LOCUS pLEW100cre-EP1 6222 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 3150..3242 /function="ALD 3' UTR" /note="Remnant of ALD 3' UTR from pLEW100" CDS 4367..5224 /function="LACTAMASE" 3'UTR complement(744..1021) /function="ACT 3' UTR" CDS complement(1082..1456) /note="BLE" promoter complement(1599..1620) /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 1629..1877 /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 1883..1923 /function="Tet Operators" 5'UTR 1927..2012 /function="GPEET SAS" /note="To SAS" 5'UTR 2013..2043 /function="GPEET 5´ UTR" 5'UTR complement(1477..1593) /function="ACT 5' UTR & SAS" conflict 588..594 /note="The Sac site that the sequence indicates is not present" CDS 2103..3134 /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 2049..2102 /note="Extraneous vector" 3'UTR 3245..3368 /function="EP-1 3' UTR Ts region" /note="Contains temperature-sensitive region that reduces expression about 10-fold at 37¡C." 3'UTR 3369..3431 /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 1548 a 1532 c 1596 g 1543 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 ATATAGCTTA TTTTATGGCA GCAACGAGAC CTTACGTAAA 1501 GCAAAACTAA ATAAGAGCGG AGACTGCAAT GCAGAGTAAA AAAAAAAAAA ACATGAATTT 1561 CAGAAGACCT TGCTGTGCCC CCGGTACGGG CTAGATCTCC CTATAGTGAG TCGTATTAAT 1621 CAGGTACCGT CATTGGGGTT AAGCGGAAAG GTGTGTGTCA GTAGGTTGTG AGGTGAAAGC 1681 GTTTTCAGAT GCATAGTGAG CTTAATGTCC TTTTCACAGT ATATCGTGTC TGATAGGTAT 1741 CTCTTATTAG TATAGTCGAA TACTAGTCAA TAGTGCGTTT TGTGCAAAAT GTCCATTTTG 1801 TGGCAGTGAT GGGGTTGTTT TATGCTATTC CGTGTCTCTG GGTGGGCGTG CATTGAAAAT 1861 AGGGGTTATC GGGTGAGGGA TCTCCCTATC AGTGATAGAG ATCTCCCTAT CAGTGATAGA 1921 GATCCCTGAG TACTGAGTTT AACATGTTCT CGTCCCGGGC TGCACGCGCC TTCGAGTTTT 1981 TTTTCCTTTT CCCCATTTTT TTCAACTTGA AGACTTCAAT TACACCAAAA AGTAAAATTC 2041 ACAAGCTCGA GGGGCAGAGC CGATCCTGTA CACTTTACTT AAAACCATTA TCTGAGTGTG 2101 AAATGTCCAA TTTACTGACC GTACACCAAA ATTTGCCTGC ATTACCGGTC GATGCaACGA 2161 GTGATGAGGT TCGCAAGAAC CTGATGGACA TGTTCAGGGA TCGCCAGGCG TTTTCTGAGC 2221 ATACCTGGAA AATGCTTCTG TCCGTTTGCC GGTCGTGGGC GGCATGGTGC AAGTTGAATA 2281 ACCGGAAATG GTTTCCCGCA GAACCTGAAG ATGTTCGCGA TTATCTTCTA TATCTTCAGG 2341 CGCGCGGTCT GGCAGTAAAA ACTATCCAGC AACATTTGGG CCAGCTAAAC ATGCTTCATC 2401 GTCGGTCCGG GCTGCCACGA CCAAGTGACA GCAATGCTGT TTCACTGGTT ATGCGGCGGA 2461 TCCGAAAAGA AAACGTTGAT GCCGGTGAAC GTGCAAAACA GGCTCTAGCG TTCGAACGCA 2521 CTGATTTCGA CCAGGTTCGT TCACTCATGG AAAATAGCGA TCGCTGCCAG GATATACGTA 2581 ATCTGGCATT TCTGGGGATT GCTTATAACA CCCTGTTACG TATAGCCGAA ATTGCCAGGA 2641 TCAGGGTTAA AGATATCTCA CGTACTGACG GTGGGAGAAT GTTAATCCAT ATTGGCAGAA 2701 CGAAAACGCT GGTTAGCACC GCAGGTGTAG AGAAGGCACT TAGCCTGGGG GTAACTAAAC 2761 TGGTCGAGCG ATGGATTTCC GTCTCTGGTG TAGCTGATGA TCCGAATAAC TACCTGTTTT 2821 GCCGGGTCAG AAAAAATGGT GTTGCCGCGC CATCTGCCAC CAGCCAGCTA TCAACTCGCG 2881 CCCTGGAAGG GATTTTTGAA GCAACTCATC GATTGATTTA CGGCGCTAAG GATGACTCTG 2941 GTCAGAGATA CCTGGCCTGG TCTGGACACA GTGCCCGTGT CGGAGCCGCG CGAGATATGG 3001 CCCGCGCTGG AGTTTCAATA CCGGAGATCA TGCAAGCTGG TGGCTGGACC AATGTAAATA 3061 TTGTCATGAA CTATATCCGT AACCTGGATA GTGAAACAGG GGCAATGGTG CGCCTGCTGG 3121 AAGATGGCGA TTAGCCATTA ACGCGGATCC TGCCCATTTA GTTGGCTTTT CCCTTGTCTC 3181 GTGTCTTTTC CGTGGAAAGG TTCCCGGAGT AATCTGATGG CACAGCAGGG AGGTGCGCCT 3241 GCAGAATGCC TTATTAACCA TCGCCTGAGA CCCACAGCCC TGTAGATTTC TGTGATGTTT 3301 CGGTTGCGTA TTCCATAATT TTAAGCGTTT CACTTCTATT TTTTTTCATT CCTTTGAATT 3361 TGGATCTTAA AATTATcATT GGTGCCTTGT GTTATTGTGC GTGCTGCGTG TGAATTTGGT 3421 GCTCTGCCTT TTTGCAGGCA TGCAAGCTAG CTTGTATTCT ATAGTGTCAC CTAAATCGTA 3481 TGTGTATGAT ACATAAGGTT ATGTATTAAT TGTAGCCGCG TTCTAACGAC AATATGTACA 3541 AGCCTAATTG TGTAGCATCT GGCTTACTGA AGCAGACCCT ATCATCTCTC TCGTAAACTG 3601 CCGTCAGAGT CGGTTTGGTT GGACGAACCT TCTGAGTTTC TGGTAACGCC GTTCCGCACC 3661 CCGGAAATGG TCAGCGAACC AATCAGCAGG GTCATCGCTA GCCAGATCCT CTACGCCGGA 3721 CGCATCGTGG CCGGCATCAC CGGCGCCACA GGTGCGGTTG CTGGCGCCTA TATCGCCGAC 3781 ATCACCGATG GGGAAGATCG GGCTCGCCAC TTCGGGCTCA TGAGCGCTTG TTTCGGCGTG 3841 GGTATGGTGG CAGGCCCCGT GGCCGGGGGA CTGTTGGGCG CCATCTCCTT GCACCATTCC 3901 TTGCGGCGGC GGTGCTCAAC GGCCTCAACC TACTACTGGG CTGCTTCCTA ATGCAGGAGT 3961 CGCATAAGGG AGAGCGTCGA TATGGTGCAC TCTCAGTACA ATCTGCTCTG ATGCCGCATA 4021 GTTAAGCCAG CCCCGACACC CGCCAACACC CGCTGACGCG CCCTGACGGG CTTGTCTGCT 4081 CCCGGCATCC GCTTACAGAC AAGCTGTGAC CGTCTCCGGG AGCTGCATGT GTCAGAGGTT 4141 TTCACCGTCA TCACCGAAAC GCGCGAGACG AAAGGGCCTC GTGATACGCC TATTTTTATA 4201 GGTTAATGTC ATGATAATAA TGGTTTCTTA GACGTCAGGT GGCACTTTTC GGGGAAATGT 4261 GCGCGGAACC CCTATTTGTT TATTTTTCTA AATACATTCA AATATGTATC CGCTCATGAG 4321 ACAATAACCC TGATAAATGC TTCAATAATA TTGAAAAAGG AAGAGTATGA GTATTCAACA 4381 TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT TTGCTCACCC 4441 AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCACGAG TGGGTTACAT 4501 CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG AACGTTTTCC 4561 AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTA TTGACGCCGG 4621 GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG AGTACTCACC 4681 AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA GTGCTGCCAT 4741 AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATCGGAG GACCGAAGGA 4801 GCTAACCGCT TTTTTGCACA GCATGGGGGA TCATGTAACT CGCCTTGATC GTTGGGAACC 4861 GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG TAGCAATGGC 4921 AACAACGTTG CGCAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC GGCAACAATT 4981 AATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG CCCTTCCGGC 5041 TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG GTATCATTGC 5101 AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA CGGGGAGTCA 5161 GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC TGATTAAGCA 5221 TTGGTAACTG TCAGACCAAG TTTACTCATA TATACTTTAG ATTGATTTAA AACTTCATTT 5281 TTAATTTAAA AGGATCTAGG TGAAGATCCT TTTTGATAAT CTCATGACCA AAATCCCTTA 5341 ACGTGAGTTT TCGTTCCACT GAGCGTCAGA CCCCGTAGAA AAGATCAAAG GATCTTCTTG 5401 AGATCCTTTT TTTCTGCGCG TAATCTGCTG CTTGCAAACA AAAAAACCAC CGCTACCAGC 5461 GGTGGTTTGT TTGCCGGATC AAGAGCTACC AACTCTTTTT CCGAAGGTAA CTGGCTTCAG 5521 CAGAGCGCAG ATACCAAATA CTGTCCTTCT AGTGTAGCCG TAGTTAGGCC ACCACTTCAA 5581 GAACTCTGTA GCACCGCCTA CATACCTCGC TCTGCTAATC CTGTTACCAG TGGCTGCTGC 5641 CAGTGGCGAT AAGTCGTGTC TTACCGGGTT GGACTCAAGA CGATAGTTAC CGGATAAGGC 5701 GCAGCGGTCG GGCTGAACGG GGGGTTCGTG CACACAGCCC AGCTTGGAGC GAACGACCTA 5761 CACCGAACTG AGATACCTAC AGCGTGAGCT ATGAGAAAGC GCCACGCTTC CCGAAGGGAG 5821 AAAGGCGGAC AGGTATCCGG TAAGCGGCAG GGTCGGAACA GGAGAGCGCA CGAGGGAGCT 5881 TCCAGGGGGA AACGCCTGGT ATCTTTATAG TCCTGTCGGG TTTCGCCACC TCTGACTTGA 5941 GCGTCGATTT TTGTGATGCT CGTCAGGGGG GCGGAGCCTA TGGAAAAACG CCAGCAACGC 6001 GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG GCCTTTTGCT CACATGTTCT TTCCTGCGTT 6061 ATCCCCTGAT TCTGTGGATA ACCGTATTAC CGCCTTTGAG TGAGCTGATA CCGCTCGCCG 6121 CAGCCGAACG ACCGAGCGCA GCGAGTCAGT GAGCGAGGAA GCGGAAGAGC GCCCAATACG 6181 CAAACCGCCT CTCCCCGCGC GTTGGCCGAT TCATTAATGC AG //