LOCUS       sequence-409764-        8611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AcrIIC1-mediated inhibition of ThermoCas9 cleavage activity in E. 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8611)
  TITLE     van der Oost E. coli CRISPR plasmids
REFERENCE   2  (bases 1 to 8611)
  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: pAcrTCas9_scr
FEATURES             Location/Qualifiers
     source          1..8611
                     /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"
     promoter        1993..2111
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     protein_bind    5778..5802
                     /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(5809..5814,5815..5831,5832..5837))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6000..6019)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6225..6302
                     /gene="lacI"
                     /label=lacI promoter
     CDS             6303..7385
                     /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(6322..6341)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        7446..7480
                     /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 ttcaccacaa ttcagcaaat tgtgaacatc atcacgttca tctttccctg
     2041 gttgccaatg gcccattttc ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta
     2101 gactggtcgt agggagacca caacggtttc cctctagaaa taattttgtt taactataag
     2161 aaggagatat acatatgaat aaaacatata aaatcggaaa aaatgcggga tatgacggct
     2221 gcgggttatg tttagccgcg atctcagaaa atgaagctat taaagttaag tatctgcgcg
     2281 atatctgtcc agactatgac ggcgacgaca aagctgagga ctggctgaga tggggaacgg
     2341 atagccgcgt gaaagctgcg gctttagaaa tggagcaata tgcttatacg tcggttggga
     2401 tggcgtcatg ttgggagttt gttgaactat gagtcgacag gattaaacaa aaatggcccg
     2461 cttcataagc aggccatttt gtctgattta taacggacgg atagtttccc cggctttcga
     2521 atgagaagat gacgccaccc caactctctt ttcccctctc actttgtaga tgttgcctag
     2581 cacatctact tggtatttct cgaatcgttt gagggttctt gaaccgatgc tgcggagcga
     2641 aaagttgtta tcatggctaa ccaaacttaa ccctccattg gaggagtcga tggtttgata
     2701 ataggcgaac agatccttaa ttttgatttc ttcccccaca gcagtcttta ttgttttttc
     2761 tcggggaaat tcgatacgga taagatcatt tgggtataga ctgaatcgga atgtatagtc
     2821 ctccgtcatt tccttccact cagagtacgg tttgttcggc tcgatcgcct tgtttggcaa
     2881 gatccctttc atcatatcta ttgtatagat agggacacaa taatatttgc catctttctc
     2941 aaagacgtcg acccgcacga tgttgctgtt gtaggcgact gttttgccat cgttgagcgg
     3001 aataacttga tttgtcgtat cgatgatttt gattgttcgg atgataggac ctagttctcc
     3061 gttcttcttc ggtttataca gaggctcttg aaacgccttt tttgggtcat tgttatgttc
     3121 aagcaaccgt tggcgaatgg cttcatatgt ccttggatcg ctttctttcc cgtacattgg
     3181 gaaatgacct gttttatcca gttggatctc ggatagtttc tttttgacga ccgtctgtat
     3241 ttttccgctc cgttcgtcga tgccgatata acgccgcaat gtttcttgat gagccgctcc
     3301 tgttatgctc cgcttcggca ttcgggagac aaaaaccggc tgcaacgatt cgagtttctc
     3361 gttatcataa tttccaagat tgagagcttt tatactctcc tttggatttt ttgataaacg
     3421 cgcctgcagt tcatcagcaa agtgcggcca aggctgcgga aactgcggat ccgtcttttt
     3481 ggacagttct ttgttttgtt cgcgccgttg atagaaggcg gtgactcggg cgatatcgct
     3541 cggcgttgtg caggcgacga tggcagcatc gacggcatga tgcaaattcg attcttcccg
     3601 gtttttgtta aaattccaac ggctgcgtaa atgggcggta atacggccgt tgaccgtgta
     3661 tactttttgt ttgtcatcgc tgtcggcgaa tttgagatgt tcgcgaataa agttagccaa
     3721 gaagcgtgag atataacggg tatcatttag attacgattt ttaaactcat tttcttcgtt
     3781 ttcatcgtaa tgaagccgaa ggagtcgatc ccgcttcttt ttcgaaaact gcttatttgt
     3841 caagacaaac gtctcgaact gttgccaacg ttctgagcct aatcctaaat attcagctgg
     3901 ggtgcggttt cctttttcac ggttctcctt tgtcaacaca agaactttat tggtatagct
     3961 atcgtccaag cttcggctgt atggaatcac atggtcgact tctgtatagc ctggttcgag
     4021 caaccgctcg atttcgatcg gttggagtga ataggcacat tttccgtttt gttcgctcca
     4081 tagtttgaat ttcacaatgt caagcccagt tggattgagc gtcagcccat attcaacaag
     4141 ttggcgaatg gcagtttcgt ttttctttcg gtttccttcc tgttctttct gcattttacg
     4201 tcgttcatca aaggattgtg atagttcccg ggccagttcg atatggatgg agaccggtga
     4261 gccgtacttt ttgataatgg cattgaccac tttgcgtgcc tgtgtcagtg cgcgcatgac
     4321 gaccggattg gcgatcggcg gaatgttcgg cagcaatacc gttttctgtt ttttctttgg
     4381 ccctgtaaat gtatatcctg ctcgttcaca agcggttgag tagacttcgc cttgttccat
     4441 atatggaagg atgttgcgaa gcgctttaag ggatagatga ccaaacttag aaaacgataa
     4501 gtttaaaagt tcttcaatca attcttcatc atagacttta tccgctagat tttccattcg
     4561 ttttccattt tgttcgtatt cgtttcgcaa gtaactgcga atgtcggtgt cgtctttaaa
     4621 catcgttaat gcgtagccaa atgtatcaaa atcaatcgga cgaaatgatt ttgctgctcc
     4681 tttgccatag acgctgtcga tcgctttccg tattttatga taggcgccga gttcaaggaa
     4741 gcgaactttc tcattttcct tcagcgtggt gtttcggtca tataaaagac ctttaaaacg
     4801 ggtgtcgtca ggcaagttaa gcaatgttcg aacatcatgg aaggtgattt tatttttatg
     4861 aaatgcttgc ttgtatataa gacgacgttc atcatcggtt agtgcccgga tgcctcccgg
     4921 ggagacaaga cgaagtttgt taatatgttc ccagacggtg aaggactgga atgtgtatgt
     4981 tgcttttggc gcgcgttttt ctttaggctc aaacgtacag aaaccgactt ttttctcgat
     5041 atcatcctta gaagcaaaag ggcgttgcga tgcccaaatg gaaatatact cgtgttcaaa
     5101 tgcttctgtg caaacgatgt tcccatattc gcgctgtttg gcgaaaatca gtttgatttc
     5161 ccgttcaaga tcgtcgcggg caacagtgtt ggtgtaatta tcctctttat tacgcttgtg
     5221 cagggaaaat ttcggatcct tgacaaccat ttctgcaacc gttcggtaac ttgaaagaat
     5281 ggattggttt tcttcaatat gtttgagcat cgtactgttt tctttgttgg tgcgctcact
     5341 cttgcggttg gatctaaatc cacgccgttt agccagatga agaaggatgc gggctaattc
     5401 atcgttattt agttttcgat ccagtgcttc aacacgaagc tgccagacgt cgatttcgtg
     5461 ctttttttca aacagcttgt tcagctcttc cttcgttaaa attccttcgc ggacgaacag
     5521 gcggcgaatg cgctccagtc gatgtttgcg acgccgcaga cgacgtcggg cggagcgggc
     5581 gaggcgacgt ggaagagcta gtgactcccc ggttttcggg ttttccgctc tgtcaaaaat
     5641 gcggacacct aaatcttcga tgcgaggaat gtccaaatta atgacagccc aaccgataga
     5701 cgtaatgccg atatcaagac cgattttata cttcatacac ggtgcctgac tgcgttagca
     5761 atttaactgt gataaacgga attgttatcc gctcacaatt cctaccgcat taaagcttat
     5821 cgatgataag ctgtcaaaca tgagaattac aacttatatc gtatggggct gacttcaggt
     5881 gctacatttg aagagataaa ttgcactgaa atctagattt cgcgggatcg agatctcgat
     5941 cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg ttgctggcgc
     6001 ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc tcatgagcgc
     6061 ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg gcgccatctc
     6121 cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac tactgggctg
     6181 cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc atcgaatggc
     6241 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     6301 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     6361 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     6421 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     6481 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     6541 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     6601 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     6661 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     6721 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     6781 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     6841 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     6901 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     6961 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggt atcgttccca
     7021 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     7081 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     7141 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     7201 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     7261 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     7321 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     7381 agtgatcaga atcgactgat ggaccaaaaa accccgccga agcggggact ttgaactagc
     7441 atacattgac agctagctca gtcctaggta taatgctagc ctagatccgc agtaacccca
     7501 tgggtcatag ttcccctgag attatcgctg tggtataatg aaagttatac cacagcaatg
     7561 atctcagggt tactatgata agggctttct gcctataggc agactgaccc gtggcgttgg
     7621 ggatcgccta tcgcccgctt tcttcgggca ttccccactc ttaggcgaac gaagaccttg
     7681 gcggaaaacg tcaaggtctt ttttatggaa gccggcggca cctcgctaac ggattcacca
     7741 ctccaagaat tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt
     7801 ggcagaacat atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca
     7861 gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     7921 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     7981 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     8041 gtgtttcgta aagtctggaa acgcggaagt cccctacgtg ctgctgaagt tgcccgcaac
     8101 agagagtgga accaaccggt gataccacga tactatgact gagagtcaac gccatgagcg
     8161 gcctcatttc ttattctgag ttacaacagt ccgcaccgct gccggtagct ccttccggtg
     8221 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
     8281 aggacaggtg ccggcagcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     8341 gccgaaacaa gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     8401 accctgtgga acacctacat ctgtattaac gaagcgctaa ccgtttttat caggctctgg
     8461 gaggcagaat aaatgatcat atcgtcaatt attacctcca cggggagagc ctgagcaaac
     8521 tggcctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     8581 aaccagcaat agacataagc ggctatttaa c
//
