LOCUS pLEW100v5b1d-BSD 7716 bp DNA circular 01-AUG-2008 For some bizarre inexplicable reason the BSD CDS, although absolutely correct at the nt level, in terms of being the correct length and being properly translated if pasted into Edit Seq (or NCBI), is being improperly translated!!! This does not seem to be happening in pLEW100 itself. I tried the 'update translations' commands, etc, as in 'help'menu. It was also as wrong in the 'socca' BSD file from Jenny, but happpend regardless of pasting from that file or from the GCK file, where the translation showed up correctly. If I highlight the exact same region, go to sequence view, then select 'partial translation ruler', I see the correct translation!!! I also found now that I can select the strands and frames to be viewed under the 'full translations' tab in the sequence view, and then I can also see the correct translation. BUT, it seems as if in other files THE CORRECT DIRECTION OF TRANSLATION IS AUTOMATIALLY SHOWN. If I tried this again, I got a reverse translation and the numbering shows clearly that the regular sequence view is showing translations in the wrong strand. Changed a C to G at position 217 in pLEW100v5b1d to eliminate the NcoI site in the rRNA spacer then cut with NcoI / RsrII and cut p52 with same set of enzyme to extract the BSD sequence. 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" 5'UTR complement(1760..1881) /function="ACT 5' UTR" promoter complement(1888..1909) /function="T7 prom" /note="T7 promoter, full-strength, constitutive" promoter 1921..2179 /function="rRNA promoter" /note="395 to 655 in AF416290 and repeated in several chromosomes, for example ChrI AL929603 792760 to 793004" protein_bind 2184..2224 /function="Tet operators" 5'UTR 2228..2313 /function="GPEET SAS" 5'UTR 2314..2348 /function="GPEET 5'UTR" CDS 2419..4068 /function="LUCIFERASE" 3'UTR 4222..4933 /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 5862..6719 /function="LACTAMASE" CDS complement(1354..1749) /function="BSR CDS" /note="Blasticidin resistance" BASE COUNT 2074 a 1753 c 1889 g 2000 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 GAACTTGCAT 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 TCGGCTCGAG TTAGCCCTCC CACACATAAC CAGAGGGCAG 1381 CAATTCACGA ATCCCAACTG CCGTCGGCTG TCCATCACTG TCCTTCACTA TGGCTTTGAT 1441 CCCAGGATGC AGATCGAGAA GCACCTGTCG GCACCGTCCG CAGGGGCTCA AGATGCCCCT 1501 GTTCTCATTT CCGATCGCGA CGATACAAGT CAGGTTGCCA GCTGCCGCAG CAGCAGCAGT 1561 GCCCAGCACC ACGAGTTCTG CACAAGGTCC CCCAGTAAAA TGATATACAT TGACACCAGT 1621 GAAGATGCGG CCGTCGCTAG AGAGAGCTGC GCTGGCGACG CTGTAGTCTT CAGAGATGGG 1681 GATGCTGTTG ATTGTAGCCG TTGCTCTTTC AATGAGGGTG GATTCTTCTT GAGACAAAGG 1741 CTTGGCCATG GTTGATAGCT TATTTTATGG CAGCAACGAG ACCTTACGTA AAGCAAAACT 1801 AAATAAGAGC GGAGACTGCA ATGCAGAGTA AAAAAAAAAA AACATGAATT TCAGAAGACC 1861 TTGCTGTGCC CCCGGTACGG GAGATCTCCC TATAGTGAGT CGTATTAATC AGGTACCTAG 1921 CTTTCCACCC AGCGCGGGTG CATTCTGGCT CTTATATATA CTTATTGTCA TGACAGAGTA 1981 TATTGTACTG TGTTGATAAG GGACGGGTAA CTGTATTGAA GAGCCGATGC TTTTGACATG 2041 TTAGATATAA TATGTTTTAT TGTAAAGTCA ATACAACACA CAATAGGATA ATAATGATAA 2101 AGTTAAAAAA GTATATATAG TAATAGAAAT ATATCTTATA TAGGAAAGAT TAAGCAGTAA 2161 AAGTAGCGCT TACGGCGTAC GGTTCCCTAT CAGTGATAGA GATCTCCCTA TCAGTGATAG 2221 AGATCCCTGA GTACTGAGTT TAACATGTTC TCGTCCCGGG CTGCACGCGC CTTCGAGTTT 2281 TTTTTCCTTT TCCCCATTTT TTTCAACTTG AAGACTTCAA TTACACCAAA AAGTAAAATT 2341 CACAAGCTTG GAATTCCTTT GTGTTACATT CTTGAATGTC GCTCGCAGTG ACATTAGCAT 2401 TCCGGTACTG TTGGTAAAAT GGAAGACGCC AAAAACATAA AGAAAGGCCC GGCGCCATTC 2461 TATCCTCTAG AGGATGGAAC CGCTGGAGAG CAACTGCATA AGGCTATGAA GAGATACGCC 2521 CTGGTTCCTG GAACAATTGC TTTTACAGAT GCACATATCG AGGTGAACAT CACGTACGCG 2581 GAATACTTCG AAATGTCCGT TCGGTTGGCA GAAGCTATGA AACGATATGG GCTGAATACA 2641 AATCACAGAA TCGTCGTATG CAGTGAAAAC TCTCTTCAAT TCTTTATGCC GGTGTTGGGC 2701 GCGTTATTTA TCGGAGTTGC AGTTGCGCCC GCGAACGACA TTTATAATGA ACGTGAATTG 2761 CTCAACAGTA TGAACATTTC GCAGCCTACC GTAGTGTTTG TTTCCAAAAA GGGGTTGCAA 2821 AAAATTTTGA ACGTGCAAAA AAAATTACCA ATAATCCAGA AAATTATTAT CATGGATTCT 2881 AAAACGGATT ACCAGGGATT TCAGTCGATG TACACGTTCG TCACATCTCA TCTACCTCCC 2941 GGTTTTAATG AATACGATTT TGTACCAGAG TCCTTTGATC GTGACAAAAC AATTGCACTG 3001 ATAATGAATT CCTCTGGATC TACTGGGTTA CCTAAGGGTG TGGCCCTTCC GCATAGAACT 3061 GCCTGCGTCA GATTCTCGCA TGCCAGAGAT CCTATTTTTG GCAATCAAAT CATTCCGGAT 3121 ACTGCGATTT TAAGTGTTGT TCCATTCCAT CACGGTTTTG GAATGTTTAC TACACTCGGA 3181 TATTTGATAT GTGGATTTCG AGTCGTCTTA ATGTATAGAT TTGAAGAAGA GCTGTTTTTA 3241 CGATCCCTTC AGGATTACAA AATTCAAAGT GCGTTGCTAG TACCAACCCT ATTTTCATTC 3301 TTCGCCAAAA GCACTCTGAT TGACAAATAC GATTTATCTA ATTTACACGA AATTGCTTCT 3361 GGGGGCGCAC CTCTTTCGAA AGAAGTCGGG GAAGCGGTTG CAAAACGCTT CCATCTTCCA 3421 GGGATACGAC AAGGATATGG GCTCACTGAG ACTACATCAG CTATTCTGAT TACACCCGAG 3481 GGGGATGATA AACCGGGCGC GGTCGGTAAA GTTGTTCCAT TTTTTGAAGC GAAGGTTGTG 3541 GATCTGGATA CCGGGAAAAC GCTGGGCGTT AATCAGAGAG GCGAATTATG TGTCAGAGGA 3601 CCTATGATTA TGTCCGGTTA TGTAAACAAT CCGGAAGCGA CCAACGCCTT GATTGACAAG 3661 GATGGATGGC TACATTCTGG AGACATAGCT TACTGGGACG AAGACGAACA CTTCTTCATA 3721 GTTGACCGCT TGAAGTCTTT AATTAAATAC AAAGGATATC AGGTGGCCCC CGCTGAATTG 3781 GAATCGATAT TGTTACAACA CCCCAACATC TTCGACGCGG GCGTGGCAGG TCTTCCCGAC 3841 GATGACGCCG GTGAACTTCC CGCCGCCGTT GTTGTTTTGG AGCACGGAAA GACGATGACG 3901 GAAAAAGAGA TCGTGGATTA CGTCGCCAGT CAAGTAACAA CCGCGAAAAG GTTGCGCGGA 3961 GGAGTTGTGT TTGTGGACGA AGTACCGAAA GGTCTTACCG GAAAACTCGA CGCAAGAAAA 4021 ATCAGAGAGA TCCTCATAAA GGCCAAGAAG GGCGGAAAGT CCAAATTGTA AAATGTAACT 4081 GTATTCAGCG ATGACGAAAT TCTTAGCTAT TGTAATATTA TATGCAAATT GATGAATGGT 4141 AATTTTGTAA TTGTGGGTCA CTGTACTATT TTAACGAATA ATAAAATCAG GTATAGGTAA 4201 CTAAAAAGGA ATTCGAGCTC GGATCCTGCC CATTTAGTTG GCTTTTCCCT TGTCTCGTGT 4261 CTTTTCCGTG GAAAGGTTCC CGGAGTAATC TGATGGCACA GCAGGGAGGT GCGCCTGCAG 4321 GTTGGTTAGG AAGGGGGGAT GATGTAAAAG AAGAAAATGG GGGGATATCT TATGTTTAAG 4381 AGGATAAATA ATGTGAAGGG GCTTTATATG CTTGCTTGGT TGTCTCGTTG GTGCATGGGG 4441 AATCTGCATG TTTGCTTTGG AGCACGCGTG GTACATCAAT GGGATCATAT CTTGCTGCCT 4501 CCCGCAGTCA CCGTGCGGAG CTGCCGGTGC CCCGTTTCTC ATAGGCGTGT GCACCTTCCG 4561 TCTGAGACCT GTAAATGGTT AAAAAGGAAT ATATAATTAC CTTTTGAAAA GTGGTAAAAA 4621 ACGAATATAT GTTTTTTTTT GGTTTTAATA CGCTCTTTTG TGTGTATGAG AGGAATAAAT 4681 AAGTGTGTGT GTGTGTGTGT GTTGTTGTTG TTGAAAAAAT GAAGGACACG AAAGCGCTCA 4741 TTAGGGGTGG GGATACACCA GGCATAATTA TCCGAAAATA TTTTGGTGCA CCAAATAAGT 4801 GAACATACTG ACGAAATCAA AGAGTTGGAG GATGGATAGG GAGGCCTCAT TGGCAGTGGT 4861 AAATTGATTT GACTTGAATA TGATACCCTC TTACTGTTTC ATTGCCACTA CAAGTTGGTT 4921 TCCTTCCCCT GCAGGCATGC AAGCTAGCTT GTATTCTATA GTGTCACCTA AATCGTATGT 4981 GTATGATACA TAAGGTTATG TATTAATTGT AGCCGCGTTC TAACGACAAT ATGTACAAGC 5041 CTAATTGTGT AGCATCTGGC TTACTGAAGC AGACCCTATC ATCTCTCTCG TAAACTGCCG 5101 TCAGAGTCGG TTTGGTTGGA CGAACCTTCT GAGTTTCTGG TAACGCCGTC CCGCACCCGG 5161 AAATGGTCAG CGAACCAATC AGCAGGGTCA TCGCTAGCCA GATCCTCTAC GCCGGACGCA 5221 TCGTGGCCGG CATCACCGGC GCCACAGGTG CGGTTGCTGG CGCCTATATC GCCGACATCA 5281 CCGATGGGGA AGATCGGGCT CGCCACTTCG GGCTCATGAG CGCTTGTTTC GGCGTGGGTA 5341 TGGTGGCAGG CCCCGTGGCC GGGGGACTGT TGGGCGCCAT CTCCTTGCAC CATTCCTTGC 5401 GGCGGCGGTG CTCAACGGCC TCAACCTACT ACTGGGCTGC TTCCTAATGC AGGAGTCGCA 5461 TAAGGGAGAG CGTCGAATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA 5521 GCCAGCCCCG ACACCCGCCA ACACCCGCTG ACGCGCCCTG ACGGGCTTGT CTGCTCCCGG 5581 CATCCGCTTA CAGACAAGCT GTGACCGTCT CCGGGAGCTG CATGTGTCAG AGGTTTTCAC 5641 CGTCATCACC GAAACGCGCG AGACGAAAGG GCCTCGTGAT ACGCCTATTT TTATAGGTTA 5701 ATGTCATGAT AATAATGGTT TCTTAGACGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG 5761 GAACCCCTAT TTGTTTATTT TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT 5821 AACCCTGATA AATGCTTCAA TAATATTGAA AAAGGAAGAG TATGAGTATT CAACATTTCC 5881 GTGTCGCCCT TATTCCCTTT TTTGCGGCAT TTTGCCTTCC TGTTTTTGCT CACCCAGAAA 5941 CGCTGGTGAA AGTAAAAGAT GCTGAAGATC AGTTGGGTGC ACGAGTGGGT TACATCGAAC 6001 TGGATCTCAA CAGCGGTAAG ATCCTTGAGA GTTTTCGCCC CGAAGAACGT TTTCCAATGA 6061 TGAGCACTTT TAAAGTTCTG CTATGTGGCG CGGTATTATC CCGTATTGAC GCCGGGCAAG 6121 AGCAACTCGG TCGCCGCATA CACTATTCTC AGAATGACTT GGTTGAGTAC TCACCAGTCA 6181 CAGAAAAGCA TCTTACGGAT GGCATGACAG TAAGAGAATT ATGCAGTGCT GCCATAACCA 6241 TGAGTGATAA CACTGCGGCC AACTTACTTC TGACAACGAT CGGAGGACCG AAGGAGCTAA 6301 CCGCTTTTTT GCACAGCATG GGGGATCATG TAACTCGCCT TGATCGTTGG GAACCGGAGC 6361 TGAATGAAGC CATACCAAAC GACGAGCGTG ACACCACGAT GCCTGTAGCA ATGGCAACAA 6421 CGTTGCGCAA ACTATTAACT GGCGAACTAC TTACTCTAGC TTCCCGGCAA CAATTAATAG 6481 ACTGGATGGA GGCGGATAAA GTTGCAGGAC CACTTCTGCG CTCGGCCCTT CCGGCTGGCT 6541 GGTTTATTGC TGATAAATCT GGAGCCGGTG AGCGTGGGTC TCGCGGTATC ATTGCAGCAC 6601 TGGGGCCAGA TGGTAAGCCC TCCCGTATCG TAGTTATCTA CACGACGGGG AGTCAGGCAA 6661 CTATGGATGA ACGAAATAGA CAGATCGCTG AGATAGGTGC CTCACTGATT AAGCATTGGT 6721 AACTGTCAGA CCAAGTTTAC TCATATATAC TTTAGATTGA TTTAAAACTT CATTTTTAAT 6781 TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC CCTTAACGTG 6841 AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT TCTTGAGATC 6901 CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA CCAGCGGTGG 6961 TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC TTCAGCAGAG 7021 CGCAGATACC AAATACTGTT CTTCTAGTGT AGCCGTAGTT AGGCCACCAC TTCAAGAACT 7081 CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT GCTGCCAGTG 7141 GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT AAGGCGCAGC 7201 GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG ACCTACACCG 7261 AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA GGGAGAAAGG 7321 CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG GAGCTTCCAG 7381 GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA CTTGAGCGTC 7441 GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC AACGCGGCCT 7501 TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT GCGTTATCCC 7561 CTGATTCTGT GGATAACCGT ATTACCGCCT TTGAGTGAGC TGATACCGCT CGCCGCAGCC 7621 GAACGACCGA GCGCAGCGAG TCAGTGAGCG AGGAAGCGGA AGAGCGCCCA ATACGCAAAC 7681 CGCCTCTCCC CGCGCGTTGG CCGATTCATT AATGCA //