pRetroX-Tight-Pur

pRetroX-Tight-Pur

pRetroX-Tight-Pur逆病毒载体基本信息：

pRetroX-Tight-Pur载体质粒图谱和多克隆位点信息：

pRetroX-Tight-Pur载体简介：

pRetroX-Tight-Pur载体描述

pRetroX-Tight-Pur is an inducible, self-inactivating, retroviral expression vector designed to express a gene of interest 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-XTM Tet-On Advanced 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 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-Pur supports high viral titers, yet eliminates potential downstream interference between the inducible PTight promoter and its adjacent viral LTR.

pRetroX-Tight-Pur 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 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 can be used as either a plasmid or retroviral expression vector. When used as a retroviral expression vector, 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 and transmit your gene of interest, 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.

pRetroX-Tet-On Advanced载体描述

pRetroX-Tet-On Advanced is a Retro-X Q retroviral vector that expresses rtTA-Advanced, an improved version of the reverse tetracycline(Tet)-controlled transactivator protein rtTA (1–4). rtTA-Advanced is more sensitive to doxycycline (Dox) and yields lower background expression than the original rtTA. The rtTA-Advanced protein is a fusion of the Tet repressor (TetR) DNA-binding domain, and three minimal “F”-type transcriptional activation domains from the herpes simplex virus VP16 protein. The gene encoding rtTA-Advanced is completely synthetic, lacks cryptic splice sites, and utilizes human codon preferences for stable expression in mammalian cells. Expression of rtTA-Advanced is driven by the powerful, constitutively active CMV promoter.

As with all of our Retro-X Q vectors, pRetroX-Tet-On Advanced uses LTR self-inactivation to eliminate promoter interference. In LTR self-inactivation, the mechanism of viral integration is used to introduce a deletion into the 5’ LTR. As a result of this mutation, the 5’ LTR CMV/MSV promoter, which drives high expression of the complete viral genome in packaging cells, is inactive in stably transduced target cells. This allows the expression of rtTA-Advanced (from PCMV IE) to proceed unimpeded (5, 6). The vector also contains a neomycin resistance gene (Neor) that allows G418 selection of stably transduced cells. rtTA-Advanced and the Neor marker are coexpressed from a bicistronic transcript containing an internal ribosome entry site (IRES).This ensures that a high frequency of G418 resistant clones express the Tet-On Advanced transactivator.

pRetroX-Tet-On Advanced contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, a packaging signal (Ψ+), and a tRNA primer binding site. The vector also contains an SV40 origin of replication for plasmid propagation in mammalian cells that express SV40 T antigen. In addition, a pUC origin of replication and an E. coli Ampr gene allow for propagation and selection in bacteria.

The pRetroX-Tet-On Advanced vector is used in conjunction with Tet response vectors to create double stable Retro-X Tet-On Advanced cell lines. Such cell lines are essentially Doxycycline (Dox)-controlled gene expression systems in which the Tet response vector expresses a gene of interest under the control of a Tet-responsive element (TRE; e.g. PTight; 7, 8). In the presence of Dox, rtTA-Advanced binds to the TRE and activates expression of the gene of interest. In the absence of Dox, rtTA-Advanced is unable to bind to the TRE, and the system is inactive. Additional information on TRE-containing vectors and protocols describing how to construct a Retro-X Tet-On Advanced cell line can be found in the Retro-X Tet-On Advanced Inducible Gene Expression System User manual (PT3958-1).

Selection of Stable Transfectants

 Selectable marker: plasmid confers resistance to G418.

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: pUC

pRetroX-Tight-Pur载体序列：

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 GCGGCCGCGC CGGCTCTAGA TCGCGAACGC GTGAATTCTA CCGGGTAGGG

 1981 GAGGCGCTTT TCCCAAGGCA GTCTGGAGCA TGCGCTTTAG CAGCCCCGCT GGGCACTTGG

 2041 CGCTACACAA GTGGCCTCTG GCCTCGCACA CATTCCACAT CCACCGGTAG GCGCCAACCG

 2101 GCTCCGTTCT TTGGTGGCCC CTTCGCGCCA CCTTCTACTC CTCCCCTAGT CAGGAAGTTC

 2161 CCCCCCGCCC CGCAGCTCGC GTCGTGCAGG ACGTGACAAA TGGAAGTAGC ACGTCTCACT

 2221 AGTCTCGTGC AGATGGACAG CACCGCTGAG CAATGGAAGC GGGTAGGCCT TTGGGGCAGC

 2281 GGCCAATAGC AGCTTTGCTC CTTCGCTTTC TGGGCTCAGA GGCTGGGAAG GGGTGGGTCC

 2341 GGGGGCGGGC TCAGGGGCGG GCTCAGGGGC GGGGCGGGCG CCCGAAGGTC CTCCGGAGGC

 2401 CCGGCATTCT GCACGCTTCA AAAGCGCACG TCTGCCGCGC TGTTCTCCTC TTCCTCATCT

 2461 CCGGGCCTTT CGACCTGCAG CCCAAGCTTA CCATGACCGA GTACAAGCCC ACGGTGCGCC

 2521 TCGCCACCCG CGACGACGTC CCCAGGGCCG TACGCACCCT CGCCGCCGCG TTCGCCGACT

 2581 ACCCCGCCAC GCGCCACACC GTCGATCCGG ACCGCCACAT CGAGCGGGTC ACCGAGCTGC

 2641 AAGAACTCTT CCTCACGCGC GTCGGGCTCG ACATCGGCAA GGTGTGGGTC GCGGACGACG

 2701 GCGCCGCGGT GGCGGTCTGG ACCACGCCGG AGAGCGTCGA AGCGGGGGCG GTGTTCGCCG

 2761 AGATCGGCCC GCGCATGGCC GAGTTGAGCG GTTCCCGGCT GGCCGCGCAG CAACAGATGG

 2821 AAGGCCTCCT GGCGCCGCAC CGGCCCAAGG AGCCCGCGTG GTTCCTGGCC ACCGTCGGCG

 2881 TCTCGCCCGA CCACCAGGGC AAGGGTCTGG GCAGCGCCGT CGTGCTCCCC GGAGTGGAGG

 2941 CGGCCGAGCG CGCCGGGGTG CCCGCCTTCC TGGAGACCTC CGCGCCCCGC AACCTCCCCT

 3001 TCTACGAGCG GCTCGGCTTC ACCGTCACCG CCGACGTCGA GGTGCCCGAA GGACCGCGCA

 3061 CCTGGTGCAT GACCCGCAAG CCCGGTGCCT GACGCCCGCC CCACGACCCG CAGCGCCCGA

 3121 CCGAAAGGAG CGCACGACCC CATGCATCGG CGTCTCGAGA TATCAGTGGT CCAGGCTCTA

 3181 GTTTTGACTC AACAATATCA CCAGCTGAAG CCTATAGAGT ACGAGCCATA GATAAAATAA

 3241 AAGATTTTAT TTAGTCTCCA GAAAAAGGGG GGAATGAAAG ACCCCACCTG TAGGTTTGGC

 3301 AAGCTAGCTT AAGTAACGCC ATTTTGCAAG GCATGGAAAA ATACATAACT GAGAATAGAG

 3361 AAGTTCAGAT CAAGGTCAGG AACAGATGGA ACAGGGTCGA CCCTAGAGAA CCATCAGATG

 3421 TTTCCAGGGT GCCCCAAGGA CCTGAAATGA CCCTGTGCCT TATTTGAACT AACCAATCAG

 3481 TTCGCTTCTC GCTTCTGTTC GCGCGCTTCT GCTCCCCGAG CTCAATAAAA GAGCCCACAA

 3541 CCCCTCACTC GGGGCGCCAG TCCTCCGATT GACTGAGTCG CCCGGGTACC CGTGTATCCA

 3601 ATAAACCCTC TTGCAGTTGC ATCCGACTTG TGGTCTCGCT GTTCCTTGGG AGGGTCTCCT

 3661 CTGAGTGATT GACTACCCGT CAGCGGGGGT CTTTCATTTG GGGGCTCGTC CGGGATCGGG

 3721 AGACCCCTGC CCAGGGACCA CCGACCCACC ACCGGGAGGT AAGCTGGCTG CCTCGCGCGT

 3781 TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT CACAGCTTGT

 3841 CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG TGTTGGCGGG

 3901 TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG GAGTGTAGAT CCGGCTGTGG

 3961 AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA

 4021 AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC

 4081 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG

 4141 CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT

 4201 TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA

 4261 GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAAAGCTTA CTGGCTTAAC TATGCGGCAT

 4321 CAGAGCAGAT TGTACTGAGA GTGCACCATA TGCGGTGTGA AATACCGCAC AGATGCGTAA

 4381 GGAGAAAATA CCGCATCAGG CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG

 4441 TCGTTCGGCT GCGGCGAGCG GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG

 4501 AATCAGGGGA TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC

 4561 GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA

 4621 AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT

 4681 TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC

 4741 TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC

 4801 TCAGTTCGGT GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC

 4861 CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT

 4921 TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG

 4981 CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTGGTA

 5041 TCTGCGCTCT GCTGAAGCCA GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA

 5101 AACAAACCAC CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA

 5161 AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG

 5221 AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC

 5281 TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG

 5341 ACAGTTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT

 5401 CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG

 5461 GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA

 5521 TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA

 5581 TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC

 5641 GCAACGTTGT TGCCATTGCT GCAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT

 5701 CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA

 5761 AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT

 5821 CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT

 5881 TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA

 5941 GTTGCTCTTG CCCGGCGTCA ACACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG

 6001 TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA

 6061 GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA

 6121 CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG

 6181 CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC

 6241 AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG

 6301 GGGTTCCGCG CACATTTCCC CGAAAAGTGC CACCTGACGT CTAAGAAACC ATTATTATCA

 6361 TGACATTAAC CTATAAAAAT AGGCGTATCA CGAGGCCCTT TCGTCTTCAA GAATTAGCTT

 6421 GGCCATTGCA TACGTTGTAT CCATATCATA ATATGTACAT TTATATTGGC TCATGTCCAA

 6481 CATTACCGCC ATGTTGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA ATTACGGGGT

 6541 CATTAGTTCA TAGCCCATAT ATGGAGT

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