LOCUS       sequence-252777-        6411 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 ScCas9, wheat codon optimized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6411)
  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 6411)
  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: pFH76
FEATURES             Location/Qualifiers
     source          1..6411
                     /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             2235..6359
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus canis 
                     Type II CRISPR/Cas system (Chatterjee et al., 2018)"
                     /label=ScCas9
                     /note="recognizes minimal 5'-NNG-3' PAM sequences"
                     /translation="MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKK
                     NLMGALLFDSGETAEATRLKRTARRRYTRRKNRIRYLQEIFANEMAKLDDSFFQRLEES
                     FLVEEDKKNERHPIFGNLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAHIIK
                     FRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESPLDEIEVDAKGILSARLSKSKRL
                     EKLIAVFPNEKKNGLFGNIIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLGQ
                     IGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKRYDEHHQDLALLKTLVR
                     QQFPEKYAEIFKDDTKNGYAGYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAEEL
                     LAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILTFRI
                     PYYVGPLARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKV
                     LPKHSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKE
                     DYFKKIECFDSVEIIGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTL
                     FEDREMIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGIRDKQSGKTILDFLK
                     SDGFSNRNFMQLIHDDSLTFKEEIEKAQVSGQGDSLHEQIADLAGSPAIKKGILQTVKI
                     VDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKELESQILKENPVEN
                     TQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSVE
                     NRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEADKAGFIKRQL
                     VETRQITKHVARILDSRMNTKRDKNDKPIREVKVITLKSKLVSDFRKDFQLYKVRDINN
                     YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKRFFY
                     SNIMNFFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFATVRKVLAMPQVNIVKKT
                     EVQTGGFSKESILSKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEKGKAKKL
                     KSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIKKELIFKLPKYSLFELENGRRRMLAS
                     ATELQKANELVLPQHLVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKY
                     ILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKYTSFGASGGFTFLDLDVKQGRLRY
                     QTVTEVLDATLIYQSITGLYETRTDLSQLGGD"
     CDS             6372..6392
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
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 tcaaatggag aagaaatact ctattgggct ggacattggc accaattccg
     2281 ttggatgggc cgtgatcact gatgattata aggtcccctc caagaagttc aaggttctgg
     2341 gtaataccaa caggaagagc ataaaaaaga atcttatggg cgctctgctc tttgatagcg
     2401 gagaaacggc ggaagctaca cggctcaagc ggacagcgag gcgccgctat accaggcgga
     2461 agaaccggat tagatacctt caggaaatct tcgcgaacga gatggccaag ctcgatgact
     2521 cattcttcca gcgccttgag gaatcgttcc tcgttgagga ggacaaaaag aacgagcgtc
     2581 accccatctt tggtaatctc gcggacgagg ttgcatacca caggaattat ccgaccatct
     2641 accacctgag gaagaagttg gcggacagcc cggagaaggc agacctgcgg ctcatctacc
     2701 tcgcgctggc gcacatcatc aaattccggg ggcatttttt gatcgaaggg aaactcaatg
     2761 ctgagaactc ggacgtggca aagctcttct atcagcttat ccaaacctat aatcagctgt
     2821 tcgaggaatc tccgttggat gagatagagg ttgacgctaa gggtatcctc tccgctcgtc
     2881 tttcgaagtc caaacgcctg gagaaactga tcgccgtttt cccgaacgag aagaagaacg
     2941 ggctcttcgg aaacataatc gcgcttgccc tgggactgac gcccaacttc aagtccaact
     3001 tcgacttgac ggaggatgcc aaacttcagc tttcaaagga cacatatgac gatgatttgg
     3061 acgagttgct cggccaaatt ggcgaccaat acgctgacct gttctctgcc gcaaaaaacc
     3121 tgtccgatgc tattctgctg agcgatattc tccggagcaa ctccgaggtg actaaggcac
     3181 cgttgtccgc cagcatggtg aaacgctacg acgagcacca ccaggacctg gctctcctca
     3241 agaccttggt taggcaacag tttcccgaga agtacgccga aatctttaag gatgacacca
     3301 agaacggcta tgccggttac gttggcatcg gaatcaagca taggaagagg accactaaac
     3361 tcgccacaca ggaggaattc tataaattta tcaagcctat tctggagaag atggacggag
     3421 ctgaggagct gctcgccaag ctgaacaggg acgatctcct gaggaagcag cggacctttg
     3481 ataatggctc gattcctcat caaatccatc tgaaggagct tcatgccata ctgcgcaggc
     3541 aagaagagtt ttacccattt ctgaaggaga atcgggagaa gatcgaaaaa atcctgacat
     3601 tccgcatacc ctactacgtg ggcccgcttg cccggggaaa ctctcgtttt gcttggctca
     3661 cgaggaagag cgaggaggcg ataacgccat ggaacttcga ggaggtcgtg gataaaggcg
     3721 ccagcgcgca atcgtttatt gagcgtatga caaacttcga cgagcagctc ccaaacaaga
     3781 aggtgttgcc aaagcacagc ctcctgtacg agtacttcac ggtttacaat gagctcacca
     3841 aagtcaagta cgttactgag cgtatgcgta agccggagtt cctctctggg gagcagaaga
     3901 aggcaatcgt tgacctgctc ttcaaaacga accgtaaggt gaccgtcaag cagctgaagg
     3961 aagattactt caaaaagatc gaatgttttg actccgtgga gattatagga gttgaggata
     4021 ggtttaatgc ctcactcggc acctaccatg acttgctcaa gatcatcaaa gacaaggatt
     4081 tcctggacaa tgaagagaac gaggatatcc ttgaggacat cgtgctcaca ctcaccctct
     4141 tcgaggaccg ggagatgatc gaggagcgcc tcaagacata cgcccacctc tttgacgaca
     4201 aggtgatgaa acaactcaag cgcaggcact acacgggttg gggacgcctc tcccgcaaga
     4261 tgattaatgg tatcagggac aaacagtccg gtaagacgat tctcgatttc ttgaagtccg
     4321 atggtttctc taatcgcaac ttcatgcagc tgatccacga cgactcactc accttcaagg
     4381 aggagattga aaaggctcaa gtgtccgggc agggagatag cctccacgag caaatcgcag
     4441 atctcgccgg atcgcctgca attaagaagg gcattcttca aactgtcaaa atcgtcgacg
     4501 agctggtcaa ggttatgggc cacaagccag aaaacatcgt tatcgagatg gctagggaga
     4561 accagaccac taccaaggga ctccaacagt cccgggagcg gaagaagagg attgaggagg
     4621 ggatcaagga actcgagtct caaattctca aggagaatcc ggtggagaac actcaactcc
     4681 agaacgagaa gctctacctg tactacctgc agaacggcag ggatatgtat gtcgatcagg
     4741 agctggacat caacaggctc tccgactacg acgtcgacca tatcgtcccg caatctttca
     4801 ttaaggacga ctcaatcgat aacaaggtcc tcacgcgctc cgtcgagaac agaggtaagt
     4861 cagacaacgt gccgtcggag gaggtggtga agaagatgaa aaactactgg cgtcaactcc
     4921 tcaacgctaa attgattacg cagagaaaat tcgataactt gaccaaagcg gaacgcggcg
     4981 gtctgtcgga ggccgacaaa gccggcttca ttaagcggca actcgtggag acacgccaga
     5041 tcacaaagca cgtcgccagg attcttgact cgcggatgaa tactaaacgc gacaagaacg
     5101 ataagccaat cagagaagtc aaggtgatta cactgaagag caaactggtc agcgacttcc
     5161 gcaaggattt ccagctttat aaggtgaggg acataaacaa ttatcaccac gcccacgacg
     5221 cgtacctcaa cgcggtggtt ggcaccgcac tcattaagaa atacccgaag ctcgagtcag
     5281 agttcgtcta cggcgactat aaagtgtacg acgttcggaa aatgatagca aagtccgagc
     5341 aagagatcgg caaggccacg gccaagagat tcttctactc caatattatg aatttcttta
     5401 agacggaggt taagttggcc aacggagaga tccggaagcg gcctctcatt gaaactaatg
     5461 gcgagacagg cgaggtggtc tggaacaagg aaaaggactt tgctaccgtc cgcaaggtcc
     5521 tcgcgatgcc gcaagtgaac attgtcaaaa agacggaagt tcagacaggc gggttctcga
     5581 aggagtcaat cttgtcgaag cgtgagtccg ccaagctcat cccacgcaag aaggggtggg
     5641 atacgcggaa gtatggaggt ttcgggtccc ccactgttgc atactcaatt ctcgtggtcg
     5701 ccaaggtgga gaagggaaaa gccaagaagc tcaaaagcgt gaaggtcctc gtgggtataa
     5761 cgataatgga aaagggtagc tacgagaagg acccaatcgg tttcctcgaa gccaaggggt
     5821 acaaagatat caaaaaggag cttatattca aactgccgaa gtactcattg tttgagctgg
     5881 agaacggcag gcggcggatg ttggcctcgg cgacagaact gcaaaaggcg aacgagctcg
     5941 tcttgcctca gcatctcgtt aggcttctct actacaccca aaatatctct gcgactacag
     6001 ggtctaacaa cctcggttac atcgagcagc atagggagga attcaaggag atctttgaga
     6061 agatcatcga cttctccgag aagtacatcc ttaagaacaa agtgaactcc aacttgaagt
     6121 cgagcttcga cgagcagttc gctgtttccg attctattct cctgtcgaac tcattcgtgt
     6181 cacttctgaa gtacacgtcg ttcggcgcat ctggcggctt taccttcctc gacctcgacg
     6241 tgaagcaagg ccggctccgc taccaaaccg tgaccgaggt tctcgacgcg acattgatct
     6301 accagtccat taccggcctg tacgaaacga ggacggacct ctcacagctc ggaggggact
     6361 ctagagcgga cccaaagaag aagcgcaagg tgtaggcttt gagaccacga a
//
