LOCUS Exported 6036 bp ds-DNA circular SYN 03-SEP-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET-Gate2 ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6036) AUTHORS Frank TITLE Direct Submission JOURNAL Exported Sep 3, 2018 from SnapGene 4.2.2 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6036 /organism="synthetic DNA construct" /note="/dnas_title=pSG0436 pET-Gate2 ccdB" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" promoter 1..17 /label=T7 promoter /note="/dnas_title=T7 promoter" /note="/vntifkey=30" protein_bind 20..44 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." CDS 20..44 /codon_start=1 /label=lac operator /note="/dnas_title=lac operator" /note="/vntifkey=4" /translation="GIVSG*QF" misc_feature 55..58 /label=RU /note="/dnas_title=RU" /note="/vntifkey=21" misc_feature 59..908 /label=outsert /note="/dnas_title=outsert" /note="/vntifkey=21" protein_bind 90..106 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 114..130 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" promoter 158..188 /label=PccdB /note="/dnas_title=PccdB" /note="/vntifkey=30" RBS 215..223 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 225..533 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MIQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSD KVSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" misc_feature 532..588 /label=MCS /note="pUC18/19 multiple cloning site" primer_bind complement(589..605) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" misc_feature 909..912 /label=RD /note="/dnas_title=RD" /note="/vntifkey=21" terminator 981..1028 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1065..1520 /direction=RIGHT /label=f1 origin /note="/dnas_title=f1 origin" /note="/vntifkey=33" CDS complement(1613..2428) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" rep_origin 2550..3138 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" rep_origin 3137 /label=ColE1 pBR322 origin /note="/dnas_title=ColE1 pBR322 origin" /note="/vntifkey=33" misc_feature 3324..3466 /label=bom /note="basis of mobility region from pBR322" CDS complement(3568..3759) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4568..5650) /codon_start=1 /gene="lacI" /product="lac repressor" /label=lacI /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" promoter complement(5651..5728) /gene="lacI" /label=lacI promoter ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gtctcgagtg 61 agaccttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 121 cagctatgac catgattacg ccaagcttga tatcgaattc ctgcagccca aactgcagtt 181 gacaacataa aaactttgtg ttatacttgt aacgtaagga ggtaatgatt cagtttaagg 241 tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta 301 ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata 361 aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga 421 ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc 481 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taagcttgca 541 tgcctgcagg tcgactctag aggatccccg ggtaccgagc tcgaattcac tggccgtcgt 601 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 661 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 721 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg 781 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 841 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 901 gggtctctcc atggtgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg 961 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg 1021 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg 1081 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 1141 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 1201 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 1261 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 1321 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 1381 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 1441 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa 1501 aatattaacg tttacaattt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 1561 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa 1621 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt 1681 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg 1741 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt 1801 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg 1861 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac 1921 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag 1981 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc 2041 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta 2101 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag 2161 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga 2221 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg 2281 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc 2341 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc 2401 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag 2461 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 2521 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 2581 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 2641 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 2701 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 2761 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 2821 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 2881 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2941 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 3001 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 3061 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 3121 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 3181 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 3241 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 3301 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt 3361 atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 3421 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc 3481 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 3541 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 3601 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg 3661 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa 3721 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg 3781 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt 3841 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg 3901 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga 3961 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt 4021 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca 4081 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat 4141 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga 4201 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc 4261 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc 4321 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa 4381 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat 4441 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc 4501 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc 4561 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 4621 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca 4681 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca 4741 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg 4801 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa 4861 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa 4921 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg 4981 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat 5041 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggt cccgctaaca 5101 gcgcgatttg ctgatgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt 5161 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg 5221 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa 5281 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga 5341 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt 5401 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa 5461 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct 5521 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca 5581 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta 5641 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc 5701 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc 5761 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat 5821 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc 5881 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg 5941 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt 6001 ccggcgtaga ggatcgagat ctcgatcccg cgaaat //