LOCUS       sequence-232257-        6708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9_3 (with intron) Golden Gate Level 0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6708)
  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 6708)
  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: BCJJ125
FEATURES             Location/Qualifiers
     source          1..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        80..184
                     /gene="bla"
                     /label=AmpR promoter
     CDS             406..1197
                     /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      1368..1956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1857..1876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2110..2127
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2208..2255
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             2262..2282
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2313..3752,3942..6605)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system"
                     /label=pcoCas9
                     /note="plant codon-optimized Cas9 gene containing the 
                     potato IV2 intron"
                     /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"
     intron          3753..3941
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             6612..6659
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(join(6702..6708,1..12))
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
       61 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
      121 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
      181 gagtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg gtaacggcgc
      241 agtggcggtt ttcatggctt gttatgactg tttttttttg gggtacagtc tatgcctcgg
      301 gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac
      361 gatgttacgc agcagggcag tcgccctaaa acaaagttaa acatcatgag ggaagcggtg
      421 atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa
      481 ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac
      541 agcgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct
      601 ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct
      661 gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
      721 gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc
      781 acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg
      841 gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
      901 ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat
      961 gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag
     1021 gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt
     1081 gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag
     1141 ttggaagaat ttgtccatta cgtaaaaggc gagatcacca aggtagtcgg caaataactg
     1201 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     1261 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     1321 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1381 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     1441 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     1501 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     1561 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     1621 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     1681 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     1741 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     1801 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     1861 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     1921 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     1981 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     2041 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     2101 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     2161 ctccccgcgc gttggccgat tcattaatca ctctgtggtc tcaaatggat tacaaggatg
     2221 atgatgataa ggattacaag gatgatgatg ataagatggc tccaaagaag aagagaaagg
     2281 ttggaatcca cggagttcca gctgctggag gtatggataa gaagtactct atcggacttg
     2341 acatcggaac caactctgtt ggatgggctg ttatcaccga tgagtacaag gttccatcta
     2401 agaagttcaa ggttcttgga aacaccgata gacactctat caagaagaac cttatcggtg
     2461 ctcttctttt cgattctgga gaaaccgctg aggctaccag attgaagaga accgctagaa
     2521 gaagatacac cagaagaaag aacagaatct gctaccttca ggaaatcttc tctaacgaga
     2581 tggctaaggt tgatgattct ttcttccaca gacttgagga gtctttcctt gttgaggagg
     2641 ataagaagca cgagagacac ccaatcttcg gaaacatcgt tgatgaggtt gcttaccacg
     2701 agaagtaccc aaccatctac caccttagaa agaagttggt tgattctacc gataaggctg
     2761 atcttagact tatctacctt gctcttgctc acatgatcaa gttcagagga cacttcctta
     2821 tcgagggaga tcttaaccca gataactctg atgttgataa gttgttcatc cagcttgttc
     2881 agacctacaa ccagcttttc gaggagaacc caatcaacgc ttctggagtt gatgctaagg
     2941 ctatcctttc tgctagactt tctaagtctc gtagacttga gaaccttatc gctcagcttc
     3001 caggagagaa gaagaacgga cttttcggaa accttatcgc tctttctctt ggacttaccc
     3061 caaacttcaa gtctaacttc gatcttgctg aggatgctaa gttgcagctt tctaaggata
     3121 cctacgatga tgatcttgat aaccttcttg ctcagatcgg agatcagtac gctgatcttt
     3181 tccttgctgc taagaacctt tctgatgcta tccttctttc tgacatcctt agagttaaca
     3241 ccgagatcac caaggctcca ctttctgctt ctatgatcaa gagatacgat gagcaccacc
     3301 aggatcttac ccttttgaag gctcttgtta gacagcagct tccagagaag tacaaggaaa
     3361 tcttcttcga tcagtctaag aacggatacg ctggatacat cgatggagga gcttctcagg
     3421 aggagttcta caagttcatc aagccaatcc ttgagaagat ggatggaacc gaggagcttc
     3481 ttgttaagtt gaacagagag gatcttctta gaaagcagag aaccttcgat aacggatcta
     3541 tcccacacca gatccacctt ggagagcttc acgctatcct tcgtagacag gaggatttct
     3601 acccattctt gaaggataac agagagaaga tcgagaagat ccttaccttc agaatcccat
     3661 actacgttgg accacttgct agaggaaact ctcgtttcgc ttggatgacc agaaagtctg
     3721 aggagacaat caccccttgg aacttcgagg aggtaagttt ctgcttctac ctttgatata
     3781 tatataataa ttatcattaa ttagtagtaa tataatattt caaatatttt tttcaaaata
     3841 aaagaatgta gtatatagca attgcttttc tgtagtttat aagtgtgtat attttaattt
     3901 ataacttttc taatatatga ccaaaatttg ttgatgtgca ggttgttgat aagggagctt
     3961 ctgctcagtc tttcatcgag agaatgacca acttcgataa gaaccttcca aacgagaagg
     4021 ttcttccaaa gcactctctt ctttacgagt acttcaccgt ttacaacgag cttaccaagg
     4081 ttaagtacgt taccgaggga atgagaaagc cagctttcct ttctggagag cagaagaagg
     4141 ctatcgttga tcttcttttc aagaccaaca gaaaggttac cgttaagcag ttgaaggagg
     4201 attacttcaa gaagatcgag tgcttcgatt ctgttgaaat ctctggagtt gaggatagat
     4261 tcaacgcttc tcttggaacc taccacgatc ttttgaagat catcaaggat aaggatttcc
     4321 ttgataacga ggagaacgag gacatccttg aggacatcgt tcttaccctt acccttttcg
     4381 aggatagaga gatgatcgag gagagactca agacctacgc tcaccttttc gatgataagg
     4441 ttatgaagca gttgaagaga agaagataca ccggatgggg tagactttct cgtaagttga
     4501 tcaacggaat cagagataag cagtctggaa agaccatcct tgatttcttg aagtctgatg
     4561 gattcgctaa cagaaacttc atgcagctta tccacgatga ttctcttacc ttcaaggagg
     4621 acatccagaa ggctcaggtt tctggacagg gagattctct tcacgagcac atcgctaacc
     4681 ttgctggatc tccagctatc aagaagggaa tccttcagac cgttaaggtt gttgatgagc
     4741 ttgttaaggt tatgggtaga cacaagccag agaacatcgt tatcgagatg gctagagaga
     4801 accagaccac ccagaaggga cagaagaact ctcgtgagag aatgaagaga atcgaggagg
     4861 gaatcaagga gcttggatct caaatcttga aggagcaccc agttgagaac acccagcttc
     4921 agaacgagaa gttgtacctt tactaccttc agaacggaag agatatgtac gttgatcagg
     4981 agcttgacat caacagactt tctgattacg atgttgatca catcgttcca cagtctttct
     5041 tgaaggatga ttctatcgat aacaaggttc ttacccgttc tgataagaac agaggaaagt
     5101 ctgataacgt tccatctgag gaggttgtta agaagatgaa gaactactgg agacagcttc
     5161 ttaacgctaa gttgatcacc cagagaaagt tcgataacct taccaaggct gagagaggag
     5221 gactttctga gcttgataag gctggattca tcaagagaca gcttgttgaa accagacaga
     5281 tcaccaagca cgttgctcag atccttgatt ctcgtatgaa caccaagtac gatgagaacg
     5341 ataagttgat cagagaggtt aaggttatca ccttgaagtc taagttggtt tctgatttca
     5401 gaaaggattt ccagttctac aaggttagag agatcaacaa ctaccaccac gctcacgatg
     5461 cttaccttaa cgctgttgtt ggaaccgctc ttatcaagaa gtacccaaag ttggagtctg
     5521 agttcgttta cggagattac aaggtttacg atgttagaaa gatgatcgct aagtctgagc
     5581 aggagatcgg aaaggctacc gctaagtact tcttctactc taacatcatg aacttcttca
     5641 agaccgagat cacccttgct aacggagaga tcagaaaaag accacttatc gagacaaacg
     5701 gagagacagg agagatcgtt tgggataagg gaagagattt cgctaccgtt agaaaggttc
     5761 tttctatgcc acaggttaac atcgttaaga aaaccgaggt tcagaccgga ggattctcta
     5821 aggagtctat ccttccaaag agaaactctg ataagttgat cgctagaaag aaggattggg
     5881 acccaaagaa gtacggagga ttcgattctc caaccgttgc ttactctgtt cttgttgttg
     5941 ctaaggttga gaagggaaag tctaagaagt tgaagtctgt taaggagctt cttggaatca
     6001 ccatcatgga gcgttcttct ttcgagaaga acccaatcga tttccttgag gctaagggat
     6061 acaaggaggt taagaaggat cttatcatca agttgccaaa gtactctctt ttcgagcttg
     6121 agaacggaag aaagagaatg cttgcttctg ctggagagct tcagaaggga aacgagcttg
     6181 ctcttccatc taagtacgtt aacttccttt accttgcttc tcactacgag aagttgaagg
     6241 gatctccaga ggataacgag cagaagcagc ttttcgttga gcagcacaag cactaccttg
     6301 atgagatcat cgagcaaatc tctgagttct ctaagagagt tatccttgct gatgctaacc
     6361 ttgataaggt tctttctgct tacaacaagc acagagataa gccaatcaga gagcaggctg
     6421 agaacatcat ccaccttttc acccttacca accttggtgc tccagctgct ttcaagtact
     6481 tcgataccac catcgataga aaaagataca cctctaccaa ggaggttctt gatgctaccc
     6541 ttatccacca gtctatcacc ggactttacg agacaagaat cgatctttct cagcttggag
     6601 gagatggtgg caagaggcca gctgctacca agaaggctgg acaggctaag aagaagaagt
     6661 gagctttgag accacgaagt ggctcttcag tggacgaaag ggcctcgt
//
