LOCUS       sequence-252673-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 xCas9 3.7.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  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 6390)
  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: pFH22
FEATURES             Location/Qualifiers
     source          1..6390
                     /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..6338
                     /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
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6351..6371
                     /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 tcaaatggac aagaagtact ccattgggct cgatatcggc acaaacagcg
     2281 tcggctgggc cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg
     2341 gcaataccga tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg
     2401 gggagacggc cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa
     2461 agaatcggat ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact
     2521 ctttcttcca taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc
     2581 acccaatctt tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat
     2641 atcatctgag gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc
     2701 tcgcgctggc gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc
     2761 cagacaacag cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt
     2821 tcgaagagaa cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc
     2881 tgtccaaatc ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg
     2941 gcctgtttgg taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact
     3001 tcgacctggc cgaagatact aagcttcaac tgagcaaaga cacctacgat gatgatctcg
     3061 acaatctgct ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc
     3121 tgtcagacgc cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc
     3181 cgctgagcgc tagtatgatc aagctgtatg atgagcacca ccaagacttg actttgctga
     3241 aggcccttgt cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta
     3301 aaaatggcta cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta
     3361 ttaagcccat cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag
     3421 aagatctgtt gcgcaaacag cgcactttcg acaatggaat catcccccac cagattcacc
     3481 tgggcgaact gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata
     3541 acagggaaaa gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg
     3601 cccggggaaa ttccagattc gcgtggatga ctcgcaaatc agaagagact atcactccct
     3661 ggaacttcga gaaggtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga
     3721 ctaactttga taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg
     3781 agtacttcac agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa
     3841 agccagcatt cctgtctgga gatcagaaga aagctatcgt ggacctcctc ttcaagacga
     3901 accggaaagt taccgtgaaa cagctcaaag aagattattt caaaaagatt gaatgtttcg
     3961 actctgttga aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg
     4021 atctcctgaa aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc
     4081 ttgaggacat tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct
     4141 tgaaaactta cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat
     4201 atacaggatg ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg
     4261 gaaagacaat cctggatttt cttaagtccg atggatttgc caaccggaac ttcatccagt
     4321 tgatccatga tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc
     4381 agggggacag tctccacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg
     4441 gaatactgca gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc
     4501 ccgagaatat cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga
     4561 acagtaggga aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc
     4621 ttaaggaaca cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc
     4681 tgcagaacgg cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact
     4741 acgacgtgga tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag
     4801 tgttgacaag atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg
     4861 tcaagaaaat gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga
     4921 agttcgataa tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct
     4981 tcatcaaaag gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg
     5041 attcacgcat gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta
     5101 ttactctgaa gtctaagctg gtttcagatt tcagaaagga ctttcagttt tataaggtga
     5161 gagagatcaa caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg
     5221 cacttatcaa aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt
     5281 acgatgttag gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt
     5341 acttctttta cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag
     5401 agattcggaa gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca
     5461 agggtaggga tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta
     5521 aaaagaccga agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca
     5581 gcgacaagct gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt
     5641 ctcctacagt cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa
     5701 aactcaaaag cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa
     5761 aaaaccccat cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca
     5821 ttaagcttcc caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta
     5881 gtgcgggcgt actgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct
     5941 tgtatctggc cagccactat gaaaagctca aaggatctcc cgaagataat gagcagaagc
     6001 agctgttcgt ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat
     6061 tctccaaaag agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata
     6121 agcacaggga taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga
     6181 ccaacttggg cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt
     6241 acacctctac aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct
     6301 atgaaacaag aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga
     6361 agaggaaggt gtaggctttg agaccacgaa
//
