LOCUS Exported 9404 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Tn7 integrating plasmid with S. pasteurianus dCas9 expressed from the Plac promoter for expression in P. aeruginosa. ACCESSION . VERSION . KEYWORDS pUC18-mini-Tn7T-Plac-dCas9 (Plac) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9404) AUTHORS Tan SZ, Reisch CR, Prather KLJ TITLE A Robust CRISPR Interference Gene Repression System in Pseudomonas. JOURNAL J Bacteriol. 2018 Mar 12;200(7). pii: JB.00575-17. doi: 10.1128/JB.00575-17. Print 2018 Apr 1. PUBMED 29311279 REFERENCE 2 (bases 1 to 9404) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9404 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 100..119 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 533..549 /label=pBluescriptKS /note="For pBluescript vector" primer_bind 534..550 /label=KS primer /note="common sequencing primer, one of multiple similar variants" terminator 578..672 /label=lambda t0 terminator /note="transcription terminator from phage lambda" terminator 775..861 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" protein_bind 907..954 /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)." CDS complement(1084..1617) /codon_start=1 /gene="aacC1" /product="gentamycin acetyltransferase" /label=GmR /note="confers resistance to gentamycin" /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR EEVMHFDIDPSTAT" promoter complement(1806..1834) /gene="intI1 (promoter lies within the coding sequence)" /label=Pc promoter /note="class 1 integron promoter" protein_bind 1875..1922 /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)." CDS complement(1965..3047) /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 3009..3028 /label=LacI-R /note="LacI, reverse primer" promoter complement(3048..3125) /gene="lacI (mutant)" /label=lacIq promoter /note="In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold." promoter 3355..3385 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 3393..3409 /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)." primer_bind 3398..3420 /label=M13/pUC Reverse /note="In lacZ gene" CDS 3461..3487 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" mobile_element 7099..7264 /mobile_element_type="transposon:Tn7" /note="mini-Tn7 element (left end of the Tn7 transposon)" rep_origin complement(7534..8122) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(7614..7633) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(8293..9153) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 8916..8935 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(9154..9258) /gene="bla" /label=AmpR promoter primer_bind 9326..9344 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(9382..9404) /label=pGEX 3' /note="pGEX vectors, reverse primer" ORIGIN 1 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 61 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac 121 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 181 tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct 241 cttcgctatt acgccagcta gaggaccagc cgcgtaacct ggcaaaatcg gttacggttg 301 agtaataaat ggatgccctg cgtaagcggg tgtgggcgga caataaagtc ttaaactgaa 361 caaaatagat ctaaactatg acaataaagt cttaaactag acagaatagt tgtaaactga 421 aatcagtcca gttatgctgt gaaaaagcat actggacttt tgttatggct aaagcaaact 481 cttcattttc tgaagtgcaa attgcccgtc gtattaaaga ggggcgtggg gttcgaggtc 541 gacggtatcg ataagctagc ttaattagct gagcttggac tcctgttgat agatccagta 601 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt 661 attggtgaga atccaagcta gactgcgatg agtggcaggg cggggcgtaa tttttttaag 721 gcagttattg gtgcccttaa acgcctgggg taatgactct ctagcttgag gcatcaaata 781 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac 841 gctctcctga gtaggacaaa tccgccgcta ggagcttgcg gcccggacga tcgaattggg 901 gatcttgaag tacctattcc gaagttccta ttctctagaa agtataggaa cttcagagcg 961 cttttgaagc tgatgtgctt aaaaacttac tcaatggaat tctagatcga attggccgcg 1021 gcgttgtgac aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa 1081 ttgttaggtg gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa 1141 ctttgtatag agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata 1201 agcaccaagc gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg 1261 cctccggtgc tcgccggaga ctgcgagatc atagatatag atctcactac gcggctgctc 1321 aaacttgggc agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc 1381 aagcgcgatg aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg 1441 ttgggagtag gtggctacgt ctccgaactc acgaccgaaa agatcaagag cagcccgcat 1501 ggatttgact tggtcagggc cgagcctaca tgtgcgaatg atgcccatac ttgagccacc 1561 taactttgtt ttagggcgac tgccctgctg cgtaacatcg ttgctgctgc gtaacatcgt 1621 tgctgctcca taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc 1681 gaggcataga ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt 1741 taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc 1801 attacagttt acgaaccgaa caggcttatg tcaattcgat ccattgctgt tgacaaaggg 1861 aatcagggga tcttgaagtt cctattccga agttcctatt ctctagaaag tataggaact 1921 tcagagcgct tttgaagcta attcgatcat gcattgcgtt gcgctcactg cccgctttcc 1981 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 2041 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga 2101 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 2161 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg 2221 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg 2281 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt aggaacgatg 2341 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc 2401 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc 2461 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat 2521 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg 2581 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc 2641 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc 2701 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac 2761 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac 2821 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc 2881 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt 2941 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa 3001 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg 3061 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg 3121 gtgtcaacgt aaatgccgct tcgccttcgc gcgcgaattg caagctgatc cgggcttatc 3181 gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct 3241 gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata 3301 atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgtttaca 3361 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 3421 aaacagaatt cgagctcctg actcaggagg tatcgatatg tacccatacg atgttccaga 3481 ttacgctatg accaacggca agatcctggg cctggccatc ggcatcgcct cggtgggcgt 3541 gggcatcatc gaggccaaga ccggcaaggt ggtgcacgcc aactcgcgcc tgttctcggc 3601 cgccaacgcc gagaacaacg ccgagcgccg cggcttccgc ggctcgcgcc gcctgaaccg 3661 ccgcaagaag caccgcgtga agcgcgtgcg cgacctgttc gagaagtacg gcatcgtgac 3721 cgacttccgc aacctgaacc tgaacccgta cgagctgcgc gtgaagggcc tgaccgagca 3781 gctgaagaac gaggagctgt tcgccgccct gcgcaccatc tcgaagcgcc gcggcatctc 3841 gtacctggac gacgccgagg acgactcgac cggctcgacc gactacgcca agtcgatcga 3901 cgagaaccgc cgcctgctga agaacaagac cccgggccag atccagctgg agcgcctgga 3961 gaagtacggc cagctgcgcg gcaacttcac cgtgtacgac gagaacggcg aggcccaccg 4021 cctgatcaac gtgttctcga cctcggacta cgagaaggag gcccgcaaga tcctggagac 4081 ccaggccgac tacaacaaga agatcaccgc cgagttcatc gacgactacg tggagatcct 4141 gacccagaag cgcaagtact accacggccc gggcaacgag aagtcgcgca ccgactacgg 4201 ccgcttccgc accgacggca ccaccctgga gaacatcttc ggcatcctga tcggcaagtg 4261 caacttctac ccggacgagt accgcgcctc gaaggcctcg tacaccgccc aggagtacaa 4321 cttcctgaac gacctgaaca acctgaaggt gtcgaccgag accggcaagc tgtcgaccga 4381 gcagaaggag tcgctggtgg agttcgccaa gaacaccgcc accctgggcc cggccaagct 4441 gctgaaggag atcgccaaga tcctggactg caaggtggac gagatcaagg gctaccgcga 4501 ggacgacaag ggcaagccgg acctgcacac cttcgagccg taccgcaagc tgaagttcaa 4561 cctggagtcg atcaacatcg acgacctgtc gcgcgaggtg atcgacaagc tggccgacat 4621 cctgaccctg aacaccgagc gcgagggcat cgaggacgcc atcaagcgca acctgccgaa 4681 ccagttcacc gaggagcaga tctcggagat catcaaggtg cgcaagtcgc agtcgaccgc 4741 cttcaacaag ggctggcact cgttctcggc caagctgatg aacgagctga tcccggagct 4801 gtacgccacc tcggacgagc agatgaccat cctgacccgc ctggagaagt tcaaggtgaa 4861 caagaagtcg tcgaagaaca ccaagaccat cgacgagaag gaggtgaccg acgagatcta 4921 caacccggtg gtggccaagt cggtgcgcca gaccatcaag atcatcaacg ccgccgtgaa 4981 gaagtacggc gacttcgaca agatcgtgat cgagatgccg cgcgacaaga acgccgacga 5041 cgagaagaag ttcatcgaca agcgcaacaa ggagaacaag aaggagaagg acgacgccct 5101 gaagcgcgcc gcctacctgt acaactcgtc ggacaagctg ccggacgagg tgttccacgg 5161 caacaagcag ctggagacca agatccgcct gtggtaccag cagggcgagc gctgcctgta 5221 ctcgggcaag ccgatctcga tccaggagct ggtgcacaac tcgaacaact tcgagatcga 5281 cgccatcctg ccgctgtcgc tgtcgttcga cgactcgctg gccaacaagg tgctggtgta 5341 cgcctggacc aaccaggaga agggccaaaa gaccccgtac caggtgatcg actcgatgga 5401 cgccgcctgg tcgttccgcg agatgaagga ctacgtgctg aagcagaagg gcctgggcaa 5461 gaagaagcgc gactacctgc tgaccaccga gaacatcgac aagatcgagg tgaagaagaa 5521 gttcatcgag cgcaacctgg tggacacccg ctacgcctcg cgcgtggtgc tgaactcgct 5581 gcagtcggcc ctgcgcgagc tgggcaagga caccaaggtg tcggtggtgc gcggccagtt 5641 cacctcgcag ctgcgccgca agtggaagat cgacaagtcg cgcgagacct accaccacca 5701 cgccgtggac gccctgatca tcgccgcctc gtcgcagctg aagctgtggg agaagcagga 5761 caacccgatg ttcgtggact acggcaagaa ccaggtggtg gacaagcaga ccggcgagat 5821 cctgtcggtg tcggacgacg agtacaagga gctggtgttc cagccgccgt accagggctt 5881 cgtgaacacc atctcgtcga agggcttcga ggacgagatc ctgttctcgt accaggtgga 5941 ctcgaagtac aaccgcaagg tgtcggacgc caccatctac tcgacccgca aggccaagat 6001 cggcaaggac aagaaggagg agacctacgt gctgggcaag atcaaggaca tctactcgca 6061 gaacggcttc gacaccttca tcaagaagta caacaaggac aagacccagt tcctgatgta 6121 ccagaaggac tcgctgacct gggagaacgt gatcgaggtg atcctgcgcg actacccgac 6181 caccaagaag tcggaggacg gcaagaacga cgtgaagtgc aacccgttcg aggagtaccg 6241 ccgcgagaac ggcctgatct gcaagtactc gaagaagggc aagggcaccc cgatcaagtc 6301 gctgaagtac tacgacaaga agctgggcaa ctgcatcgac atcaccccgg aggagtcgcg 6361 caacaaggtg atcctgcagt cgatcaaccc gtggcgcgcc gacgtgtact tcaacccgga 6421 gaccctgaag tacgagctga tgggcctgaa gtactcggac ctgtcgttcg agaagggcac 6481 cggcaactac cacatctcgc aggagaagta cgacgccatc aaggagaagg agggcatcgg 6541 caagaagtcg gagttcaagt tcaccctgta ccgcaacgac ctgatcctga tcaaggacat 6601 cgcctcgggc gagcaggaga tctaccgctt cctgtcgcgc accatgccga acgtgaacca 6661 ctacgtggag ctgaagccgt acgacaagga gaagttcgac aacgtgcagg agctggtgga 6721 ggccctgggc gaggccgaca aggtgggccg ctgcatcaag ggcctgaaca agccgaacat 6781 ctcgatctac aaggtgcgca ccgacgtgct gggcaacaag tacttcgtga agaagaaggg 6841 cgacaagccg aagctggact tcaagaacaa caagaagtaa atactctggc catttttctt 6901 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcaacgcgtg 6961 cggccgctga ttagctaagc agaaggccat cctgacggat gcctttttcg cttgctagct 7021 gcagcccggg ggatcccccg ggctgcagga attcctcgag aagcttgggc ccggtacctc 7081 gcgaaggcct tgcaggccaa ccagataagt gaaatctagt tccaaactat tttgtcattt 7141 ttaattttcg tattagctta cgacgctaca cccagttccc atctattttg tcactcttcc 7201 ctaaataatc cttaaaaact ccatttccac ccctcccagt tcccaactat tttgtccgcc 7261 cacagcgggg catttttctt cctgttatgt ttgggcgctg cattaatgaa tcggccaacg 7321 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgacccgct 7381 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 7441 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 7501 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 7561 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 7621 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 7681 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 7741 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 7801 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 7861 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 7921 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 7981 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 8041 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 8101 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 8161 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 8221 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 8281 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 8341 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 8401 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 8461 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 8521 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 8581 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 8641 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 8701 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 8761 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 8821 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 8881 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 8941 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 9001 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 9061 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 9121 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 9181 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 9241 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 9301 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg 9361 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccgg //