LOCUS       sequence-392326-        7467 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses the catalytically inactive version of ThermoCas9 
            (Thermo(d)Cas9) and its sgRNA module.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7467)
  AUTHORS   Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, 
            Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der 
            Oost J
  TITLE     Characterizing a thermostable Cas9 for bacterial genome editing and 
            silencing.
  JOURNAL   Nat Commun. 2017 Nov 21;8(1):1647. doi: 10.1038/s41467-017-01591-4.
  PUBMED    29162801
REFERENCE   2  (bases 1 to 7467)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2017-11-21"; volume: "8(1):1647. doi"; pages: " 
            10.1038/s41467-017-01591-4"
COMMENT     Sequence Label: pThermoCas9i_ctrl
FEATURES             Location/Qualifiers
     source          1..7467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(72..88)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(72..88)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(85..107)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    96..112
                     /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        complement(join(120..126,127..144,145..150))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    165..186
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1073..2077)
                     /codon_start=1
                     /gene="repB"
                     /product="RepB replication protein"
                     /label=repB
                     /note="from Enterococcus faecalis plasmid pAM-alpha-1"
                     /db_xref="GI:22652809"
                     /protein_id="AAN03827.1"
                     /translation="MGVSFNIMCPNSSIYSDEKSRVLVDKTKSGKVRPWREKKIANVDY
                     FELLHILEFKKAERVKDCAEILEYKQNRETGERKLYRVWFCKSRLCPMCNWRRAMKHGI
                     QSQKVVAEVIKQKPTVRWLFLTLTVKNVYDGEELNKSLSDMAQGFRRMMQYKKINKNLV
                     GFMRATEVTINNKDNSYNQHMHVLVCVEPTYFKNTENYVNQKQWIQFWKKAMKLDYDPN
                     VKVQMIRPKNKYKSDIQSAIDETAKYPVKDTDFMTDDEEKNLKRLSDLEEGLHRKRLIS
                     YGGLLKEIHKKLNLDDTEEGDLIHTDDDEKADEDGFSIIAMWNWERKNYFIKE"
     primer_bind     complement(2598..2615)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2769..3357)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2849..2868)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggcggaaaa cgtcaaggtc ttttttacta taaaactatt gtgcatggga gggaagcttg
       61 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      121 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
      181 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagccc
      241 ttcaaacttc ccaaaggcga gccctagtga cattagaaaa ccgactgtaa aaagtacagt
      301 cggcattatc tcatattata aaagccagtc attaggccta tctgacaatt cctgaataga
      361 gttcataaac aatcctgcat gataaccatc acaaacagaa tgatgtacct gtaaagatag
      421 cggtaaatat attgaattac ctttattaat gaattttcct gctgtaataa tgggtagaag
      481 gtaattacta ttattattga tatttaagtt aaacccagta aatgaagtcc atggaataat
      541 agaaagagaa aaagcatttt caggtatagg tgttttggga aacaatttcc ccgaaccatt
      601 atatttctct acatcagaaa ggtataaatc ataaaactct ttgaagtcat tctttacagg
      661 agtccaaata ccagagaatg ttttagatac accatcaaaa attgtataaa gtggctctaa
      721 cttatcccaa taacctaact ctccgtcgct attgtaacca gttctaaaag ctgtatttga
      781 gtttatcacc cttgtcacta agaaaataaa tgcagggtaa aatttatatc cttcttgttt
      841 tatgtttcgg tataaaacac taatatcaat ttctgtggtt atactaaaag tcgtttgttg
      901 gttcaaataa tgattaaata tctcttttct cttccaattg tctaaatcaa ttttattaaa
      961 gttcatttga tatgcctcct aaatttttat ctaaagtgaa tttaggaggc ttacttgtct
     1021 gctttcttca ttagaatcaa tcctttttta aaagtcaatc ccgtttgttg aactactctt
     1081 taataaaata atttttccgt tcccaattcc acattgcaat aatagaaaat ccatcttcat
     1141 cggctttttc gtcatcatct gtatgaatca aatcgccttc ttctgtgtca tcaaggttta
     1201 attttttatg tatttctttt aacaaaccac cataggagat taacctttta cggtgtaaac
     1261 cttcctccaa atcagacaaa cgtttcaaat tcttttcttc atcatcggtc ataaaatccg
     1321 tatcctttac aggatatttt gcagtttcgt caattgccga ttgtatatcc gatttatatt
     1381 tatttttcgg tcgaatcatt tgaactttta catttggatc atagtctaat ttcattgcct
     1441 ttttccaaaa ttgaatccat tgtttttgat tcacgtagtt ttctgtattc ttaaaataag
     1501 ttggttccac acataccaat acatgcatgt gctgattata agaattatct ttattattta
     1561 ttgtcacttc cgttgcacgc ataaaaccaa caagattttt attaattttt ttatattgca
     1621 tcattcggcg aaatccttga gccatatctg acaaactctt atttaattct tcgccatcat
     1681 aaacattttt aactgttaat gtgagaaaca accaacgaac tgttggcttt tgtttaataa
     1741 cttcagcaac aaccttttgt gactgaatgc catgtttcat tgctctcctc cagttgcaca
     1801 ttggacaaag cctggattta caaaaccaca ctcgatacaa ctttctttcg cctgtttcac
     1861 gattttgttt atactctaat atttcagcac aatcttttac tctttcagcc tttttaaatt
     1921 caagaatatg cagaagttca aagtaatcaa cattagcgat tttcttttct ctccatggtc
     1981 tcacttttcc actttttgtc ttgtccacta aaacccttga tttttcatct gaataaatgc
     2041 tactattagg acacataata ttaaaagaaa cccccatcta tttagttatt tgtttggtca
     2101 cttataactt taacagatgg ggtttttctg tgcaaccaat tttaagggtt ttcaatactt
     2161 taaaacacat acataccaac acttcaacgc acctttcagc aactaaaata aaaatgacgt
     2221 tatttctata tgtatcaaga atagaaagaa ctcgtttttc gctacgctca aaacgcaaaa
     2281 aaagcactca ttcgagtgct ttttcttatc gctccaaatc atgcgatttt ttcctctttg
     2341 cttttctttg ctcacgaagt tctcgatcac gctgcaaaac atcttgaagc gaaaaagtat
     2401 tcttcttttc ttccgatcgc tcatgctgac gcacgaaaag ccctctaggc gcataggaac
     2461 aactcctaaa tgcatgtgag gggttttctc gtccatgtga acagtcgcat acgcaatatt
     2521 ttgtttccca tactgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2581 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     2641 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     2701 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     2761 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     2821 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     2881 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     2941 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3001 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3061 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3121 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3181 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     3241 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3301 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3361 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3421 attttggtca tgagaacatc ctctttctta gtttgtatag tcaataaacg aactaaattt
     3481 tcatttttat ctttttctag acatttttat tgtatcgtat tttttattca aaaaaaatat
     3541 tgaattttaa aaatgatggt gcagtatgaa ggtgtactta gtttattggt taaacaaagt
     3601 acgcatcaat atattttttt tgatgcgatt tgaattcgct ttcattatct aaaacattca
     3661 atcgaggagg taacttaaat gaagtataaa atcggtcttg ctatcggcat tacgtctatc
     3721 ggttgggctg tcattaattt ggacattcct cgcatcgaag atttaggtgt ccgcattttt
     3781 gacagagcgg aaaacccgaa aaccggggag tcactagctc ttccacgtcg cctcgcccgc
     3841 tccgcccgac gtcgtctgcg gcgtcgcaaa catcgactgg agcgcattcg ccgcctgttc
     3901 gtccgcgaag gaattttaac gaaggaagag ctgaacaagc tgtttgaaaa aaagcacgaa
     3961 atcgacgtct ggcagcttcg tgttgaagca ctggatcgaa aactaaataa cgatgaatta
     4021 gcccgcatcc ttcttcatct ggctaaacgg cgtggattta gatccaaccg caagagtgag
     4081 cgcaccaaca aagaaaacag tacgatgctc aaacatattg aagaaaacca atccattctt
     4141 tcaagttacc gaacggttgc agaaatggtt gtcaaggatc cgaaattttc cctgcacaag
     4201 cgtaataaag aggataatta caccaacact gttgcccgcg acgatcttga acgggaaatc
     4261 aaactgattt tcgccaaaca gcgcgaatat gggaacatcg tttgcacaga agcatttgaa
     4321 cacgagtata tttccatttg ggcatcgcaa cgcccttttg cttctaagga tgatatcgag
     4381 aaaaaagtcg gtttctgtac gtttgagcct aaagaaaaac gcgcgccaaa agcaacatac
     4441 acattccagt ccttcaccgt ctgggaacat attaacaaac ttcgtcttgt ctccccggga
     4501 ggcatccggg cactaaccga tgatgaacgt cgtcttatat acaagcaagc atttcataaa
     4561 aataaaatca ccttccatga tgttcgaaca ttgcttaact tgcctgacga cacccgtttt
     4621 aaaggtcttt tatatgaccg aaacaccacg ctgaaggaaa atgagaaagt tcgcttcctt
     4681 gaactcggcg cctatcataa aatacggaaa gcgatcgaca gcgtctatgg caaaggagca
     4741 gcaaaatcat ttcgtccgat tgattttgat acatttggct acgcattaac gatgtttaaa
     4801 gacgacaccg acattcgcag ttacttgcga aacgaatacg aacaaaatgg aaaacgaatg
     4861 gaaaatctag cggataaagt ctatgatgaa gaattgattg aagaactttt aaacttatcg
     4921 ttttctaagt ttggtcatct atcccttaaa gcgcttcgca acatccttcc atatatggaa
     4981 caaggcgaag tctactcaac cgcttgtgaa cgagcaggat atacatttac agggccaaag
     5041 aaaaaacaga aaacggtatt gctgccgaac attccgccga tcgccaatcc ggtcgtcatg
     5101 cgcgcactga cacaggcacg caaagtggtc aatgccatta tcaaaaagta cggctcaccg
     5161 gtctccatcc atatcgaact ggcccgggaa ctatcacaat cctttgatga acgacgtaaa
     5221 atgcagaaag aacaggaagg aaaccgaaag aaaaacgaaa ctgccattcg ccaacttgtt
     5281 gaatatgggc tgacgctcaa tccaactggg cttgacattg tgaaattcaa actatggagc
     5341 gaacaaaacg gaaaatgtgc ctattcactc caaccgatcg aaatcgagcg gttgctcgaa
     5401 ccaggctata cagaagtcga cgctgtgatt ccatacagcc gaagcttgga cgatagctat
     5461 accaataaag ttcttgtgtt gacaaaggag aaccgtgaaa aaggaaaccg caccccagct
     5521 gaatatttag gattaggctc agaacgttgg caacagttcg agacgtttgt cttgacaaat
     5581 aagcagtttt cgaaaaagaa gcgggatcga ctccttcggc ttcattacga tgaaaacgaa
     5641 gaaaatgagt ttaaaaatcg taatctaaat gatacccgtt atatctcacg cttcttggct
     5701 aactttattc gcgaacatct caaattcgcc gacagcgatg acaaacaaaa agtatacacg
     5761 gtcaacggcc gtattaccgc ccatttacgc agccgttgga attttaacaa aaaccgggaa
     5821 gaatcgaatt tgcatcatgc cgtcgatgct gccatcgtcg cctgcacaac gccgagcgat
     5881 atcgcccgag tcaccgcctt ctatcaacgg cgcgaacaaa acaaagaact gtccaaaaag
     5941 acggatccgc agtttccgca gccttggccg cactttgctg atgaactgca ggcgcgttta
     6001 tcaaaaaatc caaaggagag tataaaagct ctcaatcttg gaaattatga taacgagaaa
     6061 ctcgaatcgt tgcagccggt ttttgtctcc cgaatgccga agcggagcat aacaggagcg
     6121 gctcatcaag aaacattgcg gcgttatatc ggcatcgacg aacggagcgg aaaaatacag
     6181 acggtcgtca aaaagaaact atccgagatc caactggata aaacaggtca tttcccaatg
     6241 tacgggaaag aaagcgatcc aaggacatat gaagccattc gccaacggtt gcttgaacat
     6301 aacaatgacc caaaaaaggc gtttcaagag cctctgtata aaccgaagaa gaacggagaa
     6361 ctaggtccta tcatccgaac aatcaaaatc atcgatacga caaatcaagt tattccgctc
     6421 aacgatggca aaacagtcgc ctacaacagc aacatcgtgc gggtcgacgt ctttgagaaa
     6481 gatggcaaat attattgtgt ccctatctat acaatagata tgatgaaagg gatcttgcca
     6541 aacaaggcga tcgagccgaa caaaccgtac tctgagtgga aggaaatgac ggaggactat
     6601 acattccgat tcagtctata cccaaatgat cttatccgta tcgaatttcc ccgagaaaaa
     6661 acaataaaga ctgctgtggg ggaagaaatc aaaattaagg atctgttcgc ctattatcaa
     6721 accatcgact cctccaatgg agggttaagt ttggttagcc atgataacaa cttttcgctc
     6781 cgcagcatcg gttcaagaac cctcaaacga ttcgagaaat accaagtaga tgtgctaggc
     6841 aacatctaca aagtgagagg ggaaaagaga gttggggtgg cgtcatcttc tcattcgaaa
     6901 gccggggaaa ctatccgtcc gttataaatc agacaaaatg gcctgcttat gaagcgggcc
     6961 atttttgttt aatcctgtcg acttgccgga attctttcac agccgcatcc ctttcttccg
     7021 caggcagcgc cttccagccc gcccagtcga gagaacggaa atcatgcagt gagtaccagc
     7081 catctagtgt ttctgctgct tcgctcattt ttctccctcc aaatgttttt ctcttccctt
     7141 actataacac agtttagaag agggagaaaa tattacggct gccgcgaaat tgtctaaaag
     7201 aagccttctt ttgcctttga ttttccggaa aaatggatta ttcttaaaat ggataccgtt
     7261 atacggatcc catgacgcta gtatccagct gggtcatagt tcccctgaga ttatcgctgt
     7321 ggtataatga aagttatacc acagcaatga tctcagggtt actatgataa gggctttctg
     7381 cctataggca gactgacccg tggcgttggg gatcgcctat cgcccgcttt cttcgggcat
     7441 tccccactct taggcgaacg aagacct
//
