LOCUS Exported 6298 bp ds-DNA circular SYN 05-SEP-2023 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pOSIP-KO (KanR, 186) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6298) 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. 2013 Sep 20;2(9):537-41. doi: 10.1021/sb400021j. Epub 2013 May 20. PUBMED 24050148 REFERENCE 2 (bases 1 to 6298) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 5, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6298 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 142..447 /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 326..345 /label=ccdB-fwd /note="ccdB gene, forward primer" rep_origin 477..1065 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 966..985 /label=pBR322ori-F /note="pBR322 origin, forward primer" 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 1276..1307 /label=tonB terminator /note="bidirectional E. coli tonB-P14 transcription terminator" protein_bind 1384..1417 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" CDS complement(1424..2137) /codon_start=1 /gene="cIts" /product="temperature-sensitive variant of the phage lambda repressor" /label=-lambda repressor (ts) /note="thermosensitivity is conferred by the A67T mutation" /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV VGKVIASQWPEETFG" 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)" RBS 2246..2268 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 2276..3286 /codon_start=1 /gene="int" /product="integrase from phage 186" /label=phage 186 integrase /translation="MTVRKNPAGGWICELYPNGAKGKRIRKKFATKGEALAFEQYTVQN PWQEEKEDRRTLKGLVDSWYSAHGITLKDGLKRQLAMHHAFECMGEPLARDFDAQMFSR YREKRLKGEYARSNRVKEVSPRTLNLELAYFRAVFNELNRLGEWKGENPLKNMRPFRTE EMEMTWLTHDQISQLLGECNRHDHPDLETVVRICLATGARWSEAESLRKSQLAKYKITY TNTKGRKNRTVPISKELYESLPDDKKGRLFSDCYGAFRSALERTGIELPAGQLTHVLRH TFASHFMMNGGNILVLQRVLGHTDIKMTMRYAHFAPDHLEDAVKLNPLVHITNSK" primer_bind complement(3449..3468) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 3460..4275 /codon_start=1 /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(3533..3552) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 4397..4785 /label=R6K gamma ori /note="gamma replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication" protein_bind 4796..4843 /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)." protein_bind 4856..5070 /gene="attP" /label=phage 186 attP /bound_moiety="phage 186 integrase" /note="attachment site of phage 186" terminator 5187..5433 /label=-lambda tL3 terminator /note="transcription terminator tL3 from phage lambda" primer_bind complement(5524..5546) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5697..5715 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" terminator 5756..5842 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 5939..6025 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6122..6208 /gene="Escherichia coli rrnB" /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 cgcgttttgt ttaactttaa gaaggagata tacatatgac 2281 cgtccgtaaa aatccggctg gcggttggat ttgtgaactc tacccaaacg gtgcaaaagg 2341 caaacgtatc agaaagaaat tcgctactaa aggcgaggct ctggcgtttg agcagtacac 2401 cgttcaaaac ccgtggcagg aagaaaagga agacaggcgc acgttaaaag ggctggttga 2461 ttcatggtat agcgctcatg gcattacact gaaagatggt ttgaaacgcc agttagccat 2521 gcaccatgct tttgagtgta tgggcgaacc actcgcacgc gatttcgatg cgcagatgtt 2581 ttcccgctac cgagaaaaac ggttaaaagg tgagtatgcc cgttcaaaca gagtgaaaga 2641 ggtatcgcct cgcacgctta atcttgagct ggcctacttc cgggcggtgt tcaatgagct 2701 aaaccgcctc ggagaatgga agggtgaaaa cccactaaaa aatatgcgcc cattccgcac 2761 agaagaaatg gaaatgactt ggctaactca cgaccaaatt tcgcaactgc tcggagagtg 2821 taaccgacat gaccaccctg atttagaaac cgtggtaaga atctgtctcg caactggcgc 2881 acggtggtct gaggccgaga gtctgagaaa aagccaactc gcgaaataca aaatcacata 2941 caccaacacg aaaggtagaa aaaatcgcac cgtcccaatc agcaaagagc tctatgagtc 3001 tctgcctgat gataaaaagg gccggttgtt cagtgattgt tatggcgcgt tccggtcagc 3061 tttggaaaga acaggtatcg aactaccggc aggacaactt acccacgttt tacgtcacac 3121 cttcgctagt cactttatga tgaatggcgg taatattttg gtgttgcagc gcgtactcgg 3181 ccatacagat ataaaaatga ccatgcgata tgcgcacttt gcacccgacc atttagagga 3241 tgcagttaaa ctaaatcctt tagttcacat tactaacagc aaataagtta tggaggttgt 3301 atgtcagttc ggctagccca tgggtatgga cagttttccc tttgatatgt aacgcacgtt 3361 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata 3421 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa 3481 acgtcttgct cccgtccgcg cttaaactcc aacatggacg ctgatttata tgggtataaa 3541 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc 3601 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat 3661 gagatggtcc gtctcaactg gctgacggag tttatgcctc tcccgaccat caagcatttt 3721 atccgtactc ctgatgatgc gtggttactc accaccgcga ttcctgggaa aacagccttc 3781 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggccgtgttc 3841 ctgcgccggt tacattcgat tcctgtttgt aattgtcctt ttaacagcga tcgtgtattt 3901 cgtcttgctc aggcgcaatc acgcatgaat aacggtttgg ttgatgcgag tgattttgat 3961 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcacaa gctcttgcca 4021 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac 4081 gaggggaaat taataggttg tattgatgtt ggacgggtcg gaatcgcaga ccgttaccag 4141 gaccttgcca ttctttggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt 4201 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc 4261 gatgagtttt tctaataata ctagcagaaa tcatccttag cgaaagctaa ggattttttt 4321 tatctgatta ccgcctttga gtgagcgtcg acctagtgcg gccgcaagat ccggccacga 4381 tgcgtccggc gtagaggatc tgaagatcag cagttcaacc tgttgatagt acgtactaag 4441 ctctcatgtt tcacgtacta agctctcatg tttaacgtac taagctctca tgtttaacga 4501 actaaaccct catggctaac gtactaagct ctcatggcta acgtactaag ctctcatgtt 4561 tcacgtacta agctctcatg tttgaacaat aaaattaata taaatcagca acttaaatag 4621 cctctaaggt tttaagtttt ataagaaaaa aaagaatata taaggctttt aaagctttta 4681 aggtttaacg gttgtggaca acaagccagg gatgtaacgc actgagaagc ccttagagcc 4741 tctcaaagca attttgagtg acacaggaac acttaacggc tgacatggga attaggaagt 4801 tcctattccg aagttcctat tctctagaaa gtataggaac ttccatatgc catgggcgat 4861 ggttctgagt aacagataat agaatggcga taaagtggcg gtagaaatgg cgaataatag 4921 gtaattattg gcaaacaatg gcaatctatg tcaatgataa ataacgcata ctattgattt 4981 tcggttgttc ttgtaggaac tcataatcgc ttggtcgctg gttcaagtcc agcaggggcc 5041 accaaattta agtagtaaat acatacagtt aggccactct ctcgagtggc ctttttgttg 5101 cctcgttttc gagtggcgat agaatggcgg tggwtttttt accgccactt tcctgaggag 5161 tgcatagcta gcttcttcgt ctgtttctac tggtattggc acaaacctga ttccaatttg 5221 agcaaggcta tgtgccatct cgatactcgt tcttaactca acagaagatg ctttgtgcat 5281 acagcccctc gtttattatt tatctcctca gccagccgct gtgctttcag tggatttcgg 5341 ataacagaaa ggccgggaaa tacccagcct cgctttgtaa cggagtagag acgaaagtga 5401 ttgcgcctac ccggatatta tcgtgaggat gcgtcatcgc cattaattca ctgatcagtg 5461 ataagctgtc aaacatgaga attgatccgg ctgcctcgcg cgtttcggtg atgacggtga 5521 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 5581 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat 5641 gacccagtca cgtagcgata gcggagtgta tgctgcacat gacattaacc tataaaaata 5701 ggcgtatcac gaggcccttt cgtcttcaag aattaattcc caattcccca ggcatcaaat 5761 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa 5821 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc 5881 cggagggtgg cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca 5941 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt 6001 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg 6061 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca 6121 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc 6181 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca 6241 acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt ggggatcg //