pRetroX-Tight-Hyg-Luc载体描述pRetroX-Tight-Hyg-Luc is a retroviral control vector (supplied with pRetroX-Tight-Hyg) that expresses fi refl y luciferase under the control of PTight, a modifi ed 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 modifi ed minimal CMV promoter (1). This vector is designed to be used as a control vector with Clontech's Advanced Inducible Expression Systems. 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-Hyg-Luc is optimized to eliminate promoter interference through LTR selfinactivation. 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, resultingin the inactivation of the 5’ LTR CMV enhancer sequence. This mechanism can reduce the phenomenon known as promoter interference (9-10) and allow more effi cient expression. Inthis 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-Hyg-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-Hyg-Luc contains a hygromycin resistance gene (Hygr) 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-Hyg-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 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: highNotes:The attached sequence fi le has been 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 sequence.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.
LOCUS pRetroX-Tight-Hyg-Luc 9052 bp DNA circular 10-NOV-2009COMMENT Created by Clontech Laboratories Inc. http://www.clontech.comCOMMENT Cat. No. 631034FEATURES Location/Qualifiers promoter 3602..4105 /label=PGK\Promoter misc_feature 4123..5148 /label=Hyg(R) LTR 5748..6184 /label=3'\MMLV\LTR\(del) CDS complement(7830..8690) /label=Amp(R) promoter 1602..1917 /label=Tight\Promoter LTR 1..727 /label=5'\LTR\(CMV/MSV) misc_feature 758..1567 /label=Packaging\Signal promoter 6463..6730 /label=SV40\Promoter rep_origin 6684..6749 /label=SV40\origin rep_origin 7071..7071 /label=pUC\origin misc_feature 1934..3586 /label=LuciferaseBASE COUNT 2184 a 2335 c 2332 g 2201 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 catcccgcca ccatgaaaaa gcctgaactc 4141 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg 4201 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat 4261 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac 4321 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggagtt cagcgagagc 4381 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc 4441 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat 4501 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca 4561 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg 4621 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag 4681 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg 4741 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa 4801 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg 4861 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg 4921 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca 4981 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt 5041 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc 5101 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagag tagatgccga 5161 ccgaacaaga gctgatttcg agaacgcctc agccagcaac tcgcgcgagc ctagcaaggc 5221 aaatgcgaga gaacggcctt acgcttggtg gcacagttct cgtccacagt tcgctaagct 5281 cgctcggctg ggtcgcggga gggccggtcg cagtgattca ggcccttctg gattgtgttg 5341 gtccccaggg cacgattgtc atgcccacgc actcgggtga tctgactgat cccgcagatt 5401 ggagatcgcc gcccgtgcct gccgattggg tgcagatccg tcgacctgca gccaagctta 5461 tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc 5521 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aatacataac 5581 tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat atgggccaaa 5641 caggatatca gtggtccagg ctctagtttt gactcaacaa tatcaccagc tgaagcctat 5701 agagtacgag ccatagataa aataaaagat tttatttagt ctccagaaaa aggggggaat 5761 gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg 5821 gaaaaataca taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagg 5881 gtcgacccta gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg 5941 tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc 6001 ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc cgattgactg 6061 agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc 6121 tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc 6181 atttgggggc tcgtccggga tcgggagacc cctgcccagg gaccaccgac ccaccaccgg 6241 gaggtaagct ggctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 6301 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 6361 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga 6421 tagcggagtg tagatccggc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg 6481 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 6541 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 6601 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 6661 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 6721 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa 6781 gcttactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg 6841 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc 6901 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 6961 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 7021 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 7081 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 7141 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 7201 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 7261 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 7321 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 7381 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 7441 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 7501 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 7561 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 7621 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 7681 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 7741 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 7801 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 7861 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 7921 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 7981 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 8041 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 8101 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg 8161 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt