LOCUS Exported 8428 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses V. cholerae TniQ, Cas8, Cas7, and Cas6 from one T7 promoter, and a CRISPR RNA from a second T7 promoter. The CRISPR array contains two BsaI sites for spacer cloning. . ACCESSION . VERSION . KEYWORDS pSL0284 (pQCascade_entry) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8428) AUTHORS Klompe SE, Vo PLH, Halpin-Healy TS, Sternberg SH TITLE Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. JOURNAL Nature. 2019 Jul;571(7764):219-225. doi: 10.1038/s41586-019-1323-z. Epub 2019 Jun 12. PUBMED 31189177 REFERENCE 2 (bases 1 to 8428) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8428 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(210..1292) /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 1254..1273 /label=LacI-R /note="LacI, reverse primer" promoter complement(1293..1370) /gene="lacI" /label=lacI promoter promoter 1416..1434 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1435..1459 /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)." promoter 1686..1704 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1705..1729 /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 complement(6527..6545) /label=T7 Term /note="T7 terminator, reverse primer" terminator 6541..6588 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(6759..7550) /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" promoter complement(7551..7642) /gene="bla" /label=AmpR promoter rep_origin complement(7690..8428) /direction=LEFT /label=CloDF13 ori /note="Plasmids containing the CloDF13 (CDF) origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins." ORIGIN 1 ttttctactg aaccgctcta gatttcagtg caatttatct cttcaaatgt agcacctgaa 61 gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga 121 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag 181 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 241 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 301 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc 361 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc 421 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc 481 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc 541 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt 601 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc 661 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca 721 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc 781 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca 841 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc 901 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc 961 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca 1021 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc 1081 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg 1141 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca 1201 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac 1261 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc 1321 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg 1381 acgctctccc ttatgcgact cctgcattag gaaattaata cgactcacta taggggaatt 1441 gtgagcggat aacaattccc ctgtagaaat aattttgttt aactttaata aggagatata 1501 ccatgggtga actgccgagt aggtagctga taacgagacc tctggtctcg tgaactgccg 1561 agtaggtagc tgataacgga tccgaattcg agctcggcgc gcctgcaggt cgacaagctt 1621 gcggccgcat aatgcttaag tcgaacagaa agtaatcgta ttgtacacgg ccgcataatc 1681 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc catcttagta 1741 tattagttaa gtataagaag gagatataca tatgtttttg caaagaccta aaccttacag 1801 cgatgaaagt ttagaaagtt tctttatccg agtggctaac aaaaatggct acggtgatgt 1861 ccatcgcttc ctagaagcca ctaaacgatt ccttcaagac attgaccata atggctatca 1921 aacctttccg actgatataa ctcggataaa cccatactca gctaaaaaca gttccagcgc 1981 acgaactgcg tcattcctga aacttgcaca attgacattt aatgaaccgc cagagctact 2041 tgggttggca attaacagaa caaacatgaa atactcgccg tcaactagcg cggttgttag 2101 aggtgcagaa gtctttcctc gcagtttact acggacgcac tccatcccct gctgtccttt 2161 gtgtctgcga gaaaatggct acgcctccta cctttggcac tttcaggggt acgaatactg 2221 ccacagccat aacgtacctt taattaccac ttgtagctgt ggtaaggagt ttgactaccg 2281 agtatctggg ttaaagggca tttgctgcaa atgcaaggag cctatcacct taaccagcag 2341 ggagaacggt catgaggcag cgtgtactgt ttcaaactgg cttgctggcc atgaatctaa 2401 acctctgcca aatcttccta aaagctaccg atggggttta gttcattggt ggatgggtat 2461 taaagatagc gagttcgatc acttttcgtt cgttcaattt ttctcaaact ggccaaggtc 2521 attccactcg ataatcgaag atgaagtaga gttcaacctt gagcatgctg ttgtcagcac 2581 gtctgaatta cgactaaaag atcttcttgg tcgattgttt ttcggttcaa ttcggttacc 2641 tgagcggaat cttcaacaca atatcatcct tggtgagctt ctctgctatt tagaaaatcg 2701 tttatggcaa gacaagggat taatcgccaa cctcaaaatg aacgcgttag aggcgactgt 2761 aatgttaaat tgtagcctcg atcagattgc atcaatggtt gaacaacgca tcttgaagcc 2821 aaatcgaaaa agcaagccca acagccctct tgatgttacc gattatctat ttcatttcgg 2881 cgatattttc tgtctttggt tagctgagtt ccaaagcgat gagtttaacc gttcgtttta 2941 tgtgtcgagg tggtaaatgc aaactctgaa agaactaatc gcatccaatc ctgacgactt 3001 aacaactgaa cttaagagag catttcgtcc actcacaccg catattgcaa ttgatggtaa 3061 tgaacttgac gcactgacga tattagtcaa tttaaccgat aagactgatg atcagaaaga 3121 cctgctcgat cgagccaaat gcaagcaaaa acttcgagat gaaaaatggt gggctagctg 3181 cataaattgc gttaattaca gacaaagcca taacccaaaa ttcccggata tacgttctga 3241 aggcgtgatt cgaacccaag ccctgggtga attaccgagt ttcctactct cgtcctcaaa 3301 aatcccacca taccattggt catatagcca tgattcaaaa tacgtcaaca aaagcgcatt 3361 cctcaccaat gagttttgtt gggatggtga gatctcatgt ttgggtgagc ttcttaaaga 3421 tgcagatcac ccactttgga atactttgaa aaagttaggt tgttctcaaa aaacttgcaa 3481 agcaatggca aaacaactag ctgatattac tctcacgact atcaatgtca cactcgcacc 3541 aaattacctg actcaaatat ctctccccga tagtgataca tcctacattt cactctctcc 3601 tgttgcatcg ctatcgatgc aaagccactt tcatcagagg ctccaagatg aaaatcggca 3661 tagcgcgata acgcggttta gccgaactac caacatgggg gtcactgcga tgacatgtgg 3721 cggcgcattt agaatgttaa agtctggcgc taagttttcc agcccccctc atcaccgatt 3781 aaacagtaaa cgaagttggt tgacgtcaga gcatgttcag tcattaaaac agtaccagcg 3841 cctcaacaaa agcctcatac ctgaaaactc tcggattgca ctccgtagaa aatacaaaat 3901 cgagcttcaa aatatggtca gatcttggtt tgcaatgcaa gaccataccc ttgattcgaa 3961 catacttatc caacatttga atcatgacct atcttactta ggagccacaa aacgttttgc 4021 atacgatcca gcgatgacca agctctttac tgagcttttg aaacgagagt tatcaaattc 4081 aatcaataat ggtgagcaac acactaatgg atcgttttta gtcctaccga atatcagggt 4141 ttgtggcgca acagctttaa gctccccggt aacggtgggg attccatcac ttacagcttt 4201 ctttggcttc gttcacgcat ttgaacggaa tataaatcgc accacctcat cgtttcgtgt 4261 tgaatccttt gcgatatgcg tccatcaact acatgtcgag aagcgaggtt tgacagcaga 4321 gtttgtggaa aaaggcgacg ggactatatc cgctcccgcg acccgggatg actggcagtg 4381 tgatgtcgta tttagcctta ttttgaacac caactttgct caacatattg accaagatac 4441 gttagttaca tcactaccaa agcgattggc tcggggttca gcaaaaattg cgattgatga 4501 ctttaaacat atcaactcat tctcgacatt agaaacagcg atcgaatctc tgccaataga 4561 agctggtagg tggttatcac tttacgcaca gtcaaacaat aatctaagtg atctattagc 4621 agccatgaca gaggaccatc agctcatggc aagctgcgtc ggttaccact tgttagaaga 4681 gcccaaagat aaaccaaact ccctcagagg ttacaaacac gctatcgccg agtgcatcat 4741 tggactcatt aactcaatca cctttagctc agagactgat cccaacacaa tcttttggtc 4801 gctaaagaac tatcaaaact acctagtggt acagccaagg agtatcaacg atgaaactac 4861 cgacaaatct agcctatgag cgctctatcg acccatcaga tgtctgtttt tttgtcgtct 4921 ggcccgatga tagaaaaaca cctttaacct acaattctcg tactctgctc gggcaaatgg 4981 aagcggcatc attagcctat gatgtctcag gtcaaccaat aaaaagtgcc accgctgagg 5041 cgttagctca agggaaccct catcaagttg atttctgcca cgttccatac ggtgcgagtc 5101 atattgaatg cagtttctcc gtctcgtttt cttctgaact acgtcaacca tataagtgta 5161 actcaagcaa agttaaacaa acgctagtgc aattagtcga gctctacgaa acgaaaatcg 5221 gctggactga gctagcaacc cgatatttga tgaacatttg caacggtaaa tggctgtgga 5281 aaaatacccg taaagcctat tgctggaaca ttgtacttac accttggcct tggaacgggg 5341 aaaaggttgg atttgaagat attcgtacta actacacctc acggcaagac tttaaaaata 5401 ataaaaattg gtctgctata gttgaaatga tcaaaaccgc attttctagt actgatgggc 5461 tggcgatatt tgaagtcagg gccaccttgc acttgccaac gaatgctatg gtgcggccaa 5521 gccaagtttt cacagaaaaa gaaagtggca gtaaaagtaa atctaaaact caaaacagtc 5581 gagtttttca gagtacaact attgatggtg aacgatcgcc aatactaggg gcctttaaaa 5641 cgggagcagc tattgcaacc attgacgact ggtatcctga agccactgag ccactaaggg 5701 tcggacggtt tggggttcat cgcgaagatg tcacttgcta ccgtcatccg tctaccggaa 5761 aagatttttt ctcgatatta caacaagcag agcactatat tgaagtgttg agcgccaaca 5821 aaactcccgc tcaagaaact atcaacgaca tgcacttttt aatggctaac ctgattaagg 5881 gtgggatgtt ccagcataaa ggagactgac tgtgaaatgg tattataaga caatcacctt 5941 tctgccagag ttgtgcaaca acgagtcact ggctgcaaag tgtctccgcg ttctgcatgg 6001 atttaactat cagtatgaga cacgaaatat cggcgtttca tttccgcttt ggtgtgatgc 6061 aacggttgga aaaaagattt catttgtcag caagaacaag atagaactcg acttactact 6121 taaacaacac tatttcgtcc aaatggaaca acttcaatac tttcatatat ccaacactgt 6181 tctcgtccca gaagattgta catacgtttc ctttagacgc tgtcaatcta tagataagct 6241 cacagcagca gggctggcaa ggaaaatcag acgcctggag aaacgtgctc tatctagagg 6301 cgagcaattt gacccatcat cttttgctca aaaagagcat actgcaatag cgcactacca 6361 ctcacttggg gagtccagca aacagacgaa ccgcaacttt cgactcaata tcaggatgct 6421 ctcggagcaa ccccgtgagg ggaactcgat ttttagtagc tatggcttat caaattcaga 6481 aaactcgttt cagcctgtac ccttaatctg acctaggctg ctgccaccgc tgagcaataa 6541 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaacctcag 6601 gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt aaaccagcaa 6661 tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc atcgtggccg 6721 gatcttgcgg cccctcggct tgaacgaatt gttagacatt atttgccgac taccttggtg 6781 atctcgcctt tcacgtagtg gacaaattct tccaactgat ctgcgcgcga ggccaagcga 6841 tcttcttctt gtccaagata agcctgtcta gcttcaagta tgacgggctg atactgggcc 6901 ggcaggcgct ccattgccca gtcggcagcg acatccttcg gcgcgatttt gccggttact 6961 gcgctgtacc aaatgcggga caacgtaagc actacatttc gctcatcgcc agcccagtcg 7021 ggcggcgagt tccatagcgt taaggtttca tttagcgcct caaatagatc ctgttcagga 7081 accggatcaa agagttcctc cgccgctgga cctaccaagg caacgctatg ttctcttgct 7141 tttgtcagca agatagccag atcaatgtcg atcgtggctg gctcgaagat acctgcaaga 7201 atgtcattgc gctgccattc tccaaattgc agttcgcgct tagctggata acgccacgga 7261 atgatgtcgt cgtgcacaac aatggtgact tctacagcgc ggagaatctc gctctctcca 7321 ggggaagccg aagtttccaa aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc 7381 cttacggtca ccgtaaccag caaatcaata tcactgtgtg gcttcaggcc gccatccact 7441 gcggagccgt acaaatgtac ggccagcaac gtcggttcga gatggcgctc gatgacgcca 7501 actacctctg atagttgagt cgatacttcg gcgatcaccg cttccctcat actcttcctt 7561 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 7621 tgtatttaga aaaataaaca aatagctagc tcactcggtc gctacgctcc gggcgtgaga 7681 ctgcggcggg cgctgcggac acatacaaag ttacccacag attccgtgga taagcagggg 7741 actaacatgt gaggcaaaac agcagggccg cgccggtggc gtttttccat aggctccgcc 7801 ctcctgccag agttcacata aacagacgct tttccggtgc atctgtggga gccgtgaggc 7861 tcaaccatga atctgacagt acgggcgaaa cccgacagga cttaaagatc cccaccgttt 7921 ccggcgggtc gctccctctt gcgctctcct gttccgaccc tgccgtttac cggatacctg 7981 ttccgccttt ctcccttacg ggaagtgtgg cgctttctca tagctcacac actggtatct 8041 cggctcggtg taggtcgttc gctccaagct gggctgtaag caagaactcc ccgttcagcc 8101 cgactgctgc gccttatccg gtaactgttc acttgagtcc aacccggaaa agcacggtaa 8161 aacgccactg gcagcagcca ttggtaactg ggagttcgca gaggatttgt ttagctaaac 8221 acgcggttgc tcttgaagtg tgcgccaaag tccggctaca ctggaaggac agatttggtt 8281 gctgtgctct gcgaaagcca gttaccacgg ttaagcagtt ccccaactga cttaaccttc 8341 gatcaaacca cctccccagg tggttttttc gtttacaggg caaaagatta cgcgcagaaa 8401 aaaaggatct caagaagatc ctttgatc //