pRetroX-PTuner

pRetroX-PTuner

pRetroX-PTuner逆病毒载体基本信息：

pRetroX-PTuner载体质粒图谱和多克隆位点信息：

pRetroX-PTuner载体简介：

pRetroX-PTuner载体描述

pRetroX-PTuner 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 allows the simultaneous expression of your DD-tagged protein of interest and puromycin acetyltransferase (PAC; for puromycin resistance (Puror)) from the same bicistronic mRNA transcript. Because PAC (i.e., Puror) is unaffected by the DD tag, it can be used as a selection marker.

Bicistronic expression of PAC 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 PAC (Puror) from an internal start site at the IRES/Purorjunction (2). 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 is derived from the pMIN series of retroviral vectors (3, 4). 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 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 also contains a ColE1 origin of replication, and an E. coli Ampr gene for propagation and selection in bacteria

pRetroX-PTuner is available in the Retro-X ProteoTuner Shield System N . It is designed to efficiently deliver and co-express your DD-tagged protein of interest and puromycin acetyltransferase (Puror) 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, the vector must be transfected into a packaging 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.

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 TCCATGGCGC CCCTCTCCCT

 1741 CCCCCCCCCC TAACGTTACT GGCCGAAGCC GCTTGGAATA AGGCCGGTGT GCGTTTGTCT

 1801 ATATGTTATT TTCCACCATA TTGCCGTCTT TTGGCAATGT GAGGGCCCGG AAACCTGGCC

 1861 CTGTCTTCTT GACGAGCATT CCTAGGGGTC TTTCCCCTCT CGCCAAAGGA ATGCAAGGTC

 1921 TGTTGAATGT CGTGAAGGAA GCAGTTCCTC TGGAAGCTTC TTGAAGACAA ACAACGTCTG

 1981 TAGCGACCCT TTGCAGGCAG CGGAACCCCC CACCTGGCGA CAGGTGCCTC TGCGGCCAAA

 2041 AGCCACGTGT ATAAGATACA CCTGCAAAGG CGGCACAACC CCAGTGCCAC GTTGTGAGTT

 2101 GGATAGTTGT GGAAAGAGTC AAATGGCTCT CCTCAAGCGT ATTCAACAAG GGGCTGAAGG

 2161 ATGCCCAGAA GGTACCCCAT TGTATGGGAT CTGATCTGGG GCCTCGGTGC ACATGCTTTA

 2221 CATGTGTTTA GTCGAGGTTA AAAAAACGTC TAGGCCCCCC GAACCACGGG GACGTGGTTT

 2281 TCCTTTGAAA AACACGATGA TAAGCTTGCC ACAACCCACA AGGAGACGAC CTTCCATGAC

 2341 CGAGTACAAG CCCACGGTGC GCCTCGCCAC CCGCGACGAC GTCCCCCGGG CCGTACGCAC

 2401 CCTCGCCGCC GCGTTCGCCG ACTACCCCGC CACGCGCCAC ACCGTCGACC CGGACCGCCA

 2461 CATCGAGCGG GTCACCGAGC TGCAAGAACT CTTCCTCACG CGCGTCGGGC TCGACATCGG

 2521 CAAGGTGTGG GTCGCGGACG ACGGCGCCGC GGTGGCGGTC TGGACCACGC CGGAGAGCGT

 2581 CGAAGCGGGG GCGGTGTTCG CCGAGATCGG CCCGCGCATG GCCGAGTTGA GCGGTTCCCG

 2641 GCTGGCCGCG CAGCAACAGA TGGAAGGCCT CCTGGCGCCG CACCGGCCCA AGGAGCCCGC

 2701 GTGGTTCCTG GCCACCGTCG GCGTCTCGCC CGACCACCAG GGCAAGGGTC TGGGCAGCGC

 2761 CGTCGTGCTC CCCGGAGTGG AGGCGGCCGA GCGCGCCGGG GTGCCCGCCT TCCTGGAGAC

 2821 CTCCGCGCCC CGCAACCTCC CCTTCTACGA GCGGCTCGGC TTCACCGTCA CCGCCGACGT

 2881 CGAGGTGCCC GAAGGACCGC GCACCTGGTG CATGACCCGC AAGCCCGGTG CCACCGGTGC

 2941 GGCTTCGAGG ATAAAATAAA AGATTTTATT TAGTCTCCAG AAAAAGGGGG GAATGAAAGA

 3001 CCCCACCTGT AGGTTTGGCA AGCTAGCTTA AGTAACGCCA TTTTGCAAGG CATGGAAAAA

 3061 TACATAACTG AGAATAGAGA AGTTCAGATC AAGGTCAGGA ACAGATGGAA CAGCTGAATA

 3121 TGGGCCAAAC AGGATATCTG TGGTAAGCAG TTCCTGCCCC GGCTCAGGGC CAAGAACAGA

 3181 TGGAACAGCT GAATATGGGC CAAACAGGAT ATCTGTGGTA AGCAGTTCCT GCCCCGGCTC

 3241 AGGGCCAAGA ACAGATGGTC CCCAGATGCG GTCCAGCCCT CAGCAGTTTC TAGAGAACCA

 3301 TCAGATGTTT CCAGGGTGCC CCAAGGACCT GAAATGACCC TGTGCCTTAT TTGAACTAAC

 3361 CAATCAGTTC GCTTCTCGCT TCTGTTCGCG CGCTTCTGCT CCCCGAGCTC AATAAAAGAG

 3421 CCCACAACCC CTCACTCGGG GCGCCAGTCC TCCGATTGAC TGAGTCGCCC GGGTACCCGT

 3481 GTATCCAATA AACCCTCTTG CAGTTGCATC CGACTTGTGG TCTCGCTGTT CCTTGGGAGG

 3541 GTCTCCTCTG AGTGATTGAC TACCCGTCAG CGGGGGTCTT TCATTGAATT CGTAATCATG

 3601 GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC

 3661 CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC

 3721 GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT

 3781 CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC

 3841 TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT

 3901 AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA

 3961 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC

 4021 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT

 4081 ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT

 4141 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG

 4201 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA

 4261 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA

 4321 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC

 4381 GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG

 4441 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG

 4501 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA

 4561 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC

 4621 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG

 4681 GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA

 4741 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT

 4801 CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG

 4861 GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC

 4921 TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC

 4981 AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC

 5041 GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC

 5101 GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC

 5161 CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA

 5221 GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC TTACTGTCAT

 5281 GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA

 5341 GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA

 5401 TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG

 5461 GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC

 5521 AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC AAAATGCCGC

 5581 AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA

 5641 TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG AATGTATTTA

 5701 GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTCTA

 5761 AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA GGCCCTTTCG

 5821 TCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT

 5881 CACAGCTTGT CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG

 5941 TGTTGGCGGG TGTCGGGGCT GGCTTAACTA TGCGGCATCA GAGCAGATTG TACTGAGAGT

 6001 GCACCATATG CGGTGTGAAA TACCGCACAG ATGCGTAAGG AGAAAATACC GCATCAGGCG

 6061 CCATTCGCCA TTCAGGCTGC GCAACTGTTG GGAAGGGCGA TCGGTGCGGG CCTCTTCGCT

 6121 ATTACGCCAG CTGGCGAAAG GGGGATGTGC TGCAAGGCGA TTAAGTTGGG TAACGCCAGG

 6181 GTTTTCCCAG TCACGACGTT GTAAAACGAC GGCCAGTGCC AAGCTTATG

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