LOCUS pLEW29 6868 bp DNA circular 22-OCT-2009 We were alerted in October 2007 by Michal Swiderski (Glasgow), there are extra EcoRI and Bam sites compared to the previously published version of the sequence such that these enzymes cut twice instead of once, releasing 2 or 3 fragments. Wirtz 1999 paper says plasmid was cut with NotI and SspI prior to transfection (NotI and PmlI would look like a better choice, assuming the PmlI site is also unique. The insert was re-sequenced in November 2007 due to alerting to other problems (which we confirmed) that tetR could not be PPCR amplified with primers designed according to the assumed sequence, so we re-sequenced the entire insert from position 6796 to 3808. There were a few insertions in the upstream flanking sequence but the main difference was that, immediately downstream of the tetR CDS, there was an insertion of about 90 bp of the 260 bp Actin 3" UTR that were not in the original (Wirtz & Clayton) or the previous"corrected" pLEW29 versions. FEATURES Location/Qualifiers misc_feature 83..881 /function="RNA PolI upstream" /note="ends 69 bp upstream of PolI CDS, probably done because looks like PolI has a weak SAS/polyY tract, so ACT SAS used instead." promoter 780..799 /function="10% T7 promoter" 5'UTR 881..990 /function="ACT 5' UTR" /note="region 1951-2063 (and 445-545) in TRBACT (M20310)" CDS 1013..1636 /function="Tet repressor" /note="CDS corrected (November 2007) to match the actually sequenced version in pLEW29, which is also a perfect match for sequences in GenBank (checked 091022)" 3'UTR 1642..1897 /function="ACT 3' UTR" /note="1691-1950 in Actin M20310 and 1376502-1375797 in TREU chr 9" 5'UTR 1919..2005 /function="PROCYCLIN 5' UTR" CDS 2025..3062 /note="HYG" CDS 3109..3432 /function="A-B TUB intergenic" /note="Entire intergenic region, 30-354 in M11748" CDS 3460..3723 /function="RNA PolI C-term" 3'UTR 3724..3808 /function="PolI 3' UTR" CDS 4833..5690 /note="LACTAMASE" BASE COUNT 1698 a 1558 c 1717 g 1895 t ORIGIN 1 GCGCGCAATT AACCCTCACT AAAGGGAACA AAAGCTGGAG CTCCACCGCG GTGGCGGCCG 61 CTCTAGAATT CGAACCCCTT CGGATCCCAT GGTTACTGCG GATGTCGCTT ACTTTTTCCA 121 TTGTTTCTTT TGTGTTCATT TCATATTTCC ATTTAAAATT TCGTTCTCTC ACTTTGTTGC 181 ACCAAACACT GTCCCATATA CCCGCAAATG GCGGACGGCT TTTGTTGTTT AGTTAGGTTC 241 GGCCCTTTCT TTCTTCTTTT TCGTTTTTTA TGTTTTTTTT TGTTGCGTGT GAAGGCTTTT 301 TCCGCCTCTG TCTGAACCAC CCATCATTGA CGACGCAACC TCGCGTCAGC ACTGACTGAG 361 CGACTGACTG GTTGGTTACT TCGAGGAAAA TGTTTGATTT CTGTCGTTTC CTCGCGTGTT 421 AACTGTTTTC TCTTCTTTCA AGTGCTGAAG CAAGCGCAAT CGTATATCCG CAAAAGGCAA 481 CAGGTGTTGT GAGTTGACGC CCCCCCCCCC GCCCGTCACG GCAATAATTG AGGCCGCATT 541 TGGTTTAAAG CTGGACTGAG GCAGTAAAAA AAAGATTAGG CGGTGTGCTG ACCACTCAGT 601 GAAAGAATAA GGAAGGGCGG GACGGGGGGG GGGAGACAAC GCTGAGCCTG CATAGCTATT 661 TTTGTGTGTT GTAAGAAGAA AGCGGAAGGA AGAAATTGTA ACTTATCAAT AACCTTAACA 721 ACAAAGGCAA AAGAAAGAAA AAGGGAGGGT GGGACACACA ACGTTGAGTA AGGGACCGGT 781 AATACGTCTC ACTATAGGGA GATCTACCGG TTACAGAACT ATTGGCTGTG ACTAATTTTT 841 CTTTGGTTTT TTTTTGTTTT GTTCACGACT CCAAGCTTGG CACAGCAAGG TCTTCTGAAA 901 TTCATGTTTT TTTTTTTTTT ACTCTGCATT GCAGTCTCCG CTCTTATTTA GTTTTGCTTT 961 ACGTAAGGTC TCGTTGCTGC CATAAAATAA GCTCTAGAAC TAGAGCTTTC ATATGTCTAG 1021 ATTAGATAAA AGTAAAGTGA TTAACAGCGC ATTAGAGCTG CTTAATGAGG TCGGAATCGA 1081 AGGTTTAACA ACCCGTAAAC TCGCCCAGAA GCTAGGTGTA GAGCAGCCTA CATTGTATTG 1141 GCATGTAAAA AATAAGCGGG CTTTGCTCGA CGCCTTAGCC ATTGAGATGT TAGATAGGCA 1201 CCATACTCAC TTTTGCCCTT TAGAAGGGGA AAGCTGGCAA GATTTTTTAC GTAATAACGC 1261 TAAAAGTTTT AGATGTGCTT TACTAAGTCA TCGCGATGGA GCAAAAGTAC ATTTAGGTAC 1321 ACGGCCTACA GAAAAACAGT ATGAAACTCT CGAAAATCAA TTAGCCTTTT TATGCCAACA 1381 AGGTTTTTCA CTAGAGAATG CATTATATGC ACTCAGCGCT GTGGGGCATT TTACTTTAGG 1441 TTGCGTATTG GAAGATCAAG AGCATCAAGT CGCTAAAGAA GAAAGGGAAA CACCTACTAC 1501 TGATAGTATG CCGCCATTAT TACGACAAGC TATCGAATTA TTTGATCACC AAGGTGCAGA 1561 GCCAGCCTTC TTATTCGGCC TTGAATTGAT CATATGCGGA TTAGAAAAAC AACTTAAATG 1621 TGAAAGTGGG TCTTAAGGAT CCCAAAATTG TTCTGTAGTC GCTGTGAGTT GACACGGCTA 1681 GTGCTTATGA TTTTCCTCGC GTGTGGTGCC TGTACTCAGC CCTATGCCTT ATTTGCAACA 1741 CATTTACGTA CAGCGCACAA GAGAAGAGAA GATCACTTGA AGATAATAAA TATAGGGTTG 1801 TAGGCATCTT GTTTAACTCA AATTTTCTCG TCTTGGTGTG TCGACATGAT TGAAATAGTG 1861 CCACCAGTTG TGTTTGATGC GTTTGTTATC TATGCAGTAT TAAGCTTGGC TGCAGGTCAC 1921 TGAGTTTAAG ATGTTCTCGT GATCGCTGCA CGCGCCTTCG AGTTTTTTTT CCTTTTACCC 1981 ATTTTTTTCA ACTTGAAGAC TTCAACGGGA CTAGTCAGCT TGATATGAAA AAGCCTGAAC 2041 TCACCGCGAC GTCTGTCGAG AAGTTTCTGA TCGAAAAGTT CGACAGCGTC TCCGACCTGA 2101 TGCAGCTCTC GGAGGGCGAA GAATCTCGTG CTTTCAGCTT CGATGTAGGA GGGCGTGGAT 2161 ATGTCCTGCG GGTAAATAGC TGCGCCGATG GTTTCTACAA AGATCGTTAT GTTTATCGGC 2221 ACTTTGCATC GGCCGCGCTC CCGATTCCGG AAGTGCTTGA CATTGGGGAA TTCAGCGAGA 2281 GCCTGACCTA TTGCATCTCC CGCCGTGCAC AGGGTGTCAC GTTGCAAGAC CTGCCTGAAA 2341 CCGAACTGCC CGCTGTTCTG CAGCCGGTCG CGGAGGCCAT GGATGCGATC GCTGCGGCCG 2401 ATCTTAGCCA GACGAGCGGG TTCGGCCCAT TCGGACCGCA AGGAATCGGT CAATACACTA 2461 CATGGCGTGA TTTCATATGC GCGATTGCTG ATCCCCATGT GTATCACTGG CAAACTGTGA 2521 TGGACGACAC CGTCAGTGCG TCCGTCGCGC AGGCTCTCGA TGAGCTGATG CTTTGGGCCG 2581 AGGACTGCCC CGAAGTCCGG CACCTCGTGC ACGCGGATTT CGGCTCCAAC AATGTCCTGA 2641 CGGACAATGG CCGCATAACA GCGGTCATTG ACTGGAGCGA GGCGATGTTC GGGGATTCCC 2701 AATACGAGGT CGCCAACATC TTCTTCTGGA GGCCGTGGTT GGCTTGTATG GAGCAGCAGA 2761 CGCGCTACTT CGAGCGGAGG CATCCGGAGC TTGCAGGATC GCCGCGGCTC CGGGCGTATA 2821 TGCTCCGCAT TGGTCTTGAC CAACTCTATC AGAGCTTGGT TGACGGCAAT TTCGATGATG 2881 CAGCTTGGGC GCAGGGTCGA TGCGACGCAA TCGTCCGATC CGGAGCCGGG ACTGTCGGGC 2941 GTACACAAAT CGCCCGCAGA AGCGCGGCCG TCTGGACCGA TGGCTGTGTA GAAGTACTCG 3001 CCGATAGTGG AAACCGACGC CCCAGCACTC GTCCGGATCG GGAGATGGGG GAGGCTAACT 3061 GAAACACGGA AGGAGCGGGA TCGACTAGTC AATTCGAGCT CGGTACCCCT AGAAAGTGTG 3121 ACAACGTCGC ACCATGTGTA GGTTTTCATT TATGTTCTTT CTTTCTTTCT TTTGTGAATT 3181 TGTTTTCTGT CTCAAATGTT TTTAATTCGC TTGGGACCTA TGTTTTTCTT GTTTTTTTGC 3241 TCACCCTTTG TGTAGGAGGC ACCCTGTCAC GTCTGTGGTT GCGTGTATGC CTTCCTTCCC 3301 CTTATTCGCT TCTTCCTGTC GTGTCACACC TCTTTCTCCC TCTCCCTTTC CGCCTTTTCT 3361 TTCAATCTTG TTTTCTCGAC CAGCCCTACT AGAGGAGAAA GAATAGTAAC CCTTTCATCA 3421 AAGAAAATAG TCGACCCGCG GCACTTGTCG CTCATCGCCG ATACGTCTAC CCACCGAGGG 3481 CGATGGGAGA ACTTCAATTT CACTGGTGTC ATATCAACGA GCGCGAGCCC ACTGTTTCAG 3541 ATGACATTTG CCTCGTCGAA GCGCTGGTTG CACAGAGCGG TGAGTCGGGG AATGAGTGAC 3601 GACCTCGAGT CCTTTAGTTC TGCCATCATG GTGGGGGAAC GCCCTCGTGT GGGAACGGCG 3661 TCCGTGCGGC TCTCGACTGA TACGGCGATT TTGCGGGATG TGCTCGAGAG GAACTTTGCA 3721 TAATCGCCTG AACCGCTATT TTAAGTATAT ATATATGTAT ATTACCTTTT TTTGTTTTTT 3781 CTTTGCGCTT TTACCTCCTC TTTTGTCTCA ACTTTTTTTA ATTTTATGAG AGGCACTTGT 3841 GTGAAATTGG ACACCATAAA GGGCTGAGGA GGGAAGCAGA GAGCAAATGA AAATATGGGA 3901 GGGTGAAAAA TACATACATA TATATTTGTG TTTGTTCGTA TTTGGAGGGA TACAAATACG 3961 ATATATGAAG ATATCAACAG TTTTTCACCT GTGGGGGAAG CGTGATTATT ATCAGTGAGT 4021 GGATGGACTG AAGAACGAAT AAATGTGTAA CAATTTCGCC CTTCATTTTT TCTTCGGTGG 4081 CAGACGGGTT TAAATCTAAT CGGATAAATA GCACTAAAGT AACGAATGTT AAAATAACCA 4141 CCGCAAAGGC ATACCTTTTC CTTTTTTGCC CTTTGTGGTG GGAAGTCAGT GGCAAAAGGA 4201 AGCGAGAGTA GCGACAGGGA AAGTCTCAGT GCCATGTGAC CGTTTCAGAA AAGAAAATGA 4261 AAAAAAGAAG GAAGGGGGAG GGCTGAATGA AGAGAGGATT TGCGGGTAAA GGAGGTGCAT 4321 AACACGAGTG TCAATGCATC GTTTCAGTGG GCGGTGCACG TGCTCGAGGG GGGCTTTCCC 4381 CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC 4441 GACCCCAAAA AACTTGATTA GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG 4501 GTTTTTCGCC CTTTGACGTT GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT 4561 GGAACAACAC TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT 4621 TCGGCCTATT GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTTTAACAAA 4681 ATATTAACGT TTACAATTTC CCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA 4741 TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT 4801 AAATGCTTCA ATAATATTGA AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC 4861 TTATTCCCTT TTTTGCGGCA TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA 4921 AAGTAAAAGA TGCTGAAGAT CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA 4981 ACAGCGGTAA GATCCTTGAG AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT 5041 TTAAAGTTCT GCTATGTGGC GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG 5101 GTCGCCGCAT ACACTATTCT CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC 5161 ATCTTACGGA TGGCATGACA GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA 5221 ACACTGCGGC CAACTTACTT CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT 5281 TGCACAACAT GGGGGATCAT GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG 5341 CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA 5401 AACTATTAAC TGGCGAACTA CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG 5461 AGGCGGATAA AGTTGCAGGA CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG 5521 CTGATAAATC TGGAGCCGGT GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG 5581 ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG 5641 AACGAAATAG ACAGATCGCT GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAG 5701 ACCAAGTTTA CTCATATATA CTTTAGATTG ATTTAAAACT TCATTTTTAA TTTAAAAGGA 5761 TCTAGGTGAA GATCCTTTTT GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT 5821 TCCACTGAGC GTCAGACCCC GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTC 5881 TGCGCGTAAT CTGCTGCTTG CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC 5941 CGGATCAAGA GCTACCAACT CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATAC 6001 CAAATACTGT CCTTCTAGTG TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC 6061 CGCCTACATA CCTCGCTCTG CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGT 6121 CGTGTCTTAC CGGGTTGGAC TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT 6181 GAACGGGGGG TTCGTGCACA CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGAT 6241 ACCTACAGCG TGAGCTATGA GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT 6301 ATCCGGTAAG CGGCAGGGTC GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACG 6361 CCTGGTATCT TTATAGTCCT GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT 6421 GATGCTCGTC AGGGGGGCGG AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGT 6481 TCCTGGCCTT TTGCTGGCCT TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG 6541 TGGATAACCG TATTACCGCC TTTGAGTGAG CTGATACCGC TCGCCGCAGC CGAACGACCG 6601 AGCGCAGCGA GTCAGTGAGC GAGGAAGCGG AAGAGCGCCC AATACGCAAA CCGCCTCTCC 6661 CCGCGCGTTG GCCGATTCAT TAATGCAGCT GGCACGACAG GTTTCCCGAC TGGAAAGCGG 6721 GCAGTGAGCG CAACGCAATT AATGTGAGTT AGCTCACTCA TTAGGCACCC CAGGCTTTAC 6781 ACTTTATGCT TCCGGCTCGT ATGTTGTGTG GAATTGTGAG CGGATAACAA TTTCACACAG 6841 GAAACAGCTA TGACCATGAT TACGCCAA //