LOCUS 40924_33922 6944 bp DNA circular SYN 18-DEC-2018 DEFINITION Chromosomal integration vector pOSIP-KT, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6944) AUTHORS St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE. TITLE One-step cloning and chromosomal integration of DNA JOURNAL ACS Synth Biol 2 (9), 537-541 (2013) PUBMED 24050148 REFERENCE 2 (bases 1 to 6944) AUTHORS St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE. TITLE Direct Submission JOURNAL Submitted (12-MAY-2013) Molecular and Biomedical Science, The University of Adelaide, Molecular Life Sciences Building, Adelaide, South Australia 5005, Australia REFERENCE 3 (bases 1 to 6944) TITLE Direct Submission REFERENCE 4 (bases 1 to 6944) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth Biol"; date: "2013"; volume: "2"; issue: "9"; pages: "537-541" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (12-MAY-2013) Molecular and Biomedical Science, The University of Adelaide, Molecular Life Sciences Building, Adelaide, South Australia 5005, Australia" COMMENT SGRef: number: 3; type: "Journal Article" FEATURES Location/Qualifiers source 1..6944 /mol_type="other DNA" /organism="synthetic DNA construct" CDS 142..444 /label=ccdB /note="CcdB, a bacterial toxin that poisons DNA gyrase" rep_origin 477..1065 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature 1125..1145 /label=BioBrick suffix /note="universal suffix for all parts" terminator 1200..1227 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator complement(1276..1307) /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" protein_bind 1384..1417 /label=FRT (minimal) /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" CDS complement(1427..2137) /label=lambda repressor (ts) /note="temperature-sensitive variant of the phage lambda repressor" RBS 2226..2234 /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 2299..3438 /label=phage phi-21 integrase /note="integrase from phage phi-21" CDS 3676..4488 /label=KanR /note="aminoglycoside phosphotransferase" rep_origin 4613..5001 /label=R6K gamma ori /note="gamma replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication" protein_bind 5012..5059 /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." misc_feature 5026..5059 /note="FRT; Flp recombinase target site" protein_bind 5139..5638 /label=phage phi-21 attP /note="attachment site of phage phi-21" terminator complement(5659..5905) /label=lambda tL3 terminator /note="transcription terminator tL3 from phage lambda" terminator 6225..6311 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6406..6492 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6587..6673 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator complement(6771..6814) /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" ORIGIN 1 gaattcgagc tcggtacccg gggatcctct agagggctta ctaaaagcca gataacagta 61 tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag 121 tatgtcaaaa agaggtatgc tatgcagttt aaggtttaca cctataaaag agagagccgt 181 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg 241 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg 301 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc 361 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc 421 attaacctga tgttctgggg aatataataa cccgtagaaa agatcaaaag atcttcttga 481 gatccttttt ttctgcgcgt aatctgctac ttgcaaacaa aaaaaccacc gctaccagcg 541 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggctttagc 601 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 661 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 721 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 781 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 841 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 901 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 961 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 1021 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 1081 gcctttttac ggttcctggc cttttgctgg ccttttgctc acattactag tagcggccgc 1141 tgcaggcatg cctcgagatg catggcgcct aacctaaact gacaggcatc aaattaagca 1201 gaaggccatc ctgacggatg gcctttttgc gtttcgaaca attgaaaaaa cctcgcgcct 1261 tacctgttga gtaatagtca aaagcctccg gtcggaggct tttgactttc tgcttactga 1321 atttcggtgg tgccgttaat taaccggtgg gccctcatga taataatggt ttcttagacg 1381 tccgaagttc ctattctcta gaaagtatag gaacttccct aggtcagcca aacgtctctt 1441 caggccactg actagcgata actttcccca caacggaaca actctcattg catgggatca 1501 ttgggtactg tgggtttagt ggttgtaaaa acacctgacc gctatccctg atcagtttct 1561 tgaaggtaaa ctcatcaccc ccaagtctgg ctatgcagaa atcacctggc tcaacagcct 1621 gctcagggtc aacgagaatt aacattccgt caggaaagct tggcttggag cctgttggtg 1681 cggtcatgga attaccttca acctcaagcc agaatgcaga atcactggct tttttggttg 1741 tgcttaccca tctctccgca tcacctttgg taaaggttct aagcttaggt gagaacatcc 1801 ctgcctgaac atgagaaaaa acagggtact catactcact tctaagtgac ggctgcatac 1861 taaccgcttc atacatctcg tagatttctc tggcgattga agggctaaat tcttcaacgc 1921 taactttgag aatttttgta agcaatgcgg cgttataagc atttaatgca ttgatgccat 1981 taaataaagc accaacgcct gactgcccca tccccatctt gtctgcgaca gattcctggg 2041 ataagccaag ttcatttttc tttttttcat aaattgcttt aaggcgacgt gcgtcctcaa 2101 gctgctcttg tgttaatggt ttcttttttg tgctcatacg ttaaatctat caccgcaagg 2161 gataaatatc taacaccgtg cgtgttgact attttacctc tggcggtgat aatggttgca 2221 tgtactaagg aggttgtata cgcgtagaac cgcaactccc aataaacgca aacccaaaac 2281 tccaacggat aatcgctgat ggctgctaga ccccgatctc acaaaatctc tatacccaat 2341 ttatattgca aattagataa gcgaaccgga aaggtatatt ggcaatacaa acatccacta 2401 tccggtcgtt ttcatagctt aggaactgat gagaatgaag caaaacaagt tgctactgaa 2461 gcaaatacca ttattgctga acaacgtacc agacaaatat taagcgtcaa tgagcgtctg 2521 gaaagaatga aaggcaggcg ctcagacatt acggtgacag aatggcttga taaatatatt 2581 tctatccagg aggacaggct gcaacataat gaactaagac ccaactccta tcggcaaaaa 2641 ggcaaaccca ttcgtctttt ccgtgagcat tgtggaatgc aacacctcaa ggatattacc 2701 gcacttgata ttgccgaaat aattgatgct gtaaaggctg aaggtcataa caggatggcg 2761 caagtcgtga gaatggtgtt gatcgacgtc ttcaaagaag cacaacacgc aggacatgtt 2821 ccgccaggat ttaacccagc gcaggcaaca aaacaaccgc gaaatcgagt aaaccgccaa 2881 agattgtcac tgctcgaatg gcaggcaata tttgaaagcg taagcagacg gcagccctat 2941 ttaaaatgcg gcatgctact tgctcttgtt actggacaac gtttaggcga tatctgcaat 3001 ttgaaattct ctgatatatg ggacgacatg ttgcacatta ctcaggaaaa aaccggttca 3061 aaacttgcta ttccgcttaa cctgaaatgc gatgctctga atattaccct tcgtgaagtt 3121 atatctcagt gcagggatgc tgttgttagt aaatatctgg tccattaccg tcacactacc 3181 tctcaagcaa acagaggaga ccaggtttct gcaaatactc tgacaacggc ttttaaaaag 3241 gccaaggaaa aatgtggcat aaaatgggag caaggaactg cgcccacatt tcatgaacag 3301 cgatctctgt cagaacgatt atatcgggaa cagggtctgg atacgcaaaa gttgttaggc 3361 cataaatcca gaaaaatgac cgaccgatac aatgatgatc gtggtaaaga ctggattatc 3421 gtagatatca aaacagcata gaaaatagcc agttttgggg aagggttttg gggaaagttt 3481 tggggaagat tttacatcat cataaaacaa cgggcgtgtt atacgcccgt tatccatggg 3541 tatggacagt tttccctttg atatgtaacg cacgttgtgt ctcaaaatct ctgatgttac 3601 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt 3661 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctcccg tccgcgctta 3721 aactccaaca tggacgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa 3781 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa 3841 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtccgtct caactggctg 3901 acggagttta tgcctctccc gaccatcaag cattttatcc gtactcctga tgatgcgtgg 3961 ttactcacca ccgcgattcc tgggaaaaca gccttccagg tattagaaga atatcctgat 4021 tcaggtgaaa atattgttga tgcgctggcc gtgttcctgc gccggttaca ttcgattcct 4081 gtttgtaatt gtccttttaa cagcgatcgt gtatttcgtc ttgctcaggc gcaatcacgc 4141 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt 4201 gaacaagtct ggaaagaaat gcacaagctc ttgccattct caccggattc agtcgtcact 4261 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt 4321 gatgttggac gggtcggaat cgcagaccgt taccaggacc ttgccattct ttggaactgc 4381 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat 4441 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta ataatactag 4501 cagaaatcat ccttagcgaa agctaaggat tttttttatc tgattaccgc ctttgagtga 4561 gcgtcgacct agtgcggccg caagatccgg ccacgatgcg tccggcgtag aggatctgaa 4621 gatcagcagt tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct 4681 ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac 4741 taagctctca tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg 4801 aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa 4861 gaaaaaaaag aatatataag gcttttaaag cttttaaggt ttaacggttg tggacaacaa 4921 gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac 4981 aggaacactt aacggctgac atgggaatta ggaagttcct attccgaagt tcctattctc 5041 tagaaagtat aggaacttcc atatgccatg gaatgaccga ccgatacaat gatgatcgtg 5101 gtaaagactg gattatcgta gatatcaaaa cagcatagaa aatagccagt tttggggaag 5161 ggttttgggg aaagttttgg ggaagatttt acatcatcat aaaacaacgg gcgtgttata 5221 cgcccgttat aatatttaac acatgtggta attacatatt tttgatgatc gcgtcaccaa 5281 actcgctgca tttcagcagt ttagcgcctt ccatcagacg ttcgaagtca taggttacgg 5341 tcttggcgtt gattgcgcct tccatacctt taacaatcag gtctgcggct tcagtccagc 5401 ccatatggcg cagcagtata taaaaattat gtattcaact cgctgatatt taatcataaa 5461 tattattttc ttgtctttgt attagccaaa atcaacttta ttcaaccgtt tgatttcatt 5521 gacgttcgtt tttattttgg ggaaagattt tctctaccat ttatatcaaa attacaagtc 5581 tacattttat taacatctca atacatgtgt ggccttgcaa gccattgcga ggccttatgc 5641 tagcttcttc gtctgtttct actggtattg gcacaaacct gattccaatt tgagcaaggc 5701 tatgtgccat ctcgatactc gttcttaact caacagaaga tgctttgtgc atacagcccc 5761 tcgtttatta tttatctcct cagccagccg ctgtgctttc agtggatttc ggataacaga 5821 aaggccggga aatacccagc ctcgctttgt aacggagtag agacgaaagt gattgcgcct 5881 acccggatat tatcgtgagg atgcgtcatc gccattaatt cactgatcag tgatagctgt 5941 caaacatgag aattgatccg gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg 6001 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 6061 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc 6121 acgtagcgat agcggagtgt atgctgcaca tgacattaac ctataaaaat aggcgtatca 6181 cgaggccctt tcgtcttcaa gaattaattc ccaattccag gcatcaaata aaacgaaagg 6241 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga 6301 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc 6361 gggcaggacg cccgccataa actgccagga attaattcca ggcatcaaat aaaacgaaag 6421 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg 6481 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg 6541 cgggcaggac gcccgccata aactgccagg aattaattcc aggcatcaaa taaaacgaaa 6601 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct 6661 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg 6721 gcgggcagga cgcccgccat aaactgccag gaattaattc caggcatcaa ataaaacgaa 6781 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc 6841 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt 6901 ggcgggcagg acgcccgcca taaactgcca ggaattgggg atcg //