LOCUS       sequence-210902-        6388 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 6388)
  TITLE     Chevalier Lab plasmids
REFERENCE   2  (bases 1 to 6388)
  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..6388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2064..2084
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2127..2145)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2213..2317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2537..3328
                     /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      3499..4087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3988..4007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4241..4258
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             join(4336..4362,4363..4365,4366..6388,1..464,465..467,
                     468..2051)
                     /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"
ORIGIN
        1 ctgatggatt cgctaacaga aacttcatgc agctcatcca cgatgattct ctcaccttta
       61 aagaggatat ccagaaggct caggtttcag gacagggtga tagtctccat gagcatatcg
      121 ctaacctcgc tggatctcct gcaatcaaga agggaatcct ccagactgtg aaggttgtgg
      181 atgagttggt gaaggtgatg ggaaggcata agcctgagaa catcgtgatc gaaatggcta
      241 gagagaacca gaccactcag aagggacaga agaactctag ggaaaggatg aagaggatcg
      301 aggaaggtat caaagagctt ggatctcaga tcctcaaaga gcaccctgtt gagaacactc
      361 agctccagaa tgagaagctc tacctctact acctccagaa cggaagggat atgtatgtgg
      421 atcaagagtt ggatatcaac aggctctctg attacgatgt tgatgctatc gtgccacagt
      481 cattcttgaa ggatgattct atcgataaca aggtgctcac caggtctgat aagaacaggg
      541 gtaagagtga taacgtgcca agtgaagagg ttgtgaagaa aatgaagaac tattggaggc
      601 agctcctcaa cgctaagctc atcactcaga gaaagttcga taacttgact aaggctgaga
      661 ggggaggact ctctgaattg gataaggcag gattcatcaa gaggcagctt gtggaaacca
      721 ggcagatcac taagcacgtt gcacagatcc tcgattctag gatgaacacc aagtacgatg
      781 agaacgataa gttgatcagg gaagtgaagg ttatcaccct caagtcaaag ctcgtgtctg
      841 atttcagaaa ggatttccaa ttctacaagg tgagggaaat caacaactac caccacgctc
      901 acgatgctta ccttaacgct gttgttggaa ccgctctcat caagaagtat cctaagctcg
      961 agtcagagtt cgtgtacggt gattacaagg tgtacgatgt gaggaagatg atcgctaagt
     1021 ctgagcaaga gatcggaaag gctaccgcta agtatttctt ctactctaac atcatgaatt
     1081 tcttcaagac cgagattacc ctcgctaacg gtgagatcag aaagaggcca ctcatcgaga
     1141 caaacggtga aacaggtgag atcgtgtggg ataagggaag ggatttcgct accgttagaa
     1201 aggtgctctc tatgccacag gtgaacatcg ttaagaaaac cgaggtgcag accggtggat
     1261 tctctaaaga gtctatcctc cctaagagga actctgataa gctcattgct aggaagaagg
     1321 attgggaccc taagaaatac ggtggtttcg attctcctac cgtggcttac tctgttctcg
     1381 ttgtggctaa ggttgagaag ggaaagagta agaagctcaa gtctgttaag gaacttctcg
     1441 gaatcactat catggaaagg tcatctttcg agaagaaccc aatcgatttc ctcgaggcta
     1501 agggatacaa agaggttaag aaggatctca tcatcaagct cccaaagtac tcactcttcg
     1561 aactcgagaa cggtagaaag aggatgctcg cttctgctgg tgagcttcaa aagggaaacg
     1621 agcttgctct cccatctaag tacgttaact ttctttacct cgcttctcac tacgagaagt
     1681 tgaagggatc tccagaagat aacgagcaga agcaactttt cgttgagcag cacaagcact
     1741 acttggatga gatcatcgag cagatctctg agttctctaa aagggtgatc ctcgctgatg
     1801 caaacctcga taaggtgttg tctgcttaca acaagcacag agataagcct atcagggaac
     1861 aggcagagaa catcatccat ctcttcaccc ttaccaacct cggtgctcct gctgctttca
     1921 agtacttcga tacaaccatc gataggaaga gatacacctc taccaaagaa gtgctcgatg
     1981 ctaccctcat ccatcagtct atcactggac tctacgagac taggatcgat ctctcacagc
     2041 tcggtggtga ttcaagggct gatcctaaga agaagaggaa ggtttgagct ttgagaccac
     2101 gaagtggctc ttcagtggac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     2161 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     2221 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2281 ctgataaatg cttcaataat attgaaaaag gaagagtatg cgctcacgca actggtccag
     2341 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     2401 ctgttttttt ggggtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt
     2461 gggtcgatgt ttgatgttat ggagcagcaa cgatgttacg cagcagggca gtcgccctaa
     2521 aacaaagtta aacatcatga gggaagcggt gatcgccgaa gtatcgactc aactatcaga
     2581 ggtagttggc gtcatcgagc gccatctcga accgacgttg ctggccgtac atttgtacgg
     2641 ctccgcagtg gatggcggcc tgaagccaca cagcgatatt gatttgctgg ttacggtgac
     2701 cgtaaggctt gatgaaacaa cgcggcgagc tttgatcaac gaccttttgg aaacttcggc
     2761 ttcccctgga gagagcgaga ttctccgcgc tgtagaagtc accattgttg tgcacgacga
     2821 catcattccg tggcgttatc cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa
     2881 tgacattctt gcaggtatct tcgagccagc cacgatcgac attgatctgg ctatcttgct
     2941 gacaaaagca agagaacata gcgttgcctt ggtaggtcca gcggcggagg aactctttga
     3001 tccggttcct gaacaggatc tatttgaggc gctaaatgaa accttaacgc tatggaactc
     3061 gccgcccgac tgggctggcg atgagcgaaa tgtagtgctt acgttgtccc gcatttggta
     3121 cagcgcagta accggcaaaa tcgcgccgaa ggatgtcgct gccgactggg caatggagcg
     3181 cctgccggcc cagtatcagc ccgtcatact tgaagctaga caggcttatc ttggacaaga
     3241 agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgtccatt acgtaaaagg
     3301 cgagatcacc aaggtagtcg gcaaataact gtcagaccaa gtttactcat atatacttta
     3361 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3421 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3481 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3541 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3601 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3661 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3721 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     3781 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     3841 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3901 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3961 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4021 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4081 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4141 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4201 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4261 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatc
     4321 actctgtggt ctcaaatgga taagaagtac tctatcggac tcgctatcgg aactaactct
     4381 gtgggatggg ctgtgatcac cgatgagtac aaggtgccat ctaagaagtt caaggttctc
     4441 ggaaacaccg ataggcactc tatcaagaaa aaccttatcg gtgctctcct cttcgattct
     4501 ggtgaaactg ctgaggctac cagactcaag agaaccgcta gaagaaggta caccagaaga
     4561 aagaacagga tctgctacct ccaagagatc ttctctaacg agatggctaa agtggatgat
     4621 tcattcttcc acaggctcga agagtcattc ctcgtggaag aagataagaa gcacgagagg
     4681 caccctatct tcggaaacat cgttgatgag gtggcatacc acgagaagta ccctactatc
     4741 taccacctca gaaagaagct cgttgattct actgataagg ctgatctcag gctcatctac
     4801 ctcgctctcg ctcacatgat caagttcaga ggacacttcc tcatcgaggg tgatctcaac
     4861 cctgataact ctgatgtgga taagttgttc atccagctcg tgcagaccta caaccagctt
     4921 ttcgaagaga accctatcaa cgcttcaggt gtggatgcta aggctatcct ctctgctagg
     4981 ctctctaagt caagaaggct tgagaacctc attgctcagc tccctggtga gaagaagaac
     5041 ggacttttcg gaaacttgat cgctctctct ctcggactca cccctaactt caagtctaac
     5101 ttcgatctcg ctgaggatgc aaagctccag ctctcaaagg atacctacga tgatgatctc
     5161 gataacctcc tcgctcagat cggagatcag tacgctgatt tgttcctcgc tgctaagaac
     5221 ctctctgatg ctatcctcct cagtgatatc ctcagagtga acaccgagat caccaaggct
     5281 ccactctcag cttctatgat caagagatac gatgagcacc accaggatct cacacttctc
     5341 aaggctcttg ttagacagca gctcccagag aagtacaaag agattttctt cgatcagtct
     5401 aagaacggat acgctggtta catcgatggt ggtgcatctc aagaagagtt ctacaagttc
     5461 atcaagccta tcctcgagaa gatggatgga accgaggaac tcctcgtgaa gctcaataga
     5521 gaggatcttc tcagaaagca gaggaccttc gataacggat ctatccctca tcagatccac
     5581 ctcggagagt tgcacgctat ccttagaagg caagaggatt tctacccatt cctcaaggat
     5641 aacagggaaa agattgagaa gattctcacc ttcagaatcc cttactacgt gggacctctc
     5701 gctagaggaa actcaagatt cgcttggatg accagaaagt ctgaggaaac catcacccct
     5761 tggaacttcg aagaggtggt ggataagggt gctagtgctc agtctttcat cgagaggatg
     5821 accaacttcg ataagaacct tccaaacgag aaggtgctcc ctaagcactc tttgctctac
     5881 gagtacttca ccgtgtacaa cgagttgacc aaggttaagt acgtgaccga gggaatgagg
     5941 aagcctgctt ttttgtcagg tgagcaaaag aaggctatcg ttgatctctt gttcaagacc
     6001 aacagaaagg tgaccgtgaa gcagctcaaa gaggattact tcaagaaaat cgagtgcttc
     6061 gattcagttg agatttctgg tgttgaggat aggttcaacg catctctcgg aacctaccac
     6121 gatctcctca agatcattaa ggataaggat ttcttggata acgaggaaaa cgaggatatc
     6181 ttggaggata tcgttcttac cctcaccctc tttgaagata gagagatgat tgaagaaagg
     6241 ctcaagacct acgctcatct cttcgatgat aaggtgatga agcagttgaa gagaagaaga
     6301 tacactggtt ggggaaggct ctcaagaaag ctcattaacg gaatcaggga taagcagtct
     6361 ggaaagacaa tccttgattt cctcaagt
//
