LOCUS Exported 7306 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pRF-BetI SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7306) 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 7306) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7306 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 3..31 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" protein_bind 37..56 /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 704..775 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 791..818 /label=T7Te terminator /note="phage T7 early transcription terminator" RBS 896..907 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 914..1633 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(959..980) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1220..1239) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1567..1588 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1673..1690) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 1843..1929 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 2021..2048 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 2067..2158 /gene="bla" /label=AmpR promoter CDS 2159..3019 /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(2377..2396) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3061..3516 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3148..3167) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind complement(4347..4364) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4481..5026) /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(4561..4580) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5098..5117 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" CDS complement(6092..7174) /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 7136..7155 /label=LacI-R /note="LacI, reverse primer" ORIGIN 1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattgcta 61 cgacttgctc atttgacaga ggataactac tagtgccgaa actgggtatg cagagcattc 121 gtcgtcgtca gctgattgat gcaaccctgg aagcaattaa tgaagttggt atgcatgatg 181 caaccattgc acagattgca cgtcgtgccg gtgttagcac cggtattatt agccattatt 241 tccgcgataa aaacggtctg ctggaagcaa ccatgcgtga tattaccagc cagctgcgtg 301 atgcagttct gaatcgtctg catgcactgc cgcagggtag cgcagaacag cgtctgcagg 361 caattgttgg tggtaatttt gatgaaaccc aggttagcag cgcagcaatg aaagcatggc 421 tggcattttg ggcaagcagc atgcatcagc cgatgctgta tcgtctgcag caggttagca 481 gtcgtcgtct gctgagcaat ctggttagcg aatttcgtcg tgaactgcct cgtgaacagg 541 cacaagaggc aggttatggt ctggcagcac tgattgatgg tctgtggctg cgtgcagcac 601 tgagcggtaa accgctggat aaaacccgtg caaatagcct gacccgtcat tttatcaccc 661 agcatctgcc gaccgattaa tcgtaaggcg agcccccagg catcaaataa aacgaaaggc 721 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta 781 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataataccg tcattcatgg 841 attcgttacc aattgacaat tgattggacg ttcaatataa tgctagctac tagagaaaga 901 ggagaaatac tagatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct 961 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg 1021 cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt 1081 gccctggccc accctcgtga ccaccttcgg ctacggcctg caatgcttcg cccgctaccc 1141 cgaccacatg aagctgcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga 1201 gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga 1261 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa 1321 catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga 1381 caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag 1441 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct 1501 gcccgacaac cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg 1561 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga 1621 gctgtacaag taataagctt ggctgttttg gcggatgaga gaagattttc agcctgatac 1681 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg 1741 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta 1801 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct 1861 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 1921 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg 1981 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat 2041 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta 2101 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagactat 2161 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 2221 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 2281 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 2341 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 2401 tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 2461 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 2521 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 2581 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 2641 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 2701 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 2761 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 2821 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 2881 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 2941 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 3001 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 3061 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 3121 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 3181 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 3241 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc 3301 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 3361 gactcttgtt ccaaacttga acaacactca accctatctc gggctattct tttgatttat 3421 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 3481 acgcgaattt taacaaaata ttaacgttta caatttaaaa ggatctaggt gaagatcctt 3541 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 3601 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 3661 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 3721 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 3781 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 3841 ctgctaatcc tgttaccagt caggcatttg agaagcacac ggtcacactg cttccggtag 3901 tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac 3961 cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc atccgcttat tatcacttat 4021 tcaggcgtag caccaggcgt ttaagggcac caataactgc cttaaaaaaa ttacgccccg 4081 ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac atggaagcca 4141 tcacagacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta 4201 taatatttgc ccatggctag cggagtgtat actggcttac tatgttggca ctgatgaggg 4261 tgtcagtgaa gtgcttcatg tggcaggaga aaaaaggctg caccggtgcg tcagcagaat 4321 atgtgataca ggatatattc cgcttcctcg ctcactgact cgctacgctc ggtcgttcga 4381 ctgcggcgag cggaaatggc ttacgaacgg ggcggagatt tcctggaaga tgccaggaag 4441 atacttaaca gggaagtgag agggccgcgg caaagccgtt tttccatagg ctccgccccc 4501 ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg acaggactat 4561 aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc 4621 ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg cctgacactc 4681 agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc cgttcagtcc 4741 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag acatgcaaaa 4801 gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga agtcatgcgc 4861 cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa gccagttacc 4921 tcggttcaaa gagttggtag ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt 4981 ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag atcatcttat 5041 taatcagata aaatatttgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt 5101 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat 5161 gcgtccggcg tagaggatct gctcatgttt gacagcttat catcgatgca taatgtgcct 5221 gtcaaatgga cgaagcaggg attctgcaaa ccctatgcta ctccctcgag ccgtcaattg 5281 tctgattcgt taccaattat taatttttaa agtatgggca atcaattgct cctgttaaaa 5341 ttgctttaga aatactttgg cagcggtttg ttgtattgag tttcatttgc gcattggtta 5401 aatggaaagt gacagtacgc tcactgcaac ctaatatttt tgaaatatcc caagagcttt 5461 ttccttcgca tgcccacgct aaacattctt tttctctttt ggttaaatcg ttgtttgatt 5521 tattatttgc tatatttatt tttcgataat tatcaactag agaaggaaca attaatggta 5581 tgttcataca cgcatgtaaa aataaactat ctatatagtt gtctttttct gaatgtgcaa 5641 aactaagcat tccgaagcca ttgttagccg tatgaatagg gaaactaaac ccagtgataa 5701 gacctgatgt tttcgcttct ttaattacat ttggagattt tttatttaca gcattgtttt 5761 caaatatatt ccaattaatt ggtgaatgat tggagttaga ataatctact ataggatcat 5821 attttattaa attagcgtca tcataatatt gcctccattt tttagggtaa ttatctagga 5881 ttgaaatatc agatttaacc atagaatgag gataaatgat cgcgagtaaa taatattcac 5941 aatgtaccat tttagtcata tcagataagc attgattaat atcattattg cttctacaag 6001 ctttaatttt attaattatt ctgtatgtgt cgtcggcatt tatgtttttc atcgaggcct 6061 ttcctgtgtg acccgctgga ataggatctt atcactgccc gctttccagt cgggaaacct 6121 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 6181 gcgccagggt ggtttttctt ttcaccagtg agactggcaa cagctgattg cccttcaccg 6241 cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat 6301 cctgtttgat ggtggttaac ggcgggatat aacatgagct atcttcggta tcgtcgtatc 6361 ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc 6421 ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca 6481 tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg 6541 gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga 6601 cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct 6661 ccacgcccag tcgcgtaccg tcctcatggg agaaaataat actgttgatg ggtgtctggt 6721 cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat 6781 cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt 6841 gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg 6901 cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg 6961 ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca 7021 cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg 7081 cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat 7141 actctgcgac atcgtataac gttactggtt tcatattcac caccctgaat tgactctctt 7201 ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggcg cgccgcttcg 7261 tcaggccaca tagctttctt gttctgatcg gaacgatcgt tggctg //