LOCUS       sequence-181776-        8095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module A, Promoter: EC1.2, Gene: Csy4-P2A-AtCas9_dead (D10A + 
            H840A), Terminator: HSP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8095)
  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 8095)
  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_A0808
FEATURES             Location/Qualifiers
     source          1..8095
                     /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             2472..2528
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(2529..2555,2556..2558,2559..5045,5046..5048,
                     5049..6632)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6645..6665
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6678..6926
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     promoter        7003..7107
                     /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 ggcgcgccac tttgataaat gttcctcgct
      901 gacgtaagaa gacattagta atggttataa tatatagctt tctatgaatg tatggtgaga
      961 aaatgtctgt tcactgattt tgagtttgga ataaaagcat ttgcgtttgg tttatcattg
     1021 cgtttataca aggacagaga tccactgagc tggaatagct taaaaccatt atcagaacaa
     1081 aataaaccat tttttgttaa gaatcagagc atagtaaaca acagaaacaa cctaagagag
     1141 gtaacttgtc caagaagata gctaattata tctattttat aaaagttatc atagtttgta
     1201 agtcacaaaa gatgcaaata acagagaaac taggagactt gagaatatac attcttgtat
     1261 atttgtattc gagattgtga aaatttgacc ataagtttaa attcttaaaa agatatatct
     1321 gatctagatg atggttatag actgtaattt taccacatgt ttaatgatgg atagtgacac
     1381 acatgacaca tcgacaacac tatagcatct tatttagatt acaacatgaa atttttctgt
     1441 aatacatgtc tttgtacata atttaaaagt aattcctaag aaatatattt atacaaggag
     1501 tttaaagaaa acatagcata aagttcaatg agtagtaaaa accatataca gtatatagca
     1561 taaagttcaa tgagtttatt acaaaagcat tggttcactt tctgtaacac gacgttaaac
     1621 cttcgtctcc aataggagcg ctactgattc aacatgccaa tatatactaa atacgtttct
     1681 acagtcaaat gctttaacgt ttcatgatta agtgactatt taccgtcaat cctttcccat
     1741 tcctcccact aatccaactt tttaattact cttaaatcac cactaagctt cgaatccatc
     1801 caaaaccaca atataaaaac agaactctcg taactcaatc atcgcaaaac aaaacaaaac
     1861 aaaacaaaaa ccccaaaaag aaagaataac ctgcaggaaa aatggatcat tatcttgata
     1921 ttagacttag acctgatcca gaatttccac cagctcaact tatgtctgtt ctttttggaa
     1981 aacttcatca agctcttgtt gctcaaggag gagatagaat tggagtttct tttcctgatc
     2041 ttgatgaatc aagatcaaga cttggagaaa gacttagaat tcatgcttct gctgatgatc
     2101 ttagagcttt gcttgctaga ccttggcttg aaggacttag agatcatctt caatttggag
     2161 aaccagctgt tgttccacat ccaactcctt atagacaagt ttcaagagtt caagctaaat
     2221 ctaatccaga aagacttaga agaagactta tgagaagaca tgatctttct gaagaagaag
     2281 ctagaaaaag aattcctgat actgttgcta gagctttgga tttgcctttt gttacactta
     2341 gatcacaatc tactggacaa cattttagac tttttattag acatggacca cttcaagtta
     2401 ctgctgaaga aggaggattt acttgttatg gactttctaa gggaggtttt gttccttggt
     2461 ttggatctgg agctactaat ttttctcttc ttaagcaagc tggagatgtt gaagaaaatc
     2521 ctggacccat ggataagaag tactctatcg gactcgctat cggaactaac tctgtgggat
     2581 gggctgtgat caccgatgag tacaaggtgc catctaagaa gttcaaggtt ctcggaaaca
     2641 ccgataggca ctctatcaag aaaaacctta tcggtgctct cctcttcgat tctggtgaaa
     2701 ctgctgaggc taccagactc aagagaaccg ctagaagaag gtacaccaga agaaagaaca
     2761 ggatctgcta cctccaagag atcttctcta acgagatggc taaagtggat gattcattct
     2821 tccacaggct cgaagagtca ttcctcgtgg aagaagataa gaagcacgag aggcacccta
     2881 tcttcggaaa catcgttgat gaggtggcat accacgagaa gtaccctact atctaccacc
     2941 tcagaaagaa gctcgttgat tctactgata aggctgatct caggctcatc tacctcgctc
     3001 tcgctcacat gatcaagttc agaggacact tcctcatcga gggtgatctc aaccctgata
     3061 actctgatgt ggataagttg ttcatccagc tcgtgcagac ctacaaccag cttttcgaag
     3121 agaaccctat caacgcttca ggtgtggatg ctaaggctat cctctctgct aggctctcta
     3181 agtcaagaag gcttgagaac ctcattgctc agctccctgg tgagaagaag aacggacttt
     3241 tcggaaactt gatcgctctc tctctcggac tcacccctaa cttcaagtct aacttcgatc
     3301 tcgctgagga tgcaaagctc cagctctcaa aggataccta cgatgatgat ctcgataacc
     3361 tcctcgctca gatcggagat cagtacgctg atttgttcct cgctgctaag aacctctctg
     3421 atgctatcct cctcagtgat atcctcagag tgaacaccga gatcaccaag gctccactct
     3481 cagcttctat gatcaagaga tacgatgagc accaccagga tctcacactt ctcaaggctc
     3541 ttgttagaca gcagctccca gagaagtaca aagagatttt cttcgatcag tctaagaacg
     3601 gatacgctgg ttacatcgat ggtggtgcat ctcaagaaga gttctacaag ttcatcaagc
     3661 ctatcctcga gaagatggat ggaaccgagg aactcctcgt gaagctcaat agagaggatc
     3721 ttctcagaaa gcagaggacc ttcgataacg gatctatccc tcatcagatc cacctcggag
     3781 agttgcacgc tatccttaga aggcaagagg atttctaccc attcctcaag gataacaggg
     3841 aaaagattga gaagattctc accttcagaa tcccttacta cgtgggacct ctcgctagag
     3901 gaaactcaag attcgcttgg atgaccagaa agtctgagga aaccatcacc ccttggaact
     3961 tcgaagaggt ggtggataag ggtgctagtg ctcagtcttt catcgagagg atgaccaact
     4021 tcgataagaa ccttccaaac gagaaggtgc tccctaagca ctctttgctc tacgagtact
     4081 tcaccgtgta caacgagttg accaaggtta agtacgtgac cgagggaatg aggaagcctg
     4141 cttttttgtc aggtgagcaa aagaaggcta tcgttgatct cttgttcaag accaacagaa
     4201 aggtgaccgt gaagcagctc aaagaggatt acttcaagaa aatcgagtgc ttcgattcag
     4261 ttgagatttc tggtgttgag gataggttca acgcatctct cggaacctac cacgatctcc
     4321 tcaagatcat taaggataag gatttcttgg ataacgagga aaacgaggat atcttggagg
     4381 atatcgttct taccctcacc ctctttgaag atagagagat gattgaagaa aggctcaaga
     4441 cctacgctca tctcttcgat gataaggtga tgaagcagtt gaagagaaga agatacactg
     4501 gttggggaag gctctcaaga aagctcatta acggaatcag ggataagcag tctggaaaga
     4561 caatccttga tttcctcaag tctgatggat tcgctaacag aaacttcatg cagctcatcc
     4621 acgatgattc tctcaccttt aaagaggata tccagaaggc tcaggtttca ggacagggtg
     4681 atagtctcca tgagcatatc gctaacctcg ctggatctcc tgcaatcaag aagggaatcc
     4741 tccagactgt gaaggttgtg gatgagttgg tgaaggtgat gggaaggcat aagcctgaga
     4801 acatcgtgat cgaaatggct agagagaacc agaccactca gaagggacag aagaactcta
     4861 gggaaaggat gaagaggatc gaggaaggta tcaaagagct tggatctcag atcctcaaag
     4921 agcaccctgt tgagaacact cagctccaga atgagaagct ctacctctac tacctccaga
     4981 acggaaggga tatgtatgtg gatcaagagt tggatatcaa caggctctct gattacgatg
     5041 ttgatgctat cgtgccacag tcattcttga aggatgattc tatcgataac aaggtgctca
     5101 ccaggtctga taagaacagg ggtaagagtg ataacgtgcc aagtgaagag gttgtgaaga
     5161 aaatgaagaa ctattggagg cagctcctca acgctaagct catcactcag agaaagttcg
     5221 ataacttgac taaggctgag aggggaggac tctctgaatt ggataaggca ggattcatca
     5281 agaggcagct tgtggaaacc aggcagatca ctaagcacgt tgcacagatc ctcgattcta
     5341 ggatgaacac caagtacgat gagaacgata agttgatcag ggaagtgaag gttatcaccc
     5401 tcaagtcaaa gctcgtgtct gatttcagaa aggatttcca attctacaag gtgagggaaa
     5461 tcaacaacta ccaccacgct cacgatgctt accttaacgc tgttgttgga accgctctca
     5521 tcaagaagta tcctaagctc gagtcagagt tcgtgtacgg tgattacaag gtgtacgatg
     5581 tgaggaagat gatcgctaag tctgagcaag agatcggaaa ggctaccgct aagtatttct
     5641 tctactctaa catcatgaat ttcttcaaga ccgagattac cctcgctaac ggtgagatca
     5701 gaaagaggcc actcatcgag acaaacggtg aaacaggtga gatcgtgtgg gataagggaa
     5761 gggatttcgc taccgttaga aaggtgctct ctatgccaca ggtgaacatc gttaagaaaa
     5821 ccgaggtgca gaccggtgga ttctctaaag agtctatcct ccctaagagg aactctgata
     5881 agctcattgc taggaagaag gattgggacc ctaagaaata cggtggtttc gattctccta
     5941 ccgtggctta ctctgttctc gttgtggcta aggttgagaa gggaaagagt aagaagctca
     6001 agtctgttaa ggaacttctc ggaatcacta tcatggaaag gtcatctttc gagaagaacc
     6061 caatcgattt cctcgaggct aagggataca aagaggttaa gaaggatctc atcatcaagc
     6121 tcccaaagta ctcactcttc gaactcgaga acggtagaaa gaggatgctc gcttctgctg
     6181 gtgagcttca aaagggaaac gagcttgctc tcccatctaa gtacgttaac tttctttacc
     6241 tcgcttctca ctacgagaag ttgaagggat ctccagaaga taacgagcag aagcaacttt
     6301 tcgttgagca gcacaagcac tacttggatg agatcatcga gcagatctct gagttctcta
     6361 aaagggtgat cctcgctgat gcaaacctcg ataaggtgtt gtctgcttac aacaagcaca
     6421 gagataagcc tatcagggaa caggcagaga acatcatcca tctcttcacc cttaccaacc
     6481 tcggtgctcc tgctgctttc aagtacttcg atacaaccat cgataggaag agatacacct
     6541 ctaccaaaga agtgctcgat gctaccctca tccatcagtc tatcactgga ctctacgaga
     6601 ctaggatcga tctctcacag ctcggtggtg attcaagggc tgatcctaag aagaagagga
     6661 aggtttgacg tcgacgatat gaagatgaag atgaaatatt tggtgtgtca aataaaaagc
     6721 ttgtgtgctt aagtttgtgt ttttttcttg gcttgttgtg ttatgaattt gtggcttttt
     6781 ctaatattaa atgaatgtaa gatcacatta taatgaataa acaaatgttt ctataatcca
     6841 ttgtgaatgt tttgttggat ctcttctgca gcatataact actgtatgtg ctatggtatg
     6901 gactatggaa tatgattaaa gataagccag agctctggtg acggacgtgg gcaggtgtga
     6961 tgaaaagcct tagggtcagg tggcactttt cggggaaatg tgcgatgaac ccctatttgt
     7021 ttatttttct aaatacattc aaatatgtat ccgctaatga gacaataacc ctgataaatg
     7081 cttcaattat attgaaaaag gaagagtatg agtatccaac atttccgtgt cgcccttatt
     7141 cccttttttg cggccttctg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     7201 aaagatgctg aagatcagtt gggtgcccga gtgggttaca ttgaactgga tctcaacagc
     7261 ggcaaaatct tagagagttt tcgccccgaa gaacgcttcc caatgatgag cactttcaaa
     7321 gttctgctat gtggcgcggt gttatcccgt attgatgctg ggcaagagca actcggtcgc
     7381 cgcatacact attctcagaa tgacttggtt gagtattcac cagtcacaga aaagcatctt
     7441 acggatggca tgacagtaag agaattatgt agtgctgcca taaccatgag tgataacact
     7501 gcggccaact tacttctgac aacgattgga ggaccgaagg agctaaccgc ttttttgcac
     7561 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     7621 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaactct ccgcaaacta
     7681 ttaactggcg aactacttac tcttgcttca cgccagcaac tcattgactg gatggaggcg
     7741 gacaaagttg caggaccact tctgcgctcg gcacttccgg ctggctggtt tattgctgat
     7801 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     7861 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     7921 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagacgaa
     7981 gggaaataaa tagtagcccg cctgatgtgc gggctttttt ttggtggtgg ttaccagtgt
     8041 cgcttagcta ccgtaggtcc tcttaggcgg tccgattagc gacatagtca tcaat
//
