LOCUS       sequence-363323-        8252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  carrying CRISPR/dCas9 system for gene knockdown in Acidithiobacillus
            ferridurans.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8252)
  AUTHORS   Chen J, Liu Y, Mahadevan R
  TITLE     Genetic Engineering of Acidithiobacillus ferridurans Using CRISPR 
            Systems To Mitigate Toxic Release in Biomining.
  JOURNAL   Environ Sci Technol. 2023 Aug 22;57(33):12315-12324. doi: 
            10.1021/acs.est.3c02492. Epub 2023 Aug 9.
  PUBMED    37556825
REFERENCE   2  (bases 1 to 8252)
  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: 
            "10.1021/acs.est.3c02492"; journalName: "Environ Sci Technol"; date:
            "2023-08-22- 22"; volume: "57"; issue: "33"; pages: "12315-12324"
COMMENT     Sequence Label: pAFi-dHdrA
FEATURES             Location/Qualifiers
     source          1..8252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(24..50,51..53,54..2540,2541..2543,2544..4130)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     terminator      4154..4225
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4241..4268
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4362..5024)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      5025..5796
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     promoter        7096..7198
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             7199..7858
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(7265..7284)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     misc_RNA        7983..8058
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      8101..8195
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(8227..8232,8233..8248,8249..8252,1..3)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
ORIGIN
        1 atgtttcacc ttatcatcaa gatatggata agaaatactc aataggttta gctatcggca
       61 caaatagcgt cggatgggcg gtgatcactg atgaatataa ggttccgtct aaaaagttca
      121 aggttctggg aaatacagac cgccacagta tcaaaaaaaa tcttataggg gctcttttat
      181 ttgacagtgg agagacagcg gaagcgactc gtctcaaacg gacagctcgt agaaggtata
      241 cacgtcggaa gaatcgtatt tgttatctac aggagatttt ttcaaatgag atggcgaaag
      301 tagatgatag tttctttcat cgacttgaag agtctttttt ggtggaagaa gacaagaagc
      361 atgaacgtca tcctattttt ggaaatatag tagatgaagt tgcttatcat gagaaatatc
      421 caactatcta tcatctgcga aaaaaattgg tagattctac tgataaagcg gatttgcgct
      481 taatctattt ggccttagcg catatgatta agtttcgtgg tcattttttg attgagggag
      541 atttaaatcc tgataatagt gatgtggaca aactatttat ccagttggta caaacctaca
      601 atcaattatt tgaagaaaac cctattaacg caagtggagt agatgctaaa gcgattcttt
      661 ctgcacgatt gagtaaatca agacgattag aaaatctcat tgctcagctc cccggtgaga
      721 agaaaaatgg cttatttggg aatctcattg ctttgtcatt gggtttgacc cctaatttta
      781 aatcaaattt tgatttggca gaagatgcta aattacagct ttcaaaagat acttacgatg
      841 atgatttaga taatttattg gcgcaaattg gagatcaata tgctgatttg tttttggcag
      901 ctaagaattt atcagatgct attttacttt cagatatcct aagagtaaat actgaaataa
      961 ctaaggctcc cctatcagct tcaatgatta aacgctacga tgaacatcat caagacttga
     1021 ctcttttaaa agctttagtt cgacaacaac ttccagaaaa gtataaagaa atcttttttg
     1081 atcaatcaaa aaacggatat gcaggttata ttgatggggg agctagccaa gaagaatttt
     1141 ataaatttat caaaccaatt ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac
     1201 taaatcgtga agatttgctg cgcaagcaac ggacctttga caacggctct attccccatc
     1261 aaattcactt gggtgagctg catgctattt tgagaagaca agaagacttt tatccatttt
     1321 taaaagacaa tcgtgagaag attgaaaaaa tcttgacttt tcgaattcct tattatgttg
     1381 gtccattggc gcgtggcaat agtcgttttg catggatgac tcggaagtct gaagaaacaa
     1441 ttaccccatg gaattttgaa gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg
     1501 aacgcatgac aaactttgat aaaaatcttc caaatgaaaa agtactacca aaacatagtt
     1561 tgctttatga gtattttacg gtttataacg aattgacaaa ggtcaaatat gttactgaag
     1621 gaatgcgaaa accagcattt ctttcaggtg aacagaagaa agccattgtt gatttactct
     1681 tcaaaacaaa tcgaaaagta accgttaagc aattaaaaga agattatttc aaaaaaatag
     1741 aatgttttga tagtgttgaa atttcaggag ttgaagatag atttaatgct tcattaggta
     1801 cctaccatga tttgctaaaa attattaaag ataaagattt tttggataat gaagaaaatg
     1861 aagatatctt agaggatatt gttttaacat tgaccttatt tgaagatagg gagatgattg
     1921 aggaaagact taaaacatat gctcacctct ttgatgataa ggtgatgaaa cagcttaaac
     1981 gtcgccgtta tactggttgg ggacgtttgt ctcgaaaatt gattaatggt attagggata
     2041 agcaatctgg caaaacaata ttagattttt tgaaatcaga tggttttgcc aatcgcaatt
     2101 ttatgcagct gatccatgat gatagtttga catttaaaga agacattcaa aaagcacaag
     2161 tgtctggaca aggcgatagt ttacatgaac atattgcaaa tttagctggt agccctgcta
     2221 ttaaaaaagg tattttacag actgtaaaag ttgttgatga attggtcaaa gtaatggggc
     2281 ggcataagcc agaaaatatc gttattgaaa tggcacgtga aaatcagaca actcaaaagg
     2341 gccagaaaaa ttcgcgagag cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa
     2401 gtcagattct taaagagcat cctgttgaaa atactcaatt gcaaaatgaa aagctctatc
     2461 tctattatct ccaaaatgga agagacatgt atgtggacca agaattagat attaatcgtt
     2521 taagtgatta tgatgtcgat gccattgttc cacaaagttt ccttaaagac gattcaatag
     2581 acaataaggt cttaacgcgt tctgataaaa atcgtggtaa atcggataac gttccaagtg
     2641 aagaagtagt caaaaagatg aaaaactatt ggagacaact tctaaacgcc aagttaatca
     2701 ctcaacgtaa gtttgataat ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata
     2761 aagctggttt tatcaaacgc caattggttg aaactcgcca aatcactaag catgtggcac
     2821 aaattttgga tagtcgcatg aatactaaat acgatgaaaa tgataaactt attcgagagg
     2881 ttaaagtgat taccttaaaa tctaaattag tttctgactt ccgaaaagat ttccaattct
     2941 ataaagtacg tgagattaac aattaccatc atgcccatga tgcgtatcta aatgccgtcg
     3001 ttggaactgc tttgattaag aaatatccaa aacttgaatc ggagtttgtc tatggtgatt
     3061 ataaagttta tgatgttcgt aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa
     3121 ccgcaaaata tttcttttac tctaatatca tgaacttctt caaaacagaa attacacttg
     3181 caaatggaga gattcgcaaa cgccctctaa tcgaaactaa tggggaaact ggagaaattg
     3241 tctgggataa agggcgagat tttgccacag tgcgcaaagt attgtccatg ccccaagtca
     3301 atattgtcaa gaaaacagaa gtacagacag gcggattctc caaggagtca attttaccaa
     3361 aaagaaattc ggacaagctt attgctcgta aaaaagactg ggatccaaaa aaatatggtg
     3421 gttttgatag tccaacggta gcttattcag tcctagtggt tgctaaggtg gaaaaaggga
     3481 aatcgaagaa gttaaaatcc gttaaagagt tactagggat cacaattatg gaaagaagtt
     3541 cctttgaaaa aaatccgatt gactttttag aagctaaagg atataaggaa gttaaaaaag
     3601 acttaatcat taaactacct aaatatagtc tttttgagtt agaaaacggt cgtaaacgga
     3661 tgctggctag tgccggagaa ttacaaaaag gaaatgagct ggctctgcca agcaaatatg
     3721 tgaatttttt atatttagct agtcattatg aaaagttgaa gggtagtcca gaagataacg
     3781 aacaaaaaca attgtttgtg gagcagcata agcattattt agatgagatt attgagcaaa
     3841 tcagtgaatt ttctaagcgt gttattttag cagatgccaa tttagataaa gttcttagtg
     3901 catataacaa acatagagac aaaccaatac gtgaacaagc agaaaatatt attcatttat
     3961 ttacgttgac gaatcttgga gctcccgctg cttttaaata ttttgataca acaattgatc
     4021 gtaaacgata tacgtctaca aaagaagttt tagatgccac tcttatccat caatccatca
     4081 ctggtcttta tgaaacacgc attgatttga gtcagctagg aggtgactaa ctcgagtaag
     4141 gatctccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     4201 ctgttgtttg tcggtgaacg ctctctacta gagtcacact ggctcacctt cgggtgggcc
     4261 tttctgcgtt tatacctagg aagcttacta gttaacttca ggacctgcag tctagtatag
     4321 tctggaacag cgcacttacg ggttgctgcg caacccaagt gctaccggcg cggcagcgtg
     4381 acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa aacacggctc atcgggcatc
     4441 ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt cggtcaaggc tggcaggtct
     4501 ggttccatgc ccggaatgcc gggctggctg ggcggctcct cgccggggcc ggtcggtagt
     4561 tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt cgccatgccc caacagcgat
     4621 tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga acaccgacag gcgcaactgg
     4681 tcgcggggct ggccccacgc cacgcggtca ttgaccacgt aggccgacac ggtgccgggg
     4741 ccgttgagct tcacgacgga gatccagcgc tcggccacca agtccttgac tgcgtattgg
     4801 accgtccgca aagaacgtcc gatgagcttg gaaagtgtct tctggctgac caccacggcg
     4861 ttctggtggc ccatctgcgc cacgaggtga tgcagcagca ttgccgccgt gggtttcctc
     4921 gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc
     4981 ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg ccatgcttat ctccatgcgg
     5041 taggggtgcc gcacggttgc ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga
     5101 caacaattat gcgttgcgta aaagtggcag tcaattacag attttcttta acctacgcaa
     5161 tgagctattg cggggggtgc cgcaatgagc tgttgcgtac cccccttttt taagttgttg
     5221 atttttaagt ctttcgcatt tcgccctata tctagttctt tggtgcccaa agaagggcac
     5281 ccctgcgggg ttcccccacg ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc
     5341 cggggcttgt tgatcgactg cgcggccttc ggccttgccc aaggtggcgc tgcccccttg
     5401 gaacccccgc actcgccgcc gtgaggctcg gggggcaggc gggcgggctt cgcccttcga
     5461 ctgcccccac tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg
     5521 gtcgctgcgc gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc
     5581 aggccgcagg ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg
     5641 caggccgcgc agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat
     5701 ccctgtggtc aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt
     5761 ccacgggccg agcgaagcga gccagccggt ggccgctcgc ggccatcgtc cacatatcca
     5821 cgggctggca agggagcgca gcgaccgcgc agggcgaagc ccggagagca agcccgtagg
     5881 gcgccgcagc cgccgtaggc ggtcacgact ttgcgaagca aagtctagtg agtatactca
     5941 agcattgagt ggcccgccgg aggcaccgcc ttgcgctgcc cccgtcgagc cggttggaca
     6001 ccaaaaggga ggggcaggca tggcggcata cgcgatcatg cgatgcaaga agctggcgaa
     6061 aatgggcaac gtggcggcca gtctcaagca cgcctaccgc gagcgcgaga cgcccaacgc
     6121 tgacgccagc aggacgccag agaacgagca ctgggcggcc agcagcaccg atgaagcgat
     6181 gggccgactg cgcgagttgc tgccagagaa gcggcgcaag gacgctgtgt tggcggtcga
     6241 gtacgtcatg acggccagcc cggaatggtg gaagtcggcc agccaagaac agcaggcggc
     6301 gttcttcgag aaggcgcaca agtggctggc ggacaagtac ggggcggatc gcatcgtgac
     6361 ggccagcatc caccgtgacg aaaccagccc gcacatgacc gcgttcgtgg tgccgctgac
     6421 gcaggacggc aggctgtcgg ccaaggagtt catcggcaac aaagcgcaga tgacccgcga
     6481 ccagaccacg tttgcggctg ctgtggccga tctagggctg caacggggca tcgagggcag
     6541 caaggcacgt cacacgcgca ttcaggcgtt ctacgaggcc ctggagcggc caccagtggg
     6601 ccacgtcacc atcagcccgc aagcggtcga gccacgcgcc tatgcaccgc agggattggc
     6661 cgaaaagctg ggaatctcaa agcgcgttga gacgccggaa gccgtggccg accggctgac
     6721 aaaagcggtt cggcaggggt atgagcctgc cctacaggcc gccgcaggag cgcgtgagat
     6781 gcgcaagaag gccgatcaag cccaagagac ggcccgagac cttcgggagc gcctgaagcc
     6841 cgttctggac gccctggggc cgttgaatcg ggatatgcag gccaaggccg ccgcgatcat
     6901 caaggccgtg ggcgaaaagc tgctgacgga acagcgggaa gtccagcgcc agaaacaggc
     6961 ccagcgccag caggaacgcg ggcgcgcaca tttccccgaa aagtgccacc tgggatgaat
     7021 gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct
     7081 tgcagtgggc ggccgtgatc ggcacgtaag aggttccaac tttcaccata atgaaataag
     7141 atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg aagctaaaat
     7201 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     7261 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     7321 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     7381 cattcttgcc cgcctgatga atgctcatcc ggagttccgt atggcaatga aagacggtga
     7441 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     7501 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     7561 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     7621 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     7681 caatatggac aacttcttcg cccccgtttt cactatgggc aaatattata cgcaaggcga
     7741 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt
     7801 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcataatt
     7861 tttttaaggc agttattggt gccctgccaa aacagctcta gacaagctcc gtctagcccg
     7921 gggatccttg acagctagct cagtcctagg tataatatgc atgtcgtagg tgcaggtcca
     7981 gcgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     8041 gtggcaccga gtcggtgctt tttttgaatt ctgtaggcta gtccgggcgc gccgaacttg
     8101 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     8161 tcggttgccg ccgggcgttt tttattggtg agaatccaga gagaattcat cctgaactta
     8221 tctagattga caattaatca tcggctcgta ta
//
