pLVX-PTuner-Green

价格:3500元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Clontech
载体名称: pLVX-PTuner-Green
质粒类型: 慢病毒载体;双顺反子载体;荧光报告载体
高拷贝/低拷贝: 高拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV
载体大小: 8536 bp
5' 测序引物及序列: CMV Forward: CGCAAATGGGCGGTAGGCGTG
3' 测序引物及序列: --
载体标签: DD tag (N-端)
载体抗性: 氨苄青霉素
筛选标记: Zsgreen1荧光筛选
克隆菌株: Stbl3
宿主细胞(系): 常规细胞系(293、CV-1、CHO等)
备注: 慢毒载体pLVX-PTuner-Green载体表达N端DD标签融合蛋白;
DD标签是一个大小为12KD的FKBP (L106P)去稳定结构域,带有DD标签的目的蛋白将很快被降解;
向培养基中加入Shield1配体后,情况将发生逆转,细胞中目的蛋白的量则迅速提高。
产品目录号: 632175
稳定性: 稳表达
组成型/诱导型: 组成型
病毒/非病毒: 慢病毒

载体质粒图谱和多克隆位点信息

pLVX-PTuner-Green载体图谱



pLVX-PTuner-Green 多克隆位点

pLVX-PTuner-Green 载体特征

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: high

Notes:
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.

References
1. 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-2009
COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT     Cat. No. 632176,632175
FEATURES             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=DD
BASE 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 
//

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