LOCUS       sequence-241144-        9439 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 enCas9-PolI3M-TBD, wheat codon optimized.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9439)
  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 9439)
  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: pFH77
FEATURES             Location/Qualifiers
     source          1..9439
                     /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             434..1225
                     /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      1396..1984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1885..1904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2138..2155
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2239..2259
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7121..7130
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             7146..7154
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
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 tttttttggg gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
      361 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
      421 aaagttaaac atcatgaggg aagcggtgat cgccgaagta tcgactcaac tatcagaggt
      481 agttggcgtc atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc
      541 cgcagtggat ggcggcctga agccacacag cgatattgat ttgctggtta cggtgaccgt
      601 aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc
      661 ccctggagag agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
      721 cattccgtgg cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga
      781 cattcttgca ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac
      841 aaaagcaaga gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc
      901 ggttcctgaa caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc
      961 gcccgactgg gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
     1021 cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct
     1081 gccggcccag tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga
     1141 agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt gtccattacg taaaaggcga
     1201 gatcaccaag gtagtcggca aataactgtc agaccaagtt tactcatata tactttagat
     1261 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     1321 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     1381 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     1441 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     1501 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     1561 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1621 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1681 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1741 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1801 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1861 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1921 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1981 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2041 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2101 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2161 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatcact
     2221 ctgtggtctc aaatggcccc aaagaagaag cgcaaggtga tggataagaa atactcaatt
     2281 ggtttggcaa ttggcacaaa ctcggtgggc tgggccgtga ttactgacga atacaaggtg
     2341 ccatcaaaga agttcaaggt gctcggcaat actgaccgcc actctatcaa gaagaatctc
     2401 atcggcgcat tgctcttcga ttcgggcgag acagcagagg ctactaggct gaagaggacc
     2461 gcaaggcgca ggtacacccg ccgcaagaat cgcatatgct accttcaaga gatcttctca
     2521 aacgagatgg ccaaggtgga cgactccttc ttccataggc tcgaagagtc ctttctggtt
     2581 gaagaggata agaagcacga gaggcatcct atattcggca acatcgtgga cgaggtggcc
     2641 tatcatgaga agtaccctac gatctaccat ctccgcaaga agctggttga cagcaccgat
     2701 aaagccgatt tgcgcctgat ctacctggca ctcgcacata tgatcaagtt ccgcgggcat
     2761 ttccttatcg agggagatct caaccccgat aactccgacg tggacaagct cttcattcag
     2821 cttgtgcaaa cctataatca gctgttcgag gagaatccaa tcaacgcgtc tggtgttgat
     2881 gcaaaggcga tcctgtccgc gcgcctgtca aaatcacgcc ggctggaaaa cttgattgcg
     2941 caacttccag gtgagaagaa gaacggtttg ttcggcaatt tgatcgcact cagcctcgga
     3001 ctgaccccca acttcaagtc caacttcgat ctggccgagg acgccaaact ccagctttcc
     3061 aaggacacct acgatgatga cctcgataac cttttggctc agattgggga ccagtacgca
     3121 gatctgttcc ttgcggcgaa gaatctctca gacgccatcc tgctttccga tatcctgagg
     3181 gtcaacaccg agatcaccaa ggcgccgctg tccgcatcca tgatcaagcg ctacgatgaa
     3241 caccaccagg atctcaccct cctcaaggcc ctggtgcgcc agcaattgcc tgaaaagtac
     3301 aaggagatct ttttcgatca gtccaagaat ggctacgcgg gctacattga cggcggagcg
     3361 agccaagagg aattctataa gttcatcaag ccgattcttg agaagatgga tggcaccgag
     3421 gagcttttgg tcaaactgaa cagagaggac ttgttgagaa agcagagaac cttcgacaat
     3481 ggctctatcc cccatcaaat tcacctcgga gagctgcacg caatcctccg tcgccaggag
     3541 gacttctatc cattccttaa agataacagg gaaaaaatcg aaaagatcct cacgttccgc
     3601 attccatact acgtgggtcc actcgcgcgg ggtaatagca gatttgcgtg gatgacccgg
     3661 aagtcggaag agacaatcac tccatggaat ttcgaggagg ttgtcgataa gggtgcgagc
     3721 gcccaatcct ttattgaacg tatgaccaac ttcgacaaga accttcccaa cgaaaaagtg
     3781 ctcccaaagc attccctgtt gtatgagtac ttcaccgttt ataatgaatt gaccaaagtt
     3841 aaatacgtca cggagggcat gcggaagccg gctttcctct cgggagagca aaagaaggca
     3901 attgtcgatc tgcttttcaa aacgaaccgt aaagtcaccg tgaagcaact caaagaggac
     3961 tatttcaaga agatcgaatg cttcgactcg gtcgaaatta gcggtgtcga ggatcggttc
     4021 aacgcctcgc tgggcactta ccacgatctc ttgaagatca taaaggacaa ggacttcctg
     4081 gataacgagg agaacgagga tattttggaa gatatagtgt tgacgctgac cctttttgag
     4141 gatcgcgaaa tgatagaaga aagacttaag acatacgccc accttttcga cgacaaggtt
     4201 atgaaacaac ttaagcggag gaggtacacc ggttggggga gactgtctcg taagttgatc
     4261 aatggcattc gcgacaagca atccggcaag accatcctgg actttttgaa gtccgacggt
     4321 tttgcgaatc gcaattttat gcaactgatc cacgacgact cactcacttt caaagaggac
     4381 attcaaaaag cgcaagtttc aggccaaggc gactcacttc atgagcacat agcaaatctt
     4441 gcaggctcac cagccatcaa aaaaggcatt ctgcaaactg tcaaggtggt tgacgagctc
     4501 gtcaaggtta tgggccggca taagcccgaa aacatcgtta tcgagatggc gcgggagaat
     4561 cagaccactc aaaaggggca gaagaactcc agagaacgga tgaagcgtat agaggaaggg
     4621 atcaaggagc tgggttccca gattctcaag gagcatccag ttgagaacac acaactccag
     4681 aatgagaagc tctatttgta ctacctgcaa aacggcaggg acatgtacgt tgaccaggaa
     4741 ctggacatta accgcttgtc agattacgac gttgaccaca tcgtgcccca gagctttctc
     4801 gcggatgatt caatcgataa caaggtgctg acccgcagcg acaagaaccg tggtaagagc
     4861 gataatgtgc cctcagaaga agtcgttaag aagatgaaaa attactggag gcaactcctg
     4921 aacgctaagt tgatcacgca acgtaagttc gacaacctga ccaaggccga gaggggcggc
     4981 ctgtccgagc ttgacaaagc tggttttata aagcgccagt tggtggagac tcgccaaata
     5041 acaaagcacg tggcacaaat ccttgactcc aggatgaata ccaagtacga cgagaacgac
     5101 aagctgatcc gcgaggtcaa agtgataact cttaagtcga agctggtgtc tgacttccgg
     5161 aaggacttcc agttttataa ggtgagggag atcaacaact accatcacgc ccacgatgca
     5221 tacctgaacg cggttgtggg cactgccctg atcaagaaat atccagccct cgaatctgag
     5281 ttcgtctacg gcgactacaa agtttacgac gtcaggaaga tgatcgcgaa gtctgaacaa
     5341 gagatcggca aggcgaccgc taagtatttc ttttactcga acatcatgaa cttcttcaag
     5401 accgaaatta cactggcgaa cggcgagatt cgcaaggccc cactcatcga aaccaacggt
     5461 gagacaggtg agatcgtctg ggataagggc agggacttcg cgaccgtgag gaaggtcctg
     5521 tccatgccac aagtgaacat agtgaagaaa accgaggtgc aaacaggagg gttctcaaag
     5581 gagagcattc tccctaagcg gaattccgac aagctgattg cccggaagaa ggactgggat
     5641 cccaagaaat atggaggttt cgactccccg acggtggcgt attctgtcct cgtcgtggcc
     5701 aaggttgaga aaggaaagag caagaagctc aagagcgtca aggagctcct cgggatcacc
     5761 attatggagc ggtcatcatt cgaaaagaac ccgatcgatt ttctggaagc caagggctac
     5821 aaggaggtca agaaggatct cataattaag ctcccaaagt actccctgtt cgagcttgaa
     5881 aacgggcgga agcgcatgct ggcctccgcc ggagagctcc agaagggaaa cgagctcgcc
     5941 ctgccttcta aatacgtcaa ctttctgtat cttgcctcac actacgagaa gctgaagggc
     6001 tcaccggagg ataacgagca aaagcagctt tttgttgaac agcacaagca ctaccttgac
     6061 gagatcattg agcaaatttc ggagttcagc aagagagtca tcctcgcaga cgccaatctt
     6121 gacaaggttc tgtcagcgta taacaagcac agagacaagc cgatacgtga gcaagctgag
     6181 aacataattc acctcttcac gcttactaat ttgggggctc cggcggcctt taagtacttc
     6241 gacactacca tagacaggaa aagatacacc tctaccaagg aggtccttga cgctacactt
     6301 atccaccaat caatcacggg gttgtacgag actcggatcg accttagcca gttgggcggc
     6361 gatggcagct ccgaaacgcc tggaacgtcc gagagcgcca cccccgaagg cgggtccgga
     6421 ggatccgggt ccgtccagat cccccagaac ccgctgatac tcgtggacgg aagctcgtac
     6481 ctgtacaggg cataccatgc gtttccacca cttacgaact ctgctggcga accaacaggg
     6541 gccatgtatg gcgtcttgaa tatgctccgg tccctgatca tgcaatacaa gccaacccat
     6601 gcagctgtgg tgttcgacgc gaaaggcaag actttccggg acgaactctt tgagcattat
     6661 aagagccata gaccacccat gccggacgat ctgcgcgccc agatagagcc actgcacgcc
     6721 atggttaagg cgatgggcct tccccttctc gcggttagcg gggtggaggc agacgacgtc
     6781 attggaacgc tcgctagaga ggccgaaaag gccggcaggc cggttctcat cagcactggg
     6841 gataaggata tggctcagct cgtgacacca aacataactc tgatcaacac catgaccaac
     6901 actatcctcg gtccagaaga agttgtgaat aagtacggcg tcccgcctga gctcatcatt
     6961 gacttccttg cgctcatggg agactcatcg gacaatatac ccggcgtgcc gggagtcggt
     7021 gaaaagaccg cccaagcgct cttgcaggga ttgggggggc tggacacctt gtacgcggag
     7081 ccagagaaga tcgcaggttt gtcgttccgc ggcgccaaga ccatggcggc caagcttgaa
     7141 cagaataagg aggtggccta tctctcctat caactcgcca cgataaagac cgacgtcgag
     7201 ctcgagctca cctgtgagca actcgaggtt cagcaaccgg cggccgagga gctgctcggc
     7261 ctgtttaaga agtacgagtt caagcgctgg acagcggatg ttgaggcagg caaatggctt
     7321 caggctaagg gagctaagcc cgcggccaag ccacaagaaa cttccgtggc cgacgaagcg
     7381 ccggaggtca ctgcgaccgt gatatcgtac gacaattacg tcacgattct tgatgaggag
     7441 acactcaagg cgtggattgc aaagttggag aaggccccgg tgtttgcatt tgatactgaa
     7501 accgactctc tggacaatat ttcggcgaac ctcgtcggac tgtcgtttgc aattgagccg
     7561 ggcgtggctg cgtacatccc agtggcgcat gactatttgg acgcccccga tcaaatcagc
     7621 cgggaaagag cgcttgaact cctcaagcca cttctcgagg atgagaaagc cctgaaggtg
     7681 gggcaaaatc tcaagtacgc ccgtgggatc ttggccaact acggcatcga gctccgcggg
     7741 atcgcattcg acaccatgtt ggagtcctac attctcaact ccgttgcggg caggcatgat
     7801 atggactccc tggccgaaag atggctgaag cacaagacga tcacattcga agagattgca
     7861 ggcaaaggca agaaccagtt gacttttaac caaatcgctc tggaggaggc gggccggtac
     7921 gccgcagagg acgctgatgt gacccttcaa ctccacctca agatgtggcc ggacctccaa
     7981 aagcataagg ggccattgaa cgtgttcgag aacattgaaa tgccgcttgt tccagtgctc
     8041 agcaggattg agcgcaacgg ggttaaaatt gaccccaagg tgcttcacaa tcactccgaa
     8101 gagctgaccc ttcggctggc agagcttgaa aaaaaagcca ctgagacatt tggctcctgg
     8161 tatcaaccga agggcggcac agaaatgttc tgccaccccc ggacggggaa gccgctgcca
     8221 aagtacccac ggatcaagac tcccaaagtt ggcgggatct ttaagaagcc gaagaacaag
     8281 gcccagcgcg agggtcgtga gccttgcgaa ttggacactc gtgagtacgt tgcaggcgcc
     8341 ccatacacgc ccgtcgagca cgtcgttttt aacttgtcat ccaccaagca gctgcagacc
     8401 atcctcttcg agaagcaggg aattaagccg ctcaagaaaa cgcccggcgg cgccccgtcc
     8461 acttctgagg aagtcctgga ggagcttgcc ctggactacc cactcccgaa ggtgatcttg
     8521 gagtacaggg gactcgccaa attgaagagc acctacacgg acaagctccc tctcatgatc
     8581 aatcctaaaa cgggccgcgt ccatacgagc taccaccaag cagtgaccgc gacaggaagg
     8641 ctgtcgtcaa ctgatccaaa tcttcaaaat atcccagtca gaaacgagga aggacggaga
     8701 atccggcagg cgttcattgc tccggaggat tacgtcatcg tttctgcaga ctactctcaa
     8761 aatgagctga ggatcatggc tcatttgtcc cgcgacaagg gcctgctgac ggccttcgcg
     8821 gagggcaagg acatccaccg tgccaccgcc gcagaggttt tcggcctccc cttggagaca
     8881 gtgacatcag agcaacgccg gtcggccaag cgtattaatt tcggcctgat atacggcatg
     8941 tccgcctttg gcctcgctcg gcagctcaac ataccaagga aggaggccca gaagtacatg
     9001 gacttgtatt tcgaacgtta ccccggcgtc ctcgagtata tggagcgtac tcgggcccaa
     9061 gctaaggagc aggggtatgt cgagacactg gatgggcgtc gcctctatct gcccgacatc
     9121 aagtcatcaa atggcgcccg tagagcggcg gccgaacgtg cagccatcaa cgcacctatg
     9181 caaggaactg ccgcggatat aattaaacgg gcaatgatcg ccgtcgatgc ctggctccaa
     9241 gcggagcaac caagggttag gatgatcatg caggtccacg atgagcttgt ctttgaggtc
     9301 cacaaggatg atgtcgacgc agtcgccaag cagattcacc agttgatgga gaattgcacc
     9361 cgcctggacg tccccctcct ggtcgaggtg gggtcgggcg agaactggga tcaggcgcat
     9421 tgagctttga gaccacgaa
//
