pIRESpuro3

pIRESpuro3

pIRESpuro3载体基本信息

pIRESpuro3载体质粒图谱和多克隆位点信息

pIRESpuro3载体简介

Description

pIRESpuro3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with puromycin, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the puromycin resistance gene downstream to a less optimal position for translation as directed by the IRES sequence (3). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in selection for cells that express the entire transcript, including the gene of interest, at high levels. The expression cassette of pIRESpuro3 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (4), the ECMV IRES followed by the gene encoding puromycin resistance (puromycin-N-acetyltransferase; 5), and the polyadenylation signal from SV40. Ribosomes can enter the bicistronic mRNA at the 5' end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker.  

Use

When using the pIRESpuro3 Vector, the antibiotic exerts selective pressure on the whole expression cassette; thus, a high dose of antibiotic will select for cells expressing a high level of the gene of interest. This selective pressure also ensures that the expression of the gene of interest will be stable  

pIRESpuro3载体序列

ORIGIN

        1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG

       61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG

      121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC

      181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT

      241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA

      301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC

      361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC

      421 ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT

      481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT

      541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA

      601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG

      661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC

      721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG

      781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA

      841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTTGGTACC

      901 GAGCTCGGAT CGATATCTGC GGCCTAGCTA GCGCTTAAGG CCTGTTAACC GGTCGTACGT

      961 CTCCGGATTC GAATTCGGAT CCGCGGCCGC ATAGATAACT GATCCAGTGT GCTGGAATTA

     1021 ATTCGCTGTC TGCGAGGGCC AGCTGTTGGG GTGAGTACTC CCTCTCAAAA GCGGGCATGA

     1081 CTTCTGCGCT AAGATTGTCA GTTTCCAAAA ACGAGGAGGA TTTGATATTC ACCTGGCCCG

     1141 CGGTGATGCC TTTGAGGGTG GCCGCGTCCA TCTGGTCAGA AAAGACAATC TTTTTGTTGT

     1201 CAAGCTTGAG GTGTGGCAGG CTTGAGATCT GGCCATACAC TTGAGTGACA ATGACATCCA

     1261 CTTTGCCTTT CTCTCCACAG GTGTCCACTC CCAGGTCCAA CTGCAGGTCG AGCATGCATC

     1321 TAGGGCGGCC AATTCCGCCC CTCTCCCCCC CCCCCCTCTC CCTCCCCCCC CCCTAACGTT

     1381 ACTGGCCGAA GCCGCTTGGA ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC

     1441 ATATTGCCGT CTTTTGGCAA TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC

     1501 ATTCCTAGGG GTCTTTCCCC TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG

     1561 GAAGCAGTTC CTCTGGAAGC TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG

     1621 CAGCGGAACC CCCCACCTGG CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT

     1681 ACACCTGCAA AGGCGGCACA ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA

     1741 GTCAAATGGC TCTCCTCAAG CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC

     1801 CATTGTATGG GATCTGATCT GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG

     1861 TTAAAAAAAC GTCTAGGCCC CCCGAACCAC GGGGACGTGG TTTTCCTTTG AAAAACACGA

     1921 TGATAAGCTT GCCACAACCC ACAAGGAGAC GACCTTCCAT GACCGAGTAC AAGCCCACGG

     1981 TGCGCCTCGC CACCCGCGAC GACGTCCCCC GGGCCGTACG CACCCTCGCC GCCGCGTTCG

     2041 CCGACTACCC CGCCACGCGC CACACCGTCG ACCCGGACCG CCACATCGAG CGGGTCACCG

     2101 AGCTGCAAGA ACTCTTCCTC ACGCGCGTCG GGCTCGACAT CGGCAAGGTG TGGGTCGCGG

     2161 ACGACGGCGC CGCGGTGGCG GTCTGGACCA CGCCGGAGAG CGTCGAAGCG GGGGCGGTGT

     2221 TCGCCGAGAT CGGCCCGCGC ATGGCCGAGT TGAGCGGTTC CCGGCTGGCC GCGCAGCAAC

     2281 AGATGGAAGG CCTCCTGGCG CCGCACCGGC CCAAGGAGCC CGCGTGGTTC CTGGCCACCG

     2341 TCGGCGTCTC GCCCGACCAC CAGGGCAAGG GTCTGGGCAG CGCCGTCGTG CTCCCCGGAG

     2401 TGGAGGCGGC CGAGCGCGCC GGGGTGCCCG CCTTCCTGGA GACCTCCGCG CCCCGCAACC

     2461 TCCCCTTCTA CGAGCGGCTC GGCTTCACCG TCACCGCCGA CGTCGAGGTG CCCGAAGGAC

     2521 CGCGCACCTG GTGCATGACC CGCAAGCCCG GTGCCTGACG CCCGCCCCAC GACCCGCAGC

     2581 GCCCGACCGA AAGGAGCGCA CGACCCCATG GCTCCGACCG AAGCCACCCG GGGCGGCCCC

     2641 GCCGACCCCG CACCCGCCCC CGAGGCCCAC CGACTCTAGA TAACTGATCA TAATCAGCCA

     2701 TACCACATTT GTAGAGGTTT TACTTGCTTT AAAAAACCTC CCACACCTCC CCCTGAACCT

     2761 GAAACATAAA ATGAATGCAA TTGTTGTTGT TAACTTGTTT ATTGCAGCTT ATAATGGTTA

     2821 CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAG

     2881 TTGTGGTTTG TCCAAACTCA TCAATGTATC TTAACGCTCG AGTGCATTCT AGTTGTGGTT

     2941 TGTCCAAACT CATCAATGTA TCTTATCATG TCTGTATACC GTCGACCTCT AGCTAGAGCT

     3001 TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC ACAATTCCAC

     3061 ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA GTGAGCTAAC

     3121 TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG TCGTGCCAGC

     3181 TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG

     3241 CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC

     3301 ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA AAGAACATGT

     3361 GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC

     3421 ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA

     3481 ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC

     3541 CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG

     3601 CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC

     3661 TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC GGTAACTATC

     3721 GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA

     3781 GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT

     3841 ACGGCTACAC TAGAAGAACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG

     3901 GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC GGTGGTTTTT

     3961 TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT CCTTTGATCT

     4021 TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA

     4081 GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA

     4141 TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC

     4201 CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA

     4261 TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA CCGCGAGACC

     4321 CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA

     4381 GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA

     4441 GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT ACAGGCATCG

     4501 TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC

     4561 GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG

     4621 TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT

     4681 CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT

     4741 CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA

     4801 ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC

     4861 GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC

     4921 CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA

     4981 GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT

     5041 TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT

     5101 TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC CGAAAAGTGC

     5161 CACCTGACGT C

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