LOCUS Exported 7220 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pOSIP-TT (TetR, P21) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7220) 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 7220) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7220 /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 2299..3441 /codon_start=1 /gene="int" /product="integrase from phage phi-21" /label=phage phi-21 integrase /translation="MAARPRSHKISIPNLYCKLDKRTGKVYWQYKHPLSGRFHSLGTDE NEAKQVATEANTIIAEQRTRQILSVNERLERMKGRRSDITVTEWLDKYISIQEDRLQHN ELRPNSYRQKGKPIRLFREHCGMQHLKDITALDIAEIIDAVKAEGHNRMAQVVRMVLID VFKEAQHAGHVPPGFNPAQATKQPRNRVNRQRLSLLEWQAIFESVSRRQPYLKCGMLLA LVTGQRLGDICNLKFSDIWDDMLHITQEKTGSKLAIPLNLKCDALNITLREVISQCRDA VVSKYLVHYRHTTSQANRGDQVSANTLTTAFKKAKEKCGIKWEQGTAPTFHEQRSLSER LYREQGLDTQKLLGHKSRKMTDRYNDDRGKDWIIVDIKTA" promoter 3587..3641 /gene="tetR" /label=tetR/tetA promoters /note="overlapping promoters for bacterial tetR and tetA" protein_bind 3622..3640 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" CDS 3649..4854 /codon_start=1 /gene="tetA from transposon Tn10" /product="tetracycline efflux protein" /label=TcR /note="confers resistance to tetracycline" /translation="MNSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGV LLALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLS GITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFI AALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQL IGQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLE FIADSSAFAFLAFISEGWLDFPVLILLAGGGIALPALQGVMSIQTKSHEQGALQGLLVS LTNATGVIGPLLFTVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQET SA" terminator 4913..5007 /label=lambda t0 terminator /note="transcription terminator from phage lambda" rep_origin 5064..5452 /label=R6K gamma ori /note="gamma replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication" protein_bind 5463..5510 /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 5590..6089 /label=phage phi-21 attP /bound_moiety="phage phi-21 integrase" /note="attachment site of phage phi-21" terminator 6110..6356 /label=-lambda tL3 terminator /note="transcription terminator tL3 from phage lambda" primer_bind complement(6446..6468) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 6619..6637 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" terminator 6678..6764 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6861..6947 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 7044..7130 /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 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 cacgttgtgc atcgatttga caccctatca 3601 ttgatagagt tattttccca ctccctatca gtgatagaga aaagtgaaat gaatagttcg 3661 acaaagatcg cattggtaat tacgttgctc gatgccatgg ggattggcct tatcatgcca 3721 gtcttgccaa cgttattacg tgaatttatt gcttcggaag atatcgctaa ccactttggc 3781 gtattgcttg cactttatgc gttaatgcag gttatctttg ctccttggct tggaaaaatg 3841 tctgaccgat ttggtcggcg cccagtgctg ttgttgtcat taataggcgc atcgctggat 3901 tacttattgc tggctttttc aagtgcgctt tggatgctgt atttaggccg tttgctttca 3961 gggatcacag gagctactgg ggctgtcgcg gcatcggtca ttgccgatac cacctcagct 4021 tctcaacgcg tgaagtggtt cggttggtta ggggcaagtt ttgggcttgg tttaatagcg 4081 gggcctatta ttggtggttt tgcaggagag atttcaccgc atagtccctt ttttatcgct 4141 gcgttgctaa atattgtcac tttccttgtg gttatgtttt ggttccgtga aaccaaaaat 4201 acacgtgata atacagatac cgaagtaggg gttgagacgc aatcaaattc ggtgtacatc 4261 actttattta aaacgatgcc cattttgttg attatttatt tttcagcgca attgataggc 4321 caaattcccg caacggtgtg ggtgctattt accgaaaatc gttttggatg gaatagcatg 4381 atggttggct tttcattagc gggtcttggt cttttacact cagtattcca agcctttgtg 4441 gcaggaagaa tagccactaa atggggcgaa aaaacggcag tactgctcga atttattgca 4501 gatagtagtg catttgcctt tttagcgttt atatctgaag gttggttaga tttccctgtt 4561 ttaattttat tggctggtgg tgggatcgct ttacctgcat tacagggagt gatgtctatc 4621 caaacaaaga gtcatgagca aggtgcttta cagggattat tggtgagcct taccaatgca 4681 accggtgtta ttggcccatt actgtttact gttatttata atcattcact accaatttgg 4741 gatggctgga tttggattat tggtttagcg ttttactgta ttattatcct gctatcaatg 4801 accttcatgt tgacccctca agctcagggg agtaaacagg agacaagtgc ttagttattt 4861 cgtcaccaaa tgaggcctcc aaaacttcat ttttaatttt tgagctcgct tggactcctg 4921 ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc 4981 cgccgggcgt tttttattgg tgagaatcca agcgtcgacc tagtgcggcc gcaagatccg 5041 gccacgatgc gtccggcgta gaggatctga agatcagcag ttcaacctgt tgatagtacg 5101 tactaagctc tcatgtttca cgtactaagc tctcatgttt aacgtactaa gctctcatgt 5161 ttaacgaact aaaccctcat ggctaacgta ctaagctctc atggctaacg tactaagctc 5221 tcatgtttca cgtactaagc tctcatgttt gaacaataaa attaatataa atcagcaact 5281 taaatagcct ctaaggtttt aagttttata agaaaaaaaa gaatatataa ggcttttaaa 5341 gcttttaagg tttaacggtt gtggacaaca agccagggat gtaacgcact gagaagccct 5401 tagagcctct caaagcaatt ttgagtgaca caggaacact taacggctga catgggaatt 5461 aggaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc catatgccat 5521 ggaatgaccg accgatacaa tgatgatcgt ggtaaagact ggattatcgt agatatcaaa 5581 acagcataga aaatagccag ttttggggaa gggttttggg gaaagttttg gggaagattt 5641 tacatcatca taaaacaacg ggcgtgttat acgcccgtta taatatttaa cacatgtggt 5701 aattacatat ttttgatgat cgcgtcacca aactcgctgc atttcagcag tttagcgcct 5761 tccatcagac gttcgaagtc ataggttacg gtcttggcgt tgattgcgcc ttccatacct 5821 ttaacaatca ggtctgcggc ttcagtccag cccatatggc gcagcagtat ataaaaatta 5881 tgtattcaac tcgctgatat ttaatcataa atattatttt cttgtctttg tattagccaa 5941 aatcaacttt attcaaccgt ttgatttcat tgacgttcgt ttttattttg gggaaagatt 6001 ttctctacca tttatatcaa aattacaagt ctacatttta ttaacatctc aatacatgtg 6061 tggccttgca agccattgcg aggccttatg ctagcttctt cgtctgtttc tactggtatt 6121 ggcacaaacc tgattccaat ttgagcaagg ctatgtgcca tctcgatact cgttcttaac 6181 tcaacagaag atgctttgtg catacagccc ctcgtttatt atttatctcc tcagccagcc 6241 gctgtgcttt cagtggattt cggataacag aaaggccggg aaatacccag cctcgctttg 6301 taacggagta gagacgaaag tgattgcgcc tacccggata ttatcgtgag gatgcgtcat 6361 cgccattaat tcactgatca gtgatagctg tcaaacatga gaattgatcc ggctgcctcg 6421 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 6481 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 6541 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatgctgcac 6601 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt 6661 cccaattccc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 6721 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt 6781 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 6841 ggaattaatt ccccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc 6901 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg 6961 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg 7021 ccaggaatta attccccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 7081 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag 7141 cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa 7201 ctgccaggaa ttggggatcg //