LOCUS       sequence-270729-        7356 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 A3A-PBE, wheat codon optimized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7356)
  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 7356)
  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: pFH79
FEATURES             Location/Qualifiers
     source          1..7356
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             79..99
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(142..160)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        228..332
                     /gene="bla"
                     /label=AmpR promoter
     CDS             554..1345
                     /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      1516..2104
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2005..2024
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2258..2275
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             join(3004..3027,3028..3030,3031..7104)
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7105..7152
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             join(7174..7356,1..66)
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
ORIGIN
        1 cccgaataca agccgtgggc tcttgtgatc caggactcca atggggagaa caagattaag
       61 atgctctctg gcggttcccc gaagaagaaa cgcaaggtgt gagctttgag accacgaagt
      121 ggctcttcag tggacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
      181 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      241 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      301 aaatgcttca ataatattga aaaaggaaga gtatgcgctc acgcaactgg tccagaacct
      361 tgaccgaacg cagcggtggt aacggcgcag tggcggtttt catggcttgt tatgactgtt
      421 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
      481 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
      541 aaagttaaac atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
      601 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
      661 cgcagtggat ggcggcctga agccacacag cgatattgat ttgctggtta cggtgaccgt
      721 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
      781 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
      841 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
      901 cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac
      961 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
     1021 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
     1081 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     1141 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     1201 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     1261 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccattacg taaaaggcga
     1321 gatcaccaag gtagtcggca aataactgtc agaccaagtt tactcatata tactttagat
     1381 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1441 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1501 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1561 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1621 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     1681 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1741 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1801 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1861 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1921 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1981 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2041 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2101 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2161 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2221 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2281 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatcact
     2341 ctgtggtctc aaatggaagc gagcccagcc tcgggcccga ggcacctgat ggacccgcac
     2401 attttcacct ccaacttcaa caacgggatc ggcaggcaca agacgtacct gtgctatgag
     2461 gttgagcgtc ttgacaatgg gaccagcgtc aagatggatc agcacagagg gttcttgcac
     2521 aaccaagcca agaatctcct ctgcgggttt tacggccgcc acgcggagct ccgtttcttg
     2581 gatctcgtcc catcactgca gctcgaccca gcccagatct atcgcgttac atggttcatc
     2641 tcgtggagcc cctgcttctc ctggggttgc gcaggtgaag tcagagcctt cctccaggaa
     2701 aatacccacg tcaggctgcg cattttcgcc gcgagaatct atgattacga tcccctctat
     2761 aaggaggccc tccagatgct ccgcgatgcc ggtgcgcagg tcagcataat gacctatgac
     2821 gagtttaagc actgctggga caccttcgtg gaccatcagg gctgtccttt ccaaccctgg
     2881 gatggactcg atgagcattc tcaggccttg agcggcaggc tgagagccat cttgcaaaat
     2941 caaggtaatt ctggctctga aacaccaggt acttctgagt ccgcaactcc tgagtcattg
     3001 aaggacaaga aatactccat tgggttggcc ataggcacca actccgttgg ctgggcagtg
     3061 atcaccgacg aatacaaggt cccgagcaaa aagtttaagg tgctgggcaa tactgatcgt
     3121 cactcaatca agaagaacct gattggagcc ctgcttttcg actctgggga aacggctgag
     3181 gcaacgcgct tgaagaggac cgcgcgcagg cggtataccc gtaggaagaa tcgtatctgc
     3241 taccttcagg agatcttttc taatgaaatg gctaaggttg atgatagctt cttccaccgc
     3301 ctggaggaat cttttctcgt cgaggaggac aagaaacacg agcgccaccc aatctttgga
     3361 aacatcgtcg acgaggttgc ctatcatgaa aaatacccaa ctatatatca tctccgtaag
     3421 aagctcgtcg acagcacaga caaagcagac ttgaggctga tatacttggc attggctcac
     3481 atgataaaat tccgtggcca ctttcttatc gagggagatc tgaatccgga caacagcgac
     3541 gttgataaac tctttatcca gctggttcag acctacaatc aattgttcga ggagaacccg
     3601 atcaatgcgt ctggcgtcga cgcaaaggcc atcctgtccg cgcggctcag caaaagccgc
     3661 agattggaga atctgatcgc acagcttccg ggtgagaaga aaaacggcct cttcggcaac
     3721 ttgatcgccc tctctctcgg ccttacccct aacttcaagt cgaacttcga cctcgccgag
     3781 gacgccaagc ttcagctctc aaaggacacc tacgacgacg acctcgataa ccttctcgca
     3841 cagatcggcg atcaatacgc cgatctcttc ctcgcggcca agaacctgag cgatgcgatt
     3901 ctgttgtccg atatcctcag agtcaacaca gagatcacca aggctcccct ctccgcgtca
     3961 atgattaagc gttatgatga gcaccatcag gatctgacac tgctcaaggc tctcgtgcgt
     4021 cagcagctcc ccgagaagta taaggagatc ttcttcgacc aaagcaagaa tggctacgcc
     4081 gggtatattg acggcggcgc gtcccaagaa gaattttata agtttatcaa gccaatcctg
     4141 gaaaagatgg acggaacgga agaactgctg gtgaaattga acagggagga cctcctcagg
     4201 aaacaacgca catttgataa cgggagcatc ccacatcaaa tacatctggg tgagcttcac
     4261 gccatcctgc gccggcagga ggacttctac cccttcctga aggacaaccg ggagaagatc
     4321 gaaaagatac tcaccttcag aatcccctac tacgtcggcc cactcgccag gggcaactct
     4381 aggttcgcct ggatgactag gaagtcagaa gaaaccatca ctccctggaa cttcgaggag
     4441 gtggtggata aaggcgccag cgcccagagc ttcatcgaga ggatgaccaa tttcgataag
     4501 aatttgccaa acgaaaaggt gttgccaaag cactccctgc tttacgaata cttcacggtc
     4561 tacaacgagc tgaccaaagt gaagtacgtg acagagggaa tgaggaagcc agccttcctg
     4621 tctggggagc aaaagaaggc cattgtggac ctcttgttta agacaaatcg caaagtgacg
     4681 gtgaagcaac tcaaggagga ttactttaaa aagattgagt gtttcgactc cgtggagatc
     4741 agcggggtcg aggacaggtt taacgccagc ctggggacat atcacgacct gctgaaaatc
     4801 attaaggaca aggacttcct ggacaacgag gagaatgagg acattctcga ggatatcgtg
     4861 ctcacgctga ctctcttcga ggacagagag atgatcgagg agagactcaa gacctacgct
     4921 catctgttcg acgacaaagt catgaaacag cttaagcgta gacgttacac cggctgggga
     4981 cgcctgtccc gtaagctgat taatgggatt cgcgataagc agagcggcaa gacaatactc
     5041 gatttcttga agtccgacgg tttcgccaat aggaacttca tgcagctcat ccatgatgat
     5101 tcgctcacct ttaaggagga tattcagaag gcgcaagttt ctggccaagg cgatagcctc
     5161 cacgaacata tcgccaacct ggcgggcagc cccgcaatca aaaagggcat cctgcaaacc
     5221 gttaaggttg tggacgaact ggttaaggtc atgggcagac ataagccgga aaacatagtc
     5281 attgaaatgg ctcgcgagaa ccagacaact cagaagggcc aaaagaattc gcgggagagg
     5341 atgaagagaa ttgaggaggg aatcaaggag ctcggatcgc agattctcaa ggagcaccct
     5401 gtggagaata ctcaacttca gaacgaaaaa ctgtacttgt actacttgca gaacgggaga
     5461 gatatgtacg ttgatcagga gcttgacatc aaccgcctga gcgactacga tgtggaccac
     5521 atagtcccac agtctttcct gaaggacgac agcatcgata ataaggtctt gacccgcagc
     5581 gacaagaatc gggggaaaag cgataacgtt ccttcggagg aggtcgtcaa gaagatgaag
     5641 aattactgga ggcaattgtt gaatgccaag cttatcacgc aaagaaagtt cgacaacctt
     5701 acgaaggctg agcgcggggg tctttccgaa ttggacaagg cggggttcat aaagcgccag
     5761 ctcgtggaga ctagacaaat caccaagcac gtggcccaga ttctggactc acggatgaac
     5821 accaagtacg acgagaacga taaactcatc cgggaggtca aggttatcac cctcaagagc
     5881 aagctggtgt ccgacttcag gaaggatttt caattctata aggtccggga aattaataat
     5941 taccaccacg cccacgatgc gtaccttaac gcggtggtgg gcacggctct gatcaagaag
     6001 tacccaaagc tggagtccga gtttgtttat ggtgactata aggtgtacga tgtccgcaaa
     6061 atgattgcca agagcgagca ggaaataggc aaggcgacag ccaagtactt cttctactca
     6121 aatatcatga atttctttaa gacagaaatc acgctggcca atggcgaaat tagaaagcgt
     6181 ccgctgattg agactaacgg agagactggc gagatagttt gggacaaagg ccgcgatttc
     6241 gctaccgtca gaaaggtgct gtctatgcca caggtcaaca tcgtcaagaa aaccgaagtc
     6301 cagaccggcg gcttctctaa ggagtctatc ctgcctaaga ggaactccga taagttgatt
     6361 gctcgcaaga aggactggga cccaaagaag tacggtggat ttgactcccc gacagtcgct
     6421 tacagcgtcc ttgtcgttgc caaggttgag aaaggcaaga gcaagaagct caagagcgtg
     6481 aaggagctgt tggggatcac cattatggag aggagctcct tcgagaagaa cccgattgac
     6541 ttcttggaag caaagggcta caaagaggtt aagaaggacc tcatcatcaa gttgccaaag
     6601 tactcccttt tcgagctcga gaacgggcgc aagagaatgc tcgcctccgc aggggaactc
     6661 cagaagggca acgaattggc ccttccttca aaatacgtga attttctgta cctggcgtcc
     6721 cattacgaaa agctgaaagg gtccccggag gacaacgaac aaaagcaact gttcgtcgag
     6781 caacataagc actatcttga cgaaataatc gagcaaatct ccgagttctc taagagggtc
     6841 atccttgctg acgccaactt ggacaaggtc ctgtcagcct acaacaagca ccgggacaag
     6901 ccgatcagag agcaagctga gaatattatt cacctcttca ccttgaccaa cttgggcgcc
     6961 cccgcggcgt tcaagtactt tgacaccacg atcgatcgta agcggtacac ctccaccaaa
     7021 gaagttctcg acgccaccct tatccaccaa tcaatcactg gcctttacga aacgaggatc
     7081 gatctgagcc aattgggagg ggataaacgg cccgcagcta ccaagaaggc ggggcaggca
     7141 aaaaagaaga aaaccaggga cagcggcgga agcactaacc ttagcgacat catcgaaaag
     7201 gagacaggaa aacagctcgt tatccaagaa agcatcctta tgctccctga ggaagttgag
     7261 gaagttatcg gaaacaagcc tgagagcgac atcctggtgc acacggcata tgacgagtcc
     7321 accgacgaga acgtgatgtt gctcactagc gacgca
//
