LOCUS       sequence-226834-        6522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9_4 Golden Gate Level 0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6522)
  AUTHORS   Castel B, Tomlinson L, Locci F, Yang Y, Jones JDG
  TITLE     Optimization of T-DNA architecture for Cas9-mediated mutagenesis in 
            Arabidopsis.
  JOURNAL   PLoS One. 2019 Jan 9;14(1):e0204778. doi: 
            10.1371/journal.pone.0204778. eCollection 2019.
  PUBMED    30625150
REFERENCE   2  (bases 1 to 6522)
  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: "PLoS One.";
            date: "2019-01-9"; volume: "14(1):e0204778. doi"; pages: " 
            10.1371/journal.pone.0204778. eCollection 2019"
COMMENT     Sequence Label: BCJJ180
FEATURES             Location/Qualifiers
     source          1..6522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        79..183
                     /gene="bla"
                     /label=AmpR promoter
     CDS             405..1196
                     /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      1367..1955
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1856..1875
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2109..2126
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2207..2272
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2279..2299
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2324..6424
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             2324..6424
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6425..6472
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(join(6515..6522,1..11))
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
       61 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
      121 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
      181 agtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg taacggcgca
      241 gtggcggttt tcatggcttg ttatgactgt ttttttttgg ggtacagtct atgcctcggg
      301 catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg
      361 atgttacgca gcagggcagt cgccctaaaa caaagttaaa catcatgagg gaagcggtga
      421 tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac
      481 cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca
      541 gcgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt
      601 tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg
      661 tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg
      721 aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca
      781 cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg
      841 taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc
      901 taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg
      961 tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg
     1021 atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg
     1081 aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt
     1141 tggaagaatt tgtccattac gtaaaaggcg agatcaccaa ggtagtcggc aaataactgt
     1201 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     1261 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     1321 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     1381 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     1441 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     1501 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     1561 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     1621 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     1681 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     1741 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     1801 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     1861 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     1921 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     1981 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2041 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2101 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     2161 tccccgcgcg ttggccgatt cattaatcac tctgtggtct caaatggact ataaggacca
     2221 cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggcccc
     2281 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccgacaaga agtacagcat
     2341 cggcctggac atcggcacca actctgtggg ctgggccgtg atcaccgacg agtacaaggt
     2401 gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca agaagaacct
     2461 gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc tgaagagaac
     2521 cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag agatcttcag
     2581 caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt ccttcctggt
     2641 ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg acgaggtggc
     2701 ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg acagcaccga
     2761 caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt tccggggcca
     2821 cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc tgttcatcca
     2881 gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca gcggcgtgga
     2941 cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa atctgatcgc
     3001 ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc tgagcctggg
     3061 cctgaccccc aacttcaaga gcaacttcga cctggccgag gatgccaaac tgcagctgag
     3121 caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg accagtacgc
     3181 cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg acatcctgag
     3241 agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaaga gatacgacga
     3301 gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta
     3361 caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg acggcggagc
     3421 cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg acggcaccga
     3481 ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga ccttcgacaa
     3541 cggcagcatc ccccaccaga tccacctggg agagctgcac gccattctgc ggcggcagga
     3601 agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc tgaccttccg
     3661 catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct ggatgaccag
     3721 aaagagcgag gaaaccatca ccccctggaa cttcgaggaa gtggtggaca agggcgcttc
     3781 cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca acgagaaggt
     3841 gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc tgaccaaagt
     3901 gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc
     3961 catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga
     4021 ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt
     4081 caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca aggacttcct
     4141 ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga cactgtttga
     4201 ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg acgacaaagt
     4261 gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc ggaagctgat
     4321 caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga agtccgacgg
     4381 cttcgccaac agaaacttca tgcagctgat ccacgacgac agcctgacct ttaaagagga
     4441 catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca ttgccaatct
     4501 ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg tggacgagct
     4561 cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg ccagagagaa
     4621 ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga tcgaagaggg
     4681 catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca cccagctgca
     4741 gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg tggaccagga
     4801 actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc agagctttct
     4861 gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc ggggcaagag
     4921 cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc ggcagctgct
     4981 gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg agagaggcgg
     5041 cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa cccggcagat
     5101 cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg acgagaatga
     5161 caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt ccgatttccg
     5221 gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg cccacgacgc
     5281 ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc tggaaagcga
     5341 gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca agagcgagca
     5401 ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga actttttcaa
     5461 gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg agacaaacgg
     5521 cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc ggaaagtgct
     5581 gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa
     5641 agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga aggactggga
     5701 ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc tggtggtggc
     5761 caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc tggggatcac
     5821 catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag ccaagggcta
     5881 caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt tcgagctgga
     5941 aaacggccgg aagagaatgc tggcctctgc cggcgaactg cagaagggaa acgaactggc
     6001 cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga agctgaaggg
     6061 ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc actacctgga
     6121 cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg acgctaatct
     6181 ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag agcaggccga
     6241 gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct tcaagtactt
     6301 tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg acgccaccct
     6361 gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc agctgggagg
     6421 cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agtaagcttt
     6481 gagaccacga agtggctctt cagtggacga aagggcctcg tg
//
