LOCUS Exported 7274 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pRF-IcaR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7274) AUTHORS Stanton BC, Nielsen AA, Tamsir A, Clancy K, Peterson T, Voigt CA TITLE Genomic mining of prokaryotic repressors for orthogonal logic gates. JOURNAL Nat Chem Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411. PUBMED 24316737 REFERENCE 2 (bases 1 to 7274) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7274 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(40..57) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 210..296 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 388..415 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 434..525 /gene="bla" /label=AmpR promoter CDS 526..1386 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(744..763) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 1428..1883 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1515..1534) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind complement(2714..2731) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2848..3393) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(2928..2947) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3465..3484 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" CDS complement(4459..5541) /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" primer_bind 5503..5522 /label=LacI-R /note="LacI, reverse primer" promoter 5676..5704 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 5710..5729 /label=lac operator (symmetric) /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). The symmetric lac operator was optimized for tight binding of lac repressor." terminator 6344..6415 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 6431..6458 /label=T7Te terminator /note="phage T7 early transcription terminator" RBS 6537..6548 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 6555..7274 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(6600..6621) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(6861..6880) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 7208..7229 /label=EGFP-C /note="EGFP, forward primer" ORIGIN 1 taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga 61 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc 121 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc 181 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact 241 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc 301 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc 361 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt 421 ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 481 caataaccct gataaatgct tcaataatat tgaaaaagga agactatgag tattcaacat 541 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 601 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 661 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 721 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg 781 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 841 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 901 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 961 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 1021 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca 1081 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 1141 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 1201 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 1261 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 1321 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 1381 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttacg cgccctgtag 1441 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 1501 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 1561 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 1621 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata 1681 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 1741 aacttgaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc 1801 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 1861 caaaatatta acgtttacaa tttaaaagga tctaggtgaa gatccttttt gataatctca 1921 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 1981 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 2041 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 2101 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 2161 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 2221 taccagtcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt 2281 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc 2341 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcac 2401 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat 2461 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca cagacggcat 2521 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca 2581 tggctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg 2641 cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga 2701 tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg 2761 aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg 2821 aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca 2881 cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc 2941 gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt 3001 cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg 3061 cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct 3121 tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag 3181 cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa 3241 actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag 3301 ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag 3361 caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa 3421 tatttgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata 3481 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag 3541 aggatctgct catgtttgac agcttatcat cgatgcataa tgtgcctgtc aaatggacga 3601 agcagggatt ctgcaaaccc tatgctactc cctcgagccg tcaattgtct gattcgttac 3661 caattattaa tttttaaagt atgggcaatc aattgctcct gttaaaattg ctttagaaat 3721 actttggcag cggtttgttg tattgagttt catttgcgca ttggttaaat ggaaagtgac 3781 agtacgctca ctgcaaccta atatttttga aatatcccaa gagctttttc cttcgcatgc 3841 ccacgctaaa cattcttttt ctcttttggt taaatcgttg tttgatttat tatttgctat 3901 atttattttt cgataattat caactagaga aggaacaatt aatggtatgt tcatacacgc 3961 atgtaaaaat aaactatcta tatagttgtc tttttctgaa tgtgcaaaac taagcattcc 4021 gaagccattg ttagccgtat gaatagggaa actaaaccca gtgataagac ctgatgtttt 4081 cgcttcttta attacatttg gagatttttt atttacagca ttgttttcaa atatattcca 4141 attaattggt gaatgattgg agttagaata atctactata ggatcatatt ttattaaatt 4201 agcgtcatca taatattgcc tccatttttt agggtaatta tctaggattg aaatatcaga 4261 tttaaccata gaatgaggat aaatgatcgc gagtaaataa tattcacaat gtaccatttt 4321 agtcatatca gataagcatt gattaatatc attattgctt ctacaagctt taattttatt 4381 aattattctg tatgtgtcgt cggcatttat gtttttcatc gaggcctttc ctgtgtgacc 4441 cgctggaata ggatcttatc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 4501 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt 4561 ttttcttttc accagtgaga ctggcaacag ctgattgccc ttcaccgcct ggccctgaga 4621 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt 4681 ggttaacggc gggatataac atgagctatc ttcggtatcg tcgtatccca ctaccgagat 4741 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg 4801 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg 4861 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt 4921 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg 4981 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg 5041 cgtaccgtcc tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag 5101 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag 5161 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt 5221 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc 5281 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt 5341 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat 5401 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct 5461 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc 5521 gtataacgtt actggtttca tattcaccac cctgaattga ctctcttccg ggcgctatca 5581 tgccataccg cgaaaggttt tgcgccattc gatggcgcgc cgcttcgtca ggccacatag 5641 ctttcttgtt ctgatcggaa cgatcgttgg ctgtgttgac aattaatcat cggctcgtat 5701 aatgtgtgga attgtgagcg ctcacaattc tatggactat gttttcacac aggggccgga 5761 tgaaagacaa aattatcgat aacgccatca ccctgtttag cgaaaaaggt tatgacggca 5821 ccaccctgga tgatattgca aaaagcgtga acatcaaaaa agccagcctg tattatcact 5881 ttgatagcaa aaaaagcatc tacgagcaga gcgttaaatg ctgtttcgat tatctgaaca 5941 acatcatcat gatgaaccag aacaaaagca actatagcat cgatgccctg tatcagtttc 6001 tgtttgagtt catcttcgat atcgaggaac gctatattcg tatgtatgtt cagctgagca 6061 acacaccgga agaattttca ggtaacattt atggccagat ccaggatctg aatcagagcc 6121 tgagcaaaga aatcgccaaa ttctatgacg aaagcaaaat caaaatgacc aaagaggact 6181 tccagaatct gattctgctg tttctggaaa gctggtatct gaaagccagc tttagccaga 6241 aatttggtgc agttgaagaa agcaaaagcc agtttaaaga tgaggtttat agcctgctga 6301 acatctttct gaagaaataa ctagggccca tacccccagg catcaaataa aacgaaaggc 6361 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta 6421 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacgacgt acggtggaag 6481 attcgttacc aattgacaat tcacctacct ttcgttaggt taggttgtta ctagagaaag 6541 aggagaaata ctagatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 6601 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 6661 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 6721 tgccctggcc caccctcgtg accaccttcg gctacggcct gcaatgcttc gcccgctacc 6781 ccgaccacat gaagctgcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 6841 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 6901 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 6961 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 7021 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 7081 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 7141 tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc aacgagaagc 7201 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 7261 agctgtacaa gtaa //