| 出品公司: | ZYbscience |
|---|---|
| 载体名称: | pRetroX-Tight-Pur-Luc |
| 质粒类型: | 逆转录病毒载体；四环素诱导载体；荧光素酶报告载体 |
| 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: | Tight |
| 载体大小: | 8201 bp |
| 5' 测序引物及序列: | -- |
| 3' 测序引物及序列: | -- |
| 载体标签: | -- |
| 载体抗性: | 氨苄青霉素 |
| 筛选标记: | 嘌呤霉素（Puromycin） |
| 克隆菌株: | DH5α 等通用菌株 |
| 宿主细胞（系）: | 常规细胞系（293、CV-1、CHO等） |
| 备注: | 逆转录病毒载体pRetroX-Tight-Pur-Luc含有荧光素酶报告基因， 是pRetroX-Tight-Pur载体的阳性对照载体。 |
| 产品目录号: | ZY632104 |
| 稳定性: | 稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 逆转录病毒 |
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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20℃低温保存
- 保质期：
三年
- 英文名：
pRetroX-Tight-Pur-Luc
- 库存：
20
- 供应商：
泽叶生物
载体基本信息
载体质粒图谱和多克隆位点信息
载体简介
pRetroX-Tight-Pur-Lu载体描述
pRetroX-Tight-Pur-Luc is a retroviral control vector that expresses firefly luciferase under the control of PTight, a modified Tet-responsive promoter. PTight consists of seven direct repeats of a 36 bp regulatory sequence that contains the 19 bp tet operator sequence (tetO), and a modified minimal CMV promoter (1). This vector is supplied with our Retro-X?Tet-On? and Tet-Off? Advanced Inducible Expression Systems (Cat. Nos. 632104 and 632105). These systems provide ready access to the inducible gene expression strategy of Gossen & Bujard, and incorporate major improvements described by Urlinger, et al. (2-6).
pRetroX-Tight-Pur-Luc is optimized to eliminate promoter interference through LTR self-inactivation. The hybrid 5’ LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This promoter drives high levels of viral genome transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (7-8) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3’ LTR enhancer region (U3). During reverse transcription of the retroviral RNA, a copy of the inactivated 3’ LTR U3 region replaces the corresponding region of the 5’ LTR, resulting in the inactivation of the 5’ LTR CMV enhancer sequence. This mechanism can reduce the phenomenon known as promoter interference (9-10) and allow more efficient expression. In this way, pRetroX-Tight-Hyg-Luc supports high viral titers, yet eliminates potential downstream interference between the inducible PTight promoter and its adjacent viral LTR.
pRetroX-Tight-Pur-Luc 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. For safety reasons, however, the vector lacks the structural genes (gag, pol, and env) necessary for retroviral particle formation and replication. pRetroX-Tight-Pur-Luc contains a puromycin resistance gene (Purr) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transfectants. In addition, the vector contains a ColE1 origin of replication and an E. coli Ampr gene for propagation and selection in bacteria.pRetroX-Tight-Pur-Luc can be used as a control vector for either plasmid or retroviral expression vectors. When used as a control for retroviral expression vectors, it must be transfected into a packaging cell line, such as GP2-293; we recommend the Retro-X Universal Packaging System (Cat. No. 631530). Packaging cell lines allow you to produce infectious, replication-incompetent retroviral particles. These retroviral particles can infect a wide range of target cells, but they 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.
Propagation in E. coli
Suitable host strains: DH5α?, DH10B?, and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: ColE1
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 retroviral vector could contain potentially hazardous recombinant virus. Due caution must be exercised in the production and
handling of recombinant retrovirus. Appropriate NIH, regional, and institutional guidelines apply.
References
1. pTRE-Tight Vectors (April 2003) Clontechniques XVIII(3):13-14.
2. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci USA 89(12):5547–5551.
3. Gossen, M., et al. (1995) Science 268(5218):1766–1769.
4. Urlinger, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97(14):7963-7968.
5. Inducible Gene Expression Systems (January 2007) Clontechniques XXII(1):1-2.
6. Tet-On Advanced Inducible Gene Expression System (2006) Clontechniques XXI(2):1-3.
7. Ory, D.S. et al. (1996) Proc. Natl. Acad. Sci. USA 93(21):11400-11406.
8. Pear, W.S. et al. (1993) Proc. Natl. Acad. Sci. USA 90(18):8392-8396.
9. Barton, G.M. & Medzhitov R. (2002) Proc. Natl. Sci. USA 99(23):14943-14945.
10. Emerman, M. & Temin, H.M. (1984) Cell 39(3 pt. 2):449-467.
载体序列
LOCUS pRetroX-Tight-Pur-Luc 8200 bp DNA circular 10-NOV-2009
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat. No. 631128,632104,631129,632105
FEATURES Location/Qualifiers
misc_feature 1..727
/label=5'\LTR\(CMV/MSV)
misc_feature 758..1567
/label=Extended\packaging\signal
misc_feature 1602..1917
/label=Tight\Promoter
misc_feature 1934..3586
/label=Luciferase
misc_feature 3602..4105
/label=PGK\Promoter
misc_feature 4126..4725
/label=Puro(R)
misc_feature 4946..5331
/label=3'\MMLV\LTR\(del)
misc_feature 5611..5878
/label=SV40\Promoter
misc_feature 5832..5897
/label=SV40\origin
misc_feature 6219..6219
/label=pUC\origin
misc_feature complement(6978..7838)
/label=Amp(R)
BASE COUNT 1943 a 2196 c 2099 g 1962 t
ORIGIN
1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
241 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
301 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
361 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
421 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
481 gtgtacggtg ggaggtctat ataagcagag ctcaataaaa gagcccacaa cccctcactc
541 ggcgcgccag tcttccgata gactgcgtcg cccgggtacc cgtattccca ataaagcctc
601 ttgctgtttg catccgaatc gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
661 tgactaccca cgacgggggt ctttcatttg ggggctcgtc cgggatttgg agacccctgc
721 ccagggacca ccgacccacc accgggaggt aagctggcca gcaacttatc tgtgtctgtc
781 cgattgtcta gtgtctatgt ttgatgttat gcgcctgcgt ctgtactagt tagctaacta
841 gctctgtatc tggcggaccc gtggtggaac tgacgagttc tgaacacccg gccgcaaccc
901 tgggagacgt cccagggact ttgggggccg tttttgtggc ccgacctgag gaagggagtc
961 gatgtggaat ccgaccccgt caggatatgt ggttctggta ggagacgaga acctaaaaca
1021 gttcccgcct ccgtctgaat ttttgctttc ggtttggaac cgaagccgcg cgtcttgtct
1081 gctgcagcgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc
1141 tgaaaattag ggccagactg ttaccactcc cttaagtttg accttaggtc actggaaaga
1201 tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt gggttacctt
1261 ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca cctttaaccg
1321 agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg gacacccaga
1381 ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc cctgggtcaa
1441 gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt ctctccccct
1501 tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca ctccttctct
1561 aggcgccgga attgaagatc tgaggccctt tcgtcttcac tcgagtttac tccctatcag
1621 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgat gtcgagttta
1681 ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt
1741 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttatc cctatcagtg
1801 atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgaggtagg
1861 cgtgtacggt gggaggccta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
1921 agaaggatcc accatggaag acgccaaaaa cataaagaaa ggcccggcgc cattctatcc
1981 gctagaggat ggaaccgctg gagagcaact gcataaggct atgaagagat acgccctggt
2041 tcctggaaca attgctttta cagatgcaca tatcgaggtg aacatcacgt acgcggaata
2101 cttcgaaatg tccgttcggt tggcagaagc tatgaaacga tatgggctga atacaaatca
2161 cagaatcgtc gtatgcagtg aaaactctct tcaattcttt atgccggtgt tgggcgcgtt
2221 atttatcgga gttgcagttg cgcccgcgaa cgacatttat aatgaacgtg aattgctcaa
2281 cagtatgaac atttcgcagc ctaccgtagt gtttgtttcc aaaaaggggt tgcaaaaaat
2341 tttgaacgtg caaaaaaaat taccaataat ccagaaaatt attatcatgg attctaaaac
2401 ggattaccag ggatttcagt cgatgtacac gttcgtcaca tctcatctac ctcccggttt
2461 taatgaatac gattttgtac cagagtcctt tgatcgtgac aaaacaattg cactgataat
2521 gaactcctct ggatctactg ggttacctaa gggtgtggcc cttccgcata gaactgcctg
2581 cgtcagattc tcgcatgcca gagatcctat ttttggcaat caaatcattc cggatactgc
2641 gattttaagt gttgttccat tccatcacgg ttttggaatg tttactacac tcggatattt
2701 gatatgtgga tttcgagtcg tcttaatgta tagatttgaa gaagagctgt ttttacgatc
2761 ccttcaggat tacaaaattc aaagtgcgtt gctagtacca accctatttt cattcttcgc
2821 caaaagcact ctgattgaca aatacgattt atctaattta cacgaaattg cttctggggg
2881 cgcacctctt tcgaaagaag tcggggaagc ggttgcaaaa cgcttccatc ttccagggat
2941 acgacaagga tatgggctca ctgagactac atcagctatt ctgattacac ccgaggggga
3001 tgataaaccg ggcgcggtcg gtaaagttgt tccatttttt gaagcgaagg ttgtggatct
3061 ggataccggg aaaacgctgg gcgttaatca gagaggcgaa ttatgtgtca gaggacctat
3121 gattatgtcc ggttatgtaa acaatccgga agcgaccaac gccttgattg acaaggatgg
3181 atggctacat tctggagaca tagcttactg ggacgaagac gaacacttct tcatagttga
3241 ccgcttgaag tctttaatta aatacaaagg ataccaggtg gcccccgctg aattggagtc
3301 gatattgtta caacacccca acatcttcga cgcgggcgtg gcaggtcttc ccgacgatga
3361 cgccggtgaa cttcccgccg ccgttgttgt tttggagcac ggaaagacga tgacggaaaa
3421 agagatcgtg gattacgtcg ccagtcaagt aacaaccgcg aaaaagttgc gcggaggagt
3481 tgtgtttgtg gacgaagtac cgaaaggtct taccggaaaa ctcgacgcaa gaaaaatcag
3541 agagatcctc ataaaggcca agaagggcgg aaagtccaaa ttgtaaaata cgcgtgaatt
3601 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
3661 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
3721 taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
3781 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
3841 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
3901 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
3961 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
4021 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
4081 ctcttcctca tctccgggcc tttcgacctg cagcccaagc ttaccatgac cgagtacaag
4141 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
4201 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
4261 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
4321 gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg
4381 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
4441 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
4501 gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
4561 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
4621 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
4681 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac
4741 ccgcagcgcc cgaccgaaag gagcgcacga ccccatgcat cggcgtctcg agatatcagt
4801 ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc
4861 atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaatga aagaccccac
4921 ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga aaaatacata
4981 actgagaata gagaagttca gatcaaggtc aggaacagat ggaacagggt cgaccctaga
5041 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga
5101 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata
5161 aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt
5221 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt
5281 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcat ttgggggctc
5341 gtccgggatc gggagacccc tgcccaggga ccaccgaccc accaccggga ggtaagctgg
5401 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
5461 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
5521 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
5581 gatccggctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
5641 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
5701 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
5761 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
5821 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
5881 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttactggctt
5941 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
6001 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
6061 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
6121 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
6181 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
6241 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
6301 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
6361 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
6421 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
6481 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
6541 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
6601 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
6661 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
6721 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
6781 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
6841 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
6901 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
6961 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
7021 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
7081 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
7141 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
7201 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
7261 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
7321 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
7381 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
7441 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
7501 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
7561 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
7621 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
7681 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
7741 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
7801 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
7861 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
7921 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
7981 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
8041 caagaattag cttggccatt gcatacgttg tatccatatc ataatatgta catttatatt
8101 ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta ttaatagtaa
8161 tcaattacgg ggtcattagt tcatagccca tatatggagt
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pRetroX-Tight-Pur-Luc载体
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