LOCUS pLEW82v4 6364 bp DNA circular 20-AUG-2008 To make this version of pLEW82 with a truncated luciferase, the aim was to have maximum expression for cytoplasmid-expressed luciferase. The truncated luciferase coding sequence (lacking the short upstream ORF that reduces expression 8-fold) was amplified from pLEW100v5b1d-trun with primers that contained 5' HindIII site and nested 3' XhoI and BamHI sites, to allow its cloning into Hind-Bam-cut pLEW82 or Hind-Xho-cut pLEW82v4. The pLEW82 version was not made. In error, it was Hind-Bam digested before cloning into Hind-Bam-digested pLEW82v4, which results in loss of the 10% T7 promoter driving BLE. This is probably not relevant as the 'improvements' made in pLEW82v4 did not appear to significantly improve its regulation. A G to A change at position 2451, comparing actual high-quality sequence with theoretical sequence in theoretical pLEW100v5b1d-trunc changes Arg to Lys. It is possible we never sequenced through luciferase in other constructs. This seems like a conservative change, but have not checked if it affects activity. We got very high-quality sequence through 3167bp of this construct (saved as sbd file plew82 luc trunc assembly contig. FEATURES Location/Qualifiers misc_feature complement(1..713) /function="rRNA Spacer" protein_bind 741..813 /function="Triple Tet Operator" promoter 720..739 /function="T7 Promoter" /note="T7 promoter & initiation region. Promoter-Op-Luc cassette orig from Lew19" 5'UTR 822..877 /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 878..908 /function="GPEET 5´ UTR" 3'UTR 2560..2712 /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 2713..2827 /function="ACT 5´ UTR" /note="actin-derived sas; used in all HD330, 430 & Lew5 descendants" CDS 2835..3206 /function="BLE" 3'UTR 3261..3548 /function="ACT 3´ UTR" /note="actin 3'UTR- same as what's used in all HD330, 430 and Lew5 descendants" terminator 3555..3687 /function="T7 Term" /note="132bp Pet3a fragment (originally PCR'd), then subcloned from published HD216, bearing T7 terminator" terminator 3688..3820 /function="T7 Term" /note="132 bp Pet3a fragment (originally PCR'd then subcloned from published HD216) bearing T7 terminator" CDS 4509..5366 /function="LACTAMASE" conflict 589..594 /note="The Sac II site indicated in the sequence is missing." misc_feature 119..3239 /note="100% verified high-quality sequence" CDS 920..2551 /function="TRUNCATED LUCIFERASE" /note="G to A change at position 2451, comparing actual high-quality sequence with theoretical sequence in theoretical pLEW100v5b1d-trunc changes an Arg to Lys. This seems like a conservative change, but have not checked if it affects activity." BASE COUNT 1621 a 1528 c 1641 g 1574 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 AGACATCCGC TCCCTATCAG 781 TGATAGAGAC GGTAGCTCCC TATCAGTGAT AGAGATCTCC CGGGCTGCAC GCGCCTTCGA 841 GTTTTTTTTC CTTTTCCCCA TTTTTTTCAA CTTGAAGACT TCAATTACAC CAAAAAGTAA 901 AATTCACAAG CTTGGTAAAA TGGAAGACGC CAAAAACATA AAGAAAGGCC CGGCGCCATt 961 CTaTCcTCTA GAGGATGGAA CCGCTGGAGA GCAACTGCAT AAGGCTATGA AGAGATACGC 1021 CCTGGTTCCT GGAACAATTG CTTTTACAGA TGCACATATC GAGGTGAACA TCACGTACGC 1081 GGAATACTTC GAAATGTCCG TTCGGTTGGC AGAAGCTATG AAACGATATG GGCTGAATAC 1141 AAATCACAGA ATCGTCGTAT GCAGTGAAAA CTCTCTTCAA TTCTTTATGC CGGTGTTGGG 1201 CGCGTTATTT ATCGGAGTTG CAGTTGCGCC CGCGAACGAC ATTTATAATG AACGTGAATT 1261 GCTCAACAGT ATGAACATTT CGCAGCCTAC CGTAGTGTTT GTTTCCAAAA AGGGGTTGCA 1321 AAAAATTTTG AACGTGCAAA AAAAATTACC AATAATCCAG AAAATTATTA TCATGGATTC 1381 TAAAACGGAT TACCAGGGAT TTCAGTCGAT GTACACGTTC GTCACATCTC ATCTACCTCC 1441 CGGTTTTAAT GAATACGATT TTGTACCAGA GTCCTTTGAT CGTGACAAAA CAATTGCACT 1501 GATAATGAAT TCCTCTGGAT CTACTGGGTt ACCTAAGGGt GtGgCCcTTC CGCATAGAAC 1561 TGCCTGCGTC AGATTCTCGC ATGCCAGAGA TCCTATTTTT GGCAATCAAA TCATTCCGGA 1621 TACTGCGATT TTAAGTGTTG TTCCATTCCA TCACGGTTTT GGAATGTTTA CTACACTCGG 1681 ATATTTGATA TGTGGATTTC GAGTCGTCTT AATGTATAGA TTTGAAGAAG AGCTGTTTTT 1741 ACGATCCCTT CAGGATTACA AAATTCAAAG TGCGTTGCTA GTACCAACCC TATTTTCATT 1801 CTTCGCCAAA AGCACTCTGA TTGACAAATA CGATTTATCT AATTTACACG AAATTGCTTC 1861 TGGGGGCGCA CCTCTTTCGA AAGAAGTCGG GGAAGCGGTT GCAAAACGCT TCCATCTTCC 1921 AGGGATACGA CAAGGATATG GGCTCACTGA GACTACATCA GCTATTCTGA TTACACCCGA 1981 GGGGGATGAT AAACCGGGCG CGGTCGGTAA AGTTGTTCCA TTTTTTGAAG CGAAGGTTGT 2041 GGATCTGGAT ACCGGGAAAA CGCTGGGCGT TAATCAGAGA GGCGAATTAT GTGTCAGAGG 2101 ACCTATGATT ATGTCCGGTT ATGTAAACAA TCCGGAAGCG ACCAACGCCT TGATTGACAA 2161 GGATGGATGG CTACATTCTG GAGACATAGC TTACTGGGAC GAAGACGAAC ACTTCTTCAT 2221 aGTTGACCGC TTGAAGTCTT TAATTAAATA CAAAGGATAT CAGGTGGCCC CCGCTGAATT 2281 GGAATCGATA TTGTTACAAC ACCCCAACAT CTTCGACGCG GGCGTGGCAG GTCTTCCCGA 2341 CGATGACGCC GGTGAACTTC CCGCCGCCGT TGTTGTTTTG GAGCACGGAA AGACGATGAC 2401 GGAAAAAGAG ATCGTGGATT ACGTCGCCAG TCAAGTAACA ACCGCGAAAA AgTtGCGCGG 2461 AGGAGTTGTG TTTGTGGACG AAGTACCGAA AGGTCTTACC GGAAAACTCG ACGCAAGAAA 2521 AATCAGAGAG ATCCTCATAA AGGCCAAGTA GGGCTCGAGG ATCCTGCCCA TTTAGTTGGC 2581 TTTTCCCTTG TCTCGTGTCT TTTCCGTGGA AAGGTTCCCG GAGTAATCTG ATGGCACAGC 2641 AGGGAGGTGC GCCTGCAGGT TGGTTAGGAA GGGGGGATGA TGTAAAAGAA GAAAATGGGG 2701 GGATTCGAGC CCGGGCACAG CAAGGTCTTC TGAAATTCAT GTTTTTTTTT TTTTTACTCT 2761 GCATTGCAGT CTCCGCTCTT ATTTAGTTTT GCTTTACGTA AGGTCTCGTT GCTGCCATAA 2821 AATAAGCTAT CACCATGGCC AAGTTGACCA GTGCCGTTCC GGTGCTCACC GCGCGCGACG 2881 TCGCCGGAGC GGTCGAGTTC TGGACCGACC GGCTCGGGTT CTCCCGGGAC TTCGTGGAGG 2941 ACGACTTCGC CGGTGTGGTC CGGGACGACG TGACCCTGTT CATCAGCGCG GTCCAGGACC 3001 AGGTGGTGCC GGACAACACC CTGGCCTGGG TGTGGGTGCG CGGCCTGGAC GAGCTGTACG 3061 CCGAGTGGTC GGAGGTCGTG TCCACGAACT TCCGGGACGC CTCCGGGCCG GCCATGACCG 3121 AGATCGGCGA GCAGCCGTGG GGGCGGGAGT TCGCCCTGCG CGACCCGGCC GGCAACTGCG 3181 TGCACTTCGT GGCCGAGGAG CAGGACTGAC CGACGCCGAC CAACACCGCC GGTCCGACGG 3241 CGGCCCACGG GTCCCAGGCC TCGGAGATCC TAACACCGGG TTGTGTGGCC AAAATTGTTC 3301 TGTAGTCGCT GTGAGTTGAC ACGGCTAGTG CTTATGATTT TCCTCGCGTG TGGTGCCTGT 3361 ACTCAGCCCT ATGCCTTATT TGCAACACAT TTACGTACAG CGCACAAGAG AAGAGAAGAT 3421 CACTTGAAGA TAATAAATAT AGGGTTGTAG GCATCTTGTT TAACTCAAAT TTTCTCGTCT 3481 TGGTGTGTCG ACATGATTGA AATAGTGCCA CCAGTTGTGT TTGATGCGTT TGTTATCTAT 3541 GCAGTATTCT GCAGGCTGCT AACAAAGCCC GAAAGGAAGC TGAGTTGGCT GCTGCCACCG 3601 CTGAGCAATA ACTAGCATAA CCCCTTGGGG CCTCTAAACG GGTCTTGAGG GGTTTTTTGC 3661 TGAAAGGAGG AACTATATCC GGATAGGGCT GCTAACAAAG CCCGAAAGGA AGCTGAGTTG 3721 GCTGCTGCCA CCGCTGAGCA ATAACTAGCA TAACCCCTTG GGGCCTCTAA ACGGGTCTTG 3781 AGGGGTTTTT TGCTGAAAGG AGGAACTATA TCCGGATAGG GCCCCTGCAG GCATGCAAGC 3841 TAGCCAGATC CTCTACGCCG GACGCATCGT GGCCGGCATC ACCGGCGCCA CAGGTGCGGT 3901 TGCTGGCGCC TATATCGCCG ACATCACCGA TGGGGAAGAT CGGGCTCGCC ACTTCGGGCT 3961 CATGAGCGCT TGTTTCGGCG TGGGTATGGT GGCAGGCCCC GTGGCCGGGG GACTGTTGGG 4021 CGCCATCTCC TTGCACCATT CCTTGCGGCG GCGGTGCTCA ACGGCCTCAA CCTACTACTG 4081 GGCTGCTTCC TAATGCAGGA GTCGCATAAG GGAGAGCGTC GATATGGTGC ACTCTCAGTA 4141 CAATCTGCTC TGATGCCGCA TAGTTAAGCC AGCCCCGACA CCCGCCAACA CCCGCTGACG 4201 CGCCCTGACG GGCTTGTCTG CTCCCGGCAT CCGCTTACAG ACAAGCTGTG ACCGTCTCCG 4261 GGAGCTGCAT GTGTCAGAGG TTTTCACCGT CATCACCGAA ACGCGCGAGA CGAAAGGGCC 4321 TCGTGATACG CCTATTTTTA TAGGTTAATG TCATGATAAT AATGGTTTCT TAGACGTCAG 4381 GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 4441 CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 4501 GGAAGAGTAT GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT 4561 GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT 4621 TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT 4681 TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA TGTGGCGCGG 4741 TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA 4801 ATGACTTGGT TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA 4861 GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA 4921 CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA 4981 CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC GAGCGTGACA 5041 CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA 5101 CTCTAGCTTC CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC 5161 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC 5221 GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG 5281 TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG ATCGCTGAGA 5341 TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT 5401 AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA 5461 ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG 5521 AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA 5581 CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA CCAACTCTTT 5641 TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC 5701 CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA 5761 TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA 5821 GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC 5881 CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG CTATGAGAAA 5941 GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA 6001 CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG 6061 GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC 6121 TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG 6181 CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT ACCGCCTTTG 6241 AGTGAGCTGA TACCGCTCGC CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG 6301 AAGCGGAAGA GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT 6361 GCAG //