LOCUS       sequence-226832-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9_2 Golden Gate Level 0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  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 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"; journalName: "PLoS One.";
            date: "2019-01-9"; volume: "14(1):e0204778. doi"; pages: " 
            10.1371/journal.pone.0204778. eCollection 2019"
COMMENT     Sequence Label: pICSL90016
FEATURES             Location/Qualifiers
     source          1..6390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..58)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        126..230
                     /gene="bla"
                     /label=AmpR promoter
     CDS             452..1243
                     /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      1414..2002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1903..1922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2156..2173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2251..6354
                     /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             2251..6354
                     /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             6367..6387
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gctttgagac cacgaagtgg ctcttcagtg gacgaaaggg cctcgtgata cgcctatttt
       61 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
      121 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      181 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgcgctcac
      241 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
      301 tggcttgtta tgactgtttt tttttggggt acagtctatg cctcgggcat ccaagcagca
      361 agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg ttacgcagca
      421 gggcagtcgc cctaaaacaa agttaaacat catgagggaa gcggtgatcg ccgaagtatc
      481 gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc
      541 cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagcg atattgattt
      601 gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct
      661 tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat
      721 tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg
      781 agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga
      841 tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc
      901 ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt
      961 aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt
     1021 gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga
     1081 ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc
     1141 ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt
     1201 ccattacgta aaaggcgaga tcaccaaggt agtcggcaaa taactgtcag accaagttta
     1261 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1321 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1381 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1441 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1501 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1561 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1621 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1681 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1741 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1801 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1861 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1921 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1981 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2041 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2101 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2161 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2221 gccgattcat taatcactct gtggtctcaa atggataaga agtactctat cggactcgat
     2281 atcggaacta actctgtggg atgggctgtg atcaccgatg agtacaaggt gccatctaag
     2341 aagttcaagg ttctcggaaa caccgatagg cactctatca agaaaaacct tatcggtgct
     2401 ctcctcttcg attctggtga aactgctgag gctaccagac tcaagagaac cgctagaaga
     2461 aggtacacca gaagaaagaa caggatctgc tacctccaag agatcttctc taacgagatg
     2521 gctaaagtgg atgattcatt cttccacagg ctcgaagagt cattcctcgt ggaagaagat
     2581 aagaagcacg agaggcaccc tatcttcgga aacatcgttg atgaggtggc ataccacgag
     2641 aagtacccta ctatctacca cctcagaaag aagctcgttg attctactga taaggctgat
     2701 ctcaggctca tctacctcgc tctcgctcac atgatcaagt tcagaggaca cttcctcatc
     2761 gagggtgatc tcaaccctga taactctgat gtggataagt tgttcatcca gctcgtgcag
     2821 acctacaacc agcttttcga agagaaccct atcaacgctt caggtgtgga tgctaaggct
     2881 atcctctctg ctaggctctc taagtcaaga aggcttgaga acctcattgc tcagctccct
     2941 ggtgagaaga agaacggact tttcggaaac ttgatcgctc tctctctcgg actcacccct
     3001 aacttcaagt ctaacttcga tctcgctgag gatgcaaagc tccagctctc aaaggatacc
     3061 tacgatgatg atctcgataa cctcctcgct cagatcggag atcagtacgc tgatttgttc
     3121 ctcgctgcta agaacctctc tgatgctatc ctcctcagtg atatcctcag agtgaacacc
     3181 gagatcacca aggctccact ctcagcttct atgatcaaga gatacgatga gcaccaccag
     3241 gatctcacac ttctcaaggc tcttgttaga cagcagctcc cagagaagta caaagagatt
     3301 ttcttcgatc agtctaagaa cggatacgct ggttacatcg atggtggtgc atctcaagaa
     3361 gagttctaca agttcatcaa gcctatcctc gagaagatgg atggaaccga ggaactcctc
     3421 gtgaagctca atagagagga tcttctcaga aagcagagga ccttcgataa cggatctatc
     3481 cctcatcaga tccacctcgg agagttgcac gctatcctta gaaggcaaga ggatttctac
     3541 ccattcctca aggataacag ggaaaagatt gagaagattc tcaccttcag aatcccttac
     3601 tacgtgggac ctctcgctag aggaaactca agattcgctt ggatgaccag aaagtctgag
     3661 gaaaccatca ccccttggaa cttcgaagag gtggtggata agggtgctag tgctcagtct
     3721 ttcatcgaga ggatgaccaa cttcgataag aaccttccaa acgagaaggt gctccctaag
     3781 cactctttgc tctacgagta cttcaccgtg tacaacgagt tgaccaaggt taagtacgtg
     3841 accgagggaa tgaggaagcc tgcttttttg tcaggtgagc aaaagaaggc tatcgttgat
     3901 ctcttgttca agaccaacag aaaggtgacc gtgaagcagc tcaaagagga ttacttcaag
     3961 aaaatcgagt gcttcgattc agttgagatt tctggtgttg aggataggtt caacgcatct
     4021 ctcggaacct accacgatct cctcaagatc attaaggata aggatttctt ggataacgag
     4081 gaaaacgagg atatcttgga ggatatcgtt cttaccctca ccctctttga agatagagag
     4141 atgattgaag aaaggctcaa gacctacgct catctcttcg atgataaggt gatgaagcag
     4201 ttgaagagaa gaagatacac tggttgggga aggctctcaa gaaagctcat taacggaatc
     4261 agggataagc agtctggaaa gacaatcctt gatttcctca agtctgatgg attcgctaac
     4321 agaaacttca tgcagctcat ccacgatgat tctctcacct ttaaagagga tatccagaag
     4381 gctcaggttt caggacaggg tgatagtctc catgagcata tcgctaacct cgctggatct
     4441 cctgcaatca agaagggaat cctccagact gtgaaggttg tggatgagtt ggtgaaggtg
     4501 atgggaaggc ataagcctga gaacatcgtg atcgaaatgg ctagagagaa ccagaccact
     4561 cagaagggac agaagaactc tagggaaagg atgaagagga tcgaggaagg tatcaaagag
     4621 cttggatctc agatcctcaa agagcaccct gttgagaaca ctcagctcca gaatgagaag
     4681 ctctacctct actacctcca gaacggaagg gatatgtatg tggatcaaga gttggatatc
     4741 aacaggctct ctgattacga tgttgatcat atcgtgccac agtcattctt gaaggatgat
     4801 tctatcgata acaaggtgct caccaggtct gataagaaca ggggtaagag tgataacgtg
     4861 ccaagtgaag aggttgtgaa gaaaatgaag aactattgga ggcagctcct caacgctaag
     4921 ctcatcactc agagaaagtt cgataacttg actaaggctg agaggggagg actctctgaa
     4981 ttggataagg caggattcat caagaggcag cttgtggaaa ccaggcagat cactaagcac
     5041 gttgcacaga tcctcgattc taggatgaac accaagtacg atgagaacga taagttgatc
     5101 agggaagtga aggttatcac cctcaagtca aagctcgtgt ctgatttcag aaaggatttc
     5161 caattctaca aggtgaggga aatcaacaac taccaccacg ctcacgatgc ttaccttaac
     5221 gctgttgttg gaaccgctct catcaagaag tatcctaagc tcgagtcaga gttcgtgtac
     5281 ggtgattaca aggtgtacga tgtgaggaag atgatcgcta agtctgagca agagatcgga
     5341 aaggctaccg ctaagtattt cttctactct aacatcatga atttcttcaa gaccgagatt
     5401 accctcgcta acggtgagat cagaaagagg ccactcatcg agacaaacgg tgaaacaggt
     5461 gagatcgtgt gggataaggg aagggatttc gctaccgtta gaaaggtgct ctctatgcca
     5521 caggtgaaca tcgttaagaa aaccgaggtg cagaccggtg gattctctaa agagtctatc
     5581 ctccctaaga ggaactctga taagctcatt gctaggaaga aggattggga ccctaagaaa
     5641 tacggtggtt tcgattctcc taccgtggct tactctgttc tcgttgtggc taaggttgag
     5701 aagggaaaga gtaagaagct caagtctgtt aaggaacttc tcggaatcac tatcatggaa
     5761 aggtcatctt tcgagaagaa cccaatcgat ttcctcgagg ctaagggata caaagaggtt
     5821 aagaaggatc tcatcatcaa gctcccaaag tactcactct tcgaactcga gaacggtaga
     5881 aagaggatgc tcgcttctgc tggtgagctt caaaagggaa acgagcttgc tctcccatct
     5941 aagtacgtta actttcttta cctcgcttct cactacgaga agttgaaggg atctccagaa
     6001 gataacgagc agaagcaact tttcgttgag cagcacaagc actacttgga tgagatcatc
     6061 gagcagatct ctgagttctc taaaagggtg atcctcgctg atgcaaacct cgataaggtg
     6121 ttgtctgctt acaacaagca cagagataag cctatcaggg aacaggcaga gaacatcatc
     6181 catctcttca cccttaccaa cctcggtgct cctgctgctt tcaagtactt cgatacaacc
     6241 atcgatagga agagatacac ctctaccaaa gaagtgctcg atgctaccct catccatcag
     6301 tctatcactg gactctacga gactaggatc gatctctcac agctcggtgg tgattcaagg
     6361 gctgatccta agaagaagag gaaggtttga
//
