LOCUS       sequence-91736-e        4140 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  2x35S promoting Glycing Max codon optimized Cas9 with hygromycin 
            plant selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4140)
  AUTHORS   Michno JM, Wang X, Liu J, Curtin SJ, Kono TJ, Stupar RM
  TITLE     CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a 
            new web-tool and a modified Cas9 enzyme.
  JOURNAL   GM Crops Food. 2015;6(4):243-52. doi: 10.1080/21645698.2015.1106063.
  PUBMED    26479970
REFERENCE   2  (bases 1 to 4140)
  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"; journalName: "GM Crops 
            Food."; date: "2015"; volume: "6(4):243-52. doi"; pages: " 
            10.1080/21645698.2015.1106063"
COMMENT     Sequence Label: Cas9 MDC32
FEATURES             Location/Qualifiers
     source          1..4140
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1..4104
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1..4104
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     RBS             4017..4025
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4111..4131
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 atggataaga agtactccat cggactggat atcggaacta actccgtggg atgggctgtg
       61 atcactgatg agtacaaggt gccatccaag aagttcaagg tgctgggaaa cactgataga
      121 cactccatca agaagaacct gatcggagct ctgctgttcg attccggaga gactgctgag
      181 gctactagac tgaagagaac tgctagaaga agatacacta gaagaaagaa cagaatctgc
      241 tacctgcaag agatcttctc caacgagatg gctaaggtgg atgattcctt cttccacaga
      301 ctggaggagt ccttcctggt ggaggaggat aagaagcacg agagacaccc aatcttcgga
      361 aacatcgtgg atgaggtggc ttaccacgag aagtacccaa ctatctacca cctgagaaag
      421 aagctggtgg attccactga taaggctgat ctgagactga tctacctggc tctggctcac
      481 atgatcaagt tcagaggaca cttcctgatc gagggagatc tgaacccaga taactccgat
      541 gtggataagc tgttcatcca actggtgcaa acttacaacc aactgttcga ggagaaccca
      601 atcaacgctt ccggagtgga tgctaaggct atcctgtccg ctagactgtc caagtccaga
      661 agactggaga acctgatcgc tcaactgcca ggagagaaga agaacggact gttcggaaac
      721 ctgatcgctc tgtccctggg actgactcca aacttcaagt ccaacttcga tctggctgag
      781 gatgctaagc tgcaactgtc caaggatact tacgatgatg atctggataa cctgctggct
      841 caaatcggag atcaatacgc tgatctgttc ctggctgcta agaacctgtc cgatgctatc
      901 ctgctgtccg atatcctgag agtgaacact gagatcacta aggctccact gtccgcttcc
      961 atgatcaaga gatacgatga gcaccaccaa gatctgactc tgctgaaggc tctggtgaga
     1021 caacaactgc cagagaagta caaggagatc ttcttcgatc aatccaagaa cggatacgct
     1081 ggatacatcg atggaggagc ttcccaagag gagttctaca agttcatcaa gccaatcctg
     1141 gagaagatgg atggaactga ggagctgctg gtgaagctga acagagagga tctgctgaga
     1201 aagcaaagaa ctttcgataa cggatccatc ccacaccaaa tccacctggg agagctgcac
     1261 gctatcctga gaagacaaga ggatttctac ccattcctga aggataacag agagaagatc
     1321 gagaagatcc tgactttcag aatcccatac tacgtgggac cactggctag aggaaactcc
     1381 agattcgctt ggatgactag aaagtccgag gagactatca ctccatggaa cttcgaggag
     1441 gtggtggata agggagcttc cgctcaatcc ttcatcgaga gaatgactaa cttcgataag
     1501 aacctgccaa acgagaaggt gctgccaaag cactccctgc tgtacgagta cttcactgtg
     1561 tacaacgagc tgactaaggt gaagtacgtg actgagggaa tgagaaagcc agctttcctg
     1621 tccggagagc aaaagaaggc tatcgtggat ctgctgttca agactaacag aaaggtgact
     1681 gtgaagcaac tgaaggagga ttacttcaag aagatcgagt gcttcgattc cgtggagatc
     1741 tccggagtgg aggatagatt caacgcttcc ctgggaactt accacgatct gctgaagatc
     1801 atcaaggata aggatttcct ggataacgag gagaacgagg atatcctgga ggatatcgtg
     1861 ctgactctga ctctgttcga ggatagagag atgatcgagg agagactgaa gacttacgct
     1921 cacctgttcg atgataaggt gatgaagcaa ctgaagagaa gaagatacac tggatgggga
     1981 agactgtcca gaaagctgat caacggaatc agagataagc aatccggaaa gactatcctg
     2041 gatttcctga agtccgatgg attcgctaac agaaacttca tgcaactgat ccacgatgat
     2101 tccctgactt tcaaggagga tatccaaaag gctcaagtgt ccggacaagg agattccctg
     2161 cacgagcaca tcgctaacct ggctggatcc ccagctatca agaagggaat cctgcaaact
     2221 gtgaaggtgg tggatgagct ggtgaaggtg atgggaagac acaagccaga gaacatcgtg
     2281 atcgagatgg ctagagagaa ccaaactact caaaagggac aaaagaactc cagagagaga
     2341 atgaagagaa tcgaggaggg aatcaaggag ctgggatccc aaatcctgaa ggagcaccca
     2401 gtggagaaca ctcaactgca aaacgagaag ctgtacctgt actacctgca aaacggaaga
     2461 gatatgtacg tggatcaaga gctggatatc aacagactgt ccgattacga tgtggatcac
     2521 atcgtgccac aatccttcct gaaggatgat tccatcgata acaaggtgct gactagatcc
     2581 gataagaaca gaggaaagtc cgataacgtg ccatccgagg aggtggtgaa gaagatgaag
     2641 aactactgga gacaactgct gaacgctaag ctgatcactc aaagaaagtt cgataacctg
     2701 actaaggctg agagaggagg actgtccgag ctggataagg ctggattcat caagagacaa
     2761 ctggtggaga ctagacaaat cactaagcac gtggctcaaa tcctggattc cagaatgaac
     2821 actaagtacg atgagaacga taagctgatc agagaggtga aggtgatcac tctgaagtcc
     2881 aagctggtgt ccgatttcag aaaggatttc caattctaca aggtgagaga gatcaacaac
     2941 taccaccacg ctcacgatgc ttacctgaac gctgtggtgg gaactgctct gatcaagaag
     3001 tacccaaagc tggagtccga gttcgtgtac ggagattaca aggtgtacga tgtgagaaag
     3061 atgatcgcta agtccgagca agagatcgga aaggctactg ctaagtactt cttctactcc
     3121 aacatcatga acttcttcaa gactgagatc actctggcta acggagagat cagaaagaga
     3181 ccactgatcg agactaacgg agagactgga gagatcgtgt gggataaggg aagagatttc
     3241 gctactgtga gaaaggtgct gtccatgcca caagtgaaca tcgtgaagaa gactgaggtg
     3301 caaactggag gattctccaa ggagtccatc ctgccaaaga gaaactccga taagctgatc
     3361 gctagaaaga aggattggga tccaaagaag tacggaggat tcgattcccc aactgtggct
     3421 tactccgtgc tggtggtggc taaggtggag aagggaaagt ccaagaagct gaagtccgtg
     3481 aaggagctgc tgggaatcac tatcatggag agatcctcct tcgagaagaa cccaatcgat
     3541 ttcctggagg ctaagggata caaggaggtg aagaaggatc tgatcatcaa gctgccaaag
     3601 tactccctgt tcgagctgga gaacggaaga aagagaatgc tggcttccgc tggagagctg
     3661 caaaagggaa acgagctggc tctgccatcc aagtacgtga acttcctgta cctggcttcc
     3721 cactacgaga agctgaaggg atccccagag gataacgagc aaaagcaact gttcgtggag
     3781 caacacaagc actacctgga tgagatcatc gagcaaatct ccgagttctc caagagagtg
     3841 atcctggctg atgctaacct ggataaggtg ctgtccgctt acaacaagca cagagataag
     3901 ccaatcagag agcaagctga gaacatcatc cacctgttca ctctgactaa cctgggagct
     3961 ccagctgctt tcaagtactt cgatactact atcgatagaa agagatacac ttccactaag
     4021 gaggtgctgg atgctactct gatccaccaa tccatcactg gactgtacga gactagaatc
     4081 gatctgtccc aactgggagg agatctgcaa ccaaagaaga agagaaaggt gggaggatga
//
