LOCUS       sequence-252774-        7308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 nSpCas9-PmCDA, Arabidopsis codon optimized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7308)
  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 7308)
  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: pFH55
FEATURES             Location/Qualifiers
     source          1..7308
                     /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             join(2235..2261,2262..2264,2265..6338)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     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"
     CDS             6564..6629
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             6636..7259
                     /codon_start=1
                     /product="cytidine deaminase from the sea lamprey 
                     Petromyzon marinus (Rogozin et al., 2007)"
                     /label=PmCDA1
                     /translation="MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGER
                     RACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI
                     LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIF
                     IQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV"
     CDS             7263..7283
                     /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 tcaaatggat aagaagtact ctatcggact cgctatcgga actaactctg
     2281 tgggatgggc tgtgatcacc gatgagtaca aggtgccatc taagaagttc aaggttctcg
     2341 gaaacaccga taggcactct atcaagaaaa accttatcgg tgctctcctc ttcgattctg
     2401 gtgaaactgc tgaggctacc agactcaaga gaaccgctag aagaaggtac accagaagaa
     2461 agaacaggat ctgctacctc caagagatct tctctaacga gatggctaaa gtggatgatt
     2521 cattcttcca caggctcgaa gagtcattcc tcgtggaaga agataagaag cacgagaggc
     2581 accctatctt cggaaacatc gttgatgagg tggcatacca cgagaagtac cctactatct
     2641 accacctcag aaagaagctc gttgattcta ctgataaggc tgatctcagg ctcatctacc
     2701 tcgctctcgc tcacatgatc aagttcagag gacacttcct catcgagggt gatctcaacc
     2761 ctgataactc tgatgtggat aagttgttca tccagctcgt gcagacctac aaccagcttt
     2821 tcgaagagaa ccctatcaac gcttcaggtg tggatgctaa ggctatcctc tctgctaggc
     2881 tctctaagtc aagaaggctt gagaacctca ttgctcagct ccctggtgag aagaagaacg
     2941 gacttttcgg aaacttgatc gctctctctc tcggactcac ccctaacttc aagtctaact
     3001 tcgatctcgc tgaggatgca aagctccagc tctcaaagga tacctacgat gatgatctcg
     3061 ataacctcct cgctcagatc ggagatcagt acgctgattt gttcctcgct gctaagaacc
     3121 tctctgatgc tatcctcctc agtgatatcc tcagagtgaa caccgagatc accaaggctc
     3181 cactctcagc ttctatgatc aagagatacg atgagcacca ccaggatctc acacttctca
     3241 aggctcttgt tagacagcag ctcccagaga agtacaaaga gattttcttc gatcagtcta
     3301 agaacggata cgctggttac atcgatggtg gtgcatctca agaagagttc tacaagttca
     3361 tcaagcctat cctcgagaag atggatggaa ccgaggaact cctcgtgaag ctcaatagag
     3421 aggatcttct cagaaagcag aggaccttcg ataacggatc tatccctcat cagatccacc
     3481 tcggagagtt gcacgctatc cttagaaggc aagaggattt ctacccattc ctcaaggata
     3541 acagggaaaa gattgagaag attctcacct tcagaatccc ttactacgtg ggacctctcg
     3601 ctagaggaaa ctcaagattc gcttggatga ccagaaagtc tgaggaaacc atcacccctt
     3661 ggaacttcga agaggtggtg gataagggtg ctagtgctca gtctttcatc gagaggatga
     3721 ccaacttcga taagaacctt ccaaacgaga aggtgctccc taagcactct ttgctctacg
     3781 agtacttcac cgtgtacaac gagttgacca aggttaagta cgtgaccgag ggaatgagga
     3841 agcctgcttt tttgtcaggt gagcaaaaga aggctatcgt tgatctcttg ttcaagacca
     3901 acagaaaggt gaccgtgaag cagctcaaag aggattactt caagaaaatc gagtgcttcg
     3961 attcagttga gatttctggt gttgaggata ggttcaacgc atctctcgga acctaccacg
     4021 atctcctcaa gatcattaag gataaggatt tcttggataa cgaggaaaac gaggatatct
     4081 tggaggatat cgttcttacc ctcaccctct ttgaagatag agagatgatt gaagaaaggc
     4141 tcaagaccta cgctcatctc ttcgatgata aggtgatgaa gcagttgaag agaagaagat
     4201 acactggttg gggaaggctc tcaagaaagc tcattaacgg aatcagggat aagcagtctg
     4261 gaaagacaat ccttgatttc ctcaagtctg atggattcgc taacagaaac ttcatgcagc
     4321 tcatccacga tgattctctc acctttaaag aggatatcca gaaggctcag gtttcaggac
     4381 agggtgatag tctccatgag catatcgcta acctcgctgg atctcctgca atcaagaagg
     4441 gaatcctcca gactgtgaag gttgtggatg agttggtgaa ggtgatggga aggcataagc
     4501 ctgagaacat cgtgatcgaa atggctagag agaaccagac cactcagaag ggacagaaga
     4561 actctaggga aaggatgaag aggatcgagg aaggtatcaa agagcttgga tctcagatcc
     4621 tcaaagagca ccctgttgag aacactcagc tccagaatga gaagctctac ctctactacc
     4681 tccagaacgg aagggatatg tatgtggatc aagagttgga tatcaacagg ctctctgatt
     4741 acgatgttga tcatatcgtg ccacagtcat tcttgaagga tgattctatc gataacaagg
     4801 tgctcaccag gtctgataag aacaggggta agagtgataa cgtgccaagt gaagaggttg
     4861 tgaagaaaat gaagaactat tggaggcagc tcctcaacgc taagctcatc actcagagaa
     4921 agttcgataa cttgactaag gctgagaggg gaggactctc tgaattggat aaggcaggat
     4981 tcatcaagag gcagcttgtg gaaaccaggc agatcactaa gcacgttgca cagatcctcg
     5041 attctaggat gaacaccaag tacgatgaga acgataagtt gatcagggaa gtgaaggtta
     5101 tcaccctcaa gtcaaagctc gtgtctgatt tcagaaagga tttccaattc tacaaggtga
     5161 gggaaatcaa caactaccac cacgctcacg atgcttacct taacgctgtt gttggaaccg
     5221 ctctcatcaa gaagtatcct aagctcgagt cagagttcgt gtacggtgat tacaaggtgt
     5281 acgatgtgag gaagatgatc gctaagtctg agcaagagat cggaaaggct accgctaagt
     5341 atttcttcta ctctaacatc atgaatttct tcaagaccga gattaccctc gctaacggtg
     5401 agatcagaaa gaggccactc atcgagacaa acggtgaaac aggtgagatc gtgtgggata
     5461 agggaaggga tttcgctacc gttagaaagg tgctctctat gccacaggtg aacatcgtta
     5521 agaaaaccga ggtgcagacc ggtggattct ctaaagagtc tatcctccct aagaggaact
     5581 ctgataagct cattgctagg aagaaggatt gggaccctaa gaaatacggt ggtttcgatt
     5641 ctcctaccgt ggcttactct gttctcgttg tggctaaggt tgagaaggga aagagtaaga
     5701 agctcaagtc tgttaaggaa cttctcggaa tcactatcat ggaaaggtca tctttcgaga
     5761 agaacccaat cgatttcctc gaggctaagg gatacaaaga ggttaagaag gatctcatca
     5821 tcaagctccc aaagtactca ctcttcgaac tcgagaacgg tagaaagagg atgctcgctt
     5881 ctgctggtga gcttcaaaag ggaaacgagc ttgctctccc atctaagtac gttaactttc
     5941 tttacctcgc ttctcactac gagaagttga agggatctcc agaagataac gagcagaagc
     6001 aacttttcgt tgagcagcac aagcactact tggatgagat catcgagcag atctctgagt
     6061 tctctaaaag ggtgatcctc gctgatgcaa acctcgataa ggtgttgtct gcttacaaca
     6121 agcacagaga taagcctatc agggaacagg cagagaacat catccatctc ttcaccctta
     6181 ccaacctcgg tgctcctgct gctttcaagt acttcgatac aaccatcgat aggaagagat
     6241 acacctctac caaagaagtg ctcgatgcta ccctcatcca tcagtctatc actggactct
     6301 acgagactag gatcgatctc tcacagctcg gtggtgattc aagggctgat cctaagaaga
     6361 agaggaaggt tggaggaggg ccaggagcag agtatgttag agcgttgttt gattttaacg
     6421 gtaacgatga agaggattta ccctttaaga aaggcgacat tctcaggatt agggataaac
     6481 ctgaagagca atggtggaat gctgaggata gtgaaggcaa acgaggaatg attttagtgc
     6541 cgtatgtgga gaaatattcg ggtgactaca aagatcatga tggtgattac aaagaccatg
     6601 acatcgacta caaggatgat gatgataagt cagggatgac agatgctgaa tatgtcagaa
     6661 tccacgaaaa gttggacatt tacacgttta agaagcagtt cttcaacaac aagaaatctg
     6721 tttcgcatag gtgctatgtg cttttcgaac taaaacgtcg tggagaaaga cgggcttgct
     6781 tttggggtta cgcggttaac aaaccacaat caggtactga acgaggaata cacgctgaaa
     6841 tcttttctat ccgaaaggtt gaggaatatc tacgtgacaa tcctggacag ttcactatca
     6901 attggtattc tagctggtca ccatgtgcag attgtgctga gaagattctc gaatggtaca
     6961 atcaagagct tagaggcaat ggacatacat tgaaaatatg ggcatgcaag ctctactacg
     7021 aaaagaatgc cagaaaccaa attgggcttt ggaacttgag ggataatgga gttgggctta
     7081 atgtcatggt ttctgagcac tatcaatgtt gtcggaagat cttcatacaa agttcccata
     7141 accagttgaa tgagaacaga tggttagaga aaacccttaa aagagccgag aagagaagat
     7201 ccgaactgag cattatgata caggtcaaaa ttctgcatac cactaagagt ccagctgtag
     7261 gtcctaagaa gaaacgtaaa gtagggccct gagctttgag accacgaa
//
