LOCUS       sequence-252042-        9441 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 9441)
  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 9441)
  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..9441
                     /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             2241..2261
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7123..7132
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             7148..7156
                     /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 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 tcaaatggcc ccaaagaaga agcgcaaggt gatggataag aaatactcaa
     2281 ttggtttggc aattggcaca aactcggtgg gctgggccgt gattactgac gaatacaagg
     2341 tgccatcaaa gaagttcaag gtgctcggca atactgaccg ccactctatc aagaagaatc
     2401 tcatcggcgc attgctcttc gattcgggcg agacagcaga ggctactagg ctgaagagga
     2461 ccgcaaggcg caggtacacc cgccgcaaga atcgcatatg ctaccttcaa gagatcttct
     2521 caaacgagat ggccaaggtg gacgactcct tcttccatag gctcgaagag tcctttctgg
     2581 ttgaagagga taagaagcac gagaggcatc ctatattcgg caacatcgtg gacgaggtgg
     2641 cctatcatga gaagtaccct acgatctacc atctccgcaa gaagctggtt gacagcaccg
     2701 ataaagccga tttgcgcctg atctacctgg cactcgcaca tatgatcaag ttccgcgggc
     2761 atttccttat cgagggagat ctcaaccccg ataactccga cgtggacaag ctcttcattc
     2821 agcttgtgca aacctataat cagctgttcg aggagaatcc aatcaacgcg tctggtgttg
     2881 atgcaaaggc gatcctgtcc gcgcgcctgt caaaatcacg ccggctggaa aacttgattg
     2941 cgcaacttcc aggtgagaag aagaacggtt tgttcggcaa tttgatcgca ctcagcctcg
     3001 gactgacccc caacttcaag tccaacttcg atctggccga ggacgccaaa ctccagcttt
     3061 ccaaggacac ctacgatgat gacctcgata accttttggc tcagattggg gaccagtacg
     3121 cagatctgtt ccttgcggcg aagaatctct cagacgccat cctgctttcc gatatcctga
     3181 gggtcaacac cgagatcacc aaggcgccgc tgtccgcatc catgatcaag cgctacgatg
     3241 aacaccacca ggatctcacc ctcctcaagg ccctggtgcg ccagcaattg cctgaaaagt
     3301 acaaggagat ctttttcgat cagtccaaga atggctacgc gggctacatt gacggcggag
     3361 cgagccaaga ggaattctat aagttcatca agccgattct tgagaagatg gatggcaccg
     3421 aggagctttt ggtcaaactg aacagagagg acttgttgag aaagcagaga accttcgaca
     3481 atggctctat cccccatcaa attcacctcg gagagctgca cgcaatcctc cgtcgccagg
     3541 aggacttcta tccattcctt aaagataaca gggaaaaaat cgaaaagatc ctcacgttcc
     3601 gcattccata ctacgtgggt ccactcgcgc ggggtaatag cagatttgcg tggatgaccc
     3661 ggaagtcgga agagacaatc actccatgga atttcgagga ggttgtcgat aagggtgcga
     3721 gcgcccaatc ctttattgaa cgtatgacca acttcgacaa gaaccttccc aacgaaaaag
     3781 tgctcccaaa gcattccctg ttgtatgagt acttcaccgt ttataatgaa ttgaccaaag
     3841 ttaaatacgt cacggagggc atgcggaagc cggctttcct ctcgggagag caaaagaagg
     3901 caattgtcga tctgcttttc aaaacgaacc gtaaagtcac cgtgaagcaa ctcaaagagg
     3961 actatttcaa gaagatcgaa tgcttcgact cggtcgaaat tagcggtgtc gaggatcggt
     4021 tcaacgcctc gctgggcact taccacgatc tcttgaagat cataaaggac aaggacttcc
     4081 tggataacga ggagaacgag gatattttgg aagatatagt gttgacgctg accctttttg
     4141 aggatcgcga aatgatagaa gaaagactta agacatacgc ccaccttttc gacgacaagg
     4201 ttatgaaaca acttaagcgg aggaggtaca ccggttgggg gagactgtct cgtaagttga
     4261 tcaatggcat tcgcgacaag caatccggca agaccatcct ggactttttg aagtccgacg
     4321 gttttgcgaa tcgcaatttt atgcaactga tccacgacga ctcactcact ttcaaagagg
     4381 acattcaaaa agcgcaagtt tcaggccaag gcgactcact tcatgagcac atagcaaatc
     4441 ttgcaggctc accagccatc aaaaaaggca ttctgcaaac tgtcaaggtg gttgacgagc
     4501 tcgtcaaggt tatgggccgg cataagcccg aaaacatcgt tatcgagatg gcgcgggaga
     4561 atcagaccac tcaaaagggg cagaagaact ccagagaacg gatgaagcgt atagaggaag
     4621 ggatcaagga gctgggttcc cagattctca aggagcatcc agttgagaac acacaactcc
     4681 agaatgagaa gctctatttg tactacctgc aaaacggcag ggacatgtac gttgaccagg
     4741 aactggacat taaccgcttg tcagattacg acgttgacca catcgtgccc cagagctttc
     4801 tcgcggatga ttcaatcgat aacaaggtgc tgacccgcag cgacaagaac cgtggtaaga
     4861 gcgataatgt gccctcagaa gaagtcgtta agaagatgaa aaattactgg aggcaactcc
     4921 tgaacgctaa gttgatcacg caacgtaagt tcgacaacct gaccaaggcc gagaggggcg
     4981 gcctgtccga gcttgacaaa gctggtttta taaagcgcca gttggtggag actcgccaaa
     5041 taacaaagca cgtggcacaa atccttgact ccaggatgaa taccaagtac gacgagaacg
     5101 acaagctgat ccgcgaggtc aaagtgataa ctcttaagtc gaagctggtg tctgacttcc
     5161 ggaaggactt ccagttttat aaggtgaggg agatcaacaa ctaccatcac gcccacgatg
     5221 catacctgaa cgcggttgtg ggcactgccc tgatcaagaa atatccagcc ctcgaatctg
     5281 agttcgtcta cggcgactac aaagtttacg acgtcaggaa gatgatcgcg aagtctgaac
     5341 aagagatcgg caaggcgacc gctaagtatt tcttttactc gaacatcatg aacttcttca
     5401 agaccgaaat tacactggcg aacggcgaga ttcgcaaggc cccactcatc gaaaccaacg
     5461 gtgagacagg tgagatcgtc tgggataagg gcagggactt cgcgaccgtg aggaaggtcc
     5521 tgtccatgcc acaagtgaac atagtgaaga aaaccgaggt gcaaacagga gggttctcaa
     5581 aggagagcat tctccctaag cggaattccg acaagctgat tgcccggaag aaggactggg
     5641 atcccaagaa atatggaggt ttcgactccc cgacggtggc gtattctgtc ctcgtcgtgg
     5701 ccaaggttga gaaaggaaag agcaagaagc tcaagagcgt caaggagctc ctcgggatca
     5761 ccattatgga gcggtcatca ttcgaaaaga acccgatcga ttttctggaa gccaagggct
     5821 acaaggaggt caagaaggat ctcataatta agctcccaaa gtactccctg ttcgagcttg
     5881 aaaacgggcg gaagcgcatg ctggcctccg ccggagagct ccagaaggga aacgagctcg
     5941 ccctgccttc taaatacgtc aactttctgt atcttgcctc acactacgag aagctgaagg
     6001 gctcaccgga ggataacgag caaaagcagc tttttgttga acagcacaag cactaccttg
     6061 acgagatcat tgagcaaatt tcggagttca gcaagagagt catcctcgca gacgccaatc
     6121 ttgacaaggt tctgtcagcg tataacaagc acagagacaa gccgatacgt gagcaagctg
     6181 agaacataat tcacctcttc acgcttacta atttgggggc tccggcggcc tttaagtact
     6241 tcgacactac catagacagg aaaagataca cctctaccaa ggaggtcctt gacgctacac
     6301 ttatccacca atcaatcacg gggttgtacg agactcggat cgaccttagc cagttgggcg
     6361 gcgatggcag ctccgaaacg cctggaacgt ccgagagcgc cacccccgaa ggcgggtccg
     6421 gaggatccgg gtccgtccag atcccccaga acccgctgat actcgtggac ggaagctcgt
     6481 acctgtacag ggcataccat gcgtttccac cacttacgaa ctctgctggc gaaccaacag
     6541 gggccatgta tggcgtcttg aatatgctcc ggtccctgat catgcaatac aagccaaccc
     6601 atgcagctgt ggtgttcgac gcgaaaggca agactttccg ggacgaactc tttgagcatt
     6661 ataagagcca tagaccaccc atgccggacg atctgcgcgc ccagatagag ccactgcacg
     6721 ccatggttaa ggcgatgggc cttccccttc tcgcggttag cggggtggag gcagacgacg
     6781 tcattggaac gctcgctaga gaggccgaaa aggccggcag gccggttctc atcagcactg
     6841 gggataagga tatggctcag ctcgtgacac caaacataac tctgatcaac accatgacca
     6901 acactatcct cggtccagaa gaagttgtga ataagtacgg cgtcccgcct gagctcatca
     6961 ttgacttcct tgcgctcatg ggagactcat cggacaatat acccggcgtg ccgggagtcg
     7021 gtgaaaagac cgcccaagcg ctcttgcagg gattgggggg gctggacacc ttgtacgcgg
     7081 agccagagaa gatcgcaggt ttgtcgttcc gcggcgccaa gaccatggcg gccaagcttg
     7141 aacagaataa ggaggtggcc tatctctcct atcaactcgc cacgataaag accgacgtcg
     7201 agctcgagct cacctgtgag caactcgagg ttcagcaacc ggcggccgag gagctgctcg
     7261 gcctgtttaa gaagtacgag ttcaagcgct ggacagcgga tgttgaggca ggcaaatggc
     7321 ttcaggctaa gggagctaag cccgcggcca agccacaaga aacttccgtg gccgacgaag
     7381 cgccggaggt cactgcgacc gtgatatcgt acgacaatta cgtcacgatt cttgatgagg
     7441 agacactcaa ggcgtggatt gcaaagttgg agaaggcccc ggtgtttgca tttgatactg
     7501 aaaccgactc tctggacaat atttcggcga acctcgtcgg actgtcgttt gcaattgagc
     7561 cgggcgtggc tgcgtacatc ccagtggcgc atgactattt ggacgccccc gatcaaatca
     7621 gccgggaaag agcgcttgaa ctcctcaagc cacttctcga ggatgagaaa gccctgaagg
     7681 tggggcaaaa tctcaagtac gcccgtggga tcttggccaa ctacggcatc gagctccgcg
     7741 ggatcgcatt cgacaccatg ttggagtcct acattctcaa ctccgttgcg ggcaggcatg
     7801 atatggactc cctggccgaa agatggctga agcacaagac gatcacattc gaagagattg
     7861 caggcaaagg caagaaccag ttgactttta accaaatcgc tctggaggag gcgggccggt
     7921 acgccgcaga ggacgctgat gtgacccttc aactccacct caagatgtgg ccggacctcc
     7981 aaaagcataa ggggccattg aacgtgttcg agaacattga aatgccgctt gttccagtgc
     8041 tcagcaggat tgagcgcaac ggggttaaaa ttgaccccaa ggtgcttcac aatcactccg
     8101 aagagctgac ccttcggctg gcagagcttg aaaaaaaagc cactgagaca tttggctcct
     8161 ggtatcaacc gaagggcggc acagaaatgt tctgccaccc ccggacgggg aagccgctgc
     8221 caaagtaccc acggatcaag actcccaaag ttggcgggat ctttaagaag ccgaagaaca
     8281 aggcccagcg cgagggtcgt gagccttgcg aattggacac tcgtgagtac gttgcaggcg
     8341 ccccatacac gcccgtcgag cacgtcgttt ttaacttgtc atccaccaag cagctgcaga
     8401 ccatcctctt cgagaagcag ggaattaagc cgctcaagaa aacgcccggc ggcgccccgt
     8461 ccacttctga ggaagtcctg gaggagcttg ccctggacta cccactcccg aaggtgatct
     8521 tggagtacag gggactcgcc aaattgaaga gcacctacac ggacaagctc cctctcatga
     8581 tcaatcctaa aacgggccgc gtccatacga gctaccacca agcagtgacc gcgacaggaa
     8641 ggctgtcgtc aactgatcca aatcttcaaa atatcccagt cagaaacgag gaaggacgga
     8701 gaatccggca ggcgttcatt gctccggagg attacgtcat cgtttctgca gactactctc
     8761 aaaatgagct gaggatcatg gctcatttgt cccgcgacaa gggcctgctg acggccttcg
     8821 cggagggcaa ggacatccac cgtgccaccg ccgcagaggt tttcggcctc cccttggaga
     8881 cagtgacatc agagcaacgc cggtcggcca agcgtattaa tttcggcctg atatacggca
     8941 tgtccgcctt tggcctcgct cggcagctca acataccaag gaaggaggcc cagaagtaca
     9001 tggacttgta tttcgaacgt taccccggcg tcctcgagta tatggagcgt actcgggccc
     9061 aagctaagga gcaggggtat gtcgagacac tggatgggcg tcgcctctat ctgcccgaca
     9121 tcaagtcatc aaatggcgcc cgtagagcgg cggccgaacg tgcagccatc aacgcaccta
     9181 tgcaaggaac tgccgcggat ataattaaac gggcaatgat cgccgtcgat gcctggctcc
     9241 aagcggagca accaagggtt aggatgatca tgcaggtcca cgatgagctt gtctttgagg
     9301 tccacaagga tgatgtcgac gcagtcgcca agcagattca ccagttgatg gagaattgca
     9361 cccgcctgga cgtccccctc ctggtcgagg tggggtcggg cgagaactgg gatcaggcgc
     9421 attgagcttt gagaccacga a
//
