LOCUS       sequence-210901-        6388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AtCAS9 nickase D10A 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: pORFE0002
FEATURES             Location/Qualifiers
     source          1..6388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(24..42)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        110..214
                     /gene="bla"
                     /label=AmpR promoter
     CDS             434..1225
                     /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      1396..1984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1885..1904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2138..2155
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             join(2233..2259,2260..2262,2263..6336)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6349..6369
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gtggctcttc agtggacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
       61 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
      121 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
      181 ataaatgctt caataatatt gaaaaaggaa gagtatgcgc tcacgcaact ggtccagaac
      241 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg
      301 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
      361 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
      421 aaagttaaac atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
      481 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
      541 cgcagtggat ggcggcctga agccacacag cgatattgat ttgctggtta cggtgaccgt
      601 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
      661 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
      721 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
      781 cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac
      841 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
      901 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
      961 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     1021 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     1081 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     1141 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccattacg taaaaggcga
     1201 gatcaccaag gtagtcggca aataactgtc agaccaagtt tactcatata tactttagat
     1261 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1321 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1381 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1441 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1501 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1561 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1621 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1681 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1741 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1801 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1861 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1921 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1981 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2041 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2101 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2161 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatcact
     2221 ctgtggtctc aaatggataa gaagtactct atcggactcg ctatcggaac taactctgtg
     2281 ggatgggctg tgatcaccga tgagtacaag gtgccatcta agaagttcaa ggttctcgga
     2341 aacaccgata ggcactctat caagaaaaac cttatcggtg ctctcctctt cgattctggt
     2401 gaaactgctg aggctaccag actcaagaga accgctagaa gaaggtacac cagaagaaag
     2461 aacaggatct gctacctcca agagatcttc tctaacgaga tggctaaagt ggatgattca
     2521 ttcttccaca ggctcgaaga gtcattcctc gtggaagaag ataagaagca cgagaggcac
     2581 cctatcttcg gaaacatcgt tgatgaggtg gcataccacg agaagtaccc tactatctac
     2641 cacctcagaa agaagctcgt tgattctact gataaggctg atctcaggct catctacctc
     2701 gctctcgctc acatgatcaa gttcagagga cacttcctca tcgagggtga tctcaaccct
     2761 gataactctg atgtggataa gttgttcatc cagctcgtgc agacctacaa ccagcttttc
     2821 gaagagaacc ctatcaacgc ttcaggtgtg gatgctaagg ctatcctctc tgctaggctc
     2881 tctaagtcaa gaaggcttga gaacctcatt gctcagctcc ctggtgagaa gaagaacgga
     2941 cttttcggaa acttgatcgc tctctctctc ggactcaccc ctaacttcaa gtctaacttc
     3001 gatctcgctg aggatgcaaa gctccagctc tcaaaggata cctacgatga tgatctcgat
     3061 aacctcctcg ctcagatcgg agatcagtac gctgatttgt tcctcgctgc taagaacctc
     3121 tctgatgcta tcctcctcag tgatatcctc agagtgaaca ccgagatcac caaggctcca
     3181 ctctcagctt ctatgatcaa gagatacgat gagcaccacc aggatctcac acttctcaag
     3241 gctcttgtta gacagcagct cccagagaag tacaaagaga ttttcttcga tcagtctaag
     3301 aacggatacg ctggttacat cgatggtggt gcatctcaag aagagttcta caagttcatc
     3361 aagcctatcc tcgagaagat ggatggaacc gaggaactcc tcgtgaagct caatagagag
     3421 gatcttctca gaaagcagag gaccttcgat aacggatcta tccctcatca gatccacctc
     3481 ggagagttgc acgctatcct tagaaggcaa gaggatttct acccattcct caaggataac
     3541 agggaaaaga ttgagaagat tctcaccttc agaatccctt actacgtggg acctctcgct
     3601 agaggaaact caagattcgc ttggatgacc agaaagtctg aggaaaccat caccccttgg
     3661 aacttcgaag aggtggtgga taagggtgct agtgctcagt ctttcatcga gaggatgacc
     3721 aacttcgata agaaccttcc aaacgagaag gtgctcccta agcactcttt gctctacgag
     3781 tacttcaccg tgtacaacga gttgaccaag gttaagtacg tgaccgaggg aatgaggaag
     3841 cctgcttttt tgtcaggtga gcaaaagaag gctatcgttg atctcttgtt caagaccaac
     3901 agaaaggtga ccgtgaagca gctcaaagag gattacttca agaaaatcga gtgcttcgat
     3961 tcagttgaga tttctggtgt tgaggatagg ttcaacgcat ctctcggaac ctaccacgat
     4021 ctcctcaaga tcattaagga taaggatttc ttggataacg aggaaaacga ggatatcttg
     4081 gaggatatcg ttcttaccct caccctcttt gaagatagag agatgattga agaaaggctc
     4141 aagacctacg ctcatctctt cgatgataag gtgatgaagc agttgaagag aagaagatac
     4201 actggttggg gaaggctctc aagaaagctc attaacggaa tcagggataa gcagtctgga
     4261 aagacaatcc ttgatttcct caagtctgat ggattcgcta acagaaactt catgcagctc
     4321 atccacgatg attctctcac ctttaaagag gatatccaga aggctcaggt ttcaggacag
     4381 ggtgatagtc tccatgagca tatcgctaac ctcgctggat ctcctgcaat caagaaggga
     4441 atcctccaga ctgtgaaggt tgtggatgag ttggtgaagg tgatgggaag gcataagcct
     4501 gagaacatcg tgatcgaaat ggctagagag aaccagacca ctcagaaggg acagaagaac
     4561 tctagggaaa ggatgaagag gatcgaggaa ggtatcaaag agcttggatc tcagatcctc
     4621 aaagagcacc ctgttgagaa cactcagctc cagaatgaga agctctacct ctactacctc
     4681 cagaacggaa gggatatgta tgtggatcaa gagttggata tcaacaggct ctctgattac
     4741 gatgttgatc atatcgtgcc acagtcattc ttgaaggatg attctatcga taacaaggtg
     4801 ctcaccaggt ctgataagaa caggggtaag agtgataacg tgccaagtga agaggttgtg
     4861 aagaaaatga agaactattg gaggcagctc ctcaacgcta agctcatcac tcagagaaag
     4921 ttcgataact tgactaaggc tgagagggga ggactctctg aattggataa ggcaggattc
     4981 atcaagaggc agcttgtgga aaccaggcag atcactaagc acgttgcaca gatcctcgat
     5041 tctaggatga acaccaagta cgatgagaac gataagttga tcagggaagt gaaggttatc
     5101 accctcaagt caaagctcgt gtctgatttc agaaaggatt tccaattcta caaggtgagg
     5161 gaaatcaaca actaccacca cgctcacgat gcttacctta acgctgttgt tggaaccgct
     5221 ctcatcaaga agtatcctaa gctcgagtca gagttcgtgt acggtgatta caaggtgtac
     5281 gatgtgagga agatgatcgc taagtctgag caagagatcg gaaaggctac cgctaagtat
     5341 ttcttctact ctaacatcat gaatttcttc aagaccgaga ttaccctcgc taacggtgag
     5401 atcagaaaga ggccactcat cgagacaaac ggtgaaacag gtgagatcgt gtgggataag
     5461 ggaagggatt tcgctaccgt tagaaaggtg ctctctatgc cacaggtgaa catcgttaag
     5521 aaaaccgagg tgcagaccgg tggattctct aaagagtcta tcctccctaa gaggaactct
     5581 gataagctca ttgctaggaa gaaggattgg gaccctaaga aatacggtgg tttcgattct
     5641 cctaccgtgg cttactctgt tctcgttgtg gctaaggttg agaagggaaa gagtaagaag
     5701 ctcaagtctg ttaaggaact tctcggaatc actatcatgg aaaggtcatc tttcgagaag
     5761 aacccaatcg atttcctcga ggctaaggga tacaaagagg ttaagaagga tctcatcatc
     5821 aagctcccaa agtactcact cttcgaactc gagaacggta gaaagaggat gctcgcttct
     5881 gctggtgagc ttcaaaaggg aaacgagctt gctctcccat ctaagtacgt taactttctt
     5941 tacctcgctt ctcactacga gaagttgaag ggatctccag aagataacga gcagaagcaa
     6001 cttttcgttg agcagcacaa gcactacttg gatgagatca tcgagcagat ctctgagttc
     6061 tctaaaaggg tgatcctcgc tgatgcaaac ctcgataagg tgttgtctgc ttacaacaag
     6121 cacagagata agcctatcag ggaacaggca gagaacatca tccatctctt cacccttacc
     6181 aacctcggtg ctcctgctgc tttcaagtac ttcgatacaa ccatcgatag gaagagatac
     6241 acctctacca aagaagtgct cgatgctacc ctcatccatc agtctatcac tggactctac
     6301 gagactagga tcgatctctc acagctcggt ggtgattcaa gggctgatcc taagaagaag
     6361 aggaaggttt gagctttgag accacgaa
//
