pLVX-PTuner-Green

载体基本信息

载体质粒图谱和多克隆位点信息

载体简介

pLVX-PTuner-Green载体描述pLVX-PTuner Green is a bicistronic, HIV-1-based, lentiviral expression vector that allows you to precisely regulate the amount of your protein of interest in virtually any mammalian cell type, including primary 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, causing rapid degradation of the fusion protein. Degradation of the DD-tagged protein can be prevented 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.pLVX-PTuner Green allows the simultaneous expression of your DD-tagged protein of interest and the fluorescent protein ZsGreen1 from the same bicistronic mRNA transcript. ZsGreen1 is a human codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein, ZsGreen (2). Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the DD tag coding sequence. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), positioned between the MCS and the ZsGreen1 gene (see above), facilitates cap-independent translation of ZsGreen1 from an internal start site at the IRES/ZsGreen1 junction (3). Because ZsGreen1 is unaffected by the DD tag, it can be used as an indicator of transfection and transduction efficiency, as well as a marker for selection and cell sorting.pLVX-PTuner Green contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral and transgene RNA (4), leading to increased viral titers from packaging cells and enhanced expression of your gene of interest in target cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (5). Finally, pLVX-PTuner Green also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (6). The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.pLVX-PTuner Green is available in the Lenti-X ProteoTuner Shield System N (w/ ZsGreen1) [Cat. No. 632175]. The vector is designed to constitutively coexpress a DD-tagged protein of interest and ZsGreen1 from PCMV IE when transduced into mammalian cells. In order to create your N-terminally 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. Before it can be transduced into target cells, the vector must be cotransfected into 293T cells with our Lenti-X HTX Packaging System (Cat. Nos. 631247 and 631249) and packaged into viral particles. This packaging system allows the safe production of high titer, infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including non-dividing and primary cells (7).ZsGreen1 is the brightest commercially available green fluorescent protein. The presence of this protein allows the selection of stable transductants by flow cytometry (or other detection methods) with standard FITC filter sets (ZsGreen1 has an excitation maximum of 493 nm and 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 the ligand 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: pUC Copy number: highNotes:The vector sequence was compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by Clontech. This vector has not been completely sequenced.The viral supernatants produced by this lentiviral vector could contain potentially hazardous recombinant virus. Due caution must be exercised in the production and handling of recombinant lentivirus. Appropriate NIH, regional, and institutional guidelines apply.References1. Banaszynski, L. et al. (2006) Cell 126(5):995–1004.2. BD Living Colors pIRES2-ZsGreen1 Vector (January 2005) Clontechniques XX(1):8.3. Jang, S. K. et al. (1988) J. Virol. 62(8):2636–2643.4. Zufferey, R. et al. (1999) J. Virol. 73(4):2886–2892.5. Cochrane, A. W. et al. (1990) Proc. Natl. Acad. Sci. USA 87(3):1198–1202.6. Zennou, V. et al. (2000) Cell 101(2):173–185.7. Wu, X. et al. (2000) Mol. Ther. 2(1):47–55.

载体序列

LOCUS pLVX-PTuner_Green 8536 bp DNA circular 8-NOV-2009COMMENT Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT Cat. No. 632176,632175FEATURES Location/Qualifiers CDS 3749..4444 /label=ZsGreen1 misc_feature 3174..3748 /label=IRES LTR 1..635 /label=5'\LTR misc_feature 685..822 /label=packaging\signal misc_feature 2028..2151 /label=cPPT/CTS\(DNA\flap) promoter 2185..2787 /label=CMV\IE\Promoter misc_feature 4458..5049 /label=WPRE rep_origin complement(6357..7030) /label=pUC\origin CDS complement(7175..8171) /label=Amp(R) misc_binding 1303..1536 /label=RRE misc_binding 636..653 /label=PBS LTR 5252..5888 /label=3'\LTR CDS 2805..3128 /label=DDBASE COUNT 2220 a 2097 c 2180 g 2039 t ORIGIN 1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2041 gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2101 aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2161 acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 2221 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2281 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2341 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2401 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2461 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2521 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2581 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2641 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2701 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 2761 tccatagaag acaccgactc tactagagga tctatttaaa taccatggga gtgcaggtgg 2821 aaaccatctc cccaggagac gggcgcacct tccccaagcg cggccagacc tgtgtggtgc 2881 actacaccgg gatgcttgaa gatggaaaga aagtcgattc ctcccgggac agaaacaagc 2941 cctttaagtt tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc 3001 agatgagtgt gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca 3061 ctgggcaccc aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa 3121 aaccggaaac cggtgaattc ctcgagacta gttctagagc ggccgcggat cccgcccctc 3181 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt 3241 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc 3301 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca 3361 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac 3421 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg 3481 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt 3541 gagttggata gttgtggaaa gagtcaaatg gctcacctca agcgtattca acaaggggct 3601 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg 3661 ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca cggggacgtg 3721 gttttccttt gaaaaacacg atgataatat ggcccagtcc aagcacggcc tgaccaagga 3781 gatgaccatg aagtaccgca tggagggctg cgtggacggc cacaagttcg tgatcaccgg 3841 cgagggcatc ggctacccct tcaagggcaa gcaggccatc aacctgtgcg tggtggaggg 3901 cggccccttg cccttcgccg aggacatctt gtccgccgcc ttcatgtacg gcaaccgcgt 3961 gttcaccgag tacccccagg acatcgtcga ctacttcaag aactcctgcc ccgccggcta 4021 cacctgggac cgctccttcc tgttcgagga cggcgccgtg tgcatctgca acgccgacat 4081 caccgtgagc gtggaggaga actgcatgta ccacgagtcc aagttctacg gcgtgaactt 4141 ccccgccgac ggccccgtga tgaagaagat gaccgacaac tgggagccct cctgcgagaa 4201 gatcatcccc gtgcccaagc agggcatctt gaagggcgac gtgagcatgt acctgctgct 4261 gaaggacggt ggccgcttgc gctgccagtt cgacaccgtg tacaaggcca agtccgtgcc 4321 ccgcaagatg cccgactggc acttcatcca gcacaagctg acccgcgagg accgcagcga 4381 cgccaagaac cagaagtggc acctgaccga gcacgccatc gcctccggct ccgccttgcc 4441 ctgaacgcgt ctggaacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta 4501 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc 4561 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt 4621 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg 4681 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt 4741 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct 4801 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt 4861 cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct 4921 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc 4981 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct 5041 ccccgcctgg aattaattct gcagtcgaga cctagaaaaa catggagcaa tcacaagtag 5101 caatacagca gctaccaatg ctgattgtgc ctggctagaa gcacaagagg aggaggaggt 5161 gggttttcca gtcacacctc aggtaccttt aagaccaatg acttacaagg cagctgtaga 5221 tcttagccac tttttaaaag aaaagagggg actggaaggg ctaattcact cccaacgaag 5281 acaagatatc cttgatctgt ggatctacca cacacaaggc tacttccctg attagcagaa 5341 ctacacacca gggccagggg tcagatatcc actgaccttt ggatggtgct acaagctagt 5401 accagttgag ccagataagg tagaagaggc caataaagga gagaacacca gcttgttaca 5461 ccctgtgagc ctgcatggga tggatgaccc ggagagagaa gtgttagagt ggaggtttga 5521 cagccgccta gcatttcatc acgtggcccg agagctgcat ccggagtact tcaagaactg 5581 ctgatatcga gcttgctaca agggactttc cgctggggac tttccaggga ggcgtggcct 5641 gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg ctttttgcct 5701 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga 5761 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc 5821 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct 5881 ctagcagtag tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa 5941 tatcagagag tgagaggcct tgacattgct agcgtttacc gtcgacctct agctagagct 6001 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 6061 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 6121 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 6181 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 6241 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 6301 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 6361 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 6421 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 6481 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 6541 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 6601 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 6661 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 6721 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 6781 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 6841 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 6901 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 6961 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 7021 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 7081 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 7141 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 7201 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 7261 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 7321 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 7381 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 7441 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 7501 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 7561 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 7621 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 7681 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 7741 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 7801 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 7861 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 7921 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 7981 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 8041 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 8101 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 8161 cacctgacgt cgacggatcg ggagatcaac ttgtttattg cagcttataa tggttacaaa 8221 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 8281 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat aactcaagct 8341 aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa ttacctgtgg 8401 tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag aaattgtatt 8461 tgttaaatat gtactacaaa cttagtagtt tttaaagaaa ttgtatttgt taaatatgta 8521 ctacaaactt agtagt