LOCUS       sequence-258519-        6561 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 SpCas9, wheat codon optimized (version 2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6561)
  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 6561)
  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: pFH24
FEATURES             Location/Qualifiers
     source          1..6561
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..66
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             150..170
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             183..248
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             273..4376
                     /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             273..4376
                     /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"
     primer_bind     complement(4494..4512)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4580..4684
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4906..5697
                     /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      5868..6456
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6357..6376
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
       61 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      121 cattaatcac tctgtggtct caaatggcac cgaagaagaa gcgcaaagtg gcccacgtga
      181 tggactataa ggaccacgat ggcgactaca aggaccatga cattgactat aaggatgacg
      241 acgataagaa cgcccagagc ggcgccagcg tcatggacaa gaagtacagc atcggcctgg
      301 acatcgggac caacagcgtg gggtgggcgg ttatcacgga cgagtacaag gtgccaagca
      361 agaagttcaa ggtgctgggc aacaccgacc gccactccat caagaagaac ctcatcggag
      421 ccctcctctt cgacagcggc gaaacggctg aggctaccag gctcaagagg accgctaggc
      481 gcaggtacac ccgcaggaag aacaggatct gctacctcca agaaatcttc agcaacgaga
      541 tggcgaaggt ggacgactcc ttcttccacc gcctggagga gagcttcctc gtcgaggaag
      601 ataagaagca cgagaggcac ccaatcttcg gcaacatcgt ggacgaggtc gcctaccacg
      661 agaagtaccc aaccatctac cacctgagga agaagctcgt ggacagcacc gacaaggccg
      721 acctccgcct gatctacctc gccctggccc acatgattaa gttcaggggc cacttcctga
      781 tcgagggcga cctcaaccca gacaactccg acgtggacaa gctgttcatc caactcgtcc
      841 agacctacaa ccaactcttc gaggagaacc caatcaacgc ttccggcgtg gacgctaagg
      901 ctatcctgag cgccaggctc tccaagtccc gcaggctgga gaacctgatc gcccagctcc
      961 caggcgagaa gaagaacggc ctgttcggca acctcatcgc tctctccctg ggcctcaccc
     1021 caaacttcaa gagcaacttc gacctcgctg aggacgccaa gctgcaactc agcaaggaca
     1081 cctacgacga cgacctcgac aacctcctgg cccagatcgg cgaccaatac gccgacctgt
     1141 tcctcgccgc caagaacctg tccgacgcca tcctcctgag cgacatcctc cgcgtgaaca
     1201 ccgagatcac caaggcccca ctctccgcca gcatgatcaa acgctacgac gagcaccacc
     1261 aggacctgac cctcctgaag gccctggtca ggcaacagct cccagagaag tacaaggaaa
     1321 tcttcttcga ccagtccaag aacggctacg ctggctacat cgacggcgga gcgagccaag
     1381 aggagttcta caagttcatc aagccaatcc tggagaagat ggacggcacc gaggagctgc
     1441 tggtgaagct caacagggag gacctcctga ggaagcagcg caccttcgac aacggctcca
     1501 tcccacacca aatccacctc ggcgagctgc acgctatcct ccgcaggcaa gaggacttct
     1561 acccattcct caaggacaac agggagaaga tcgagaagat cctgaccttc cgcatcccat
     1621 actacgtggg gccactcgcc aggggcaact cccgcttcgc ttggatgacc cgcaagagcg
     1681 aggaaacgat caccccgtgg aacttcgagg aagtggtgga caagggcgct tccgctcaga
     1741 gcttcatcga gaggatgacc aacttcgaca agaacctgcc aaacgagaag gtgctcccaa
     1801 agcacagcct cctgtacgaa tacttcaccg tctacaacga gctgaccaag gtgaagtatg
     1861 tgaccgaggg catgaggaaa ccagccttcc tgtccggcga gcagaagaag gccatcgtgg
     1921 acctcctgtt caagaccaac aggaaggtga ccgtcaagca actcaaggaa gattacttca
     1981 agaagatcga gtgcttcgac tccgtggaga tcagcggcgt cgaggacagg ttcaacgcca
     2041 gcctcggcac ctaccacgac ctcctgaaga tcatcaagga caaggacttc ctggacaacg
     2101 aggagaacga ggacatcctg gaggacatcg tgctgaccct caccctgttc gaggacaggg
     2161 agatgatcga ggagcgcctc aagacctacg cccacctctt cgacgacaaa gttatgaagc
     2221 aactgaagcg caggcgctac accggctggg gcaggctgtc ccgcaagctc atcaacggca
     2281 tccgcgacaa gcagtccggc aagaccatcc tcgacttcct gaagagcgac ggcttcgcca
     2341 acaggaactt catgcaactg atccacgacg actccctcac cttcaaggaa gatattcaga
     2401 aggctcaagt ctccggccag ggcgacagcc tgcacgagca catcgctaac ctcgctggct
     2461 ccccagccat caagaagggc atcctgcaaa ccgtgaaagt cgtggacgag ctggtgaagg
     2521 tcatgggcag gcacaagcca gagaacatcg tcatcgagat ggcccgcgag aaccaaacca
     2581 cccagaaggg ccaaaagaac agcagggagc gcatgaagcg catcgaggaa ggcatcaagg
     2641 agctgggctc ccaaatcctc aaggagcacc cagtcgagaa cacccaactc cagaacgaga
     2701 agctctacct gtactacctc cagaacggca gggatatgta tgtggaccaa gagctggaca
     2761 tcaaccgcct cagcgactac gacgtggacc acatcgtccc acagtccttc ctgaaggacg
     2821 acagcatcga caacaaggtg ctcaccaggt ccgacaagaa ccggggcaag tccgacaacg
     2881 tcccaagcga ggaagtggtc aagaagatga agaactactg gaggcagctc ctgaacgcca
     2941 agctcatcac ccaaaggaag ttcgacaacc tcaccaaggc tgagcgcggc ggcctcagcg
     3001 agctggacaa ggcgggcttc atcaagaggc agctcgtgga aacccgccaa atcaccaagc
     3061 acgtcgccca aatcctcgac tcccgcatga acaccaagta cgacgagaac gacaagctga
     3121 ttagggaggt gaaggtcatc accctgaagt ccaagctcgt gagcgacttc aggaaggact
     3181 tccagttcta caaggtccgc gagatcaaca actaccacca cgcccacgac gcttacctca
     3241 acgctgtggt gggcaccgcc ctcatcaaga agtacccaaa gctggagtcc gagttcgtgt
     3301 acggcgacta caaggtgtac gacgttcgca agatgatcgc caagagcgag caagagatcg
     3361 gcaaggccac cgccaaatac ttcttctact ccaacattat gaacttcttc aagaccgaga
     3421 tcaccctggc taacggcgag atcaggaagc gcccactcat cgaaacgaac ggcgagactg
     3481 gcgagatcgt gtgggacaag ggcagggact tcgccaccgt ccgcaaggtc ctctccatgc
     3541 cacaggtgaa catcgtcaag aaaaccgagg tccagaccgg cggcttctcc aaggagagca
     3601 tcctgccaaa gaggaactcc gacaagctca tcgcccgcaa gaaggactgg gacccaaaga
     3661 agtacggcgg attcgactcc ccaaccgtgg cctacagcgt cctggtggtc gccaaggtgg
     3721 agaagggcaa gtccaagaag ctcaagagcg tcaaggagct gctgggcatc accatcatgg
     3781 agaggagcag cttcgagaag aacccaatcg acttcctgga ggccaagggc tacaaggaag
     3841 tgaagaagga cctgattatc aagctcccaa agtattccct cttcgagctg gagaacggca
     3901 ggaagcgcat gttggcttcc gctggcgagc tgcaaaaggg caacgagctg gccctgccat
     3961 ccaagtatgt gaacttcctc tacctggcct cccactacga gaagctcaag ggcagcccag
     4021 aggacaacga gcaaaagcag ctgttcgtcg agcagcacaa gcactacctc gacgagatca
     4081 tcgagcaaat ctccgagttc tccaagcgcg tgatcctcgc cgacgccaac ctggacaagg
     4141 tcctcagcgc ctacaacaag cacagggaca agccaatccg cgagcaggcc gagaacatca
     4201 tccacctctt caccctgacc aacctcggcg ctccagcggc cttcaaatac ttcgacacca
     4261 ccatcgacag gaaacgctac acctccacca aggaagtgct cgacgccacc ctcatccatc
     4321 aaagcatcac cgggctctac gaaacgagaa tcgacctgtc acaactgggc ggcgacagtt
     4381 ctctaaacgc caccagcggg aagcgcgtgc ctaaggaggc cggagccggg accgacaaga
     4441 agaagaaaag ttaagctttg agaccacgaa gtggctcttc agtggacgaa agggcctcgt
     4501 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     4561 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     4621 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     4681 gagtatgcgc tcacgcaact ggtccagaac cttgaccgaa cgcagcggtg gtaacggcgc
     4741 agtggcggtt ttcatggctt gttatgactg tttttttttg gggtacagtc tatgcctcgg
     4801 gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac
     4861 gatgttacgc agcagggcag tcgccctaaa acaaagttaa acatcatgag ggaagcggtg
     4921 atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa
     4981 ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac
     5041 agcgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct
     5101 ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct
     5161 gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
     5221 gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc
     5281 acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg
     5341 gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
     5401 ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat
     5461 gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag
     5521 gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt
     5581 gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag
     5641 ttggaagaat ttgtccatta cgtaaaaggc gagatcacca aggtagtcgg caaataactg
     5701 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5761 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5821 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5881 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5941 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6001 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6061 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6121 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6181 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6241 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6301 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6361 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6421 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6481 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6541 tctgtggata accgtattac c
//
