LOCUS p2T7TAblue 6740 bp DNA circular 29-JUL-2008 This vector was designed by David Horn and is the most widely used workhorse for RNAi in bloodstream-form trypanosomes (or any stage cell that does not already contain the HYG marker. A doubly inducible system for RNA interference and rapid RNAi plasmid construction in Trypanosoma brucei (2005) Alibu VP, Storm L, Haile S, Clayton C & Horn D. Mol Biochem Parasitol 139:75-82. A gene fragment is cloned between the AhdI (or isoschizomer Eam11051) sites (or other adjacent sites, if necessary) and, by destroying the LacZ coding region enables blue-white selection. The plasmid is linearlized at the NotI or EcoRV sites for transfection. This file is based on original data from David Horn website, with improved annotations. FEATURES Location/Qualifiers source 5400..6246 /note="lacZ" /note=" Region changed from previous version of 2T7 from david's version my file dated august 2003. What vector was this fragment cloned from? pGEM series, probably" misc_feature 1..585 /note="rRNA spacer" terminator 6467..6727 /note="2 T7 terminators" terminator complement(4884..5144) /note="2 T7 terminators" misc_feature 6343..6456 /note="rRNA spacer fragment" promoter complement(6313..6332) /note="T7 promoter" protein_bind complement(6289..6307) /note="Tet operator" promoter complement(4567..4857) /note="rRNA promoter" 5'UTR complement(4436..4566) /note="ACT 5' UTR" CDS complement(3413..4435) /note="HYG" 3'UTR complement(3117..3412) /note="ACT 3' UTR" misc_feature 5145..5212 /note="ACT intergenic fragment" misc_feature 5213..5322 /note="rRNA spacer fragment" promoter 5339..5358 /note="T7 promoter" protein_bind 5364..5382 /note="Tet operator" CDS complement(5741..6106) /note="lacZ" CDS complement(1587..2447) /note="LACTAMASE" modified_base 1665..1665 /note="C-->G mutation David introduced a C>G mutation to eliminate the Eam11051 site in beta Lactamase CDS" BASE COUNT 1674 a 1737 c 1701 g 1628 t ORIGIN 1 GAATTCGATG CGAGGCGAAT CGCTCAGTAG TGTTAGTGCA GGCATTGTTT CTCTTTTCCA 61 TTTCGGCTCG CTCTCTGTGC CAGAAAGGCT TTTGGAGTCT TGGTTACTCG TGGGACTAGG 121 GCAAGCGTCG CTTCCCTAAT AATTGTTGCT AACGTTTCAC CGTGCGTGTG TGGATATCTT 181 CCAAGTTGTC ATTTTCTCTA CCCCTCTCTT TTCCGGCGCG GTCTAACTTC CTCCAACTAG 241 CGCATTTCCA TGATAACGGG GTCCACATAG TCCGTTGTAG TGAAATTGGC ATGCCATCAG 301 GGCCAAGGCA TACATCGCGG CCGCGATAAA AATGATACGA TGATCTCCGT GTGGAGCAGA 361 CCGTGGCGAC CAGTTTGTAC CATGGAAGTT CTTCGATAGA GTTATATCGA GTGCGGAATT 421 GATGCTACGG TCACAGTGTC GTATCGTGTG GTCTCCCATG TGTTTTTGAC GGTGTACCAA 481 TTATCTTTGC ACTAATGGGC TATGGAATCT TTCCTTTTAA TCAGGGGGAC ATGTTTATCC 541 CACGCCAGTA GTCTGGAATT AGAATACATA CCAACTATAT GAGCTCGAAT TCTGCATTAA 601 TGAATCGGCC AACGCGCGGG GAGAGGCGGT TTGCGTATTG GGCGCTCTTC CGCTTCCTCG 661 CTCACTGACT CGCTGCGCTC GGTCGTTCGG CTGCGGCGAG CGGTATCAGC TCACTCAAAG 721 GCGGTAATAC GGTTATCCAC AGAATCAGGG GATAACGCAG GAAAGAACAT GTGAGCAAAA 781 GGCCAGCAAA AGGCCAGGAA CCGTAAAAAG GCCGCGTTGC TGGCGTTTTT CCATAGGCTC 841 CGCCCCCCTG ACGAGCATCA CAAAAATCGA CGCTCAAGTC AGAGGTGGCG AAACCCGACA 901 GGACTATAAA GATACCAGGC GTTTCCCCCT GGAAGCTCCC TCGTGCGCTC TCCTGTTCCG 961 ACCCTGCCGC TTACCGGATA CCTGTCCGCC TTTCTCCCTT CGGGAAGCGT GGCGCTTTCT 1021 CATAGCTCAC GCTGTAGGTA TCTCAGTTCG GTGTAGGTCG TTCGCTCCAA GCTGGGCTGT 1081 GTGCACGAAC CCCCCGTTCA GCCCGACCGC TGCGCCTTAT CCGGTAACTA TCGTCTTGAG 1141 TCCAACCCGG TAAGACACGA CTTATCGCCA CTGGCAGCAG CCACTGGTAA CAGGATTAGC 1201 AGAGCGAGGT ATGTAGGCGG TGCTACAGAG TTCTTGAAGT GGTGGCCTAA CTACGGCTAC 1261 ACTAGAAGAA CAGTATTTGG TATCTGCGCT CTGCTGAAGC CAGTTACCTT CGGAAAAAGA 1321 GTTGGTAGCT CTTGATCCGG CAAACAAACC ACCGCTGGTA GCGGTGGTTT TTTTGTTTGC 1381 AAGCAGCAGA TTACGCGCAG AAAAAAAGGA TCTCAAGAAG ATCCTTTGAT CTTTTCTACG 1441 GGGTCTGACG CTCAGTGGAA CGAAAACTCA CGTTAAGGGA TTTTGGTCAT GAGATTATCA 1501 AAAAGGATCT TCACCTAGAT CCTTTTAAAT TAAAAATGAA GTTTTAAATC AATCTAAAGT 1561 ATATATGAGT AAACTTGGTC TGACAGTTAC CAATGCTTAA TCAGTGAGGC ACCTATCTCA 1621 GCGATCTGTC TATTTCGTTC ATCCATAGTT GCCTGACTCC CCGTGGTGTA GATAACTACG 1681 ATACGGGAGG GCTTACCATC TGGCCCCAGT GCTGCAATGA TACCGCGAGA CCCACGCTCA 1741 CCGGCTCCAG ATTTATCAGC AATAAACCAG CCAGCCGGAA GGGCCGAGCG CAGAAGTGGT 1801 CCTGCAACTT TATCCGCCTC CATCCAGTCT ATTAATTGTT GCCGGGAAGC TAGAGTAAGT 1861 AGTTCGCCAG TTAATAGTTT GCGCAACGTT GTTGCCATTG CTACAGGCAT CGTGGTGTCA 1921 CGCTCGTCGT TTGGTATGGC TTCATTCAGC TCCGGTTCCC AACGATCAAG GCGAGTTACA 1981 TGATCCCCCA TGTTGTGCAA AAAAGCGGTT AGCTCCTTCG GTCCTCCGAT CGTTGTCAGA 2041 AGTAAGTTGG CCGCAGTGTT ATCACTCATG GTTATGGCAG CACTGCATAA TTCTCTTACT 2101 GTCATGCCAT CCGTAAGATG CTTTTCTGTG ACTGGTGAGT ACTCAACCAA GTCATTCTGA 2161 GAATAGTGTA TGCGGCGACC GAGTTGCTCT TGCCCGGCGT CAATACGGGA TAATACCGCG 2221 CCACATAGCA GAACTTTAAA AGTGCTCATC ATTGGAAAAC GTTCTTCGGG GCGAAAACTC 2281 TCAAGGATCT TACCGCTGTT GAGATCCAGT TCGATGTAAC CCACTCGTGC ACCCAACTGA 2341 TCTTCAGCAT CTTTTACTTT CACCAGCGTT TCTGGGTGAG CAAAAACAGG AAGGCAAAAT 2401 GCCGCAAAAA AGGGAATAAG GGCGACACGG AAATGTTGAA TACTCATACT CTTCCTTTTT 2461 CAATATTATT GAAGCATTTA TCAGGGTTAT TGTCTCATGA GCGGATACAT ATTTGAATGT 2521 ATTTAGAAAA ATAAACAAAT AGGGGTTCCG CGCACATTTC CCCGAAAAGT GCCACCTGAC 2581 GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA ATAGGCGTAT CACGAGGCCC 2641 TTTCGTCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT GACACATGCA GCTCCCGGAG 2701 ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA 2761 GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT AACTATGCGG CATCAGAGCA GATTGTACTG 2821 AGAGTGCACC ATTCGACGCT CTCCCTTATG CGACTCCTGC ATTAGGAAGC AGCCCAGTAG 2881 TAGGTTGAGG CCGTTGAGCA CCGCCGCCGC AAGGAATGGT GCAAGGAGAT GGCGCCCAAC 2941 AGTCCCCCGG CCACGGGGCC TGCCACCATA CCCACGCCGA AACAAGCGCT CATGAGCCCG 3001 AAGTGGCGAG CCCGATCTTC CCCATCGGTG ATGTCGGCGA TATAGGCGCC AGCAACCGCA 3061 CCTGTGGCGC CGGTGATGCC GGCCACGATG CGTCCGGCGT AGAGGATCTG GCTAGGAATA 3121 CTGCATAGAT AACAAACGCA TCAAACACAA CTGGTGGCAC TATTTCAATC ATGTCGACAC 3181 ACCAAGACGA GAAAATTTGA GTTAAACAAG ATGCCTACAA CCCTATATTT ATTATCTTCA 3241 AGTGATCTTC TCTTCTCTTG TGCGCTGTAC GTAAATGTGT TGCAAATAAG GCATAGGGCT 3301 GAGTACAGGC ACCACACGCG AGGAAAATCA TAAGCACTAG CCGTGTCAAC TCACAGCGAC 3361 TACAGAACAA TTTTGGCCAC ACAACCCGGT GTTAGGATCT CCGAGGCCTC TATTCCTTTG 3421 CCCTCGGACG AGTGCTGGGG CGTCGGTTTC CACTATCGGC GAGTACTTCT ACACAGCCAT 3481 CGGTCCAGAC GGCCGCGCTT CTGCGGGCGA TTTGTGTACG CCCGACAGTC CCGGCTCCGG 3541 ATCGGACGAT TGCGTCGCAT CGACCCTGCG CCCAAGCTGC ATCATCGAAA TTGCCGTCAA 3601 CCAAGCTCTG ATAGAGTTGG TCAAGACCAA TGCGGAGCAT ATACGCCCGG AGCCGCGGCG 3661 ATCCTGCAAG CTCCGGATGC CTCCGCTCGA AGTAGCGCGT CTGCTGCTCC ATACAAGCCA 3721 ACCACGGCCT CCAGAAGAAG ATGTTGGCGA CCTCGTATTG GGAATCCCCG AACATCGCCT 3781 CGCTCCAGTC AATGACCGCT GTTATGCGGC CATTGTCCGT CAGGACATTG TTGGAGCCGA 3841 AATCCGCGTG CACGAGGTGC CGGACTTCGG GGCAGTCCTC GGCCCAAAGC ATCAGCTCAT 3901 CGAGAGCCTG CGCGACGGAC GCACTGACGG TGTCGTCCAT CACAGTTTGC CAGTGATACA 3961 CATGGGGATC AGCAATCGCG CATATGAAAT CACGCCATGT AGTGTATTGA CCGATTCCTT 4021 GCGGTCCGAA TGGGCCGAAC CCGCTCGTCT GGCTAAGATC GGCCGCAGCG ATCGCATCCA 4081 TGGCCTCCGC GACCGGCTGC AGAACAGCGG GCAGTTCGGT TTCAGGCAGG TCTTGCAACG 4141 TGACACCCTG TGCACGGCGG GAGATGCAAT AGGTCAGGCT CTCGCTGAAT TCCCCAATGT 4201 CAAGCACTTC CGGAATCGGG AGCGCGGCCG ATGCAAAGTG CCGATAAACA TAACGATCTT 4261 TGTAGAAACC ATCGGCGCAG CTATTTACCC GCAGGACATA TCCACGCCCT CCTACATCGA 4321 AGCTGAAAGC ACGAGATTCT TCGCCCTCCG AGAGCTGCAT CAGGTCGGAG ACGCTGTCGA 4381 ACTTTTCGAT CAGAAACTTC TCGACAGACG TCGCGGTGAG TTCAGGCTTT TTCATCACTA 4441 GTTCTAGAGC TTATTTTATG GCAGCAACGA GACCTTACGT AAAGCAAAAC TAAATAAGAG 4501 CGGAGACTGC AATGCAGAGT AAAAAAAAAA AAAACATGAA TTTCAGAAGA CCTTGCTGTG 4561 CCCGGGCTCT GAGAGCGGTC AGTTGCTCTC CTGCTCCGTA CGCCGTAAGC GCTACTTTTA 4621 CTGCTTAATC TTTCCTATAT AAGATATATT TCTATTACTA TATATACTTT TTTAACTTTA 4681 TCATTATTAT CCTATTGTGT GTTGTATTGA CTTTACAATA AAACATATTA TATCTAACAT 4741 GTCAAAAGCA TCGGCTCTTC AATACAGTTA CCCGTCCCTT ATCAACACAG TACAATATAC 4801 TCTGTCATGA CAATAAGTAT ATATAAGAGC CAGAATGCAC CCGCGCTGGG TGGAAAGCTA 4861 GCTTGCATGC CTGCAGGGGC CCAATCCGGA TATAGTTCTC CTTTCAGCAA AAAACCCCTC 4921 AAGACCCGTT TAGAGGCCCC AAGGGGTTAT GCTAGTTATT GCTCAGCGGT GGCAGCAGCC 4981 AACTCAGCTT CCTTTCGGGC TTTGTTAGCA GCCCTATCCG GATATAGTTC TCCTTTCAGC 5041 AAAAAACCCC TCAAGACCCG TTTAGAGGCC CCAAGGGGTT ATGCTAGTTA TTGCTCAGCG 5101 GTGGCAGCAG CCAACTCAGC TTCCTTTCGG GCTTTGTTAG CAGCCTGCAG AATACTGCAT 5161 AGATAACAAA CGCATCAAAC ACAACTGGTG GCACTATTTC AATCATGTCG AGGAGGACCG 5221 AATACTAATA ACGACACTTG CGGTCAAAAA GTAGAAGAAC AAATGCTCAA CGATGAGTGA 5281 ATCAGGTTAG GGTAGTCGGA AAATTATACA GAATGTCTTT GGCAACACAC CGGCTGATTA 5341 ATACGACTCA CTATAGGGAG ATCTCCCTAT CAGTGATAGA GATCTCCCCG GGCCCCCCCT 5401 CGAGTTAACG ACCTTGCGTC CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT 5461 CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCGAT 5521 GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG CCGTAAAGCA 5581 CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT GACGGGGAAA GCCGGCGAAC 5641 GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA 5701 GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG 5761 TCCATTCGCC ATTCAGGCTG CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC 5821 TATTACGCCA GCTGGCGAAA GGGGGATGTG CTGCAAGGCG ATTAAGTTGG GTAACGCCAG 5881 GGTTTTCCCA GTCACGACGT TGTAAAACGA CGGCCAGTGA ATTGTAATAC GACTCACTAT 5941 AGGGCGAATT GGGCCCGACG TCGCATGCTC CCGGCCGCCA TGGCCGCGGG ATATCACTAG 6001 TGCGGCCGCC TGCAGGTCGA CCATATGGGA GAGCTCCCAA CGCGTTGGAT GCATAGCTTG 6061 AGTATTCTAT AGTGTCACCT AAATAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG 6121 TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA 6181 GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCGA CGCAAGGTCG 6241 TTAACGGATC CACTAGTTCT AGAGCGGCCG GCCGCCACCG CGGCTAGATC TCTATCACTG 6301 ATAGGGAGAT CTCCCTATAG TGAGTCGTAT TAATCAGCCG GTGTGTTGCC AAAGACATTC 6361 TGTATAATTT TCCAACTACC CTAACCTGAT TCACTCATCG TTGAGCATTT GTTCTTCTAC 6421 TTTTTGACCG CAAGTGTCGT TATTAGTATT CGGTCCTGGG CTGCAGGCTG CTAACAAAGC 6481 CCGAAAGGAA GCTGAGTTGG CTGCTGCCAC CGCTGAGCAA TAACTAGCAT AACCCCTTGG 6541 GGCCTCTAAA CGGGTCTTGA GGGGTTTTTT GCTGAAAGGA GAACTATATC CGGATAGGGC 6601 TGCTAACAAA GCCCGAAAGG AAGCTGAGTT GGCTGCTGCC ACCGCTGAGC AATAACTAGC 6661 ATAACCCCTT GGGGCCTCTA AACGGGTCTT GAGGGGTTTT TTGCTGAAAG GAGAACTATA 6721 TCCGGATTGG GCCCCTGCAG //