| 出品公司: | Clontech |
|---|---|
| 载体名称: | pLVX-PTuner |
| 质粒类型: | 慢病毒载体；双顺反子载体 |
| 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: | 8473 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: | 嘌呤霉素（Puromycin） |
| 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: | 632173 |
| 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载体质粒基本信息
pLVX-PTuner质粒图谱载体图谱和pLVX-PTuner载体序列质粒序列多克隆位点信息
pLVX-PTuner质粒载体简介
pLVX-PTuner载体描述
pLVX-PTuner 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 allows the simultaneous expression of your DD-tagged protein of interest and a puromycin resistance marker (Puror) from the same bicistronic mRNA transcript. Expression of this 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 Puror (above), facilitates cap-independent translation of Puror from an internal start site at the IRES/Puror junction (2). Because Puror is unaffected by the DD tag, puromycin resistance can be used as an indicator of transduction efficiency and a marker for selection.
pLVX-PTuner 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 (3), 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 (4). Finally, pLVX-PTuner 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 (5). 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 is available in the Lenti-X ProteoTuner Shield System N (Cat. No. 632173). The vector is designed to constitutively coexpress a DD-tagged protein of interest and puromycin resistance 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 (6).
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. Jang, S. K. et al. (1988) J. Virol. 62(8):2636–2643.
3. Zufferey, R. et al. (1999) J. Virol. 73(4):2886–2892.
4. Cochrane, A. W. et al. (1990) Proc. Natl. Acad. Sci. USA 87(3):1198–1202.
5. Zennou, V. et al. (2000) Cell 101(2):173–185.
6. Wu, X. et al. (2000) Mol. Ther. 2(1):47–55.
pLVX-PTuner质粒序列载体序列
LOCUS pLVX-PTuner 8472 bp DNA circular 8-NOV-2009
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat. No. 632173,632174
FEATURES Location/Qualifiers
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 4394..4985
/label=WPRE
rep_origin complement(6293..6966)
/label=pUC\origin
CDS complement(7111..8107)
/label=Amp(R)
misc_binding 1303..1536
/label=RRE
splicing_signal 744..745
/label=SD
splicing_signal 1883..1884
/label=SA
misc_binding 636..653
/label=PBS
LTR 5188..5824
/label=3'\LTR
CDS 2805..3128
/label=DD
misc_feature 3173..3780
/label=IRES
CDS 3781..4380
/label=Puro(R)
BASE COUNT 2170 a 2109 c 2188 g 2005 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 gctctcctca agcgtattca acaaggggct
3601 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
3661 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
3721 ggttttcctt tgaaaaacac gatgataagc ttgccacaac ccacaaggag acgaccttcc
3781 atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta
3841 cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac
3901 cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac
3961 atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag
4021 agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt
4081 tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag
4141 cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa gggtctgggc
4201 agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg
4261 gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc
4321 gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctag
4381 acgcgtctgg aacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
4441 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
4501 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
4561 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
4621 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
4681 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
4741 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt
4801 tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
4861 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
4921 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
4981 gcctggaatt aattctgcag tcgagaccta gaaaaacatg gagcaatcac aagtagcaat
5041 acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt
5101 tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
5161 agccactttt taaaagaaaa gaggggactg gaagggctaa ttcactccca acgaagacaa
5221 gatatccttg atctgtggat ctaccacaca caaggctact tccctgatta gcagaactac
5281 acaccagggc caggggtcag atatccactg acctttggat ggtgctacaa gctagtacca
5341 gttgagccag ataaggtaga agaggccaat aaaggagaga acaccagctt gttacaccct
5401 gtgagcctgc atgggatgga tgacccggag agagaagtgt tagagtggag gtttgacagc
5461 cgcctagcat ttcatcacgt ggcccgagag ctgcatccgg agtacttcaa gaactgctga
5521 tatcgagctt gctacaaggg actttccgct ggggactttc cagggaggcg tggcctgggc
5581 gggactgggg agtggcgagc cctcagatcc tgcatataag cagctgcttt ttgcctgtac
5641 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
5701 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
5761 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
5821 cagtagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc
5881 agagagtgag aggccttgac attgctagcg tttaccgtcg acctctagct agagcttggc
5941 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
6001 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
6061 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
6121 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
6181 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
6241 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
6301 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
6361 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
6421 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
6481 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
6541 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
6601 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
6661 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
6721 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
6781 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
6841 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
6901 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
6961 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
7021 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
7081 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
7141 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
7201 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
7261 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
7321 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
7381 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
7441 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
7501 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
7561 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
7621 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
7681 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
7741 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
7801 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
7861 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
7921 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
7981 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
8041 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
8101 tgacgtcgac ggatcgggag atcaacttgt ttattgcagc ttataatggt tacaaataaa
8161 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
8221 tgtccaaact catcaatgta tcttatcatg tctggatcaa ctggataact caagctaacc
8281 aaaatcatcc caaacttccc accccatacc ctattaccac tgccaattac ctgtggtttc
8341 atttactcta aacctgtgat tcctctgaat tattttcatt ttaaagaaat tgtatttgtt
8401 aaatatgtac tacaaactta gtagttttta aagaaattgt atttgttaaa tatgtactac
8461 aaacttagta gt
//
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