LOCUS       sequence-255017-        8445 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 encodes a non-targeting guide RNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8445)
  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 8445)
  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: pSL0762 (pQCascade_crRNA-nt)
FEATURES             Location/Qualifiers
     source          1..8445
                     /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        1703..1721
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1722..1746
                     /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(6544..6562)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6558..6605
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(6776..7567)
                     /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(7568..7659)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7707..8445)
                     /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 taacgttgtc tgacacttgt cacaaaccgc
     1561 taggaggtga actgccgagt aggtagctga taacggatcc gaattcgagc tcggcgcgcc
     1621 tgcaggtcga caagcttgcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg
     1681 tacacggccg cataatcgaa attaatacga ctcactatag gggaattgtg agcggataac
     1741 aattccccat cttagtatat tagttaagta taagaaggag atatacatat gtttttgcaa
     1801 agacctaaac cttacagcga tgaaagttta gaaagtttct ttatccgagt ggctaacaaa
     1861 aatggctacg gtgatgtcca tcgcttccta gaagccacta aacgattcct tcaagacatt
     1921 gaccataatg gctatcaaac ctttccgact gatataactc ggataaaccc atactcagct
     1981 aaaaacagtt ccagcgcacg aactgcgtca ttcctgaaac ttgcacaatt gacatttaat
     2041 gaaccgccag agctacttgg gttggcaatt aacagaacaa acatgaaata ctcgccgtca
     2101 actagcgcgg ttgttagagg tgcagaagtc tttcctcgca gtttactacg gacgcactcc
     2161 atcccctgct gtcctttgtg tctgcgagaa aatggctacg cctcctacct ttggcacttt
     2221 caggggtacg aatactgcca cagccataac gtacctttaa ttaccacttg tagctgtggt
     2281 aaggagtttg actaccgagt atctgggtta aagggcattt gctgcaaatg caaggagcct
     2341 atcaccttaa ccagcaggga gaacggtcat gaggcagcgt gtactgtttc aaactggctt
     2401 gctggccatg aatctaaacc tctgccaaat cttcctaaaa gctaccgatg gggtttagtt
     2461 cattggtgga tgggtattaa agatagcgag ttcgatcact tttcgttcgt tcaatttttc
     2521 tcaaactggc caaggtcatt ccactcgata atcgaagatg aagtagagtt caaccttgag
     2581 catgctgttg tcagcacgtc tgaattacga ctaaaagatc ttcttggtcg attgtttttc
     2641 ggttcaattc ggttacctga gcggaatctt caacacaata tcatccttgg tgagcttctc
     2701 tgctatttag aaaatcgttt atggcaagac aagggattaa tcgccaacct caaaatgaac
     2761 gcgttagagg cgactgtaat gttaaattgt agcctcgatc agattgcatc aatggttgaa
     2821 caacgcatct tgaagccaaa tcgaaaaagc aagcccaaca gccctcttga tgttaccgat
     2881 tatctatttc atttcggcga tattttctgt ctttggttag ctgagttcca aagcgatgag
     2941 tttaaccgtt cgttttatgt gtcgaggtgg taaatgcaaa ctctgaaaga actaatcgca
     3001 tccaatcctg acgacttaac aactgaactt aagagagcat ttcgtccact cacaccgcat
     3061 attgcaattg atggtaatga acttgacgca ctgacgatat tagtcaattt aaccgataag
     3121 actgatgatc agaaagacct gctcgatcga gccaaatgca agcaaaaact tcgagatgaa
     3181 aaatggtggg ctagctgcat aaattgcgtt aattacagac aaagccataa cccaaaattc
     3241 ccggatatac gttctgaagg cgtgattcga acccaagccc tgggtgaatt accgagtttc
     3301 ctactctcgt cctcaaaaat cccaccatac cattggtcat atagccatga ttcaaaatac
     3361 gtcaacaaaa gcgcattcct caccaatgag ttttgttggg atggtgagat ctcatgtttg
     3421 ggtgagcttc ttaaagatgc agatcaccca ctttggaata ctttgaaaaa gttaggttgt
     3481 tctcaaaaaa cttgcaaagc aatggcaaaa caactagctg atattactct cacgactatc
     3541 aatgtcacac tcgcaccaaa ttacctgact caaatatctc tccccgatag tgatacatcc
     3601 tacatttcac tctctcctgt tgcatcgcta tcgatgcaaa gccactttca tcagaggctc
     3661 caagatgaaa atcggcatag cgcgataacg cggtttagcc gaactaccaa catgggggtc
     3721 actgcgatga catgtggcgg cgcatttaga atgttaaagt ctggcgctaa gttttccagc
     3781 ccccctcatc accgattaaa cagtaaacga agttggttga cgtcagagca tgttcagtca
     3841 ttaaaacagt accagcgcct caacaaaagc ctcatacctg aaaactctcg gattgcactc
     3901 cgtagaaaat acaaaatcga gcttcaaaat atggtcagat cttggtttgc aatgcaagac
     3961 catacccttg attcgaacat acttatccaa catttgaatc atgacctatc ttacttagga
     4021 gccacaaaac gttttgcata cgatccagcg atgaccaagc tctttactga gcttttgaaa
     4081 cgagagttat caaattcaat caataatggt gagcaacaca ctaatggatc gtttttagtc
     4141 ctaccgaata tcagggtttg tggcgcaaca gctttaagct ccccggtaac ggtggggatt
     4201 ccatcactta cagctttctt tggcttcgtt cacgcatttg aacggaatat aaatcgcacc
     4261 acctcatcgt ttcgtgttga atcctttgcg atatgcgtcc atcaactaca tgtcgagaag
     4321 cgaggtttga cagcagagtt tgtggaaaaa ggcgacggga ctatatccgc tcccgcgacc
     4381 cgggatgact ggcagtgtga tgtcgtattt agccttattt tgaacaccaa ctttgctcaa
     4441 catattgacc aagatacgtt agttacatca ctaccaaagc gattggctcg gggttcagca
     4501 aaaattgcga ttgatgactt taaacatatc aactcattct cgacattaga aacagcgatc
     4561 gaatctctgc caatagaagc tggtaggtgg ttatcacttt acgcacagtc aaacaataat
     4621 ctaagtgatc tattagcagc catgacagag gaccatcagc tcatggcaag ctgcgtcggt
     4681 taccacttgt tagaagagcc caaagataaa ccaaactccc tcagaggtta caaacacgct
     4741 atcgccgagt gcatcattgg actcattaac tcaatcacct ttagctcaga gactgatccc
     4801 aacacaatct tttggtcgct aaagaactat caaaactacc tagtggtaca gccaaggagt
     4861 atcaacgatg aaactaccga caaatctagc ctatgagcgc tctatcgacc catcagatgt
     4921 ctgttttttt gtcgtctggc ccgatgatag aaaaacacct ttaacctaca attctcgtac
     4981 tctgctcggg caaatggaag cggcatcatt agcctatgat gtctcaggtc aaccaataaa
     5041 aagtgccacc gctgaggcgt tagctcaagg gaaccctcat caagttgatt tctgccacgt
     5101 tccatacggt gcgagtcata ttgaatgcag tttctccgtc tcgttttctt ctgaactacg
     5161 tcaaccatat aagtgtaact caagcaaagt taaacaaacg ctagtgcaat tagtcgagct
     5221 ctacgaaacg aaaatcggct ggactgagct agcaacccga tatttgatga acatttgcaa
     5281 cggtaaatgg ctgtggaaaa atacccgtaa agcctattgc tggaacattg tacttacacc
     5341 ttggccttgg aacggggaaa aggttggatt tgaagatatt cgtactaact acacctcacg
     5401 gcaagacttt aaaaataata aaaattggtc tgctatagtt gaaatgatca aaaccgcatt
     5461 ttctagtact gatgggctgg cgatatttga agtcagggcc accttgcact tgccaacgaa
     5521 tgctatggtg cggccaagcc aagttttcac agaaaaagaa agtggcagta aaagtaaatc
     5581 taaaactcaa aacagtcgag tttttcagag tacaactatt gatggtgaac gatcgccaat
     5641 actaggggcc tttaaaacgg gagcagctat tgcaaccatt gacgactggt atcctgaagc
     5701 cactgagcca ctaagggtcg gacggtttgg ggttcatcgc gaagatgtca cttgctaccg
     5761 tcatccgtct accggaaaag attttttctc gatattacaa caagcagagc actatattga
     5821 agtgttgagc gccaacaaaa ctcccgctca agaaactatc aacgacatgc actttttaat
     5881 ggctaacctg attaagggtg ggatgttcca gcataaagga gactgactgt gaaatggtat
     5941 tataagacaa tcacctttct gccagagttg tgcaacaacg agtcactggc tgcaaagtgt
     6001 ctccgcgttc tgcatggatt taactatcag tatgagacac gaaatatcgg cgtttcattt
     6061 ccgctttggt gtgatgcaac ggttggaaaa aagatttcat ttgtcagcaa gaacaagata
     6121 gaactcgact tactacttaa acaacactat ttcgtccaaa tggaacaact tcaatacttt
     6181 catatatcca acactgttct cgtcccagaa gattgtacat acgtttcctt tagacgctgt
     6241 caatctatag ataagctcac agcagcaggg ctggcaagga aaatcagacg cctggagaaa
     6301 cgtgctctat ctagaggcga gcaatttgac ccatcatctt ttgctcaaaa agagcatact
     6361 gcaatagcgc actaccactc acttggggag tccagcaaac agacgaaccg caactttcga
     6421 ctcaatatca ggatgctctc ggagcaaccc cgtgagggga actcgatttt tagtagctat
     6481 ggcttatcaa attcagaaaa ctcgtttcag cctgtaccct taatctgacc taggctgctg
     6541 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     6601 ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata
     6661 aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga
     6721 ccgggtcatc gtggccggat cttgcggccc ctcggcttga acgaattgtt agacattatt
     6781 tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg
     6841 cgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga
     6901 cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg
     6961 cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct
     7021 catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa
     7081 atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa
     7141 cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct
     7201 cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag
     7261 ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga
     7321 gaatctcgct ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc
     7381 gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct
     7441 tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat
     7501 ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt
     7561 ccctcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7621 gcggatacat atttgaatgt atttagaaaa ataaacaaat agctagctca ctcggtcgct
     7681 acgctccggg cgtgagactg cggcgggcgc tgcggacaca tacaaagtta cccacagatt
     7741 ccgtggataa gcaggggact aacatgtgag gcaaaacagc agggccgcgc cggtggcgtt
     7801 tttccatagg ctccgccctc ctgccagagt tcacataaac agacgctttt ccggtgcatc
     7861 tgtgggagcc gtgaggctca accatgaatc tgacagtacg ggcgaaaccc gacaggactt
     7921 aaagatcccc accgtttccg gcgggtcgct ccctcttgcg ctctcctgtt ccgaccctgc
     7981 cgtttaccgg atacctgttc cgcctttctc ccttacggga agtgtggcgc tttctcatag
     8041 ctcacacact ggtatctcgg ctcggtgtag gtcgttcgct ccaagctggg ctgtaagcaa
     8101 gaactccccg ttcagcccga ctgctgcgcc ttatccggta actgttcact tgagtccaac
     8161 ccggaaaagc acggtaaaac gccactggca gcagccattg gtaactggga gttcgcagag
     8221 gatttgttta gctaaacacg cggttgctct tgaagtgtgc gccaaagtcc ggctacactg
     8281 gaaggacaga tttggttgct gtgctctgcg aaagccagtt accacggtta agcagttccc
     8341 caactgactt aaccttcgat caaaccacct ccccaggtgg ttttttcgtt tacagggcaa
     8401 aagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatc
//
