LOCUS       sequence-409757-        8171 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dThermoCas9-mediated silencing of genomic gene in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8171)
  TITLE     van der Oost E. coli CRISPR plasmids
REFERENCE   2  (bases 1 to 8171)
  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"
COMMENT     Sequence Label: pdTCas9_scr
FEATURES             Location/Qualifiers
     source          1..8171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(127..786)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     701..720
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(787..889)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1281..1298)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1415..1960)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1495..1514)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5338..5362
                     /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(5369..5374,5375..5391,5392..5397))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5560..5579)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5785..5862
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5863..6945
                     /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     complement(5882..5901)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        7006..7040
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
ORIGIN
        1 gaccctgccc tgaaccgacg accgggtcga atttgctttc gaatttctgc cattcatccg
       61 cttattatca cttattcagg cgtagcaacc aggcgtttaa gggcaccaat aactgcctta
      121 aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
      181 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
      241 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
      301 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
      361 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
      421 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
      481 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
      541 cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag
      601 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
      661 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
      721 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
      781 ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag
      841 tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt
      901 tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc
      961 tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc
     1021 caacttactg atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta
     1081 tcagctgtcc ctcctgttca gctactgacg gggtggtgcg taacggcaaa agcaccgccg
     1141 gacatcagcg ctagcggagt gtatactggc ttactatgtt ggcactgatg agggtgtcag
     1201 tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga
     1261 tacaggatat attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg
     1321 cgagcggaaa tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt
     1381 aacagggaag tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca
     1441 agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat
     1501 accaggcgtt tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta
     1561 ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc
     1621 gggtaggcag ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc
     1681 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca
     1741 ctggcagcag ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta
     1801 aggctaaact gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt
     1861 caaagagttg gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt
     1921 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca
     1981 gataaaatat ttgtcgacag gattaaacaa aaatggcccg cttcataagc aggccatttt
     2041 gtctgattta taacggacgg atagtttccc cggctttcga atgagaagat gacgccaccc
     2101 caactctctt ttcccctctc actttgtaga tgttgcctag cacatctact tggtatttct
     2161 cgaatcgttt gagggttctt gaaccgatgc tgcggagcga aaagttgtta tcatggctaa
     2221 ccaaacttaa ccctccattg gaggagtcga tggtttgata ataggcgaac agatccttaa
     2281 ttttgatttc ttcccccaca gcagtcttta ttgttttttc tcggggaaat tcgatacgga
     2341 taagatcatt tgggtataga ctgaatcgga atgtatagtc ctccgtcatt tccttccact
     2401 cagagtacgg tttgttcggc tcgatcgcct tgtttggcaa gatccctttc atcatatcta
     2461 ttgtatagat agggacacaa taatatttgc catctttctc aaagacgtcg acccgcacga
     2521 tgttgctgtt gtaggcgact gttttgccat cgttgagcgg aataacttga tttgtcgtat
     2581 cgatgatttt gattgttcgg atgataggac ctagttctcc gttcttcttc ggtttataca
     2641 gaggctcttg aaacgccttt tttgggtcat tgttatgttc aagcaaccgt tggcgaatgg
     2701 cttcatatgt ccttggatcg ctttctttcc cgtacattgg gaaatgacct gttttatcca
     2761 gttggatctc ggatagtttc tttttgacga ccgtctgtat ttttccgctc cgttcgtcga
     2821 tgccgatata acgccgcaat gtttcttgat gagccgctcc tgttatgctc cgcttcggca
     2881 ttcgggagac aaaaaccggc tgcaacgatt cgagtttctc gttatcataa tttccaagat
     2941 tgagagcttt tatactctcc tttggatttt ttgataaacg cgcctgcagt tcatcagcaa
     3001 agtgcggcca aggctgcgga aactgcggat ccgtcttttt ggacagttct ttgttttgtt
     3061 cgcgccgttg atagaaggcg gtgactcggg cgatatcgct cggcgttgtg caggcgacga
     3121 tggcagcatc gacggcatga tgcaaattcg attcttcccg gtttttgtta aaattccaac
     3181 ggctgcgtaa atgggcggta atacggccgt tgaccgtgta tactttttgt ttgtcatcgc
     3241 tgtcggcgaa tttgagatgt tcgcgaataa agttagccaa gaagcgtgag atataacggg
     3301 tatcatttag attacgattt ttaaactcat tttcttcgtt ttcatcgtaa tgaagccgaa
     3361 ggagtcgatc ccgcttcttt ttcgaaaact gcttatttgt caagacaaac gtctcgaact
     3421 gttgccaacg ttctgagcct aatcctaaat attcagctgg ggtgcggttt cctttttcac
     3481 ggttctcctt tgtcaacaca agaactttat tggtatagct atcgtccaag cttcggctgt
     3541 atggaatcac agcgtcgact tctgtatagc ctggttcgag caaccgctcg atttcgatcg
     3601 gttggagtga ataggcacat tttccgtttt gttcgctcca tagtttgaat ttcacaatgt
     3661 caagcccagt tggattgagc gtcagcccat attcaacaag ttggcgaatg gcagtttcgt
     3721 ttttctttcg gtttccttcc tgttctttct gcattttacg tcgttcatca aaggattgtg
     3781 atagttcccg ggccagttcg atatggatgg agaccggtga gccgtacttt ttgataatgg
     3841 cattgaccac tttgcgtgcc tgtgtcagtg cgcgcatgac gaccggattg gcgatcggcg
     3901 gaatgttcgg cagcaatacc gttttctgtt ttttctttgg ccctgtaaat gtatatcctg
     3961 ctcgttcaca agcggttgag tagacttcgc cttgttccat atatggaagg atgttgcgaa
     4021 gcgctttaag ggatagatga ccaaacttag aaaacgataa gtttaaaagt tcttcaatca
     4081 attcttcatc atagacttta tccgctagat tttccattcg ttttccattt tgttcgtatt
     4141 cgtttcgcaa gtaactgcga atgtcggtgt cgtctttaaa catcgttaat gcgtagccaa
     4201 atgtatcaaa atcaatcgga cgaaatgatt ttgctgctcc tttgccatag acgctgtcga
     4261 tcgctttccg tattttatga taggcgccga gttcaaggaa gcgaactttc tcattttcct
     4321 tcagcgtggt gtttcggtca tataaaagac ctttaaaacg ggtgtcgtca ggcaagttaa
     4381 gcaatgttcg aacatcatgg aaggtgattt tatttttatg aaatgcttgc ttgtatataa
     4441 gacgacgttc atcatcggtt agtgcccgga tgcctcccgg ggagacaaga cgaagtttgt
     4501 taatatgttc ccagacggtg aaggactgga atgtgtatgt tgcttttggc gcgcgttttt
     4561 ctttaggctc aaacgtacag aaaccgactt ttttctcgat atcatcctta gaagcaaaag
     4621 ggcgttgcga tgcccaaatg gaaatatact cgtgttcaaa tgcttctgtg caaacgatgt
     4681 tcccatattc gcgctgtttg gcgaaaatca gtttgatttc ccgttcaaga tcgtcgcggg
     4741 caacagtgtt ggtgtaatta tcctctttat tacgcttgtg cagggaaaat ttcggatcct
     4801 tgacaaccat ttctgcaacc gttcggtaac ttgaaagaat ggattggttt tcttcaatat
     4861 gtttgagcat cgtactgttt tctttgttgg tgcgctcact cttgcggttg gatctaaatc
     4921 cacgccgttt agccagatga agaaggatgc gggctaattc atcgttattt agttttcgat
     4981 ccagtgcttc aacacgaagc tgccagacgt cgatttcgtg ctttttttca aacagcttgt
     5041 tcagctcttc cttcgttaaa attccttcgc ggacgaacag gcggcgaatg cgctccagtc
     5101 gatgtttgcg acgccgcaga cgacgtcggg cggagcgggc gaggcgacgt ggaagagcta
     5161 gtgactcccc ggttttcggg ttttccgctc tgtcaaaaat gcggacacct aaatcttcga
     5221 tgcgaggaat gtccaaatta atgacagccc aaccgataga cgtaatgccg atagcaagac
     5281 cgattttata cttcatacac ggtgcctgac tgcgttagca atttaactgt gataaacgga
     5341 attgttatcc gctcacaatt cctaccgcat taaagcttat cgatgataag ctgtcaaaca
     5401 tgagaattac aacttatatc gtatggggct gacttcaggt gctacatttg aagagataaa
     5461 ttgcactgaa atctagattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
     5521 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     5581 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
     5641 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
     5701 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
     5761 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
     5821 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     5881 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     5941 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     6001 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     6061 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     6121 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     6181 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     6241 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     6301 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     6361 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     6421 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     6481 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     6541 caacaaacca tgcaaatgct gaatgagggt atcgttccca ctgcgatgct ggttgccaac
     6601 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     6661 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     6721 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     6781 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     6841 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     6901 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgatcaga atcgactgat
     6961 ggaccaaaaa accccgccga agcggggact ttgaactagc atacattgac agctagctca
     7021 gtcctaggta taatgctagc ctagatccgc agtaacccca tgggtcatag ttcccctgag
     7081 attatcgctg tggtataatg aaagttatac cacagcaatg atctcagggt tactatgata
     7141 agggctttct gcctataggc agactgaccc gtggcgttgg ggatcgccta tcgcccgctt
     7201 tcttcgggca ttccccactc ttaggcgaac gaagaccttg gcggaaaacg tcaaggtctt
     7261 ttttatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat
     7321 caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg
     7381 tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg
     7441 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     7501 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     7561 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     7621 acgcggaagt cccctacgtg ctgctgaagt tgcccgcaac agagagtgga accaaccggt
     7681 gataccacga tactatgact gagagtcaac gccatgagcg gcctcatttc ttattctgag
     7741 ttacaacagt ccgcaccgct gccggtagct ccttccggtg ggcgcggggc atgactatcg
     7801 tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
     7861 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgccctgca
     7921 ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat
     7981 ctgtattaac gaagcgctaa ccgtttttat caggctctgg gaggcagaat aaatgatcat
     8041 atcgtcaatt attacctcca cggggagagc ctgagcaaac tggcctcagg catttgagaa
     8101 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc
     8161 ggctatttaa c
//
