LOCUS       sequence-258521-        6534 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 SpCas9, wheat codon optimized (version 3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6534)
  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 6534)
  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: pFH25
FEATURES             Location/Qualifiers
     source          1..6534
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     109..126
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             210..230
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      279..288
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             285..4388
                     /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             285..4388
                     /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             4392..4418
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4398..4424
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YDVPDYASL"
     CDS             4437..4457
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4464..4484
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4527..4545)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4613..4717
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4939..5730
                     /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      5901..6489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6390..6409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
       61 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      121 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      181 cattaatcac tctgtggtct caaatggggc cgaagaagaa gaggaaggtg ggtagggtgt
      241 gtcggattag cagccttcgg tacaggggtc ctggcattgc caccatggac aagaagtact
      301 ctatcgggtt ggatatagga acgaatagtg tgggttgggc tgtcatcact gacgagtaca
      361 aggtgccatc gaagaaattt aaagtgctgg gcaacaccga caggcactcc atcaagaaga
      421 acctaatagg tgcgttgctc tttgattccg gggagacagc cgaagcgacc cgcctcaagc
      481 gcacagcaag aaggcgctat acgcgtagaa agaacaggat ctgctaccta caagagatct
      541 tttccaacga aatggcgaaa gtggatgact ccttcttcca tcggctcgag gaaagcttcc
      601 tcgtagaaga ggacaagaag cacgaacgcc atccgatatt cgggaacatt gtcgacgaag
      661 tcgcatacca cgagaagtat cctaccatct atcacctgag gaagaagctc gtagatagca
      721 ctgacaaggc tgatttgcgc ctcatctatt tggccctggc ccatatgatt aagttccgtg
      781 ggcacttcct catcgaaggc gaccttaatc cggacaattc cgatgttgac aaactcttca
      841 tccaactggt ccagacatac aatcagctct ttgaagagaa ccccatcaac gcgtcaggag
      901 ttgatgcgaa ggcaatcttg agtgctcggc tgtcgaaaag ccggaggctc gaaaatctga
      961 tagcccaact gccaggcgaa aagaagaacg gattgtttgg caaccttatt gcccttagtc
     1021 tagggctcac ccctaacttc aagtctaact ttgacctggc cgaggatgcc aagctccaac
     1081 tgtccaaaga cacgtacgac gacgatctcg acaatctgct tgcccaaatc ggcgaccagt
     1141 atgcggacct tttccttgca gcgaagaact tgtcagatgc aatcctcttg tccgatattc
     1201 tgcgcgttaa cactgagatt accaaggctc ctctttcggc atccatgatc aaacggtacg
     1261 acgaacatca ccaggatctc acgctcctca aggctctcgt cagacaacag ctgccggaga
     1321 agtacaaaga gatcttcttc gatcaatcaa agaacgggta tgctgggtac atcgacggtg
     1381 gcgcgtcaca ggaggagttc tacaagttta tcaagcccat tctcgagaag atggatggca
     1441 ctgaagagct cctggttaag ctcaatcgcg aggatctact ccgcaagcag cgtaccttcg
     1501 acaatggaag cattccacac caaatccatc tgggcgagct tcacgccata cttcgtaggc
     1561 aagaggactt ttacccattc ctcaaagaca accgggagaa aatcgagaag atcctcacat
     1621 tcagaatccc ctactatgtg ggcccattgg caaggggcaa ctctaggttc gcgtggatga
     1681 ctagaaaatc ggaggaaacg attaccccat ggaacttcga ggaggtggtg gacaagggcg
     1741 cttctgccca aagcttcatc gagaggatga ccaacttcga caagaatctg ccaaacgaga
     1801 aggtcctccc caaacattcc ctactgtacg agtacttcac cgtctacaac gagctgacca
     1861 aggtcaagta cgttactgag ggtatgagga aaccggcctt tctgtcagga gagcagaaga
     1921 aggccatagt ggatctcctt ttcaagacca atcgcaaggt cacagtcaag cagctaaagg
     1981 aggactactt taagaaaatc gagtgctttg actctgtgga gataagcggc gttgaggatc
     2041 gcttcaacgc ctcgctggga acgtaccacg acttgctcaa gatcattaag gataaagact
     2101 tcctcgataa cgaagagaac gaggacatct tggaggacat cgttctgacg cttactctct
     2161 tcgaggaccg cgagatgatc gaagagaggc tcaagacgta tgcgcacctc tttgacgaca
     2221 aagtgatgaa gcagctgaag cgcaggcgct acacaggttg gggcaggctg agcaggaagc
     2281 tgatcaatgg cattcgcgat aagcagtcgg gcaagaccat cctcgacttc ctcaagtccg
     2341 atgggttcgc aaacaggaac tttatgcagc taatccacga tgactccttg accttcaagg
     2401 aggacattca gaaggctcag gtttcaggcc aaggcgattc gctccacgag catatcgcga
     2461 atttggccgg atctccagcc atcaagaagg gtatacttca aacagtgaag gttgtcgatg
     2521 agctggtcaa agtgatgggt agacataagc ccgagaatat cgtgattgag atggctcggg
     2581 agaaccaaac cacacagaag gggcagaaga actccaggga aaggatgaag agaatcgaag
     2641 aaggcatcaa ggagctcgga tcacaaattc tcaaggagca tccggtcgaa aacacccaac
     2701 tgcaaaacga gaagctctac ctctactacc tccaaaacgg acgcgatatg tacgtggacc
     2761 aggagctcga catcaatcgc ctatctgact acgatgtcga tcatatagta ccacaaagct
     2821 tcctcaaaga cgactctatc gacaacaagg tgctgaccag gtccgacaag aaccgcggga
     2881 agtcagacaa cgtgccttct gaggaagtcg tcaagaagat gaagaactac tggaggcaac
     2941 tcctcaatgc aaagctcatt acacaaagga agttcgacaa ccttaccaag gcggagagag
     3001 gcggcctcag tgagctagat aaggccggat ttatcaagcg ccaactggtt gagacacggc
     3061 aaatcaccaa gcacgttgca cagatcctgg actcacgcat gaacacaaaa tacgatgaga
     3121 atgacaagct aatccgggaa gtcaaagtta ttactctgaa atctaaactg gtgtccgact
     3181 ttcggaagga cttccagttc tataaggtcc gtgagatcaa caactaccat cacgcccatg
     3241 atgcctatct caatgctgtg gttggcactg cactcatcaa gaaatatccc aagcttgagt
     3301 cggagttcgt ctacggtgac tacaaggttt atgacgtaag gaagatgatt gcgaagagtg
     3361 agcaggaaat cggcaaggcc accgccaagt acttctttta cagcaacata atgaacttct
     3421 tcaagactga gatcaccctg gccaatggag agatcagaaa acgtccgctg atagagacta
     3481 acggagaaac tggtgagata gtgtgggaca aggggagaga ctttgctact gtccggaagg
     3541 tgctaagcat gccacaagtc aacatcgtca agaaaacgga agtccaaacc ggcggcttct
     3601 cgaaagagtc aatccttccg aagcgcaata gcgataagct tatagcccgt aagaaagact
     3661 gggatccaaa gaagtacggt ggctttgaca gtcctacagt ggcgtactcc gtgctggttg
     3721 ttgccaaggt ggaaaagggg aagtcgaaga agctgaagag cgtaaaggag ttgctcggga
     3781 ttaccattat ggagcgttcc agctttgaga agaatcccat tgatttcttg gaggcgaaag
     3841 gatacaagga agtcaagaaa gatctcatca taaagttgcc gaaatacagc ttgttcgagc
     3901 tggagaatgg gagaaagagg atgcttgcca gcgcaggcga gttgcaaaag gggaacgagc
     3961 tggcactccc atccaagtac gtcaacttcc tgtatctcgc ctcacactat gagaaactta
     4021 agggttcccc agaggacaat gagcaaaagc agctgtttgt ggagcaacac aagcactacc
     4081 tcgatgagat catcgaacag atcagtgagt tctcgaagag ggtgattctt gctgacgcta
     4141 accttgacaa ggtgttgtcc gcctacaaca agcatcggga caaacccatc cgggagcagg
     4201 cagaaaacat catacacttg ttcacgttga cgaaccttgg cgctcctgcg gcgttcaaat
     4261 attttgacac gacgattgat aggaagcgct ataccagcac caaagaagta ctcgatgcca
     4321 ccctaattca ccagtctatt acaggcctgt acgaaacgag gatcgacttg tcgcaactag
     4381 gaggagatgc ctatccgtat gacgtgccag attacgcctc ccttgggtct ggttccccta
     4441 agaagaagcg gaaagtggag gatcccaaga agaagcgcaa agtctaagct ttgagaccac
     4501 gaagtggctc ttcagtggac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     4561 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     4621 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     4681 ctgataaatg cttcaataat attgaaaaag gaagagtatg cgctcacgca actggtccag
     4741 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     4801 ctgttttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc
     4861 gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct
     4921 aaaacaaagt taaacatcat gagggaagcg gtgatcgccg aagtatcgac tcaactatca
     4981 gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac
     5041 ggctccgcag tggatggcgg cctgaagcca cacagcgata ttgatttgct ggttacggtg
     5101 accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg
     5161 gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac
     5221 gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc
     5281 aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct ggctatcttg
     5341 ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga ggaactcttt
     5401 gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac gctatggaac
     5461 tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc ccgcatttgg
     5521 tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg ggcaatggag
     5581 cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta tcttggacaa
     5641 gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca ttacgtaaaa
     5701 ggcgagatca ccaaggtagt cggcaaataa ctgtcagacc aagtttactc atatatactt
     5761 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5821 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5881 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5941 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6001 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6061 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6121 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6181 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6241 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6301 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6361 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6421 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6481 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctgg
//
