pRetroX-PTuner-IRES

pRetroX-PTuner-IRES

pRetroX-PTuner-IRES逆病毒载体基本信息：

pRetroX-PTuner-IRES载体质粒图谱和多克隆位点信息：

pRetroX-PTuner-IRES载体简介：

pRetroX-PTuner-IRES 载体描述

pRetroX-PTuner IRES is a bicistronic, retroviral expression vector that allows you to precisely regulate the amount of your protein of interest in stably transduced mammalian cells. The vector encodes a 12 kDa, FKBP (L106P) destabilization domain (DD; 1) that is expressed as an N-terminal tag on your protein of interest; this domain causes the rapid degradation of any protein to which it is fused. Once expressed, the amount of DD-tagged protein present in the cell can be rapidly increased by the addition of Shield1 stabilizing ligand to the medium. Shield1 is a membrane permeable molecule that binds to the DD tag, 'shielding' the fusion protein from proteasomal degradation. pRetroX-PTuner IRES allows the simultaneous expression of your DD-tagged protein of interest and ZsGreen1 fluorescent protein from the same

bicistronic mRNA transcript. Because ZsGreen1 is unaffected by the DD tag, it can be used as an indicator of transfection efficiency, as well as a marker for selection and cell sorting.

ZsGreen1 is a human codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein, ZsGreen (2). Bicistronic expression of ZsGreen1 and the DD-tagged protein of interest is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). This IRES allows cap-independent translation of ZsGreen1 from an internal start site at the IRES/ZsGreen1 junction (3). A gene cloned into the multiple cloning site (MCS), located between the DD and the IRES sequences, is expressed as a bicistronic message transcribed from the 5’ LTR.

pRetroX-PTuner IRES is derived from the pMIN series of retroviral vectors (4, 5). These optimized vectors have the ability to produce high viral titers, and high levels of recombinant protein. In addition, because they lack retroviral structural genes (gag, pol, and env) necessary for retroviral particle formation and

replication, these vectors exhibit improved safety profiles. pRetroX-PTuner IRES 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. pRetroX-PTuner IRES also contains a ColE1 origin of replication, and an E. coli Ampr gene for propagation and selection in bacteria.

pRetroX-PTuner IRES is available in the RetroX-ProteoTuner Shield System N (w/ ZsGreen1) [Cat. No. 632167]. It is designed to efficiently deliver and co-express your DD-tagged protein of interest and ZsGreen1 in any mitotically active mammalian cell. In order to create your DD-tagged protein of interest, your gene of interest must be cloned into the MCS in the same reading frame as the DD tag sequence, and it must contain a stop codon at the end of its coding sequence.

In order to infect mammalian cells with pRetroX-PTuner IRES, the vector must be transfected into apackaging cell line, such as the RetroPack PT67 Cell line, AmphoPack-293, EcoPack2-293 , Pantropic Retroviral Expression System , or Retro-X Universal Packaging System . These cell lines package RNA transcribed from the vector into infectious, replication-incompetent, retroviral particles. Such retroviral particles can transduce 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.

Stably transduced mammalian cells express ZsGreen1, the brightest commercially available green fluorescent protein. The presence of this protein allows the selection of stable transductants by flow cytometry (orother detection methods) with standard FITC filter sets (ZsGreen1 has an excitation maximum of 493 nmand an emission maximum of 505 nm).

When cells expressing a DD-tagged protein of interest are grown in medium containing Shield1, the ligand binds to the DD tag and protects the fusion protein from degradation. As a result, the protein quickly accumulates inside the cells in amounts that are directly proportional to the concentration of Shield1 in the medium. If the cells are subsequently grown in medium lacking Shield1, the DD tag is no longer stabilized, and the fusion protein is rapidly degraded. Because the effects of Shield1 are concentration-dependent and reversible, it is possible to fine-tune the amount of fusion protein present in the cells simply by adjusting the concentration of Shield1 in the medium (1).

Propagation in E. coli

 Suitable host strains: Stellar Competent Cells.

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

 E. coli replication origin: ColE1

 Copy number: high

pRetroX-PTuner-IRES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 CCCGCGGATG GGAGTGCAGG

 1381 TGGAAACCAT CTCCCCAGGA GACGGGCGCA CCTTCCCCAA GCGCGGCCAG ACCTGTGTGG

 1441 TGCACTACAC CGGGATGCTT GAAGATGGAA AGAAAGTCGA TTCCTCCCGG GACAGAAACA

 1501 AGCCCTTTAA GTTTATGCTA GGCAAGCAGG AGGTGATCCG AGGCTGGGAA GAAGGGGTTG

 1561 CCCAGATGAG TGTGGGTCAG AGAGCCAAAC TGACTATATC TCCAGATTAT GCCTATGGTG

 1621 CCACTGGGCA CCCAGGCATC ATCCCACCAC ATGCCACTCT CGTCTTCGAT GTGGAGCTTC

 1681 TAAAACCGGA ACCGCGGCCG CAGATCTATC GATGGATCCG TGCCCCTCTC CCTCCCCCCC

 1741 CCCTAACGTT ACTGGCCGAA GCCGCTTGGA ATAAGGCCGG TGTGCGTTTG TCTATATGTT

 1801 ATTTTCCACC ATATTGCCGT CTTTTGGCAA TGTGAGGGCC CGGAAACCTG GCCCTGTCTT

 1861 CTTGACGAGC ATTCCTAGGG GTCTTTCCCC TCTCGCCAAA GGAATGCAAG GTCTGTTGAA

 1921 TGTCGTGAAG GAAGCAGTTC CTCTGGAAGC TTCTTGAAGA CAAACAACGT CTGTAGCGAC

 1981 CCTTTGCAGG CAGCGGAACC CCCCACCTGG CGACAGGTGC CTCTGCGGCC AAAAGCCACG

 2041 TGTATAAGAT ACACCTGCAA AGGCGGCACA ACCCCAGTGC CACGTTGTGA GTTGGATAGT

 2101 TGTGGAAAGA GTCAAATGGC TCACCTCAAG CGTATTCAAC AAGGGGCTGA AGGATGCCCA

 2161 GAAGGTACCC CATTGTATGG GATCTGATCT GGGGCCTCGG TGCACATGCT TTACATGTGT

 2221 TTAGTCGAGG TTAAAAAACG TCTAGGCCCC CCGAACCACG GGGACGTGGT TTTCCTTTGA

 2281 AAAACACGAT GATAATATGG CCCAGTCCAA GCACGGCCTG ACCAAGGAGA TGACCATGAA

 2341 GTACCGCATG GAGGGCTGCG TGGACGGCCA CAAGTTCGTG ATCACCGGCG AGGGCATCGG

 2401 CTACCCCTTC AAGGGCAAGC AGGCCATCAA CCTGTGCGTG GTGGAGGGCG GCCCCTTGCC

 2461 CTTCGCCGAG GACATCTTGT CCGCCGCCTT CATGTACGGC AACCGCGTGT TCACCGAGTA

 2521 CCCCCAGGAC ATCGTCGACT ACTTCAAGAA CTCCTGCCCC GCCGGCTACA CCTGGGACCG

 2581 CTCCTTCCTG TTCGAGGACG GCGCCGTGTG CATCTGCAAC GCCGACATCA CCGTGAGCGT

 2641 GGAGGAGAAC TGCATGTACC ACGAGTCCAA GTTCTACGGC GTGAACTTCC CCGCCGACGG

 2701 CCCCGTGATG AAGAAGATGA CCGACAACTG GGAGCCCTCC TGCGAGAAGA TCATCCCCGT

 2761 GCCCAAGCAG GGCATCTTGA AGGGCGACGT GAGCATGTAC CTGCTGCTGA AGGACGGTGG

 2821 CCGCTTGCGC TGCCAGTTCG ACACCGTGTA CAAGGCCAAG TCCGTGCCCC GCAAGATGCC

 2881 CGACTGGCAC TTCATCCAGC ACAAGCTGAC CCGCGAGGAC CGCAGCGACG CCAAGAACCA

 2941 GAAGTGGCAC CTGACCGAGC ACGCCATCGC CTCCGGCTCC GCCTTGCCCT GAGCGGCTTC

 3001 GAGGATAAAA TAAAAGATTT TATTTAGTCT CCAGAAAAAG GGGGGAATGA AAGACCCCAC

 3061 CTGTAGGTTT GGCAAGCTAG CTTAAGTAAC GCCATTTTGC AAGGCATGGA AAAATACATA

 3121 ACTGAGAATA GAGAAGTTCA GATCAAGGTC AGGAACAGAT GGAACAGCTG AATATGGGCC

 3181 AAACAGGATA TCTGTGGTAA GCAGTTCCTG CCCCGGCTCA GGGCCAAGAA CAGATGGAAC

 3241 AGCTGAATAT GGGCCAAACA GGATATCTGT GGTAAGCAGT TCCTGCCCCG GCTCAGGGCC

 3301 AAGAACAGAT GGTCCCCAGA TGCGGTCCAG CCCTCAGCAG TTTCTAGAGA ACCATCAGAT

 3361 GTTTCCAGGG TGCCCCAAGG ACCTGAAATG ACCCTGTGCC TTATTTGAAC TAACCAATCA

 3421 GTTCGCTTCT CGCTTCTGTT CGCGCGCTTC TGCTCCCCGA GCTCAATAAA AGAGCCCACA

 3481 ACCCCTCACT CGGGGCGCCA GTCCTCCGAT TGACTGAGTC GCCCGGGTAC CCGTGTATCC

 3541 AATAAACCCT CTTGCAGTTG CATCCGACTT GTGGTCTCGC TGTTCCTTGG GAGGGTCTCC

 3601 TCTGAGTGAT TGACTACCCG TCAGCGGGGG TCTTTCATTG AATTCGTAAT CATGGTCATA

 3661 GCTGTTTCCT GTGTGAAATT GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG

 3721 CATAAAGTGT AAAGCCTGGG GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG

 3781 CTCACTGCCC GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA

 3841 ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC

 3901 GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG

 3961 GTTATCCACA GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA

 4021 GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA

 4081 CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG

 4141 ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT

 4201 TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG

 4261 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC

 4321 CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT

 4381 AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA

 4441 TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGAAC

 4501 AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC

 4561 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT

 4621 TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC

 4681 TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT

 4741 CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA

 4801 AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT

 4861 ATTTCGTTCA TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG

 4921 CTTACCATCT GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA

 4981 TTTATCAGCA ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT

 5041 ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT

 5101 TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT

 5161 TGGTATGGCT TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT

 5221 GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC

 5281 CGCAGTGTTA TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC

 5341 CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT

 5401 GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG

 5461 AACTTTAAAA GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT

 5521 ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC

 5581 TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA

 5641 GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG

 5701 AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA

 5761 TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCTAAGAAAC

 5821 CATTATTATC ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTCTCGC

 5881 GCGTTTCGGT GATGACGGTG AAAACCTCTG ACACATGCAG CTCCCGGAGA CGGTCACAGC

 5941 TTGTCTGTAA GCGGATGCCG GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG

 6001 CGGGTGTCGG GGCTGGCTTA ACTATGCGGC ATCAGAGCAG ATTGTACTGA GAGTGCACCA

 6061 TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAAAA TACCGCATCA GGCGCCATTC

 6121 GCCATTCAGG CTGCGCAACT GTTGGGAAGG GCGATCGGTG CGGGCCTCTT CGCTATTACG

 6181 CCAGCTGGCG AAAGGGGGAT GTGCTGCAAG GCGATTAAGT TGGGTAACGC CAGGGTTTTC

 6241 CCAGTCACGA CGTTGTAAAA CGACGGCCAG TGCCAAGCTT ATG

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