LOCUS pLEW100v5b1d-trunc 7685 bp DNA circular 01-AUG-2008 This construct was primarily made to see if cytoplasmically located Luciferase gave a higher signal for whole-cell imaging, on the hypothesis that impermeability of the glycosomal membrane to luciferin substrate was the cause of essentially background signal in whole cells. A single A to T change at position 4017 causes premature termination of LUCIFERASE, causing it to loose its original C-terminal peroxisomal (glycosomal) targeting sequence. In addition, the glycosomal location of luciferase was hypothesized (by me) to be the cause of its apparent toxicity when expressed at high levels. We noticed the toxicity when attempting to grow in mice a cell line in which full-length luciferase had been integrated into an active VSG expression site. The potential use of this construct for live-cell imaging was discontinued after discussions with Filip Claes (Institute of Tropical Medicine Antwerp) who showed in 2007 that Renilla luciferase which does not appear to have a peroxisomal targeting sequence) worked very well for in-vivo imaging of T. brucei. After making a similar truncated version in pLEW82v4, using a PCR-amplified LUC from this construct, we found an G to A change close to the C-terminus that changes Arg to Lys, comparing actual high-quality sequence with sequence in this theoretical pLEW100v5b1d-trunc. 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(23..714) /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." terminator complement(730..860) /function="T7 term" /note="Actual sequence 9/26/5" terminator complement(862..992) /function="T7 term" /note="Actual sequence 9/26/5" 3'UTR complement(1006..1294) /function="ACT 3' UTR" CDS complement(1348..1719) /function="BLE" 5'UTR complement(1729..1850) /function="ACT 5' UTR" promoter complement(1857..1878) /function="T7 prom" /note="T7 promoter, full-strength, constitutive" promoter 1890..2148 /function="rRNA promoter" /note="395 to 655 in AF416290 and repeated in several chromosomes, for example ChrI AL929603 792760 to 793004" protein_bind 2153..2193 /function="Tet operators" 5'UTR 2197..2282 /function="GPEET SAS" 5'UTR 2283..2317 /function="GPEET 5'UTR" CDS 2388..4019 /function="TRUNCATED LUCIFERASE" 3'UTR 4191..4902 /function="ALD 3' UTR" /note="long aldolase 3' UTR from pHD103 for optimized BF expression (about 6x higher than the short ALD UTR used in pLew79 & pLew82)." /note="Note that there are no T7 terminators downstream of T7 transcription unit in this construct, which could be a problem in some insertion sites!" CDS 5831..6688 /function="LACTAMASE" BASE COUNT 2042 a 1782 c 1899 g 1962 t ORIGIN 1 GAATTCGAGC TCCAGCTGAG CTCATATAGT TGGTATGTAT TCTAATTCCA GACTACTGGC 61 GTGGATAAAC ATGTCCCCTG ATTAAAAGGA AAGATTCCAT AGCCCATTAG TGCAAAGATA 121 ATTGGTACAC CGTCAAAAAC ACATGGGAGA CCACACGATA CGACACTGTG ACCGTAGCAT 181 CAATTCCGCA CTCGATATAA CTCTATCGAA GAACTTCCAT GGTACAAACT GGTCGCCACG 241 GTCTGCTCCA CACGGAGATC ATCGTATCAT TTTTATCGAT AGCGGCCGCT ATCGATGTAT 301 GCCTTGGCCC TGATGGCATG CCAATTTCAC TACAACGGAC TATGTGGACC CCGTTATCAT 361 GGAAATGCGC TAGTTGGAGG AAGTTAGACC GCGCCGGAAA AGAGAGGGGT AGAGAAAATG 421 ACAACTTGGA AGATATCCAC ACACGCACGG TGAAACGTTA GCAACAATTA TTAGGGAAGC 481 ACGCTTGCCC TAGTCCCACG AGTAACCAAG ACTCCAAAAG CCTTTCTGGC ACAGAGAGCG 541 AGCCGAAATG GAAAAGAGAA ACAATGCCTG CACTAACACT ACTGAGCGAT TCGCCTCGCC 601 GCGGAGGACC GAATACTAAT AACGACACTT GCGGTCAAAA AGTAGAAGAA CAAATGCTCA 661 ACGATGAGTG AATCAGGTTA GGGTAGTTGG AAAATTATAC AGAATGTCTT TGGCAACACA 721 CCGCTTAAGC CTATCCGGAT ATAGTTCTCC TTTCAGCAAA AAACCCCTCA AGACCCGTTT 781 AGAGGCCCCA AGGGGTTATG CTAGTTATTG CTCAGCGGTG GCAGCAGCCA ACTCAGCTTC 841 CTTTCGGGCT TTGTTAGCAG CCCTATCCGG ATATAGTTCT CCTTTCAGCA AAAAACCCCT 901 CAAGACCCGT TTAGAGGCCC CAAGGGGTTA TGCTAGTTAT TGCTCAGCGG TGGCAGCAGC 961 CAACTCAGCT TCCTTTCGGG CTTTGTTAGC AGCATTTAAA TGCAGAATAC TGCATAGATA 1021 ACAAACGCAT CAAACACAAC TGGTGGCACT ATTTCAATCA TGTCGACACA CCAAGACGAG 1081 AAAATTTGAG TTAAACAAGA TGCCTACAAC CCTATATTTA TTATCTTCAA GTGATCTTCT 1141 CTTCTCTTGT GCGCTGTACG TAAATGTGTT GCAAATAAGG CATAGGGCTG AGTACAGGCA 1201 CCACACGCGT GGAAAATCAT AAGCACTAGC CGTGTCAACT CACAGCGACT ACAGAACAAT 1261 TTTGGCCACA CAACCCGGTG TTAGGATCTC CGAGGCCTCG GACCCGTCGG GCCGCGTCGG 1321 ACCGGCGGTG TTGGTCGGCG TCGGTCAGTC CTGCTCCTCG GCCACGAAGT GCACGCAGTT 1381 GCCGGCCGGG TCGCGCAGGG CGAACTCCCG CCCCCACGGC TGCTCGCCGA TCTCGGTCAT 1441 GGCCGGCCCG GAGGCGTCCC GGAAGTTCGT GGACACGACC TCCGACCACT CGGCGTACAG 1501 CTCGTCCAGG CCGCGCACCC ACACCCAGGC CAGGGTGTTG TCCGGCACCA CCTGGTCCTG 1561 GACCGCGCTG ATGAACAGGG TCACGTCGTC CCGGACCACA CCGGCGAAGT CGTCCTCCAC 1621 GAAGTCCCGG GAGAACCCGA GCCGGTCGGT CCAGAACTCG ACCGCTCCGG CGACGTCGCG 1681 CGCGGTGAGC ACCGGAACGG CACTGGTCAA CTTGGCCATG GTGATAGCTT ATTTTATGGC 1741 AGCAACGAGA CCTTACGTAA AGCAAAACTA AATAAGAGCG GAGACTGCAA TGCAGAGTAA 1801 AAAAAAAAAA ACATGAATTT CAGAAGACCT TGCTGTGCCC CCGGTACGGG AGATCTCCCT 1861 ATAGTGAGTC GTATTAATCA GGTACCTAGC TTTCCACCCA GCGCGGGTGC ATTCTGGCTC 1921 TTATATATAC TTATTGTCAT GACAGAGTAT ATTGTACTGT GTTGATAAGG GACGGGTAAC 1981 TGTATTGAAG AGCCGATGCT TTTGACATGT TAGATATAAT ATGTTTTATT GTAAAGTCAA 2041 TACAACACAC AATAGGATAA TAATGATAAA GTTAAAAAAG TATATATAGT AATAGAAATA 2101 TATCTTATAT AGGAAAGATT AAGCAGTAAA AGTAGCGCTT ACGGCGTACG GTTCCCTATC 2161 AGTGATAGAG ATCTCCCTAT CAGTGATAGA GATCCCTGAG TACTGAGTTT AACATGTTCT 2221 CGTCCCGGGC TGCACGCGCC TTCGAGTTTT TTTTCCTTTT CCCCATTTTT TTCAACTTGA 2281 AGACTTCAAT TACACCAAAA AGTAAAATTC ACAAGCTTGG AATTCCTTTG TGTTACATTC 2341 TTGAATGTCG CTCGCAGTGA CATTAGCATT CCGGTACTGT TGGTAAAATG GAAGACGCCA 2401 AAAACATAAA GAAAGGCCCG GCGCCATTCT ATCCTCTAGA GGATGGAACC GCTGGAGAGC 2461 AACTGCATAA GGCTATGAAG AGATACGCCC TGGTTCCTGG AACAATTGCT TTTACAGATG 2521 CACATATCGA GGTGAACATC ACGTACGCGG AATACTTCGA AATGTCCGTT CGGTTGGCAG 2581 AAGCTATGAA ACGATATGGG CTGAATACAA ATCACAGAAT CGTCGTATGC AGTGAAAACT 2641 CTCTTCAATT CTTTATGCCG GTGTTGGGCG CGTTATTTAT CGGAGTTGCA GTTGCGCCCG 2701 CGAACGACAT TTATAATGAA CGTGAATTGC TCAACAGTAT GAACATTTCG CAGCCTACCG 2761 TAGTGTTTGT TTCCAAAAAG GGGTTGCAAA AAATTTTGAA CGTGCAAAAA AAATTACCAA 2821 TAATCCAGAA AATTATTATC ATGGATTCTA AAACGGATTA CCAGGGATTT CAGTCGATGT 2881 ACACGTTCGT CACATCTCAT CTACCTCCCG GTTTTAATGA ATACGATTTT GTACCAGAGT 2941 CCTTTGATCG TGACAAAACA ATTGCACTGA TAATGAATTC CTCTGGATCT ACTGGGTTAC 3001 CTAAGGGTGT GGCCCTTCCG CATAGAACTG CCTGCGTCAG ATTCTCGCAT GCCAGAGATC 3061 CTATTTTTGG CAATCAAATC ATTCCGGATA CTGCGATTTT AAGTGTTGTT CCATTCCATC 3121 ACGGTTTTGG AATGTTTACT ACACTCGGAT ATTTGATATG TGGATTTCGA GTCGTCTTAA 3181 TGTATAGATT TGAAGAAGAG CTGTTTTTAC GATCCCTTCA GGATTACAAA ATTCAAAGTG 3241 CGTTGCTAGT ACCAACCCTA TTTTCATTCT TCGCCAAAAG CACTCTGATT GACAAATACG 3301 ATTTATCTAA TTTACACGAA ATTGCTTCTG GGGGCGCACC TCTTTCGAAA GAAGTCGGGG 3361 AAGCGGTTGC AAAACGCTTC CATCTTCCAG GGATACGACA AGGATATGGG CTCACTGAGA 3421 CTACATCAGC TATTCTGATT ACACCCGAGG GGGATGATAA ACCGGGCGCG GTCGGTAAAG 3481 TTGTTCCATT TTTTGAAGCG AAGGTTGTGG ATCTGGATAC CGGGAAAACG CTGGGCGTTA 3541 ATCAGAGAGG CGAATTATGT GTCAGAGGAC CTATGATTAT GTCCGGTTAT GTAAACAATC 3601 CGGAAGCGAC CAACGCCTTG ATTGACAAGG ATGGATGGCT ACATTCTGGA GACATAGCTT 3661 ACTGGGACGA AGACGAACAC TTCTTCATAG TTGACCGCTT GAAGTCTTTA ATTAAATACA 3721 AAGGATATCA GGTGGCCCCC GCTGAATTGG AATCGATATT GTTACAACAC CCCAACATCT 3781 TCGACGCGGG CGTGGCAGGT CTTCCCGACG ATGACGCCGG TGAACTTCCC GCCGCCGTTG 3841 TTGTTTTGGA GCACGGAAAG ACGATGACGG AAAAAGAGAT CGTGGATTAC GTCGCCAGTC 3901 AAGTAACAAC CGCGAAAAGG TTGCGCGGAG GAGTTGTGTT TGTGGACGAA GTACCGAAAG 3961 GTCTTACCGG AAAACTCGAC GCAAGAAAAA TCAGAGAGAT CCTCATAAAG GCCAAGTAGG 4021 GCGGAAAGTC CAAATTGTAA AATGTAACTG TATTCAGCGA TGACGAAATT CTTAGCTATT 4081 GTAATATTAT ATGCAAATTG ATGAATGGTA ATTTTGTAAT TGTGGGTCAC TGTACTATTT 4141 TAACGAATAA TAAAATCAGG TATAGGTAAC TAAAAAGGAA TTCGAGCTCG GATCCTGCCC 4201 ATTTAGTTGG CTTTTCCCTT GTCTCGTGTC TTTTCCGTGG AAAGGTTCCC GGAGTAATCT 4261 GATGGCACAG CAGGGAGGTG CGCCTGCAGG TTGGTTAGGA AGGGGGGATG ATGTAAAAGA 4321 AGAAAATGGG GGGATATCTT ATGTTTAAGA GGATAAATAA TGTGAAGGGG CTTTATATGC 4381 TTGCTTGGTT GTCTCGTTGG TGCATGGGGA ATCTGCATGT TTGCTTTGGA GCACGCGTGG 4441 TACATCAATG GGATCATATC TTGCTGCCTC CCGCAGTCAC CGTGCGGAGC TGCCGGTGCC 4501 CCGTTTCTCA TAGGCGTGTG CACCTTCCGT CTGAGACCTG TAAATGGTTA AAAAGGAATA 4561 TATAATTACC TTTTGAAAAG TGGTAAAAAA CGAATATATG TTTTTTTTTG GTTTTAATAC 4621 GCTCTTTTGT GTGTATGAGA GGAATAAATA AGTGTGTGTG TGTGTGTGTG TTGTTGTTGT 4681 TGAAAAAATG AAGGACACGA AAGCGCTCAT TAGGGGTGGG GATACACCAG GCATAATTAT 4741 CCGAAAATAT TTTGGTGCAC CAAATAAGTG AACATACTGA CGAAATCAAA GAGTTGGAGG 4801 ATGGATAGGG AGGCCTCATT GGCAGTGGTA AATTGATTTG ACTTGAATAT GATACCCTCT 4861 TACTGTTTCA TTGCCACTAC AAGTTGGTTT CCTTCCCCTG CAGGCATGCA AGCTAGCTTG 4921 TATTCTATAG TGTCACCTAA ATCGTATGTG TATGATACAT AAGGTTATGT ATTAATTGTA 4981 GCCGCGTTCT AACGACAATA TGTACAAGCC TAATTGTGTA GCATCTGGCT TACTGAAGCA 5041 GACCCTATCA TCTCTCTCGT AAACTGCCGT CAGAGTCGGT TTGGTTGGAC GAACCTTCTG 5101 AGTTTCTGGT AACGCCGTCC CGCACCCGGA AATGGTCAGC GAACCAATCA GCAGGGTCAT 5161 CGCTAGCCAG ATCCTCTACG CCGGACGCAT CGTGGCCGGC ATCACCGGCG CCACAGGTGC 5221 GGTTGCTGGC GCCTATATCG CCGACATCAC CGATGGGGAA GATCGGGCTC GCCACTTCGG 5281 GCTCATGAGC GCTTGTTTCG GCGTGGGTAT GGTGGCAGGC CCCGTGGCCG GGGGACTGTT 5341 GGGCGCCATC TCCTTGCACC ATTCCTTGCG GCGGCGGTGC TCAACGGCCT CAACCTACTA 5401 CTGGGCTGCT TCCTAATGCA GGAGTCGCAT AAGGGAGAGC GTCGAATGGT GCACTCTCAG 5461 TACAATCTGC TCTGATGCCG CATAGTTAAG CCAGCCCCGA CACCCGCCAA CACCCGCTGA 5521 CGCGCCCTGA CGGGCTTGTC TGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC 5581 CGGGAGCTGC ATGTGTCAGA GGTTTTCACC GTCATCACCG AAACGCGCGA GACGAAAGGG 5641 CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA ATAATGGTTT CTTAGACGTC 5701 AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT TCTAAATACA 5761 TTCAAATATG TATCCGCTCA TGAGACAATA ACCCTGATAA ATGCTTCAAT AATATTGAAA 5821 AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT 5881 TTGCCTTCCT GTTTTTGCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA 5941 GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG 6001 TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC 6061 GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA 6121 GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT 6181 AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT 6241 GACAACGATC GGAGGACCGA AGGAGCTAAC CGCTTTTTTG CACAGCATGG GGGATCATGT 6301 AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA 6361 CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG GCGAACTACT 6421 TACTCTAGCT TCCCGGCAAC AATTAATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC 6481 ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA 6541 GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT 6601 AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA 6661 GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT CATATATACT 6721 TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA TCCTTTTTGA 6781 TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT CAGACCCCGT 6841 AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG CGCGTAATCT GCTGCTTGCA 6901 AACAAAAAAA CCACCGCTAC CAGCGGTGGT TTGTTTGCCG GATCAAGAGC TACCAACTCT 6961 TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTTC TTCTAGTGTA 7021 GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC TCGCTCTGCT 7081 AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG GGTTGGACTC 7141 AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT CGTGCACACA 7201 GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG AGCTATGAGA 7261 AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG GCAGGGTCGG 7321 AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT ATAGTCCTGT 7381 CGGGTTTCGC CACCTCTGAC TTGAGCGTCG ATTTTTGTGA TGCTCGTCAG GGGGGCGGAG 7441 CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT GCTGGCCTTT 7501 TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA TTACCGCCTT 7561 TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT CAGTGAGCGA 7621 GGAAGCGGAA GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA 7681 ATGCA //