| 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 |
买家导航
pLVX-PTuner-Green载体质粒基本信息
pLVX-PTuner-Green质粒图谱载体图谱和pLVX-PTuner-Green载体序列质粒序列多克隆位点信息
pLVX-PTuner-Green质粒载体简介
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.
pLVX-PTuner-Green质粒序列载体序列
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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