pRetroX-IRES-DsRedExpress

pRetroX-IRES-DsRedExpress

pRetroX-IRES-DsRedExpress逆病毒载体基本信息：

pRetroX-IRES-DsRedExpress载体质粒图谱和多克隆位点信息：

pRetroX-IRES-DsRedExpress载体简介：

pRetroX-IRES-DsRedExpress载体描述

pRetroX-IRES-DsRedExpress is a bicistronic, fl uorescent, retroviral vector that allows both a gene of interest and the DsRed-Express gene to be translated from a single bicistronic mRNA.pRetroX-IRES-DsRedExpress is designed for effi cient delivery and selection (by fl ow cytometry or other methods) of stably transduced mammalian cells expressing the DsRed-Express fl uorescent protein and the protein of interest. This vector can be used to obtain stable cell lines without time-consuming drug and clonal selection. DsRed-Express is a rapidly maturing variant of Discosoma sp. red fl uorescent protein (DsRed) and is easily detected with standard Rhodamine/propidium iodide fi lter sets (1). Bicistronic expression from this vector is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). This IRES facilitates cap-independent translation from an internal start site at the IRES/DsRed-Express junction (2). DsRed-Express contains nine amino acid substitutions that enhance its solubility, reduce its green emission, and accelerate its maturation (3). This retroviral vector is derived from the pMIN series of vectors (4, 5). These optimized vectors have the ability to produce high viral titers, express genes at high levels, and, due to the absence of retroviral coding sequences, exhibit improved safety profi les. The multiple cloning site (MCS) in pRetroX-IRES-DsRedExpress is between the 5’ MMLV LTR and the IRES sequence. Genes cloned into the MCS are expressed as a bicistronic message transcribed from the 5’ LTR.

pRetroX-IRES-DsRedExpress contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, the packaging signal (ψ), and the tRNA primer-binding site. For safety reasons, however, the vector lacks the structural genes (gag, pol, and env) necessary for retroviral particle formation and replication. pRetroX-IRES-DsRedExpress contains a ColE1 origin of replication, and an E. coli Ampr gene for propagation and selection in bacteria.

pRetroX-IRES-DsRedExpress is designed to effi ciently deliver and co-express your gene of interest and DsRed-Express in any mitotically-active mammalian cell. When cloning into pRetroX-IRES-DsRedExpress, your gene of interest should contain an initiation codon (ATG) and a stop codon. The vector can be infected or transfected into mammalian cells. If required, stable transformants can be selected by fl ow cytometry or limiting dilution.

In order to infect mammalian cells with pRetroX-IRES-DsRedExpress, the vector must be transfected into a packaging cell line, such as the RetroPack PT67 Cell line (631510), AmphoPack-293 (631505), EcoPack2- 293 (631507), Pantropic Expression System (631512), or Retro-X Universal Packaging System (631530). These cell lines package RNA from the vector into infectious, replication-incompetent, retroviral particles. Such retroviral particles can infect target cells and transmit the gene of interest, but cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chance of producing replication-competent virus due to recombination events during cell proliferation.

Propagation in E. coli

 Suitable host strains: DH5α, Fusion Blue, and other general purpose strains.

 Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.

 E. coli replication origin: ColE1

 Copy number: high

pRetroX-IRES-DsRedExpress载体序列：

ORIGIN

    1 TGAAAGACCC CACCTGTAGG TTTGGCAAGC TAGCTTAAGT AACGCCATTT TGCAAGGCAT

   61 GGAAAAATAC ATAACTGAGA ATAGAAAAGT TCAGATCAAG GTCAGGAACA GATGGAACAG

  121 CTGAATATGG GCCAAACAGG ATATCTGTGG TAAGCAGTTC CTGCCCCGGC TCAGGGCCAA

  181 GAACAGATGG AACAGCTGAA TATGGGCCAA ACAGGATATC TGTGGTAAGC AGTTCCTGCC

  241 CCGGCTCAGG GCCAAGAACA GATGGTCCCC AGATGCGGTC CAGCCCTCAG CAGTTTCTAG

  301 AGAACCATCA GATGTTTCCA GGGTGCCCCA AGGACCTGAA ATGACCCTGT GCCTTATTTG

  361 AACTAACCAA TCAGTTCGCT TCTCGCTTCT GTTCGCGCGC TTCTGCTCCC CGGGCTCAAT

  421 AAAAGAGCCC ACAACCCCTC ACTCGGCGCG CCAGTCCTCC GATTGACTGA GTCGCCCGGG

  481 TACCCGTGTA TCCAATAAAC CCTCTTGCAG TTGCATCCGA CTTGTGGCCT CGCTGTTCCT

  541 TGGGAGGGTC TCCTCTGAGT GATTGACTAC CCGTCAGCGG GGGTCTTTCA TTTGGGGGCT

  601 CGTCCGGGAT CGGGAGACCC CTGCCCAGGG ACCACCGACC CACCACCGGG AGGTAAGCTG

  661 GCCAGCAACT TATCTGTGTC TGTCCGATTG TCTAGTGTCT ATGACTGATT TTATGCGCCT

  721 GCGTCGGTAC TAGTTAGCTA ACTAGCTCTG TATCTGGCGG ACCCGTGGTG GAACTGACGA

  781 GTTCGGAACA CCCGGCCGCA ACCCTGGGAG ACGTCCCAGG GACTTCGGGG GCCGTTTTTG

  841 TGGCCCGACC TGAGTCCAAA AATCCCGATC GTTTTGGACT CTTTGGTGCA CCCCCCTTAG

  901 AGGAGGGATA TGTGGTTCTG GTAGGAGACG AGAACCTAAA ACAGTTCCCG CCTCCGTCTG

  961 AATTTTTGCT TTCGGTTTGG GACCGAAGCC GCGCCGCGCG TCTTGTCTGC TGCAGCATCG

 1021 TTCTGTGTTG TCTCTGTCTG ACTGTGTTTC TGTATTTGTC TGAAAATACG CGTGTCGAGC

 1081 TTTTGGAGTA CGTCGTCTTT AGGTTGGGGG GAGGGGTTTT ATGCGATGGA GTTTCCCCAC

 1141 ACTGAGTGGG TGGAGACTGA AGTTAGGCCA GCTTGGCACT TGATGTAATT CTCCTTGGAA

 1201 TTTGCCCTTT TTGAGTTTGG ATCTTGGTTC ATTCTCAAGC CTCAGACAGT GGTTCAAAGT

 1261 TTTTTTCTTC CATTTCAGCC GTCGTGAAAA CTACCCCTAA AAGCCAAAAA AATCGAATTC

 1321 CCAAACTTAA GCTTGGTACC GAGCTCGGAT CTCACGTGGG CCCGCGGCCG CAGATCTATC

 1381 GATGGATCCG TGCCCCTCTC CCTCCCCCCC CCCTAACGTT ACTGGCCGAA GCCGCTTGGA

 1441 ATAAGGCCGG TGTGCGTTTG TCTATATGTT ATTTTCCACC ATATTGCCGT CTTTTGGCAA

 1501 TGTGAGGGCC CGGAAACCTG GCCCTGTCTT CTTGACGAGC ATTCCTAGGG GTCTTTCCCC

 1561 TCTCGCCAAA GGAATGCAAG GTCTGTTGAA TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC

 1621 TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG CAGCGGAACC CCCCACCTGG

 1681 CGACAGGTGC CTCTGCGGCC AAAAGCCACG TGTATAAGAT ACACCTGCAA AGGCGGCACA

 1741 ACCCCAGTGC CACGTTGTGA GTTGGATAGT TGTGGAAAGA GTCAAATGGC TCACCTCAAG

 1801 CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC CATTGTATGG GATCTGATCT

 1861 GGGGCCTCGG TGCACATGCT TTACATGTGT TTAGTCGAGG TTAAAAAACG TCTAGGCCCC

 1921 CCGAACCACG GGGACGTGGT TTTCCTTTGA AAAACACGAT GATAATATGG CCTCCTCCGA

 1981 GGACGTCATC AAGGAGTTCA TGCGCTTCAA GGTGCGCATG GAGGGCTCCG TGAACGGCCA

 2041 CGAGTTCGAG ATCGAGGGCG AGGGCGAGGG CCGCCCCTAC GAGGGCACCC AGACCGCCAA

 2101 GCTGAAGGTG ACCAAGGGCG GCCCCCTGCC CTTCGCCTGG GACATCCTGT CCCCCCAGTT

 2161 CCAGTACGGC TCCAAGGTGT ACGTGAAGCA CCCCGCCGAC ATCCCCGACT ACAAGAAGCT

 2221 GTCCTTCCCC GAGGGCTTCA AGTGGGAGCG CGTGATGAAC TTCGAGGACG GCGGCGTGGT

 2281 GACCGTGACC CAGGACTCCT CCCTGCAGGA CGGCTCCTTC ATCTACAAGG TGAAGTTCAT

 2341 CGGCGTGAAC TTCCCCTCCG ACGGCCCCGT AATGCAGAAG AAGACTATGG GCTGGGAGGC

 2401 CTCCACCGAG CGCCTGTACC CCCGCGACGG CGTGCTGAAG GGCGAGATCC ACAAGGCCCT

 2461 GAAGCTGAAG GACGGCGGCC ACTACCTGGT GGAGTTCAAG TCTATCTATA TGGCCAAGAA

 2521 GCCCGTGCAG CTGCCCGGCT ACTACTACGT GGACTCCAAG CTGGACATCA CCTCCCACAA

 2581 CGAGGACTAC ACCATCGTGG AGCAGTACGA GCGCGCCGAG GGCCGCCACC ACCTGTTCCT

 2641 GTAGAATGCT CGAGGATAAA ATAAAAGATT TTATTTAGTC TCCAGAAAAA GGGGGGAATG

 2701 AAAGACCCCA CCTGTAGGTT TGGCAAGCTA GCTTAAGTAA CGCCATTTTG CAAGGCATGG

 2761 AAAAATACAT AACTGAGAAT AGAGAAGTTC AGATCAAGGT CAGGAACAGA TGGAACAGCT

 2821 GAATATGGGC CAAACAGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC AGGGCCAAGA

 2881 ACAGATGGAA CAGCTGAATA TGGGCCAAAC AGGATATCTG TGGTAAGCAG TTCCTGCCCC

 2941 GGCTCAGGGC CAAGAACAGA TGGTCCCCAG ATGCGGTCCA GCCCTCAGCA GTTTCTAGAG

 3001 AACCATCAGA TGTTTCCAGG GTGCCCCAAG GACCTGAAAT GACCCTGTGC CTTATTTGAA

 3061 CTAACCAATC AGTTCGCTTC TCGCTTCTGT TCGCGCGCTT CTGCTCCCCG AGCTCAATAA

 3121 AAGAGCCCAC AACCCCTCAC TCGGGGCGCC AGTCCTCCGA TTGACTGAGT CGCCCGGGTA

 3181 CCCGTGTATC CAATAAACCC TCTTGCAGTT GCATCCGACT TGTGGTCTCG CTGTTCCTTG

 3241 GGAGGGTCTC CTCTGAGTGA TTGACTACCC GTCAGCGGGG GTCTTTCATT GAATTCGTAA

 3301 TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC ACACAACATA

 3361 CGAGCCGGAA GCATAAAGTG TAAAGCCTGG GGTGCCTAAT GAGTGAGCTA ACTCACATTA

 3421 ATTGCGTTGC GCTCACTGCC CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA

 3481 TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG

 3541 CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG

 3601 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA

 3661 GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC

 3721 CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA

 3781 GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG

 3841 ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT

 3901 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT

 3961 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG

 4021 TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC

 4081 AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC

 4141 ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA

 4201 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC

 4261 AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG

 4321 GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA

 4381 AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT

 4441 ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA

 4501 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTCGTGTA GATAACTACG

 4561 ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA

 4621 CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT

 4681 CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT

 4741 AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA

 4801 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA

 4861 TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA

 4921 AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT

 4981 GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA

 5041 GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG

 5101 CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC

 5161 TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA

 5221 TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT

 5281 GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT

 5341 CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT

 5401 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC

 5461 GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC

 5521 TTTCGTCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG

 5581 ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA

 5641 GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG

 5701 AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG TAAGGAGAAA ATACCGCATC

 5761 AGGCGCCATT CGCCATTCAG GCTGCGCAAC TGTTGGGAAG GGCGATCGGT GCGGGCCTCT

 5821 TCGCTATTAC GCCAGCTGGC GAAAGGGGGA TGTGCTGCAA GGCGATTAAG TTGGGTAACG

 5881 CCAGGGTTTT CCCAGTCACG ACGTTGTAAA ACGACGGCCA GTGCCAAGCT TATG

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