LOCUS       sequence-255022-        8428 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses V. cholerae CAST 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    .
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 Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-019-1323-z"; journalName: "Nature"; date: "2019-07";
            volume: "571"; issue: "7764"; pages: "219-225"
COMMENT     Sequence Label: pSL0284 (pQCascade_entry)
FEATURES             Location/Qualifiers
     source          1..8428
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
//
