LOCUS       sequence-217628-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AtDeadCAS9 D10A H840A module CDS1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  TITLE     Chevalier Lab plasmids
REFERENCE   2  (bases 1 to 6390)
  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: pORFE0003
FEATURES             Location/Qualifiers
     source          1..6390
                     /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"
     primer_bind     complement(4180..4198)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4266..4370
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4592..5383
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5554..6142
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6043..6062
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6296..6313
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 atggataaga agtactctat cggactcgct atcggaacta actctgtggg atgggctgtg
       61 atcaccgatg agtacaaggt gccatctaag aagttcaagg ttctcggaaa caccgatagg
      121 cactctatca agaaaaacct tatcggtgct ctcctcttcg attctggtga aactgctgag
      181 gctaccagac tcaagagaac cgctagaaga aggtacacca gaagaaagaa caggatctgc
      241 tacctccaag agatcttctc taacgagatg gctaaagtgg atgattcatt cttccacagg
      301 ctcgaagagt cattcctcgt ggaagaagat aagaagcacg agaggcaccc tatcttcgga
      361 aacatcgttg atgaggtggc ataccacgag aagtacccta ctatctacca cctcagaaag
      421 aagctcgttg attctactga taaggctgat ctcaggctca tctacctcgc tctcgctcac
      481 atgatcaagt tcagaggaca cttcctcatc gagggtgatc tcaaccctga taactctgat
      541 gtggataagt tgttcatcca gctcgtgcag acctacaacc agcttttcga agagaaccct
      601 atcaacgctt caggtgtgga tgctaaggct atcctctctg ctaggctctc taagtcaaga
      661 aggcttgaga acctcattgc tcagctccct ggtgagaaga agaacggact tttcggaaac
      721 ttgatcgctc tctctctcgg actcacccct aacttcaagt ctaacttcga tctcgctgag
      781 gatgcaaagc tccagctctc aaaggatacc tacgatgatg atctcgataa cctcctcgct
      841 cagatcggag atcagtacgc tgatttgttc ctcgctgcta agaacctctc tgatgctatc
      901 ctcctcagtg atatcctcag agtgaacacc gagatcacca aggctccact ctcagcttct
      961 atgatcaaga gatacgatga gcaccaccag gatctcacac ttctcaaggc tcttgttaga
     1021 cagcagctcc cagagaagta caaagagatt ttcttcgatc agtctaagaa cggatacgct
     1081 ggttacatcg atggtggtgc atctcaagaa gagttctaca agttcatcaa gcctatcctc
     1141 gagaagatgg atggaaccga ggaactcctc gtgaagctca atagagagga tcttctcaga
     1201 aagcagagga ccttcgataa cggatctatc cctcatcaga tccacctcgg agagttgcac
     1261 gctatcctta gaaggcaaga ggatttctac ccattcctca aggataacag ggaaaagatt
     1321 gagaagattc tcaccttcag aatcccttac tacgtgggac ctctcgctag aggaaactca
     1381 agattcgctt ggatgaccag aaagtctgag gaaaccatca ccccttggaa cttcgaagag
     1441 gtggtggata agggtgctag tgctcagtct ttcatcgaga ggatgaccaa cttcgataag
     1501 aaccttccaa acgagaaggt gctccctaag cactctttgc tctacgagta cttcaccgtg
     1561 tacaacgagt tgaccaaggt taagtacgtg accgagggaa tgaggaagcc tgcttttttg
     1621 tcaggtgagc aaaagaaggc tatcgttgat ctcttgttca agaccaacag aaaggtgacc
     1681 gtgaagcagc tcaaagagga ttacttcaag aaaatcgagt gcttcgattc agttgagatt
     1741 tctggtgttg aggataggtt caacgcatct ctcggaacct accacgatct cctcaagatc
     1801 attaaggata aggatttctt ggataacgag gaaaacgagg atatcttgga ggatatcgtt
     1861 cttaccctca ccctctttga agatagagag atgattgaag aaaggctcaa gacctacgct
     1921 catctcttcg atgataaggt gatgaagcag ttgaagagaa gaagatacac tggttgggga
     1981 aggctctcaa gaaagctcat taacggaatc agggataagc agtctggaaa gacaatcctt
     2041 gatttcctca agtctgatgg attcgctaac agaaacttca tgcagctcat ccacgatgat
     2101 tctctcacct ttaaagagga tatccagaag gctcaggttt caggacaggg tgatagtctc
     2161 catgagcata tcgctaacct cgctggatct cctgcaatca agaagggaat cctccagact
     2221 gtgaaggttg tggatgagtt ggtgaaggtg atgggaaggc ataagcctga gaacatcgtg
     2281 atcgaaatgg ctagagagaa ccagaccact cagaagggac agaagaactc tagggaaagg
     2341 atgaagagga tcgaggaagg tatcaaagag cttggatctc agatcctcaa agagcaccct
     2401 gttgagaaca ctcagctcca gaatgagaag ctctacctct actacctcca gaacggaagg
     2461 gatatgtatg tggatcaaga gttggatatc aacaggctct ctgattacga tgttgatgct
     2521 atcgtgccac agtcattctt gaaggatgat tctatcgata acaaggtgct caccaggtct
     2581 gataagaaca ggggtaagag tgataacgtg ccaagtgaag aggttgtgaa gaaaatgaag
     2641 aactattgga ggcagctcct caacgctaag ctcatcactc agagaaagtt cgataacttg
     2701 actaaggctg agaggggagg actctctgaa ttggataagg caggattcat caagaggcag
     2761 cttgtggaaa ccaggcagat cactaagcac gttgcacaga tcctcgattc taggatgaac
     2821 accaagtacg atgagaacga taagttgatc agggaagtga aggttatcac cctcaagtca
     2881 aagctcgtgt ctgatttcag aaaggatttc caattctaca aggtgaggga aatcaacaac
     2941 taccaccacg ctcacgatgc ttaccttaac gctgttgttg gaaccgctct catcaagaag
     3001 tatcctaagc tcgagtcaga gttcgtgtac ggtgattaca aggtgtacga tgtgaggaag
     3061 atgatcgcta agtctgagca agagatcgga aaggctaccg ctaagtattt cttctactct
     3121 aacatcatga atttcttcaa gaccgagatt accctcgcta acggtgagat cagaaagagg
     3181 ccactcatcg agacaaacgg tgaaacaggt gagatcgtgt gggataaggg aagggatttc
     3241 gctaccgtta gaaaggtgct ctctatgcca caggtgaaca tcgttaagaa aaccgaggtg
     3301 cagaccggtg gattctctaa agagtctatc ctccctaaga ggaactctga taagctcatt
     3361 gctaggaaga aggattggga ccctaagaaa tacggtggtt tcgattctcc taccgtggct
     3421 tactctgttc tcgttgtggc taaggttgag aagggaaaga gtaagaagct caagtctgtt
     3481 aaggaacttc tcggaatcac tatcatggaa aggtcatctt tcgagaagaa cccaatcgat
     3541 ttcctcgagg ctaagggata caaagaggtt aagaaggatc tcatcatcaa gctcccaaag
     3601 tactcactct tcgaactcga gaacggtaga aagaggatgc tcgcttctgc tggtgagctt
     3661 caaaagggaa acgagcttgc tctcccatct aagtacgtta actttcttta cctcgcttct
     3721 cactacgaga agttgaaggg atctccagaa gataacgagc agaagcaact tttcgttgag
     3781 cagcacaagc actacttgga tgagatcatc gagcagatct ctgagttctc taaaagggtg
     3841 atcctcgctg atgcaaacct cgataaggtg ttgtctgctt acaacaagca cagagataag
     3901 cctatcaggg aacaggcaga gaacatcatc catctcttca cccttaccaa cctcggtgct
     3961 cctgctgctt tcaagtactt cgatacaacc atcgatagga agagatacac ctctaccaaa
     4021 gaagtgctcg atgctaccct catccatcag tctatcactg gactctacga gactaggatc
     4081 gatctctcac agctcggtgg tgattcaagg gctgatccta agaagaagag gaaggtttga
     4141 gctttgagac cacgaagtgg ctcttcagtg gacgaaaggg cctcgtgata cgcctatttt
     4201 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4261 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4321 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgcgctcac
     4381 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     4441 tggcttgtta tgactgtttt tttttggggt acagtctatg cctcgggcat ccaagcagca
     4501 agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg ttacgcagca
     4561 gggcagtcgc cctaaaacaa agttaaacat catgagggaa gcggtgatcg ccgaagtatc
     4621 gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc
     4681 cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagcg atattgattt
     4741 gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct
     4801 tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat
     4861 tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg
     4921 agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga
     4981 tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc
     5041 ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt
     5101 aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt
     5161 gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga
     5221 ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc
     5281 ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt
     5341 ccattacgta aaaggcgaga tcaccaaggt agtcggcaaa taactgtcag accaagttta
     5401 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5461 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5521 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5581 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5641 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5701 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5761 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5821 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5881 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5941 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6001 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6061 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6121 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6181 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6241 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     6301 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     6361 gccgattcat taatcactct gtggtctcaa
//
