LOCUS       sequence-173256-        8783 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expression of dcas9 D10A H840A from a TetR-regulated uvtetO 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8783)
  AUTHORS   Singh AK, Carette X, Potluri LP, Sharp JD, Xu R, Prisic S, Husson RN
  TITLE     Investigating essential gene function in Mycobacterium tuberculosis 
            using an efficient CRISPR interference system.
  JOURNAL   Nucleic Acids Res. 2016 Jul 12. pii: gkw625.
  PUBMED    27407107
REFERENCE   2  (bases 1 to 8783)
  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: "Nucleic 
            Acids Res. 2016 Jul 12. pii: gkw625."
COMMENT     Sequence Label: pRH2502
FEATURES             Location/Qualifiers
     source          1..8783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..27,28..30,31..2517,2518..2520,2521..4104)
                     /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"
     CDS             4117..4137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4158..4292
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4195..4214)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4249..4268
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4488..5076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4568..4587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5407..6222)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPHAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6130..6149
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6214..6233
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(8170..8216)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     regulatory      8405..8414
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggacaaga agtacagcat cggcctggcg atcggcacga actcggtggg ctgggcggtc
       61 atcacggacg agtacaaggt cccctccaag aagttcaagg tcctgggcaa caccgaccgg
      121 cacagcatca agaagaacct gatcggcgcc ctgctgttcg actccggcga gaccgccgag
      181 gcgacgcgcc tgaagcggac cgcgcggcgc cgatacacgc ggcgcaagaa ccgcatctgc
      241 tacctgcagg aaatcttctc gaacgagatg gccaaggtgg acgacagctt cttccaccgg
      301 ctggaggagt ccttcctggt ggaggaggac aagaagcacg agcgccaccc gatcttcggc
      361 aacatcgtgg acgaggtggc ctaccacgag aagtacccca cgatctacca cctgcgcaag
      421 aagctggtgg acagcaccga caaggcggac ctgcggctga tctacctggc cctggcgcac
      481 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaacccgga caactcggac
      541 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc
      601 atcaacgcct ccggcgtgga cgccaaggcg atcctgtcgg cgcgcctgtc caagtcgcgg
      661 cgcctggaga acctgatcgc ccagctgccg ggcgagaaga agaacggcct gttcggcaac
      721 ctgatcgcgc tgagcctggg cctgacgccc aacttcaagt ccaacttcga cctggccgag
      781 gacgcgaagc tgcagctgtc gaaggacacc tacgacgacg acctggacaa cctgctggcc
      841 cagatcggcg accagtacgc ggacctgttc ctggccgcca agaacctgag cgacgccatc
      901 ctgctgtccg acatcctgcg ggtcaacacc gagatcacga aggccccgct gagcgcgtcc
      961 atgatcaagc ggtacgacga gcaccaccag gacctgaccc tgctgaaggc gctggtgcgc
     1021 cagcagctgc ccgagaagta caaggaaatc ttcttcgacc agtcgaagaa cggctacgcc
     1081 ggctacatcg acggcggcgc gagccaggag gagttctaca agttcatcaa gccgatcctg
     1141 gagaagatgg acggcacgga ggagctgctg gtcaagctga accgcgagga cctgctgcgc
     1201 aagcagcgga ccttcgacaa cggctcgatc ccgcaccaga tccacctggg cgagctgcac
     1261 gccatcctgc ggcgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc
     1321 gagaagatcc tgaccttccg catcccgtac tacgtgggcc ccctggcccg cggcaactcg
     1381 cggttcgcgt ggatgacccg gaagagcgag gagaccatca cgccgtggaa cttcgaggag
     1441 gtcgtggaca agggcgcctc ggcgcagagc ttcatcgagc gcatgaccaa cttcgacaag
     1501 aacctgccga acgagaaggt cctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     1561 tacaacgagc tgacgaaggt caagtacgtg accgagggca tgcggaagcc ggccttcctg
     1621 tcgggcgagc agaagaaggc gatcgtggac ctgctgttca agaccaaccg caaggtcacg
     1681 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gtttcgactc ggtcgagatc
     1741 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc
     1801 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtc
     1861 ctgaccctga cgctgttcga ggaccgcgag atgatcgagg agcggctgaa gacgtacgcc
     1921 cacctgttcg acgacaaggt gatgaagcag ctgaagcggc gccgatacac cggctggggc
     1981 cgcctgtcgc ggaagctgat caacggcatc cgggacaagc agtccggcaa gaccatcctg
     2041 gacttcctga agtcggacgg cttcgcgaac cgcaacttca tgcagctgat ccacgacgac
     2101 agcctgacct tcaaggagga catccagaag gcccaagtga gcggccaggg cgactccctg
     2161 cacgagcaca tcgccaacct ggcgggctcg ccggcgatca agaagggcat cctgcagacc
     2221 gtcaaggtcg tggacgagct ggtcaaggtg atgggccgcc acaagcccga gaacatcgtg
     2281 atcgagatgg cccgggagaa ccagaccacg cagaagggcc agaagaacag ccgcgagcgg
     2341 atgaagcgca tcgaggaggg catcaaggag ctgggctcgc agatcctgaa ggagcacccg
     2401 gtcgagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc
     2461 gacatgtacg tggaccagga gctggacatc aaccggctga gcgactacga cgtggacgcc
     2521 atcgtgccgc agtcgttcct gaaggacgac agcatcgaca acaaggtcct gacccgctcg
     2581 gacaagaacc ggggcaagtc ggacaacgtg cccagcgagg aggtcgtgaa gaagatgaag
     2641 aactactggc gccagctgct gaacgccaag ctgatcacgc agcgcaagtt cgacaacctg
     2701 accaaggccg agcggggcgg cctgagcgag ctggacaagg cgggcttcat caagcgccag
     2761 ctggtcgaga cccggcagat cacgaagcac gtggcgcaga tcctggactc gcggatgaac
     2821 acgaagtacg acgagaacga caagctgatc cgcgaggtca aggtgatcac cctgaagtcc
     2881 aagctggtct cggacttccg caaggacttc cagttctaca aggtccggga gatcaacaac
     2941 taccaccacg cccacgacgc gtacctgaac gccgtcgtgg gcaccgcgct gatcaagaag
     3001 tacccgaagc tggagtcgga gttcgtctac ggcgactaca aggtctacga cgtgcgcaag
     3061 atgatcgcca agagcgagca ggagatcggc aaggccaccg cgaagtactt cttctactcc
     3121 aacatcatga acttcttcaa gaccgagatc acgctggcca acggcgagat ccgcaagcgc
     3181 ccgctgatcg agaccaacgg cgagacgggc gagatcgtct gggacaaggg ccgcgacttc
     3241 gcgaccgtcc ggaaggtgct gagcatgccg caggtcaaca tcgtgaagaa gaccgaggtg
     3301 cagacgggcg gcttctccaa ggagtcgatc ctgcccaagc gcaactcgga caagctgatc
     3361 gcccggaaga aggactggga cccgaagaag tacggcggct tcgactcccc caccgtcgcc
     3421 tactcggtgc tggtcgtggc gaaggtcgag aagggcaagt ccaagaagct gaagtcggtg
     3481 aaggagctgc tgggcatcac catcatggag cgctcgtcgt tcgagaagaa cccgatcgac
     3541 ttcctggagg cgaagggcta caaggaggtc aagaaggacc tgatcatcaa gctgcccaag
     3601 tacagcctgt tcgagctgga gaacggccgc aagcggatgc tggcctccgc gggcgagctg
     3661 cagaagggca acgagctggc cctgccgtcg aagtacgtca acttcctgta cctggcgtcg
     3721 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag
     3781 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagcgggtc
     3841 atcctggccg acgcgaacct ggacaaggtg ctgtcggcct acaacaagca ccgcgacaag
     3901 ccgatccggg agcaggcgga gaacatcatc cacctgttca ccctgacgaa cctgggcgcc
     3961 cccgccgcgt tcaagtactt cgacaccacg atcgaccgca agcggtacac ctccacgaag
     4021 gaggtgctgg acgcgaccct gatccaccag tcgatcaccg gcctgtacga gacgcgcatc
     4081 gacctgagcc agctgggcgg cgactcccgg gcggacccga agaagaagcg gaaggtgtga
     4141 ttaattaaca aactcggaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     4201 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     4261 aactcatcaa tgtatcttat catgtctgga tcctctagag tcgacctgca ggtttaaacg
     4321 cggccgcacg cgttgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4381 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4441 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4501 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4561 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4621 accctgccgc ttaccggata cctgtccgcc tttctcccct cgggaagcgt ggcgctttct
     4681 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4741 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4801 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4861 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4921 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4981 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5041 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5101 gggtctgacg ctcatggaac tagtgaggtc tgcctcgtga agaaggtgtt gctgactcat
     5161 accaggcctg aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttggtgagag
     5221 ctttgttgta ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg
     5281 cgttgtcggg aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac
     5341 aaagccgccg tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa
     5401 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     5461 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     5521 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     5581 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     5641 gacgactgaa tccggtgaga atggcaaaag attatgcatt tctttccaga cttgttcaac
     5701 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     5761 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     5821 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     5881 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     5941 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     6001 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     6061 cagaaacaac tctggcgcat ggggcttccc atacaagcga tagattgtcg cacctgattg
     6121 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     6181 tcgcggcctc gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     6241 gtttatgtaa gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta
     6301 acatcagaga ttttgagaca caacgtggct ttcccccccc cccgacgtca ggtggctagc
     6361 tggcgggaac ggcaatgctc ctggcggcgg cggggctggt gcccaggtct ccgcacagag
     6421 cggcggtgct ggcgctcgcg gccaggcgtg gttcttcgcg tactgacaag aaacccccct
     6481 ctttaggact cagtgtcctt gggagggggg ctttttgcgt ttcaggaggt cttggccagc
     6541 ttggacatcg cctcagcgat agcctcgtcg cgggcctcag acgccatctg gtacttcatc
     6601 gccatcctag gagtcgtgtg accgagacgg gccatcagct ccttggtcgt cgcacctgcc
     6661 tgagcggcga acgtagcgcc gacagcgcgg aggtcgtgga tgcggagttc cggccgaccg
     6721 atcttggcgt agccacgctt cagcgacttg gtgaacgcgg acttcgacag ccggttgccc
     6781 tgcgtcgtgg tcaccaggaa tgcctcgggg cccttgttca tcttcgtacg gtccttcatg
     6841 tgcgctcgga tcatctccgc gacgtgaggc ggaaccgtca caggacgctt cgaccggacg
     6901 gtcttggcgt tgccaacgac gatcttgttc cccacgcggg aagcgccacg gcgcacccgg
     6961 agcttcatcg tcatgccgtc gtccacgatg tccttgcggc gaagctcgat cagctctccg
     7021 aaccggaggc tcgtccacgc caggatgtat gccgcgatcc ggtagtgcac gaagatctca
     7081 gcggcgacga tgtccagctc ctcaggcgtc agcgcctcta cgtcgcgctc atcggctgcc
     7141 ttctgctcga tccggcacgg gttctctgcg atcagcttgt cctcgaccgc tgtgttcatc
     7201 accgcccgga ggacgttgta ggcatgccgg cgggcagtcg ggtgcttcct acccatcccg
     7261 gcccaccacg cacgcaccag agctggcgtc atctctgtga ccgccacttc acctagcacc
     7321 gggtagatgc ggcgctccgc gtgcccgctg tacagatccc tggtgccgtc tgcgaggtcg
     7381 cgctccacga gccacttccg ggtgtactcc tccagcgtga tggcgctggc ggctgccttc
     7441 ttcgcccggt cctgtggagg ggtccaggtc tccatctcga tgagccgctt ctcgcccgcg
     7501 agccaggctt cggcgtccat cttgttgtcg taggtctgca gcgcgtagta cctcacaccg
     7561 tcctgcgggt tgacgtatga ggcttggatc ctcccgctgc gctgagtctt cagcgatccc
     7621 catccgcgac gtgccaacta ggtctcctct cgtcgtgaac aaggctaccg ggttgcaact
     7681 cctgtgcaac tctcaggctt caacgcgctt ctacgacctg caatttcttt ccacttagag
     7741 gatgcagccg agagggggta aaaacctatc ttgaccggcc catatatgtg gtcggcagac
     7801 acccattctt ccaaactagc tacgcgggtt cgattcccgt cgcccgctcc gctggtcaga
     7861 gggtgttttc gccctctggc catttttctt tccaggggtc tgcaactctt gtgcgactct
     7921 tctgacctgg gcatacgcgg ttgcaacgca tccctgatct ggctactttc gatgctgaca
     7981 aacgaataga gccccccgcc tgcgcgaaca gacgaggggc attcacacca gattggagct
     8041 ggtgcagtga agagaataga ccgggacaag gttgcaccgg gagttgcagc ggtcggaacc
     8101 ctcgccgtcg gcgggctggc gttcgccctg tcgttcacgg ctctagctga tcaccgcggc
     8161 catgatggca taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     8221 ctggcagtcg atcgtacgct agttaactac gtcgacatcg atttaatcga tcgatatgca
     8281 tcaattgatt taaatactag tgttaactat ttaattgggg accctagagg tccccttttt
     8341 tattttaaaa attttttcaa ggggacctct agggtcccca gctggctagc gagtcatgag
     8401 gtcaccatgg tggccatcta gacatcatcg gtaccaggcc tagatctact agaaatattg
     8461 gatcgtcggc accgtcacgg ccgtgggagg cggcacgatc cgcgacgtga tgatcggccg
     8521 catccccacg gtgctgcgca gtgagctcta cgccatcccg gcgttgatct gtgcgttcgc
     8581 acgcacaggc ccggtgtgag aagggtctct gacgagcggg agaactccct atcagtgata
     8641 gagtttgtcc tccctatcag tgatagatag gctctgggag tacccgtgtg tacgaccagc
     8701 acggcataca tcatttcgac gccgagagat tcgccgcccg aaatgagcac gatccgctcg
     8761 aggcatgcga aggagatata cat
//
