pTet-Off-Advanced expresses an improved version of the Tet (tetracycline)-controlled transactivator protein called tTA-Advanced (1–4). It is expressed to higher levels than the original tTA used in the Tet-Off? System (2). The tTA-Advanced protein is a fusion of amino acids 1–207 of the Tet repressor (TetR) and 39 amino acids containing three minimal "F"-type transcriptional activation domains (ADs) from the VP16 protein of herpes simplex virus. The gene is fully synthetic, lacks cryptic splice sites in the mRNA, and utilizes human codon preferences for stable expression in mammalian cells.

載體應(yīng)用

The pTet-Off-Advanced Vector is used to develop stable Tet-Off Advanced cell lines, which are hosts for a tetracycline-controlled gene expression system. Once a vector containing a gene of interest under the control of a Tet-responsive element (e.g., TRE-Tight or TRE2) is transfected into a Tet-Off Advanced stable cell line, tTA-Advanced binds to the TRE in the absence of doxycycline (Dox, a tetracycline derivative), and activates transcription of the gene of interest to a very high level. In the presence of Dox, tTA-Advanced is unable to bind the TRE in a Tet-responsive promoter and the system is inactive. Additional information on TRE-containing vectors and protocols describing how to construct a Tet-Off Advanced cell line can be found in the Tet-Off Advanced Inducible Gene Expression System User Manual (PT3945-1).

0001 CTCGAGGAGC TTGGCCCATT GCATACGTTG TATCCATATC ATAATATGTA CATTTATATT 
0061 GGCTCATGTC CAACATTACC GCCATGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA 
0121 TCAATTACGG GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG 
0181 GTAAATGGCC CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG 
0241 TATGTTCCCA TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA 
0301 CGGTAAACTG CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT 
0361 GACGTCAATG ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC 
0421 TTTCCTACTT GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT 
0481 TGGCAGTACA TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC 
0541 CCCATTGACG TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT 
0601 CGTAACAACT CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT 
0661 ATAAGCAGAG CTCGTTTAGT GAACCGTCAG ATCGCCTGGA GACGCCATCC ACGCTGTTTT 
0721 GACCTCCATA GAAGACACCG GGACCGATCC AGCCTCCGCG GCCCCGAATT CACCATGTCT 
0781 AGACTGGACA AGAGCAAAGT CATAAACTCT GCTCTGGAAT TACTCAATGA AGTCGGTATC 
0841 GAAGGCCTGA CGACAAGGAA ACTCGCTCAA AAGCTGGGAG TTGAGCAGCC TACCCTGTAC 
0901 TGGCACGTGA AGAACAAGCG GGCCCTGCTC GATGCCCTGG CAATCGAGAT GCTGGACAGG 
0961 CATCATACCC ACTTCTGCCC CCTGGAAGGC GAGTCATGGC AAGACTTTCT GCGGAACAAC 
1021 GCCAAGTCAT TCCGCTGTGC TCTCCTCTCA CATCGCGACG GGGCTAAAGT GCATCTCGGC 
1081 ACCCGCCCAA CAGAGAAACA GTACGAAACC CTGGAAAATC AGCTCGCGTT CCTGTGTCAG 
1141 CAAGGCTTCT CCCTGGAGAA CGCACTGTAC GCTCTGTCCG CCGTGGGCCA CTTTACACTG 
1201 GGCTGCGTAT TGGAGGATCA GGAGCATCAA GTAGCAAAAG AGGAAAGAGA GACACCTACC 
1261 ACCGATTCTA TGCCCCCACT TCTGAGACAA GCAATTGAGC TGTTCGACCA TCAGGGAGCC 
1321 GAACCTGCCT TCCTTTTCGG CCTGGAACTA ATCATATGTG GCCTGGAGAA ACAGCTAAAG 
1381 TGCGAAAGCG GCGGGCCGGC CGACGCCCTT GACGATTTTG ACTTAGACAT GCTCCCAGCC 
1441 GATGCCCTTG ACGACTTTGA CCTTGATATG CTGCCTGCTG ACGCTCTTGA CGATTTTGAC 
1501 CTTGACATGC TCCCCGGGTA ACTAAGTAAG GATCCAGACA TGATAAGATA CATTGATGAG 
1561 TTTGGACAAA CCACAACTAG AATGCAGTGA AAAAAATGCT TTATTTGTGA AATTTGTGAT 
1621 GCTATTGCTT TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA CAACAATTGC 
1681 ATTCATTTTA TGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTAAAG CAAGTAAAAC 
1741 CTCTACAAAT GTGGTATGGC TGATTATGAT CCTGCAAGCC TCGTCGTCCT GGCCGGACCA 
1801 CGCTATCTGT GCAAGGTCCC CGGCCCCGGA CGCGCGCTCC ATGAGCAGAG CGCCCGCCGC 
1861 CGAGGCGAAG ACTCGGGCGG CGCCCTGCCC GTCCCACCAG GTCAACAGGC GGTAACCGGC 
1921 CTCTTCATCG GGAATGCGCG CGACCTTCAG CATCGCCGGC ATGTCCCCCT GGCGGACGGG 
1981 AAGTATCCAG CTCGACCAAG CTTGGCGAGA TTTTCAGGAG CTAAGGAAGC TAAAATGGAG 
2041 AAAAAAATCA CTGGATATAC CACCGTTGAT ATATCCCAAT GGCATCGTAA AGAACATTTT 
2101 GAGGCATTTC AGTCAGTTGC TCAATGTACC TATAACCAGA CCGTTCAGCT GCATTAATGA 
2161 ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC 
2221 ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG 
2281 GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC 
2341 CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 
2401 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA 
2461 CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 
2521 CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT 
2581 AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 
2641 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC 
2701 AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA 
2761 GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT 
2821 AGAAGAACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 
2881 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG 
2941 CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG 
3001 TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA 
3061 AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 
3121 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG 
3181 ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA 
3241 CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC CGCGAGACCC ACGCTCACCG 
3301 GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 
3361 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT 
3421 TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC 
3481 TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA 
3541 TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT 
3601 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC 
3661 ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA 
3721 TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA CACGGGATAA TACCGCGCCA 
3781 CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 
3841 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT 
3901 TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC 
3961 GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA 
4021 TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT 
4081 TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCC ACCTGACGTC 
4141 TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA GGCGTATCAC GAGGCCCTTT 
4201 CGTCTTCACT CGAGGTCGAG GGATCCAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA 
4261 ACCACAACTA GAATGCAGTG AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT 
4321 TTATTTGTAA CCATTATAAG CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT 
4381 ATGTTTCAGG TTCAGGGGGA GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA 
4441 TGTGGTATGG CTGATTATGA TCTCTAGTCA AGGCACTATA CATCAAATAT TCCTTATTAA 
4501 CCCCTTTACA AATTAAAAAG CTAAAGGTAC ACAATTTTTG AGCATAGTTA TTAATAGCAG 
4561 ACACTCTATG CCTGTGTGGA GTAAGAAAAA ACAGTATGTT ATGATTATAA CTGTTATGCC 
4621 TACTTATAAA GGTTACAGAA TATTTTTCCA TAATTTTCTT GTATAGCAGT GCAGCTTTTT 
4681 CCTTTGTGGT GTAAATAGCA AAGCAAGCAA GAGTTCTATT ACTAAACACA GCATGACTCA 
4741 AAAAACTTAG CAATTCTGAA GGAAAGTCCT TGGGGTCTTC TACCTTTCTC TTCTTTTTTG 
4801 GAGGAGTAGA ATGTTGAGAG TCAGCAGTAG CCTCATCATC ACTAGATGGC ATTTCTTCTG 
4861 AGCAAAACAG GTTTTCCTCA TTAAAGGCAT TCCACCACTG CTCCCATTCA TCAGTTCCAT 
4921 AGGTTGGAAT CTAAAATACA CAAACAATTA GAATCAGTAG TTTAACACAT TATACACTTA 
4981 AAAATTTTAT ATTTACCTTA GAGCTTTAAA TCTCTGTAGG TAGTTTGTCC AATTATGTCA 
5041 CACCACAGAA GTAAGGTTCC TTCACAAAGA TCCGGACCAA AGCGGCCATC GTGCCTCCCC 
5101 ACTCCTGCAG TTCGGGGGCA TGGATGCGCG GATAGCCGCT GCTGGTTTCC TGGATGCCGA 
5161 CGGATTTGCA CTGCCGGTAG AACTCCGCGA GGTCGTCCAG CCTCAGGCAG CAGCTGAACC 
5221 AACTCGCGAG GGGATCGAGC CCGGGGTGGG CGAAGAACTC CAGCATGAGA TCCCCGCGCT 
5281 GGAGGATCAT CCAGCCGGCG TCCCGGAAAA CGATTCCGAA GCCCAACCTT TCATAGAAGG 
5341 CGGCGGTGGA ATCGAAATCT CGTGATGGCA GGTTGGGCGT CGCTTGGTCG GTCATTTCGA 
5401 ACCCCAGAGT CCCGCTCAGA AGAACTCGTC AAGAAGGCGA TAGAAGGCGA TGCGCTGCGA 
5461 ATCGGGAGCG GCGATACCGT AAAGCACGAG GAAGCGGTCA GCCCATTCGC CGCCAAGCTC 
5521 TTCAGCAATA TCACGGGTAG CCAACGCTAT GTCCTGATAG CGGTCCGCCA CACCCAGCCG 
5581 GCCACAGTCG ATGAATCCAG AAAAGCGGCC ATTTTCCACC ATGATATTCG GCAAGCAGGC 
5641 ATCGCCATGG GTCACGACGA GATCCTCGCC GTCGGGCATG CGCGCCTTGA GCCTGGCGAA 
5701 CAGTTCGGCT GGCGCGAGCC CCTGATGCTC TTCGTCCAGA TCATCCTGAT CGACAAGACC 
5761 GGCTTCCATC CGAGTACGTG CTCGCTCGAT GCGATGTTTC GCTTGGTGGT CGAATGGGCA 
5821 GGTAGCCGGA TCAAGCGTAT GCAGCCGCCG CATTGCATCA GCCATGATGG ATACTTTCTC 
5881 GGCAGGAGCA AGGTGAGATG ACAGGAGATC CTGCCCCGGC ACTTCGCCCA ATAGCAGCCA 
5941 GTCCCTTCCC GCTTCAGTGA CAACGTCGAG CACAGCTGCG CAAGGAACGC CCGTCGTGGC 
6001 CAGCCACGAT AGCCGCGCTG CCTCGTCCTG CAGTTCATTC AGGGCACCGG ACAGGTCGGT 
6061 CTTGACAAAA AGAACCGGGC GCCCCTGCGC TGACAGCCGG AACACGGCGG CATCAGAGCA 
6121 GCCGATTGTC TGTTGTGCCC AGTCATAGCC GAATAGCCTC TCCACCCAAG CGGCCGGAGA 
6181 ACCTGCGTGC AATCCATCTT GTTCAATCAT GCGAAACGAT CCTCATCCTG TCTCTTGATC 
6241 AGATCTTGAT CCCCTGCGCC ATCAGATCCT TGGCGGCAAG AAAGCCATCC AGTTTACTTT 
6301 GCAGGGCTTC CCAACCTTAC CAGAGGGCGC CCCAGCTGGC AATTCCGGTT CGCTTGCTGT 
6361 CCATAAAACC GCCCAGTCTA GCTATCGCCA TGTAAGCCCA CTGCAAGCTA CCTGCTTTCT 
6421 CTTTGCGCTT GCGTTTTCCC TTGTCCAGAT AGCCCAGTAG CTGACATTCA TCCGGGGTCA 
6481 GCACCGTTTC TGCGGACTGG CTTTCTACGT GTTCCGCTTC CTTTAGCAGC CCTTGCGCCC 
6541 TGAGTGCTTG CGGCAGCGTG AAGCTAGCTT TTTGCAAAAG CCTAGGCCTC CAAAAAAGCC 
6601 TCCTCACTAC TTCTGGAATA GCTCAGAGGC CGAGGCGGCC TCGGCCTCTG CATAAATAAA 
6661 AAAAATTAGT CAGCCATGGG GCGGAGAATG GGCGGAACTG GGCGGAGTTA GGGGCGGGAT 
6721 GGGCGGAGTT AGGGGCGGGA CTATGGTTGC TGACTAATTG AGATGCATGC TTTGCATACT 
6781 TCTGCCTGCT GGGGAGCCTG GGGACTTTCC ACACCTGGTT GCTGACTAAT TGAGATGCAT 
6841 GCTTTGCATA CTTCTGCCTG CTGGGGAGCC TGGGGACTTT CCACACCCTA ACTGACACAC 
6901 ATTCCACAGC TGCCTCGCGC GTTTCGGTGA TGACGGTGAA AACCTCTGAC ACATGCAGCT 
6961 CCCGGAGACG GTCACAGCTT GTCTGTAAGC GGATGCCGGG AGCAGACAAG CCCGTCAGGG 
7021 CGCGTCAGCG GGTGTTGGCG GGTGTCGGGG CGCAGCCATG ACCCAGTCAC GTAGCGATAG 
7081 CGGAGTGTAT ACTGGCTTAA CTATGCGGCA TCAGAGCAGA TTGTACTGAG AGTGCACCTC