LOCUS Exported 8578 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION LuxCDABE operon (R6K ori) under T7, in loxP and lox5171 sites.. ACCESSION . VERSION . KEYWORDS pW34 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8578) AUTHORS Wang G, Zhao Z, Ke J, Engel Y, Shi YM, Robinson D, Bingol K, Zhang Z, Bowen B, Louie K, Wang B, Evans R, Miyamoto Y, Cheng K, Kosina S, De Raad M, Silva L, Luhrs A, Lubbe A, Hoyt DW, Francavilla C, Otani H, Deutsch S, Washton NM, Rubin EM, Mouncey NJ, Visel A, Northen T, Cheng JF, Bode HB, Yoshikuni Y TITLE CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria. JOURNAL Nat Microbiol. 2019 Dec;4(12):2498-2510. doi: 10.1038/s41564-019-0573-8. Epub 2019 Oct 14. PUBMED 31611640 REFERENCE 2 (bases 1 to 8578) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8578 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 42..64 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 56..73 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 57..73 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(98..116) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" oriT 161..269 /note="incP origin of transfer" rep_origin 425..781 /label=R6K gamma ori /note="gamma replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication" protein_bind 824..857 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 907..923 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 907..923 /label=pBluescriptSK /note="For pBluescript vector" primer_bind 973..992 /label=T7 /note="T7 promoter, forward primer" promoter 973..991 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 992..1016 /label=lac operator /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)." RBS 1031..1053 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1061..2503 /codon_start=1 /gene="luxC" /product="LuxC fatty acid reductase" /label=LuxC /note="from the Photorhabdus luminescens bioluminescence operon" /translation="MTKKISFIINGQVEIFPEGDDLVQSINFGDNSVYLPILNDSHVKN IIDCNGNNELRLHNIVNFLYTVGQRWKNEEYSRRRTYIRDLKKYMGYSEEMAKLEANWI SMILCSKGGLYDVVENELGSRHIMDEWLPQDESYVRAFPKGKSVHLLAGNVPLSGIMSI LRAILTKNQCIIKTSSTDPFTANALALSFIDVDPNHPITRSLSVIYWPHQGDTSLAKEI MQHADVIVAWGGPDAINWAVEHAPSYADVIKFGSKKSLCIIDNPVDLTSAATGAAHDVC FYDQRACFSAQNIYYMGNHYEEFKLALIEKLNLYAHILPNAKKDFDEKAAYSLVQKESL FAGLKVEVDIHQRWMIIESNAGVEFNQPLGRCVYLHHVDNIEQILPYVQKNKTQTISIF PWESSFKYRDALALKGAERIVEAGMNNIFRVGGSHDGMRPLQRLVTYISHERPSNYTAK DVAVEIEQTRFLEEDKFLVFVP" CDS 2516..3439 /codon_start=1 /gene="luxD" /product="LuxD acyltransferase" /label=LuxD /note="from the Photorhabdus luminescens bioluminescence operon" /translation="MENESKYKTIDHVICVEGNKKIHVWETLPEENSPKRKNAIIIASG FARRMDHFAGLAEYLSRNGFHVIRYDSLHHVGLSSGTIDEFTMSIGKQSLLAVVDWLTT RKINNFGMLASSLSARIAYASLSEINASFLITAVGVVNLRYSLERALGFDYLSLPINEL PNNLDFEGHKLGAEVFARDCLDFGWEDLASTINNMMYLDIPFIAFTANNDNWVKQDEVI TLLSNIRSNRCKIYSLLGSSHDLSENLVVLRNFYQSVTKAAIAMDNDHLDIDVDITEPS FEHLTIATVNERRMRIEIENQAISLS" CDS 3488..4570 /codon_start=1 /gene="luxA" /product="LuxA luciferase subunit" /label=LuxA /note="from the Photorhabdus luminescens bioluminescence operon" /translation="MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHH FTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGRFRFG ICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAY SRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNEKKAQLELYNEVAQEYGHDIHNI DHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFV LKGHKDTNRRIDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASM KLFQSDVMPFLKEKQRSLLY" CDS 4585..5568 /codon_start=1 /gene="luxB" /product="LuxB luciferase subunit" /label=LuxB /note="from the Photorhabdus luminescens bioluminescence operon" /translation="MKFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENH FSDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILG FSDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAY TPGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEI DHQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECIT AAKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHMEYT" CDS 5747..6856 /codon_start=1 /gene="luxE" /product="LuxE" /label=LuxE /note="from the Photorhabdus luminescens bioluminescence operon" /translation="MTSYVDKQEITASSEIDDLIFSSDPLVWSYDEQEKIRKKLVLDAF RNHYKHCREYRHYCQAHKVDDNITEIDDIPVFPTSVFKFTRLLTSQENEIESWFTSSGT NGLKSQVARDRLSIERLLGSVSYGMKYVGSWFDHQIELVNLGPDRFNAHNIWFKYVMSL VELLYPTTFTVTEERIDFVKTLNSLERIKNQGKDLCLIGSPYFIYLLCHYMKDKKISFS GDKSLYIITGGGWKSYEKESLKRDDFNHLLFDTFNLSDISQIRDIFNQVELNTCFFEDE MQRKHVPPWVYARALDPETLKPVPDGTPGLMSYMDASATSYPAFIVTDDVGIISREYGK YPGVLVEILRRVNTRTQKGCALSLTEAFDS" primer_bind 6954..6974 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" primer_bind complement(7198..7216) /label=T7 Term /note="T7 terminator, reverse primer" terminator 7212..7259 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" primer_bind complement(7348..7364) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(7349..7365) /label=pBluescriptKS /note="For pBluescript vector" protein_bind complement(8329..8362) /label=lox5171 /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GTACACAT). lox5171 sites are compatible with each other, but incompatible with loxP or loxN sites (Lee and Saito, 1988)." ORIGIN 1 aatttttgcg gccgctatgt gttgggtaac gccagggttt tcccagtcac gacgttgtaa 61 aacgacggcc agtgaattaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta 121 taggttaatg tcatgataat aatggtttct tagagcttat cggccagcct cgcagagcag 181 gattcccgtt gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa cacccgctcg 241 cgggtgggcc tacttcacct atcctgcccg gctgacgccg ttggatacac caaggaaagt 301 ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga tataccgaaa 361 aaatcgctat aatgaccccg aagcagggtt atgcagcgaa gatccgcagt tcaacctgtt 421 gatagtacgt actaagctct catgtttcac gtactaagct ctcatgttta acgtactaag 481 ctctcatgtt taacgaacta aaccctcatg gctaacgtac taagctctca tggctaacgt 541 actaagctct catgtttcac gtactaagct ctcatgtttg aacaataaaa ttaatataaa 601 tcagcaactt aaatagcctc taaggtttta agttttataa gaaaaaaaag aatatataag 661 gcttttaaag cttttaaggt ttaacggttg tggacaacaa gccagggatg taacgcactg 721 agaagccctt agagcctctc aaagcaattt tgagtgacac aggaacactt aacggctgac 781 atgggaatta gccatggcaa agcctcgcaa tccagtgcaa aggataactt cgtatagcat 841 acattatacg aagttatgat aaccgtatta ccgcctttga gtgagctcca ccgcggtggc 901 ggccgctcta gaactagtgg atccccatgc gtccggcgta gaggatcgag atcgatctcg 961 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc 1021 tagaaataat tttgtttaac tttaagaagg agatatacat atgactaaaa aaatttcatt 1081 cattattaac ggccaggttg aaatctttcc cgaaggtgat gatttagtgc aatccattaa 1141 ttttggtgat aatagtgttt acctgccaat attgaatgac tctcatgtaa aaaacattat 1201 tgattgtaat ggaaataacg aattacggtt gcataacatt gtcaattttc tctatacggt 1261 agggcaaaga tggaaaaatg aagaatactc aagacgcagg acatacattc gtgacttaaa 1321 aaaatatatg ggatattcag aagaaatggc taagctagag gccaattgga tatctatgat 1381 tttatgttct aaaggcggcc tttatgatgt tgtagaaaat gaacttggtt ctcgccatat 1441 catggatgaa tggctacctc aggatgaaag ttatgttcgg gcttttccga aaggtaaatc 1501 tgtacatctg ttggcaggta atgttccatt atctgggatc atgtctatat tacgcgcaat 1561 tttaactaag aatcagtgta ttataaaaac atcgtcaacc gatcctttta ccgctaatgc 1621 attagcgtta agttttattg atgtagaccc taatcatccg ataacgcgct ctttatctgt 1681 tatatattgg ccccaccaag gtgatacatc actcgcaaaa gaaattatgc aacatgcgga 1741 tgttattgtc gcttggggag ggccagatgc gattaattgg gcggtagagc atgcgccatc 1801 ttatgctgat gtgattaaat ttggttctaa aaagagtctt tgcattatcg ataatcctgt 1861 tgatttgacg tccgcagcga caggtgcggc tcatgatgtt tgtttttacg atcagcgagc 1921 ttgtttttct gcccaaaaca tatattacat gggaaatcat tatgaggaat ttaagttagc 1981 gttgatagaa aaacttaatc tatatgcgca tatattaccg aatgccaaaa aagattttga 2041 tgaaaaggcg gcctattctt tagttcaaaa agaaagcttg tttgctggat taaaagtaga 2101 ggtggatatt catcaacgtt ggatgattat tgagtcaaat gcaggtgtgg aatttaatca 2161 accacttggc agatgtgtgt accttcatca cgtcgataat attgagcaaa tattgcctta 2221 tgttcaaaaa aataagacgc aaaccatatc tatttttcct tgggagtcat catttaaata 2281 tcgagatgcg ttagcattaa aaggtgcgga aaggattgta gaagcaggaa tgaataacat 2341 atttcgagtt ggtggatctc atgacggaat gagaccgttg caacgattag tgacatatat 2401 ttctcatgaa aggccatcta actatacggc taaggatgtt gcggttgaaa tagaacagac 2461 tcgattcctg gaagaagata agttccttgt atttgtccca taataggtaa aaagtatgga 2521 aaatgaatca aaatataaaa ccatcgacca cgttatttgt gttgaaggaa ataaaaaaat 2581 tcatgtttgg gaaacgctgc cagaagaaaa cagcccaaag agaaagaatg ccattattat 2641 tgcgtctggt tttgcccgca ggatggatca ttttgctggt ctggcggaat atttatcgcg 2701 gaatggattt catgtgatcc gctatgattc gcttcaccac gttggattga gttcagggac 2761 aattgatgaa tttacaatgt ctataggaaa gcagagcttg ttagcagtgg ttgattggtt 2821 aactacacga aaaataaata acttcggtat gttggcttca agcttatctg cgcggatagc 2881 ttatgcaagc ctatctgaaa tcaatgcttc gtttttaatc accgcagtcg gtgttgttaa 2941 cttaagatat tctcttgaaa gagctttagg gtttgattat ctcagtctac ccattaatga 3001 attgccgaat aatctagatt ttgaaggcca taaattgggt gctgaagtct ttgcgagaga 3061 ttgtcttgat tttggttggg aagatttagc ttctacaatt aataacatga tgtatcttga 3121 tataccgttt attgctttta ctgcaaataa cgataattgg gtcaagcaag atgaagttat 3181 cacattgtta tcaaatattc gtagtaatcg atgcaagata tattctttgt taggaagttc 3241 gcatgacttg agtgaaaatt tagtggtcct gcgcaatttt tatcaatcgg ttacgaaagc 3301 cgctatcgcg atggataatg atcatctgga tattgatgtt gatattactg aaccgtcatt 3361 tgaacattta actattgcga cagtcaatga acgccgaatg agaattgaga ttgaaaatca 3421 agcaatttct ctgtcttaaa atctattgag atattctatc actcaaatag caatataagg 3481 actctctatg aaatttggaa actttttgct tacataccaa cctccccaat tttctcaaac 3541 agaggtaatg aaacgtttgg ttaaattagg tcgcatctct gaggagtgtg gttttgatac 3601 cgtatggtta ctggagcatc atttcacgga gtttggtttg cttggtaacc cttatgtcgc 3661 tgctgcatat ttacttggcg cgactaaaaa attgaatgta ggaactgccg ctattgttct 3721 tcccacagcc catccagtac gccaacttga agatgtgaat ttattggatc aaatgtcaaa 3781 aggacgattt cggtttggta tttgccgagg gctttacaac aaggactttc gcgtattcgg 3841 cacagatatg aataacagtc gcgccttagc ggaatgctgg tacgggctga taaagaatgg 3901 catgacagag ggatatatgg aagctgataa tgaacatatc aagttccata aggtaaaagt 3961 aaaccccgcg gcgtatagca gaggtggcgc accggtttat gtggtggctg aatcagcttc 4021 gacgactgag tgggctgctc aatttggcct accgatgata ttaagttgga ttataaatac 4081 taacgaaaag aaagcacaac ttgagcttta taatgaagtg gctcaagaat atgggcacga 4141 tattcataat atcgaccatt gcttatcata tataacatct gtagatcatg actcaattaa 4201 agcgaaagag atttgccgga aatttctggg gcattggtat gattcttatg tgaatgctac 4261 gactattttt gatgattcag accaaacaag aggttatgat ttcaataaag ggcagtggcg 4321 tgactttgta ttaaaaggac ataaagatac taatcgccgt attgattaca gttacgaaat 4381 caatcccgtg ggaacgccgc aggaatgtat tgacataatt caaaaagaca ttgatgctac 4441 aggaatatca aatatttgtt gtggatttga agctaatgga acagtagacg aaattattgc 4501 ttccatgaag ctcttccagt ctgatgtcat gccatttctt aaagaaaaac aacgttcgct 4561 attatattag ctaaggagaa agaaatgaaa tttggattgt tcttccttaa cttcatcaat 4621 tcaacaactg ttcaagaaca aagtatagtt cgcatgcagg aaataacgga gtatgttgat 4681 aagttgaatt ttgaacagat tttagtgtat gaaaatcatt tttcagataa tggtgttgtc 4741 ggcgctcctc tgactgtttc tggttttctg ctcggtttaa cagagaaaat taaaattggt 4801 tcattaaatc acatcattac aactcatcat cctgtccgca tagcggagga agcttgctta 4861 ttggatcagt taagtgaagg gagatttatt ttagggttta gtgattgcga aaaaaaagat 4921 gaaatgcatt tttttaatcg cccggttgaa tatcaacagc aactatttga agagtgttat 4981 gaaatcatta acgatgcttt aacaacaggc tattgtaatc cagataacga tttttatagc 5041 ttccctaaaa tatctgtaaa tccccatgct tatacgccag gcggacctcg gaaatatgta 5101 acagcaacca gtcatcatat tgttgagtgg gcggccaaaa aaggtattcc tctcatcttt 5161 aagtgggatg attctaatga tgttagatat gaatatgctg aaagatataa agccgttgcg 5221 gataaatatg acgttgacct atcagagata gaccatcagt taatgatatt agttaactat 5281 aacgaagata gtaataaagc taaacaagag acgcgtgcat ttattagtga ttatgttctt 5341 gaaatgcacc ctaatgaaaa tttcgaaaat aaacttgaag aaataattgc agaaaacgct 5401 gtcggaaatt atacggagtg tataactgcg gctaagttgg caattgaaaa gtgtggtgcg 5461 aaaagtgtat tgctgtcctt tgaaccaatg aatgatttga tgagccaaaa aaatgtaatc 5521 aatattgttg atgataatat taagaagtac cacatggaat atacctaata gatttcgagt 5581 tgcagcgagg cggcaagtga acgaatcccc aggagcatag ataactatgt gactggggtg 5641 agtgaaagca gccaacaaag cagcagcttg aaagatgaag ggtataaaag agtatgacag 5701 cagtgctgcc atactttcta atattatctt gaggagtaaa acaggtatga cttcatatgt 5761 tgataaacaa gaaattacag caagctcaga aattgatgat ttgatttttt cgagcgatcc 5821 attagtgtgg tcttacgacg agcaggaaaa aatcagaaag aaacttgtgc ttgatgcatt 5881 tcgtaatcat tataaacatt gtcgagaata tcgtcactac tgtcaggcac acaaagtaga 5941 tgacaatatt acggaaattg atgacatacc tgtattccca acatcggttt ttaagtttac 6001 tcgcttatta acttctcagg aaaacgagat tgaaagttgg tttaccagta gcggcacgaa 6061 tggtttaaaa agtcaggtgg cgcgtgacag attaagtatt gagagactct taggctctgt 6121 gagttatggc atgaaatatg ttggtagttg gtttgatcat caaatagaat tagtcaattt 6181 gggaccagat agatttaatg ctcataatat ttggtttaaa tatgttatga gtttggtgga 6241 attgttatat cctacgacat ttaccgtaac agaagaacga atagattttg ttaaaacatt 6301 gaatagtctt gaacgaataa aaaatcaagg gaaagatctt tgtcttattg gttcgccata 6361 ctttatttat ttactctgcc attatatgaa agataaaaaa atctcatttt ctggagataa 6421 aagcctttat atcataaccg gaggcggctg gaaaagttac gaaaaagaat ctctgaaacg 6481 tgatgatttc aatcatcttt tatttgatac tttcaatctc agtgatatta gtcagatccg 6541 agatatattt aatcaagttg aactcaacac ttgtttcttt gaggatgaaa tgcagcgtaa 6601 acatgttccg ccgtgggtat atgcgcgagc gcttgatcct gaaacgttga aacctgtacc 6661 tgatggaacg ccggggttga tgagttatat ggatgcgtca gcaaccagtt atccagcatt 6721 tattgttacc gatgatgtcg ggataattag cagagaatat ggtaagtatc ccggcgtgct 6781 cgttgaaatt ttacgtcgcg tcaatacgag gacgcagaaa gggtgtgctt taagcttaac 6841 cgaagcgttt gatagttgca ggcatgagct tgacggccgc gggccccatg gatcgatagc 6901 tggtcgacac aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt 6961 cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaataatga 7021 gctagcccgc ctaatgagcg ggcttttttt tctcggccta ggagatactt aacagggaag 7081 tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga 7141 aatctgacgc tcaaatcagt ggcaaagccc gaaaggaagc tgagttggct gctgccaccg 7201 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc 7261 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag 7321 gctgcaggaa ttcgatatca agcttatcga taccgtcgac ctcgaggggg ggcccggtac 7381 cctccacgtg ttgccccagc aatcagcgcg accttgcccc tccaacgtca tctcgttctc 7441 cgctcatgag ctcagccaat cgactggcga gcggcatcgc attcttcgca tcccgcctct 7501 ggcggatgca ggaagatcaa cggatctcgg cccagttgac ccagggctgt cgccacaatg 7561 tcgcgggagc ggatcaaccg agcaaaggca tgaccgactg gaccttcctt ctgaaggctc 7621 ttctccttga gccacctgtc cgccaaggca aagcgctcac agcagtggtc attctcgaga 7681 taatcgacgc gtaccaactt gccatcctga agaatggtgc agtgtctcgg caccccatag 7741 ggaacctttg ccatcaactc ggcaagatgc agcgtcgtgt tggcatcgtg tcccacgccg 7801 aggagaagta cctgcccatc gagttcatgg acacgggcga ccgggcttgc aggcgagtga 7861 ggtggcaggg gcaatggatc agagatgatc tgctctgcct gtggccccgc tgccgcaaag 7921 gcaaatggat gggcgctgcg ctttacattt ggcaggcgcc agaatgtgtc agagacaact 7981 ccaaggtccg gtgtaacggg cgacgtggca ggatcgaacg gctcgtcgtc cagacctgac 8041 cacgagggca tgacgagcgt ccctcccgga cccagcgcag cacgcagggc ctcgatcagt 8101 ccaagtggcc catcttcgag gggccggacg ctacggaagg agctgtggac cagcagcaca 8161 ccgccggggg taaccccaag gttgagaagc tgaccgatga gctcggcttt tcgccattcg 8221 tattgcattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt 8281 agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgaat aacttcgtat 8341 aatgtgtact atacgaagtt attcaacgtc tcattttcgc caaaagttgg cccagggctt 8401 cccggtatca acagggacac caggatttat ttattctgcg aagtgatctt ccgtcacagg 8461 tatttattcg cgataagctc atggagcggc gtaaccgtcg cacaggaagg acagagaaag 8521 cgcggatctg ggaagtgacg gacagaacgg tcaggacctg gagcggccgc ttttttcc //