3961 AATATCTTTA TTTTCATTAC ATCTGTGTGT TGGTTTTTTG TGTGAATCGA TAGCGATAAG

     4021 GATCCGCGTA TGGTGCACTC TCAGTACAAT CTGCTCTGAT GCCGCATAGT TAAGCCAGCC

     4081 CCGACACCCG CCAACACCCG CTGACGCGCC CTGACGGGCT TGTCTGCTCC CGGCATCCGC

     4141 TTACAGACAA GCTGTGACCG TCTCCGGGAG CTGCATGTGT CAGAGGTTTT CACCGTCATC

     4201 ACCGAAACGC GCGAGACGAA AGGGCCTCGT GATACGCCTA TTTTTATAGG TTAATGTCAT

     4261 GATAATAATG GTTTCTTAGA CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC

     4321 TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG

     4381 ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTATGAGT ATTCAACATT TCCGTGTCGC

     4441 CCTTATTCCC TTTTTTGCGG CATTTTGCCT TCCTGTTTTT GCTCACCCAG AAACGCTGGT

     4501 GAAAGTAAAA GATGCTGAAG ATCAGTTGGG TGCACGAGTG GGTTACATCG AACTGGATCT

     4561 CAACAGCGGT AAGATCCTTG AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA TGATGAGCAC

     4621 TTTTAAAGTT CTGCTATGTG GCGCGGTATT ATCCCGTATT GACGCCGGGC AAGAGCAACT

     4681 CGGTCGCCGC ATACACTATT CTCAGAATGA CTTGGTTGAG TACTCACCAG TCACAGAAAA

     4741 GCATCTTACG GATGGCATGA CAGTAAGAGA ATTATGCAGT GCTGCCATAA CCATGAGTGA

     4801 TAACACTGCG GCCAACTTAC TTCTGACAAC GATCGGAGGA CCGAAGGAGC TAACCGCTTT

     4861 TTTGCACAAC ATGGGGGATC ATGTAACTCG CCTTGATCGT TGGGAACCGG AGCTGAATGA

     4921 AGCCATACCA AACGACGAGC GTGACACCAC GATGCCTGTA GCAATGGCAA CAACGTTGCG

     4981 CAAACTATTA ACTGGCGAAC TACTTACTCT AGCTTCCCGG CAACAATTAA TAGACTGGAT

     5041 GGAGGCGGAT AAAGTTGCAG GACCACTTCT GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT

     5101 TGCTGATAAA TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT ATCATTGCAG CACTGGGGCC

     5161 AGATGGTAAG CCCTCCCGTA TCGTAGTTAT CTACACGACG GGGAGTCAGG CAACTATGGA

     5221 TGAACGAAAT AGACAGATCG CTGAGATAGG TGCCTCACTG ATTAAGCATT GGTAACTGTC

     5281 AGACCAAGTT TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT AATTTAAAAG

     5341 GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC GTGAGTTTTC

     5401 GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT

     5461 TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT

     5521 GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT

     5581 ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA ACTCTGTAGC

     5641 ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA

     5701 GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG

     5761 CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG

     5821 ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG

     5881 GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA

     5941 CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT

     6001 GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG

     6061 GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGGCTCGA CAGATCT

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