LOCUS Exported 6224 bp ds-DNA circular SYN 22-APR-2020 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pOSIP-CH (CamR, HK022) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6224) 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 6224) AUTHORS . TITLE Direct Submission JOURNAL Exported Apr 22, 2020 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6224 /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)" CDS 2270..3343 /codon_start=1 /gene="int" /product="integrase from phage HK022" /label=HK022 integrase /translation="MGRRRSHERRDLPPNLYIRNNGYYCYRDPRTGKEFGLGRDRRIAI TEAIQANIELLSGNRRESLIDRIKGADAITLHAWLDRYETILSERGIRPKTLLDYASKI RAIRRKLPDKPLADISTKEVAAMLNTYVAEGKSASAKLIRSTLVDVFREAIAEGHVATN PVTATRTAKSEVRRSRLTANEYVAIYHAAEPLPIWLRLAMDLAVVTGQRVGDLCRMKWS DINDNHLHIEQSKTGAKLAIPLTLTIDALNISLADTLQQCREASSSETIIASKHHDPLS PKTVSKYFTKARNASGLSFDGNPPTFHELRSLSARLYRNQIGDKFAQRLLGHKSDSMAA RYRDSRGREWDKIEIDK" promoter 3442..3544 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" CDS 3545..4204 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(3611..3630) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" terminator 4225..4319 /label=lambda t0 terminator /note="transcription terminator from phage lambda" rep_origin 4376..4764 /label=R6K gamma ori /note="gamma replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication" protein_bind 4775..4822 /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 4852..5078 /gene="attP" /label=HK022 attP /bound_moiety="HK022 integrase" /note="attachment site of phage HK022" terminator 5113..5359 /label=-lambda tL3 terminator /note="transcription terminator tL3 from phage lambda" primer_bind complement(5450..5472) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5623..5641 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" terminator 5682..5768 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 5865..5951 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6048..6134 /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 cgcgtgtagc cttttgaaga ggatcagaaa tgggaagaag 2281 gcgaagtcat gagcgccggg atttaccccc taacctttat ataagaaaca atggatatta 2341 ctgctacagg gacccaagga cgggtaaaga gtttggatta ggccgagaca ggcgaatcgc 2401 aatcactgaa gctatacagg ccaacattga gttgctatcc gggaacaggc gtgagtcact 2461 gatagacaga attaaaggcg ctgacgcaat cactcttcat gcgtggcttg accgatatga 2521 aacaatcctc agcgagaggg gtatcaggcc gaaaactcta ctcgactacg ccagcaaaat 2581 cagagcaatc cgaagaaaat tgccggacaa accgctcgct gacatatcaa cgaaagaggt 2641 ggcagcaatg ctaaacacct acgtagcaga aggcaaatcg gcttccgcaa aattgatcag 2701 gtcaaccctt gttgacgttt ttcgtgaggc aatagccgag gggcatgtgg ctacgaatcc 2761 ggtaacagca acccgcacag caaaatcaga agtaaggcgt tcaagactga cagctaatga 2821 atatgtcgcg atataccatg cagccgaacc gctcccaatc tggctgagac tggcaatgga 2881 tttggctgtc gttacagggc agagagtagg cgatttgtgc agaatgaaat ggtcagacat 2941 aaacgacaac catcttcaca ttgaacagag taaaacaggg gctaaactcg ccattccact 3001 aacgctaacg attgacgcgc tcaatatctc attggctgat acactacagc aatgcaggga 3061 ggccagcagc agtgaaacta taatcgcatc aaagcatcac gatccgcttt ccccgaaaac 3121 agtatcaaag tattttacaa aggcgagaaa tgcatctgga ctctcatttg atggaaaccc 3181 gccaacattc catgaactgc gtagcctgtc agcgaggcta taccggaacc agattggcga 3241 taagtttgct caacgtcttc tcgggcataa atcagattca atggcggcgc ggtataggga 3301 cagccgtgga cgggaatggg acaaaattga aatcgacaaa tgattttatt ttgactaata 3361 atgacctact taccatgggt atggacagtt ttccctttga tatgtaacgc acgttgtgga 3421 tatctggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga 3481 aataagatca ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc 3541 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa 3601 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct 3661 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt 3721 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga 3781 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac 3841 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat 3901 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat 3961 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa 4021 cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca 4081 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt 4141 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc 4201 gtaatttgat atcgagctcg cttggactcc tgttgataga tccagtaatg acctcagaac 4261 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc 4321 caagcgtcga cctagtgcgg ccgcaagatc cggccacgat gcgtccggcg tagaggatct 4381 gaagatcagc agttcaacct gttgatagta cgtactaagc tctcatgttt cacgtactaa 4441 gctctcatgt ttaacgtact aagctctcat gtttaacgaa ctaaaccctc atggctaacg 4501 tactaagctc tcatggctaa cgtactaagc tctcatgttt cacgtactaa gctctcatgt 4561 ttgaacaata aaattaatat aaatcagcaa cttaaatagc ctctaaggtt ttaagtttta 4621 taagaaaaaa aagaatatat aaggctttta aagcttttaa ggtttaacgg ttgtggacaa 4681 caagccaggg atgtaacgca ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga 4741 cacaggaaca cttaacggct gacatgggaa ttaggaagtt cctattccga agttcctatt 4801 ctctagaaag tataggaact tccatatgcc atgggacaaa attgaaatcg acaaatgatt 4861 ttattttgac taataatgac ctacttacat taatttactg ataattaaag agattttaaa 4921 tatacaactt attcacctaa aggatgacaa aataacatta atcacttaaa aatcatcgca 4981 ttacactaat ctgtggttaa atgatagact acataatgcg acaaaacgca acatatccag 5041 tcactatgaa tcaactactt agatagtatt agtgacctga gacagagcat tagctagctt 5101 cttcgtctgt ttctactggt attggcacaa acctgattcc aatttgagca aggctatgtg 5161 ccatctcgat actcgttctt aactcaacag aagatgcttt gtgcatacag cccctcgttt 5221 attatttatc tcctcagcca gccgctgtgc tttcagtgga tttcggataa cagaaaggcc 5281 gggaaatacc cagcctcgct ttgtaacgga gtagagacga aagtgattgc gcctacccgg 5341 atattatcgt gaggatgcgt catcgccatt aattcactga tcagtgataa gctgtcaaac 5401 atgagaattg atccggctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 5461 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 5521 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 5581 gcgatagcgg agtgtatgct gcacatgaca ttaacctata aaaataggcg tatcacgagg 5641 ccctttcgtc ttcaagaatt aattcccaat tccccaggca tcaaataaaa cgaaaggctc 5701 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta 5761 ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg 5821 caggacgccc gccataaact gccaggaatt aattccccag gcatcaaata aaacgaaagg 5881 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga 5941 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc 6001 gggcaggacg cccgccataa actgccagga attaattccc caggcatcaa ataaaacgaa 6061 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc 6121 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt 6181 ggcgggcagg acgcccgcca taaactgcca ggaattgggg atcg //