LOCUS       sequence-252664-        6456 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 SpCas9, wheat codon optimized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6456)
  AUTHORS   Hahn F, Korolev A, Sanjurjo Loures L, Nekrasov V
  TITLE     A modular cloning toolkit for genome editing in plants.
  JOURNAL   BMC Plant Biol. 2020 Apr 23;20(1):179. doi: 
            10.1186/s12870-020-02388-2.
  PUBMED    32326901
REFERENCE   2  (bases 1 to 6456)
  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: "BMC Plant 
            Biol."; date: "2020-04-23"; volume: "20(1):179. doi"; pages: " 
            10.1186/s12870-020-02388-2"
COMMENT     Sequence Label: pFH13
FEATURES             Location/Qualifiers
     source          1..6456
                     /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             436..1227
                     /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      1398..1986
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1887..1906
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2140..2157
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2241..2261
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2286..6389
                     /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             2286..6389
                     /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             6390..6437
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
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 tttttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
      361 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
      421 acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
      481 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
      541 tccgcagtgg atggcggcct gaagccacac agcgatattg atttgctggt tacggtgacc
      601 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
      661 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
      721 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
      781 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
      841 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
      901 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
      961 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     1021 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     1081 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     1141 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccatta cgtaaaaggc
     1201 gagatcacca aggtagtcgg caaataactg tcagaccaag tttactcata tatactttag
     1261 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     1321 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     1381 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     1441 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     1501 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     1561 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     1621 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     1681 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     1741 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     1801 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     1861 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     1921 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     1981 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2041 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2101 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2161 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatca
     2221 ctctgtggtc tcaaatggca cctaaaaaaa agaggaaggt ggggatacat ggggtgccag
     2281 ctgcaatgga taaaaagtac tctatcgggc tcgatatcgg cactaactcc gtggggtggg
     2341 ccgtcatcac agacgaatac aaggtcccat cgaaaaaatt taaagttttg ggcaataccg
     2401 acagacactc tatcaaaaag aacctgattg gagcgctttt gtttgattct ggagaaacgg
     2461 ccgaagccac ccggctcaag cggaccgccc gtcggcgcta tactcgtcgg aagaaccgca
     2521 tttgttacct tcaggagatc ttttcaaatg agatggctaa ggtggacgat tctttctttc
     2581 accggctgga ggagtccttc cttgtcgaag aggacaaaaa acacgagagg catccaatat
     2641 tcggcaacat tgtggatgaa gtggcatatc acgagaagta ccccaccata taccatctta
     2701 gaaagaaact tgtcgactcc acggacaagg ccgaccttcg tctgatttac ctcgcgctcg
     2761 cccatatgat caagtttcgg ggccatttcc tcatcgaggg agacttgaat cctgacaatt
     2821 ctgacgtcga caaattgttt atccaactcg tgcaaacgta caaccaactc tttgaggaga
     2881 acccaataaa cgcctccgga gttgatgcaa aagctatcct gagcgcgcgc ctttcgaagt
     2941 cccggaggct ggagaacttg atcgcccaac tcccaggcga aaagaagaac ggcttgtttg
     3001 gcaatcttat tgccctgagc ctcgggctta cacccaactt taagtcaaac ttcgacctgg
     3061 cagaggatgc taagctccag ctctcgaagg acacctacga cgacgatttg gacaacctct
     3121 tggcccaaat cggcgatcag tatgccgatt tgttcctcgc cgccaaaaac ctctcggatg
     3181 cgattctgct ctctgacatc ttgcgcgtga acaccgagat aacgaaggcg ccgctgtctg
     3241 cgtccatgat caagcggtac gacgagcacc atcaggacct gaccctcctt aaggcgttgg
     3301 tgcgccaaca attgcccgag aagtataagg aaatcttctt cgatcaatcc aagaacggct
     3361 acgccggata catagacgga ggggcctccc aggaggagtt ctataagttt atcaagccta
     3421 ttcttgaaaa gatggatggc accgaggagc tcctcgttaa acttaacagg gaggaccttc
     3481 tccgtaagca aagaaccttc gataatgggt caatacccca ccaaatccac ctcggagagc
     3541 tccacgcgat cctcagacgg caggaggatt tttatccatt cctcaaagat aaccgtgaga
     3601 aaatcgaaaa gatcctgacc ttccgcatcc catactacgt cgggcctttg gcgcggggta
     3661 acagcagatt cgcctggatg accaggaaat cagaagagac aataaccccg tggaacttcg
     3721 aagaggttgt tgacaagggt gcgtccgcac agtctttcat cgaaaggatg actaactttg
     3781 acaaaaacct tcccaatgag aaagtgctcc ctaagcattc cctgttgtat gagtacttca
     3841 ccgtctacaa cgagttgacc aaagtcaagt acgtcacaga ggggatgcgc aagccagcct
     3901 ttctcagcgg cgagcaaaag aaggctattg ttgacctctt gttcaagacg aacaggaagg
     3961 tcaccgtgaa gcagctgaag gaggattact ttaagaagat agagtgcttc gactcggtgg
     4021 agatttcagg ggtcgaggac cgcttcaatg cctcacttgg gacataccat gacctgctta
     4081 agattatcaa ggacaaggac tttctggaca atgaagagaa cgaggacatc ttggaggaca
     4141 ttgtcctcac actcacgctc ttcgaggacc gggaaatgat tgaagagaga ctcaagactt
     4201 atgcgcactt gtttgatgac aaggtgatga agcaactcaa gaggcgcagg tacacaggct
     4261 ggggccgcct ctcccgtaag ctcatcaatg gcatccgtga caagcagtcc ggaaagacta
     4321 tattggactt tctgaagtcc gacggtttcg ccaaccggaa tttcatgcaa cttatccacg
     4381 atgactctct gacattcaag gaagatatcc aaaaggccca ggtgagcgga cagggcgact
     4441 ctctgcatga gcacatcgcg aaccttgccg gatccccggc catcaagaag ggaatccttc
     4501 agaccgtgaa ggttgtcgat gagcttgtta aggtgatggg caggcacaag cctgagaaca
     4561 ttgtcataga gatggcccgt gagaatcaga caacgcagaa gggccagaag aactccagag
     4621 agcgtatgaa aagaattgaa gaagggatta aggagttggg ctcacaaatc ctgaaggagc
     4681 acccagtcga gaatacgcag ctgcagaacg agaaactgta tctgtattac ctgcagaacg
     4741 gacgcgacat gtatgtcgac caggaactcg atatcaatag actcagcgat tatgacgtcg
     4801 accatatcgt gcctcaatcg ttcctcaagg atgactccat cgataacaag gtgctcacga
     4861 gatcggacaa gaatcgcggc aaatccgaca acgtcccgag cgaggaggtt gtcaagaaaa
     4921 tgaagaatta ctggaggcaa ctgcttaacg caaagctgat cacccagaga aagttcgaca
     4981 atctcacgaa ggcagagcgg ggtggacttt ctgaactgga taaggcaggg tttattaaac
     5041 gccagctggt ggagacacgt cagatcacaa agcatgtggc gcagattctg gacagccgga
     5101 tgaatacgaa gtatgacgag aacgataagc tgatcaggga ggtgaaagtt atcacattga
     5161 agtccaagct cgtgtccgat tttcggaagg acttccagtt ttataaggtt agggagatta
     5221 acaactatca ccacgctcac gacgcctatc tgaatgcggt tgttggcacg gcactcatta
     5281 aaaagtaccc caagctggag tccgagttcg tgtacggtga ctacaaggtg tacgacgtgc
     5341 gcaagatgat tgctaagtct gaacaagaga taggcaaggc aaccgccaag tactttttct
     5401 actccaacat aatgaacttt ttcaaaaccg agataactct tgcgaatggc gagatccgca
     5461 aacggccact tatcgagacg aatggtgaga ctggagagat cgtgtgggac aagggacggg
     5521 attttgcgac tgttaggaag gtgctttcca tgccgcaagt gaacatagtg aagaaaacag
     5581 aggttcaaac gggaggcttt agcaaggaat ccatcctccc caagaggaat tccgataagc
     5641 tcatagctag aaaaaaggac tgggacccaa agaagtatgg cgggtttgac tcaccgaccg
     5701 tcgcgtactc ggttctcgtc gttgcgaagg ttgaaaaggg taagtctaaa aagctcaaat
     5761 cagtgaagga gctcctgggc attaccatca tggagaggtc atcatttgag aagaatccaa
     5821 tcgatttcct tgaggccaag gggtacaagg aggtgaagaa ggacctgata atcaagctgc
     5881 ctaagtattc actcttcgaa ctcgaaaacg ggaggaagag gatgctggca tccgctggag
     5941 agcttcagaa gggaaatgaa ctcgcccttc catccaagta cgtcaacttc ctttacctcg
     6001 cctcgcatta cgagaagctc aagggttccc ctgaagataa tgagcaaaaa cagctcttcg
     6061 tggagcaaca taagcattac ctggacgaga ttatcgaaca gatctccgag ttttccaagc
     6121 gtgtcatcct cgctgacgct aatctcgaca aggttctctc cgcctacaac aaacaccggg
     6181 acaagccaat ccgggagcaa gccgaaaata ttatccacct cttcaccctt actaatctcg
     6241 gggcacctgc ggcgtttaaa tactttgaca ccacgataga ccgcaagcgc tatacgtcca
     6301 ccaaggaggt tcttgacgcg accctcattc accagagcat cacaggcctc tacgaaacga
     6361 ggatcgacct cagccagctg ggtggggata aaagacccgc ggcaactaag aaagccggcc
     6421 aagcgaaaaa gaagaaatga gctttgagac cacgaa
//
