LOCUS pLEW100cre-EP1-6G 6220 bp DNA circular 20-JUL-2008 FEATURES Location/Qualifiers misc_feature complement(8..712) /function="rRNA spacer" /note="Complement of 36 to 694 of accession number Z46903 and complement of 809632 to 810308 on chromosome 1 (AL929603) (the entire 'intergenic region' on chromosome 1 is about 10 kb." 3'UTR 3147..3239 /function="ALD 3' UTR" /note="Remnant of ALD 3' UTR from pLEW100" CDS 4365..5222 /function="LACTAMASE" 3'UTR complement(744..1021) /function="ACT 3' UTR" CDS complement(1082..1456) /note="BLE" promoter complement(1595..1616) /function="T7 Promoter" /note="T7 promoter, full-strength, constitutive. Note that there are no T7 terminators downstream of T7 transcription unit in this construct, which could be a problem in some insertion sites!" promoter 1625..1873 /function="GPEET Promoter" /note="Best match with GPEET2 promoter on chromosome 6: only 2 nt differ in 249 BLASTed. Originally Christine's PARP-A promoter region." protein_bind 1879..1919 /function="Tet Operators" 5'UTR 1923..2009 /function="GPEET poisoned SAS" /note="To SAS: actual sequence after inserting 6 G residues to severely poison the polyY SAS region and reduce expression level." 5'UTR 2010..2040 /function="GPEET 5´ UTR" 5'UTR complement(1475..1589) /function="ACT 5' UTR & SAS" conflict 588..594 /note="The Sac site that the sequence indicates is not present" CDS 2100..3131 /function="CRE RECOMBINASE" /note="Cre was cloned into pLEW100 as a Hind-Bam fragment, but a one-nt change destroyed the HindIII site. Not clear how used Bam, as there is an internal site in Cre. On John's receommendation when he supplied the plasmid, we re-sequenced the gene. Ultimately, when making variants of it, we resequenced other bits, which explains why a few nt in this sequence differ from that given for the parental pLEW100 on this web site. Donelson's paper says Cre is the sequence from GenBank X03453, but they truncated that sequence in their vector. This ORF matches the ORF in X03453 and AF234173. The 5'UTR comes from whatever vector they used." misc_feature 2046..2099 /note="Extraneous vector" 3'UTR 3242..3365 /function="EP-1 3' UTR Ts region" /note="Contains temperature-sensitive region that reduces expression about 10-fold at 37¡C." 3'UTR 3366..3428 /function="GPEET PAS" /note="Note from Mike: presumed polyA sequence of pGAPRONE, aligns with homologous region of pSGL33 GPEET 3'UTR except for final 3 bases, TTT. Sequence from pGAPRONE with adjustment (cyan base) based on my sequence data - 9 Feb 07, MDS." BASE COUNT 1547 a 1531 c 1602 g 1537 t 3 others ORIGIN 1 GAATTCGAGC TCATATAGTT GGTATGTATT CTAATTCCAG ACTACTGGCG TGGATAAACA 61 TGTCCCCTGA TTAAAAGGAA AGATTCCATA GCCCATTAGT GCAAAGATAA TTGGTACACC 121 GTCAAAAACA CATGGGAGAC CACACGATAC GACACTGTGA CCGTAGCATC AATTCCGCAC 181 TCGATATAAC TCTATCGAAG AACTTCCATG GTACAAACTG GTCGCCACGG TCTGCTCCAC 241 ACGGAGATCA TCGTATCATT TTTATCGATA GCGGCCGCTA TCGATGTATG CCTTGGCCCT 301 GATGGCATGC CAATTTCACT ACAACGGACT ATGTGGACCC CGTTATCATG GAAATGCGCT 361 AGTTGGAGGA AGTTAGACCG CGCCGGAAAA GAGAGGGGTA GAGAAAATGA CAACTTGGAA 421 GATATCCACA CACGCACGGT GAAACGTTAG CAACAATTAT TAGGGAAGCA CGCTTGCCCT 481 AGTCCCACGA GTAACCAAGA CTCCAAAAGC CTTTCTGGCA CAGAGAGCGA GCCGAAATGG 541 AAAAGAGAAA CAATGCCTGC ACTAACACTA CTGAGCGATT CGCCTCGCCG CGGAGGACCG 601 AATACTAATA ACGACACTTG CGGTCAAAAA GTAGAAGAAC AAATGCTCAA CGATGAGTGA 661 ATCAGGTTAG GGTAGTTGGA AAATTATACA GAATGTCTTT GGCAACACAC CGGCnnnCTT 721 GCATGCCTGC AAGGCCTTGC AGAATACTGC ATAGATAACA AACGCATCAA ACACAACTGG 781 TGGCACTATT TCAATCATGT CGACACACCA AGACGAGAAA ATTTGAGTTA AACAAGATGC 841 CTACAACCCT ATATTTATTA TCTTCAAGTG ATCTTCTCTT CTCTTGTGCG CTGTACGTAA 901 ATGTGTTGCA AATAAGGCAT AGGGCTGAGT ACAGGCACCA CACGCGAGGA AAATCATAAG 961 CACTAGCCGT GTCAACTCAC AGCGACTACA GAACAATTTT GGCCACACAA CCCGGTGTTA 1021 GGATCTCCGA GGCCTGGGAC CCGTGGGCCG CCGTCGGACC GGCGGTGTTG GTCGGCGTCG 1081 GTCAGTCCTG CTCCTCGGCC ACGAAGTGCA CGCAGTTGCC GGCCGGGTCG CGCAGGGCGA 1141 ACTCCCGCCC CCACGGCTGC TCGCCGATCT CGGTCATGGC CGGCCCGGAG GCGTCCCGGA 1201 AGTTCGTGGA CACGACCTCC GACCACTCGG CGTACAGCTC GTCCAGGCCG CGCACCCACA 1261 CCCAGGCCAG GGTGTTGTCC GGCACCACCT GGTCCTGGAC CGCGCTGATG AACAGGGTCA 1321 CGTCGTCCCG GACCACACCG GCGAAGTCGT CCTCCACGAA GTCCCGGGAG AACCCGAGCC 1381 GGTCGGTCCA GAACTCGACC GCTCCGGCGA CGTCGCGCGC GGTGAGCACC GGAACGGCAC 1441 TGGTCAACTT GGCCATGGTG ATAGCTTATT TTATGGCAGC AACGAGACCT TACGTAAAGC 1501 AAAACTAAAT AAGAGCGGAG ACTGCAATGC AGAGTAAAAA AAAAAAAAAC ATGAATTTCA 1561 GAAGACCTTG CTGTGCCCCC GGTACGGGAG ATCTCCCTAT AGTGAGTCGT ATTAATCAGG 1621 TACCGTCATT GGGGTTAAGC GGAAAGGTGT GTGTCAGTAG GTTGTGAGGT GAAAGCGTTT 1681 TCAGATGCAT AGTGAGCTTA ATGTCCTTTT CACAGTATAT CGTGTCTGAT AGGTATCTCT 1741 TATTAGTATA GTCGAATACT AGTCAATAGT GCGTTTTGTG CAAAATGTCC ATTTTGTGGC 1801 AGTGATGGGG TTGTTTTATG CTATTCCGTG TCTCTGGGTG GGCGTGCATT GAAAATAGGG 1861 GTTATCGGGT GAGGGATCTC CCTATCAGTG ATAGAGATCT CCCTATCAGT GATAGAGATC 1921 CCTGAGTACT GAGTTTAACA TGTTCTCGTC CCGGGCTGCA CGCGCCTTCG AGTTTTggTT 1981 gCcTTTtgCC CATTTggTTC AACTTGAAGA CTTCAATTAC ACCAAAAAGT AAAATTCACA 2041 AGCTCGAGGG GCAGAGCCGA TCCTGTACAC TTTACTTAAA ACCATTATCT GAGTGTGAAA 2101 TGTCCAATTT ACTGACCGTA CACCAAAATT TGCCTGCATT ACCGGTCGAT GCaACGAGTG 2161 ATGAGGTTCG CAAGAACCTG ATGGACATGT TCAGGGATCG CCAGGCGTTT TCTGAGCATA 2221 CCTGGAAAAT GCTTCTGTCC GTTTGCCGGT CGTGGGCGGC ATGGTGCAAG TTGAATAACC 2281 GGAAATGGTT TCCCGCAGAA CCTGAAGATG TTCGCGATTA TCTTCTATAT CTTCAGGCGC 2341 GCGGTCTGGC AGTAAAAACT ATCCAGCAAC ATTTGGGCCA GCTAAACATG CTTCATCGTC 2401 GGTCCGGGCT GCCACGACCA AGTGACAGCA ATGCTGTTTC ACTGGTTATG CGGCGGATCC 2461 GAAAAGAAAA CGTTGATGCC GGTGAACGTG CAAAACAGGC TCTAGCGTTC GAACGCACTG 2521 ATTTCGACCA GGTTCGTTCA CTCATGGAAA ATAGCGATCG CTGCCAGGAT ATACGTAATC 2581 TGGCATTTCT GGGGATTGCT TATAACACCC TGTTACGTAT AGCCGAAATT GCCAGGATCA 2641 GGGTTAAAGA TATCTCACGT ACTGACGGTG GGAGAATGTT AATCCATATT GGCAGAACGA 2701 AAACGCTGGT TAGCACCGCA GGTGTAGAGA AGGCACTTAG CCTGGGGGTA ACTAAACTGG 2761 TCGAGCGATG GATTTCCGTC TCTGGTGTAG CTGATGATCC GAATAACTAC CTGTTTTGCC 2821 GGGTCAGAAA AAATGGTGTT GCCGCGCCAT CTGCCACCAG CCAGCTATCA ACTCGCGCCC 2881 TGGAAGGGAT TTTTGAAGCA ACTCATCGAT TGATTTACGG CGCTAAGGAT GACTCTGGTC 2941 AGAGATACCT GGCCTGGTCT GGACACAGTG CCCGTGTCGG AGCCGCGCGA GATATGGCCC 3001 GCGCTGGAGT TTCAATACCG GAGATCATGC AAGCTGGTGG CTGGACCAAT GTAAATATTG 3061 TCATGAACTA TATCCGTAAC CTGGATAGTG AAACAGGGGC AATGGTGCGC CTGCTGGAAG 3121 ATGGCGATTA GCCATTAACG CGGATCCTGC CCATTTAGTT GGCTTTTCCC TTGTCTCGTG 3181 TCTTTTCCGT GGAAAGGTTC CCGGAGTAAT CTGATGGCAC AGCAGGGAGG TGCGCCTGCA 3241 GAATGCCTTA TTAACCATCG CCTGAGACCC ACAGCCCTGT AGATTTCTGT GATGTTTCGG 3301 TTGCGTATTC CATAATTTTA AGCGTTTCAC TTCTATTTTT TTTCATTCCT TTGAATTTGG 3361 ATCTTAAAAT TATcATTGGT GCCTTGTGTT ATTGTGCGTG CTGCGTGTGA ATTTGGTGCT 3421 CTGCCTTTTC TGCAGGCATG CAAGCTAGCT TGTATTCTAT AGTGTCACCT AAATCGTATG 3481 TGTATGATAC ATAAGGTTAT GTATTAATTG TAGCCGCGTT CTAACGACAA TATGTACAAG 3541 CCTAATTGTG TAGCATCTGG CTTACTGAAG CAGACCCTAT CATCTCTCTC GTAAACTGCC 3601 GTCAGAGTCG GTTTGGTTGG ACGAACCTTC TGAGTTTCTG GTAACGCCGT TCCGCACCCC 3661 GGAAATGGTC AGCGAACCAA TCAGCAGGGT CATCGCTAGC CAGATCCTCT ACGCCGGACG 3721 CATCGTGGCC GGCATCACCG GCGCCACAGG TGCGGTTGCT GGCGCCTATA TCGCCGACAT 3781 CACCGATGGG GAAGATCGGG CTCGCCACTT CGGGCTCATG AGCGCTTGTT TCGGCGTGGG 3841 TATGGTGGCA GGCCCCGTGG CCGGGGGACT GTTGGGCGCC ATCTCCTTGC ACCATTCCTT 3901 GCGGCGGCGG TGCTCAACGG CCTCAACCTA CTACTGGGCT GCTTCCTAAT GCAGGAGTCG 3961 CATAAGGGAG AGCGTCGATA TGGTGCACTC TCAGTACAAT CTGCTCTGAT GCCGCATAGT 4021 TAAGCCAGCC CCGACACCCG CCAACACCCG CTGACGCGCC CTGACGGGCT TGTCTGCTCC 4081 CGGCATCCGC TTACAGACAA GCTGTGACCG TCTCCGGGAG CTGCATGTGT CAGAGGTTTT 4141 CACCGTCATC ACCGAAACGC GCGAGACGAA AGGGCCTCGT GATACGCCTA TTTTTATAGG 4201 TTAATGTCAT GATAATAATG GTTTCTTAGA CGTCAGGTGG CACTTTTCGG GGAAATGTGC 4261 GCGGAACCCC TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC 4321 AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTATGAGT ATTCAACATT 4381 TCCGTGTCGC CCTTATTCCC TTTTTTGCGG CATTTTGCCT TCCTGTTTTT GCTCACCCAG 4441 AAACGCTGGT GAAAGTAAAA GATGCTGAAG ATCAGTTGGG TGCACGAGTG GGTTACATCG 4501 AACTGGATCT CAACAGCGGT AAGATCCTTG AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA 4561 TGATGAGCAC TTTTAAAGTT CTGCTATGTG GCGCGGTATT ATCCCGTATT GACGCCGGGC 4621 AAGAGCAACT CGGTCGCCGC ATACACTATT CTCAGAATGA CTTGGTTGAG TACTCACCAG 4681 TCACAGAAAA GCATCTTACG GATGGCATGA CAGTAAGAGA ATTATGCAGT GCTGCCATAA 4741 CCATGAGTGA TAACACTGCG GCCAACTTAC TTCTGACAAC GATCGGAGGA CCGAAGGAGC 4801 TAACCGCTTT TTTGCACAGC ATGGGGGATC ATGTAACTCG CCTTGATCGT TGGGAACCGG 4861 AGCTGAATGA AGCCATACCA AACGACGAGC GTGACACCAC GATGCCTGTA GCAATGGCAA 4921 CAACGTTGCG CAAACTATTA ACTGGCGAAC TACTTACTCT AGCTTCCCGG CAACAATTAA 4981 TAGACTGGAT GGAGGCGGAT AAAGTTGCAG GACCACTTCT GCGCTCGGCC CTTCCGGCTG 5041 GCTGGTTTAT TGCTGATAAA TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT ATCATTGCAG 5101 CACTGGGGCC AGATGGTAAG CCCTCCCGTA TCGTAGTTAT CTACACGACG GGGAGTCAGG 5161 CAACTATGGA TGAACGAAAT AGACAGATCG CTGAGATAGG TGCCTCACTG ATTAAGCATT 5221 GGTAACTGTC AGACCAAGTT TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT 5281 AATTTAAAAG GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC 5341 GTGAGTTTTC GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG 5401 ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG 5461 TGGTTTGTTT GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA 5521 GAGCGCAGAT ACCAAATACT GTCCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA 5581 ACTCTGTAGC ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA 5641 GTGGCGATAA GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC 5701 AGCGGTCGGG CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA 5761 CCGAACTGAG ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA 5821 AGGCGGACAG GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC 5881 CAGGGGGAAA CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC 5941 GTCGATTTTT GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG 6001 CCTTTTTACG GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT 6061 CCCCTGATTC TGTGGATAAC CGTATTACCG CCTTTGAGTG AGCTGATACC GCTCGCCGCA 6121 GCCGAACGAC CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC GGAAGAGCGC CCAATACGCA 6181 AACCGCCTCT CCCCGCGCGT TGGCCGATTC ATTAATGCAG //