LOCUS pLEW82 6540 bp DNA circular 28-JUL-2008 Purpose: T7-driven insert expression in BF or PF. Uses truncated aldolase 3'UTR originally derived from pHD330 (about 6x lower BF expression than the full-length ALD UTR in pLew100, but no difference in expression levels in PF, between long and short UTRs). FEATURES Location/Qualifiers misc_feature complement(1..713) /function="rRNA Spacer" promoter 720..739 /function="T7 Promoter" /note="T7 promoter & initiation region. Promoter-Op-Luc cassette orig from Lew19" 5'UTR 770..825 /function="GPEET upstream" /note="to splice site. Note that this is truncated at the upstream SmaI site compared to the GPEET upstream region in pLew20" 5'UTR 826..856 /function="GPEET 5´ UTR" CDS 888..929 /note="Extraneous CDS" CDS 931..2580 /function="LUCIFERASE" 3'UTR 2734..2886 /function="ALD 'short' 3´ UTR" /note="Perfect match with 3480 to 3612 in X52586" /note="truncated aldolase 3'UTR originally derived from pHD330 (about 6x lower BF expression than the full-length ALD UTR in pLew100, but no difference in expression levels in PF, between long and short UTRs) " 5'UTR 2887..3001 /function="ACT 5´ UTR" /note="actin-derived sas; used in all HD330, 430 & Lew5 descendants" CDS 3011..3382 /function="BLE" 3'UTR 3437..3724 /function="ACT 3´ UTR" /note="actin 3'UTR- same as what's used in all HD330, 430 and Lew5 descendants" terminator 3731..3863 /function="T7 Term" /note="132bp Pet3a fragment (originally PCR'd), then subcloned from published HD216, bearing T7 terminator" terminator 3864..3996 /function="T7 Term" /note="132 bp Pet3a fragment (originally PCR'd then subcloned from published HD216) bearing T7 terminator" CDS 4685..5542 /function="LACTAMASE" conflict 766..773 /note="From a restriction digest of a pLew111 derivative, an SrfI site (GCCCGGGC) originally designated here was absent, according to Shawn Motyka (Barbara Sollner Webb lab) email 9/8/3. This was confirmed by George from 2 seq trace files of Fabian's pLew111-bsd construct." protein_bind 745..763 /function="Tet Operator" BASE COUNT 1682 a 1544 c 1674 g 1640 t 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 TAGGGAAGCG ACGCTTGCCC 481 TAGTCCCACG AGTAACCAAG ACTCCAAAAG CCTTTCTGGC ACAGAGAGCG AGCCGAAATG 541 GAAAAGAGAA ACAATGCCTG CACTAACACT ACTGAGCGAT TCGCCTCGCC GCGGAGGACC 601 GAATACTAAT AACGACACTT GCGGTCAAAA AGTAGAAGAA CAAATGCTCA ACGATGAGTG 661 AATCAGGTTA GGGTAGTTGG AAAATTATAC AGAATGTCTT TGGCAACACA CCGGCTGATT 721 AATACGACTC ACTATAGGGA GATCTCCCTA TCAGTGATAG AGATCTCCCG GGCTGCACGC 781 GCCTTCGAGT TTTTTTTCCT TTTCCCCATT TTTTTCAACT TGAAGACTTC AATTACACCA 841 AAAAGTAAAA TTCACAAGCT TGGAATTCCT TTGTGTTACA TTCTTGAATG TCGCTCGCAG 901 TGACATTAGC ATTCCGGTAC TGTTGGTAAA ATGGAAGACG CCAAAAACAT AAAGAAAGGC 961 CCGGCGCCAT TCTATCCTCT AGAGGATGGA ACCGCTGGAG AGCAACTGCA TAAGGCTATG 1021 AAGAGATACG CCCTGGTTCC TGGAACAATT GCTTTTACAG ATGCACATAT CGAGGTGAAC 1081 ATCACGTACG CGGAATACTT CGAAATGTCC GTTCGGTTGG CAGAAGCTAT GAAACGATAT 1141 GGGCTGAATA CAAATCACAG AATCGTCGTA TGCAGTGAAA ACTCTCTTCA ATTCTTTATG 1201 CCGGTGTTGG GCGCGTTATT TATCGGAGTT GCAGTTGCGC CCGCGAACGA CATTTATAAT 1261 GAACGTGAAT TGCTCAACAG TATGAACATT TCGCAGCCTA CCGTAGTGTT TGTTTCCAAA 1321 AAGGGGTTGC AAAAAATTTT GAACGTGCAA AAAAAATTAC CAATAATCCA GAAAATTATT 1381 ATCATGGATT CTAAAACGGA TTACCAGGGA TTTCAGTCGA TGTACACGTT CGTCACATCT 1441 CATCTACCTC CCGGTTTTAA TGAATACGAT TTTGTACCAG AGTCCTTTGA TCGTGACAAA 1501 ACAATTGCAC TGATAATGAA TTCCTCTGGA TCTACTGGGT TACCTAAGGG TGTGGCCCTT 1561 CCGCATAGAA CTGCCTGCGT CAGATTCTCG CATGCCAGAG ATCCTATTTT TGGCAATCAA 1621 ATCATTCCGG ATACTGCGAT TTTAAGTGTT GTTCCATTCC ATCACGGTTT TGGAATGTTT 1681 ACTACACTCG GATATTTGAT ATGTGGATTT CGAGTCGTCT TAATGTATAG ATTTGAAGAA 1741 GAGCTGTTTT TACGATCCCT TCAGGATTAC AAAATTCAAA GTGCGTTGCT AGTACCAACC 1801 CTATTTTCAT TCTTCGCCAA AAGCACTCTG ATTGACAAAT ACGATTTATC TAATTTACAC 1861 GAAATTGCTT CTGGGGGCGC ACCTCTTTCG AAAGAAGTCG GGGAAGCGGT TGCAAAACGC 1921 TTCCATCTTC CAGGGATACG ACAAGGATAT GGGCTCACTG AGACTACATC AGCTATTCTG 1981 ATTACACCCG AGGGGGATGA TAAACCGGGC GCGGTCGGTA AAGTTGTTCC ATTTTTTGAA 2041 GCGAAGGTTG TGGATCTGGA TACCGGGAAA ACGCTGGGCG TTAATCAGAG AGGCGAATTA 2101 TGTGTCAGAG GACCTATGAT TATGTCCGGT TATGTAAACA ATCCGGAAGC GACCAACGCC 2161 TTGATTGACA AGGATGGATG GCTACATTCT GGAGACATAG CTTACTGGGA CGAAGACGAA 2221 CACTTCTTCA TAGTTGACCG CTTGAAGTCT TTAATTAAAT ACAAAGGATA TCAGGTGGCC 2281 CCCGCTGAAT TGGAATCGAT ATTGTTACAA CACCCCAACA TCTTCGACGC GGGCGTGGCA 2341 GGTCTTCCCG ACGATGACGC CGGTGAACTT CCCGCCGCCG TTGTTGTTTT GGAGCACGGA 2401 AAGACGATGA CGGAAAAAGA GATCGTGGAT TACGTCGCCA GTCAAGTAAC AACCGCGAAA 2461 AAGTTGCGCG GAGGAGTTGT GTTTGTGGAC GAAGTACCGA AAGGTCTTAC CGGAAAACTC 2521 GACGCAAGAA AAATCAGAGA GATCCTCATA AAGGCCAAGA AGGGCGGAAA GTCCAAATTG 2581 TAAAATGTAA CTGTATTCAG CGATGACGAA ATTCTTAGCT ATTGTAATAT TATATGCAAA 2641 TTGATGAATG GTAATTTTGT AATTGTGGGT CACTGTACTA TTTTAACGAA TAATAAAATC 2701 AGGTATAGGT AACTAAAAAG GAATTCGAGC TCGGATCCTG CCCATTTAGT TGGCTTTTCC 2761 CTTGTCTCGT GTCTTTTCCG TGGAAAGGTT CCCGGAGTAA TCTGATGGCA CAGCAGGGAG 2821 GTGCGCCTGC AGGTTGGTTA GGAAGGGGGG ATGATGTAAA AGAAGAAAAT GGGGGGATTC 2881 GAGCCCGGGC ACAGCAAGGT CTTCTGAAAT TCATGTTTTT TTTTTTTTTA CTCTGCATTG 2941 CAGTCTCCGC TCTTATTTAG TTTTGCTTTA CGTAAGGTCT CGTTGCTGCC ATAAAATAAG 3001 CTATATCACC ATGGCCAAGT TGACCAGTGC CGTTCCGGTG CTCACCGCGC GCGACGTCGC 3061 CGGAGCGGTC GAGTTCTGGA CCGACCGGCT CGGGTTCTCC CGGGACTTCG TGGAGGACGA 3121 CTTCGCCGGT GTGGTCCGGG ACGACGTGAC CCTGTTCATC AGCGCGGTCC AGGACCAGGT 3181 GGTGCCGGAC AACACCCTGG CCTGGGTGTG GGTGCGCGGC CTGGACGAGC TGTACGCCGA 3241 GTGGTCGGAG GTCGTGTCCA CGAACTTCCG GGACGCCTCC GGGCCGGCCA TGACCGAGAT 3301 CGGCGAGCAG CCGTGGGGGC GGGAGTTCGC CCTGCGCGAC CCGGCCGGCA ACTGCGTGCA 3361 CTTCGTGGCC GAGGAGCAGG ACTGACCGAC GCCGACCAAC ACCGCCGGTC CGACGGCGGC 3421 CCACGGGTCC CAGGCCTCGG AGATCCTAAC ACCGGGTTGT GTGGCCAAAA TTGTTCTGTA 3481 GTCGCTGTGA GTTGACACGG CTAGTGCTTA TGATTTTCCT CGCGTGTGGT GCCTGTACTC 3541 AGCCCTATGC CTTATTTGCA ACACATTTAC GTACAGCGCA CAAGAGAAGA GAAGATCACT 3601 TGAAGATAAT AAATATAGGG TTGTAGGCAT CTTGTTTAAC TCAAATTTTC TCGTCTTGGT 3661 GTGTCGACAT GATTGAAATA GTGCCACCAG TTGTGTTTGA TGCGTTTGTT ATCTATGCAG 3721 TATTCTGCAG GCTGCTAACA AAGCCCGAAA GGAAGCTGAG TTGGCTGCTG CCACCGCTGA 3781 GCAATAACTA GCATAACCCC TTGGGGCCTC TAAACGGGTC TTGAGGGGTT TTTTGCTGAA 3841 AGGAGGAACT ATATCCGGAT AGGGCTGCTA ACAAAGCCCG AAAGGAAGCT GAGTTGGCTG 3901 CTGCCACCGC TGAGCAATAA CTAGCATAAC CCCTTGGGGC CTCTAAACGG GTCTTGAGGG 3961 GTTTTTTGCT GAAAGGAGGA ACTATATCCG GATAGGGCCC CTGCAGGCAT GCAAGCTAGC 4021 CAGATCCTCT ACGCCGGACG CATCGTGGCC GGCATCACCG GCGCCACAGG TGCGGTTGCT 4081 GGCGCCTATA TCGCCGACAT CACCGATGGG GAAGATCGGG CTCGCCACTT CGGGCTCATG 4141 AGCGCTTGTT TCGGCGTGGG TATGGTGGCA GGCCCCGTGG CCGGGGGACT GTTGGGCGCC 4201 ATCTCCTTGC ACCATTCCTT GCGGCGGCGG TGCTCAACGG CCTCAACCTA CTACTGGGCT 4261 GCTTCCTAAT GCAGGAGTCG CATAAGGGAG AGCGTCGATA TGGTGCACTC TCAGTACAAT 4321 CTGCTCTGAT GCCGCATAGT TAAGCCAGCC CCGACACCCG CCAACACCCG CTGACGCGCC 4381 CTGACGGGCT TGTCTGCTCC CGGCATCCGC TTACAGACAA GCTGTGACCG TCTCCGGGAG 4441 CTGCATGTGT CAGAGGTTTT CACCGTCATC ACCGAAACGC GCGAGACGAA AGGGCCTCGT 4501 GATACGCCTA TTTTTATAGG TTAATGTCAT GATAATAATG GTTTCTTAGA CGTCAGGTGG 4561 CACTTTTCGG GGAAATGTGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA TACATTCAAA 4621 TATGTATCCG CTCATGAGAC AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA 4681 GAGTATGAGT ATTCAACATT TCCGTGTCGC CCTTATTCCC TTTTTTGCGG CATTTTGCCT 4741 TCCTGTTTTT GCTCACCCAG AAACGCTGGT GAAAGTAAAA GATGCTGAAG ATCAGTTGGG 4801 TGCACGAGTG GGTTACATCG AACTGGATCT CAACAGCGGT AAGATCCTTG AGAGTTTTCG 4861 CCCCGAAGAA CGTTTTCCAA TGATGAGCAC TTTTAAAGTT CTGCTATGTG GCGCGGTATT 4921 ATCCCGTATT GACGCCGGGC AAGAGCAACT CGGTCGCCGC ATACACTATT CTCAGAATGA 4981 CTTGGTTGAG TACTCACCAG TCACAGAAAA GCATCTTACG GATGGCATGA CAGTAAGAGA 5041 ATTATGCAGT GCTGCCATAA CCATGAGTGA TAACACTGCG GCCAACTTAC TTCTGACAAC 5101 GATCGGAGGA CCGAAGGAGC TAACCGCTTT TTTGCACAAC ATGGGGGATC ATGTAACTCG 5161 CCTTGATCGT TGGGAACCGG AGCTGAATGA AGCCATACCA AACGACGAGC GTGACACCAC 5221 GATGCCTGTA GCAATGGCAA CAACGTTGCG CAAACTATTA ACTGGCGAAC TACTTACTCT 5281 AGCTTCCCGG CAACAATTAA TAGACTGGAT GGAGGCGGAT AAAGTTGCAG GACCACTTCT 5341 GCGCTCGGCC CTTCCGGCTG GCTGGTTTAT TGCTGATAAA TCTGGAGCCG GTGAGCGTGG 5401 GTCTCGCGGT ATCATTGCAG CACTGGGGCC AGATGGTAAG CCCTCCCGTA TCGTAGTTAT 5461 CTACACGACG GGGAGTCAGG CAACTATGGA TGAACGAAAT AGACAGATCG CTGAGATAGG 5521 TGCCTCACTG ATTAAGCATT GGTAACTGTC AGACCAAGTT TACTCATATA TACTTTAGAT 5581 TGATTTAAAA CTTCATTTTT AATTTAAAAG GATCTAGGTG AAGATCCTTT TTGATAATCT 5641 CATGACCAAA ATCCCTTAAC GTGAGTTTTC GTTCCACTGA GCGTCAGACC CCGTAGAAAA 5701 GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT TGCAAACAAA 5761 AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA CTCTTTTTCC 5821 GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTCCTTCTAG TGTAGCCGTA 5881 GTTAGGCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC TGCTAATCCT 5941 GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG ACTCAAGACG 6001 ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA CACAGCCCAG 6061 CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT GAGAAAGCGC 6121 CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG TCGGAACAGG 6181 AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC CTGTCGGGTT 6241 TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC GGAGCCTATG 6301 GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA 6361 CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG CCTTTGAGTG 6421 AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC 6481 GGAAGAGCGC CCAATACGCA AACCGCCTCT CCCCGCGCGT TGGCCGATTC ATTAATGCAG //