LOCUS       sequence-182179-        7796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module A, Promoter: AtUbi10, Gene: AtCas9_H840A nickase, Terminator:
            HSP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7796)
  AUTHORS   Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, 
            Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF
  TITLE     A multi-purpose toolkit to enable advanced genome engineering in 
            plants.
  JOURNAL   Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 
            10.1105/tpc.16.00922.
  PUBMED    28522548
REFERENCE   2  (bases 1 to 7796)
  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: "Plant Cell.
            2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922."
COMMENT     Sequence Label: pMOD_A0302
FEATURES             Location/Qualifiers
     source          1..7796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      80..668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     569..588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     822..839
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             join(2230..4746,4747..4749,4750..6333)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6346..6366
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6379..6627
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     promoter        6704..6808
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gccttgacgg caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
       61 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      121 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ctaactcttt
      181 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      241 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      301 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      361 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgctcacagc
      421 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      481 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      541 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      601 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      661 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
      721 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
      781 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
      841 aagcggaaga gcgcccacct gctcatgatc ggcgcgccgt cgagctgcag gtcaacggat
      901 caggatattc ttgtttaaga tgttgaactc tatggaggtt tgtatgaact gatgatctag
      961 gaccggataa gttcccttct tcatagcgaa cttattcaaa gaatgttttg tgtatcattc
     1021 ttgttacatt gttattaatg aaaaaatatt attggtcatt ggactgaaca cgagtgttaa
     1081 atatggacca ggccccaaat aagatccatt gatatatgaa ttaaataaca agaataaatc
     1141 gagtcaccaa accacttgcc ttttttaacg agacttgttc accaacttga tacaaaagtc
     1201 attatcctat gcaaatcaat aatcatacaa aaatatccaa taacactaaa aaattaaaag
     1261 aaatggataa tttcacaata tgttatacga taaagaagtt acttttccaa gaaattcact
     1321 gattttataa gcccacttgc attagataaa tggcaaaaaa aaacaaaaag gaaaagaaat
     1381 aaagcacgaa gaattctaga aaatacgaaa tacgcttcaa tgcagtggga cccacggttc
     1441 aattattgcc aattttcagc tccaccgtat atttaaaaaa taaaacgata atgctaaaaa
     1501 aatataaatc gtaacgatcg ttaaatctca acggctggat cttatgacga ccgttagaaa
     1561 ttgtggttgt cgacgagtca gtaataaacg gcgtcaaagt ggttgcagcc ggcacacacg
     1621 agtcgtgttt atcaactcaa agcacaaata cttttcctca acctaaaaat aaggcaatta
     1681 gccaaaaaca actttgcgtg taaacaacgc tcaatacacg tgtcatttta ttattagcta
     1741 ttgcttcacc gccttagctt tctcgtgacc tagtcgtcct cgtcttttct tcttcttctt
     1801 ctataaaaca atacccaaag agctcttctt cttcacaatt cagatttcaa tttctcaaaa
     1861 tcttaaaaac tttctctcaa ttctctctac cgtgatcaag gtaaatttct gtgttcctta
     1921 ttctctcaaa atcttcgatt ttgttttcgt tcgatcccaa tttcgtatat gttctttggt
     1981 ttagattctg ttaatcttag atcgaacacg attttctggg tttgatcgtt agatatcatc
     2041 ttaattctcg attagggttt catagatatc atccgatttg ttcaaataat ttgagttttg
     2101 tcgaataatt actcttcgat ttgtgatttc tatctagatc tggtgttagt ttctagtttg
     2161 tgcgatcgaa tttgtcgatt aatctgagtt tttctgatta acaggcctgc aggtagatcg
     2221 ctcgtcgaca tggataagaa gtactctatc ggactcgata tcggaactaa ctctgtggga
     2281 tgggctgtga tcaccgatga gtacaaggtg ccatctaaga agttcaaggt tctcggaaac
     2341 accgataggc actctatcaa gaaaaacctt atcggtgctc tcctcttcga ttctggtgaa
     2401 actgctgagg ctaccagact caagagaacc gctagaagaa ggtacaccag aagaaagaac
     2461 aggatctgct acctccaaga gatcttctct aacgagatgg ctaaagtgga tgattcattc
     2521 ttccacaggc tcgaagagtc attcctcgtg gaagaagata agaagcacga gaggcaccct
     2581 atcttcggaa acatcgttga tgaggtggca taccacgaga agtaccctac tatctaccac
     2641 ctcagaaaga agctcgttga ttctactgat aaggctgatc tcaggctcat ctacctcgct
     2701 ctcgctcaca tgatcaagtt cagaggacac ttcctcatcg agggtgatct caaccctgat
     2761 aactctgatg tggataagtt gttcatccag ctcgtgcaga cctacaacca gcttttcgaa
     2821 gagaacccta tcaacgcttc aggtgtggat gctaaggcta tcctctctgc taggctctct
     2881 aagtcaagaa ggcttgagaa cctcattgct cagctccctg gtgagaagaa gaacggactt
     2941 ttcggaaact tgatcgctct ctctctcgga ctcaccccta acttcaagtc taacttcgat
     3001 ctcgctgagg atgcaaagct ccagctctca aaggatacct acgatgatga tctcgataac
     3061 ctcctcgctc agatcggaga tcagtacgct gatttgttcc tcgctgctaa gaacctctct
     3121 gatgctatcc tcctcagtga tatcctcaga gtgaacaccg agatcaccaa ggctccactc
     3181 tcagcttcta tgatcaagag atacgatgag caccaccagg atctcacact tctcaaggct
     3241 cttgttagac agcagctccc agagaagtac aaagagattt tcttcgatca gtctaagaac
     3301 ggatacgctg gttacatcga tggtggtgca tctcaagaag agttctacaa gttcatcaag
     3361 cctatcctcg agaagatgga tggaaccgag gaactcctcg tgaagctcaa tagagaggat
     3421 cttctcagaa agcagaggac cttcgataac ggatctatcc ctcatcagat ccacctcgga
     3481 gagttgcacg ctatccttag aaggcaagag gatttctacc cattcctcaa ggataacagg
     3541 gaaaagattg agaagattct caccttcaga atcccttact acgtgggacc tctcgctaga
     3601 ggaaactcaa gattcgcttg gatgaccaga aagtctgagg aaaccatcac cccttggaac
     3661 ttcgaagagg tggtggataa gggtgctagt gctcagtctt tcatcgagag gatgaccaac
     3721 ttcgataaga accttccaaa cgagaaggtg ctccctaagc actctttgct ctacgagtac
     3781 ttcaccgtgt acaacgagtt gaccaaggtt aagtacgtga ccgagggaat gaggaagcct
     3841 gcttttttgt caggtgagca aaagaaggct atcgttgatc tcttgttcaa gaccaacaga
     3901 aaggtgaccg tgaagcagct caaagaggat tacttcaaga aaatcgagtg cttcgattca
     3961 gttgagattt ctggtgttga ggataggttc aacgcatctc tcggaaccta ccacgatctc
     4021 ctcaagatca ttaaggataa ggatttcttg gataacgagg aaaacgagga tatcttggag
     4081 gatatcgttc ttaccctcac cctctttgaa gatagagaga tgattgaaga aaggctcaag
     4141 acctacgctc atctcttcga tgataaggtg atgaagcagt tgaagagaag aagatacact
     4201 ggttggggaa ggctctcaag aaagctcatt aacggaatca gggataagca gtctggaaag
     4261 acaatccttg atttcctcaa gtctgatgga ttcgctaaca gaaacttcat gcagctcatc
     4321 cacgatgatt ctctcacctt taaagaggat atccagaagg ctcaggtttc aggacagggt
     4381 gatagtctcc atgagcatat cgctaacctc gctggatctc ctgcaatcaa gaagggaatc
     4441 ctccagactg tgaaggttgt ggatgagttg gtgaaggtga tgggaaggca taagcctgag
     4501 aacatcgtga tcgaaatggc tagagagaac cagaccactc agaagggaca gaagaactct
     4561 agggaaagga tgaagaggat cgaggaaggt atcaaagagc ttggatctca gatcctcaaa
     4621 gagcaccctg ttgagaacac tcagctccag aatgagaagc tctacctcta ctacctccag
     4681 aacggaaggg atatgtatgt ggatcaagag ttggatatca acaggctctc tgattacgat
     4741 gttgatgcta tcgtgccaca gtcattcttg aaggatgatt ctatcgataa caaggtgctc
     4801 accaggtctg ataagaacag gggtaagagt gataacgtgc caagtgaaga ggttgtgaag
     4861 aaaatgaaga actattggag gcagctcctc aacgctaagc tcatcactca gagaaagttc
     4921 gataacttga ctaaggctga gaggggagga ctctctgaat tggataaggc aggattcatc
     4981 aagaggcagc ttgtggaaac caggcagatc actaagcacg ttgcacagat cctcgattct
     5041 aggatgaaca ccaagtacga tgagaacgat aagttgatca gggaagtgaa ggttatcacc
     5101 ctcaagtcaa agctcgtgtc tgatttcaga aaggatttcc aattctacaa ggtgagggaa
     5161 atcaacaact accaccacgc tcacgatgct taccttaacg ctgttgttgg aaccgctctc
     5221 atcaagaagt atcctaagct cgagtcagag ttcgtgtacg gtgattacaa ggtgtacgat
     5281 gtgaggaaga tgatcgctaa gtctgagcaa gagatcggaa aggctaccgc taagtatttc
     5341 ttctactcta acatcatgaa tttcttcaag accgagatta ccctcgctaa cggtgagatc
     5401 agaaagaggc cactcatcga gacaaacggt gaaacaggtg agatcgtgtg ggataaggga
     5461 agggatttcg ctaccgttag aaaggtgctc tctatgccac aggtgaacat cgttaagaaa
     5521 accgaggtgc agaccggtgg attctctaaa gagtctatcc tccctaagag gaactctgat
     5581 aagctcattg ctaggaagaa ggattgggac cctaagaaat acggtggttt cgattctcct
     5641 accgtggctt actctgttct cgttgtggct aaggttgaga agggaaagag taagaagctc
     5701 aagtctgtta aggaacttct cggaatcact atcatggaaa ggtcatcttt cgagaagaac
     5761 ccaatcgatt tcctcgaggc taagggatac aaagaggtta agaaggatct catcatcaag
     5821 ctcccaaagt actcactctt cgaactcgag aacggtagaa agaggatgct cgcttctgct
     5881 ggtgagcttc aaaagggaaa cgagcttgct ctcccatcta agtacgttaa ctttctttac
     5941 ctcgcttctc actacgagaa gttgaaggga tctccagaag ataacgagca gaagcaactt
     6001 ttcgttgagc agcacaagca ctacttggat gagatcatcg agcagatctc tgagttctct
     6061 aaaagggtga tcctcgctga tgcaaacctc gataaggtgt tgtctgctta caacaagcac
     6121 agagataagc ctatcaggga acaggcagag aacatcatcc atctcttcac ccttaccaac
     6181 ctcggtgctc ctgctgcttt caagtacttc gatacaacca tcgataggaa gagatacacc
     6241 tctaccaaag aagtgctcga tgctaccctc atccatcagt ctatcactgg actctacgag
     6301 actaggatcg atctctcaca gctcggtggt gattcaaggg ctgatcctaa gaagaagagg
     6361 aaggtttgac gtcgacgata tgaagatgaa gatgaaatat ttggtgtgtc aaataaaaag
     6421 cttgtgtgct taagtttgtg tttttttctt ggcttgttgt gttatgaatt tgtggctttt
     6481 tctaatatta aatgaatgta agatcacatt ataatgaata aacaaatgtt tctataatcc
     6541 attgtgaatg ttttgttgga tctcttctgc agcatataac tactgtatgt gctatggtat
     6601 ggactatgga atatgattaa agataagcca gagctctggt gacggacgtg ggcaggtgtg
     6661 atgaaaagcc ttagggtcag gtggcacttt tcggggaaat gtgcgatgaa cccctatttg
     6721 tttatttttc taaatacatt caaatatgta tccgctaatg agacaataac cctgataaat
     6781 gcttcaatta tattgaaaaa ggaagagtat gagtatccaa catttccgtg tcgcccttat
     6841 tccctttttt gcggccttct gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     6901 aaaagatgct gaagatcagt tgggtgcccg agtgggttac attgaactgg atctcaacag
     6961 cggcaaaatc ttagagagtt ttcgccccga agaacgcttc ccaatgatga gcactttcaa
     7021 agttctgcta tgtggcgcgg tgttatcccg tattgatgct gggcaagagc aactcggtcg
     7081 ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag aaaagcatct
     7141 tacggatggc atgacagtaa gagaattatg tagtgctgcc ataaccatga gtgataacac
     7201 tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg cttttttgca
     7261 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     7321 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaactc tccgcaaact
     7381 attaactggc gaactactta ctcttgcttc acgccagcaa ctcattgact ggatggaggc
     7441 ggacaaagtt gcaggaccac ttctgcgctc ggcacttccg gctggctggt ttattgctga
     7501 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     7561 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     7621 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacga
     7681 agggaaataa atagtagccc gcctgatgtg cgggcttttt tttggtggtg gttaccagtg
     7741 tcgcttagct accgtaggtc ctcttaggcg gtccgattag cgacatagtc atcaat
//
