LOCUS       sequence-181775-        7926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module A, Promoter: 35S, 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 7926)
  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 7926)
  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_A0801
FEATURES             Location/Qualifiers
     source          1..7926
                     /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"
     promoter        1366..1711
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     1654..1674
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             2303..2359
                     /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(2360..2386,2387..2389,2390..4876,4877..4879,
                     4880..6463)
                     /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             6476..6496
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6509..6757
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     promoter        6834..6938
                     /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 ggcgcgccag atttgccttt tcaatttcag
      901 aaagaatgct aacccacaga tggttagaga ggcttacgca gcaggtatca tcaagacgat
      961 ctacccgagc aataatctcc aggaaatcaa ataccttccc aagaaggtta aagatgcagt
     1021 caaaagattc aggactaact gcatcaagaa cacagagaaa gatatatttc tcaagatcag
     1081 aagtactatt ccagtatgga cgattcaagg cttgcttcac aaaccaaggc aagtaataga
     1141 gattggagtc tctaaaaagg tagttcccac tgaatcaaag gccatggagt caaagattca
     1201 aatagaggac ctaacagaac tcgccgtaaa gactggcgaa cagttcatac agagtctctt
     1261 acgactcaat gacaagaaga aaatcttcgt caacatggtg gagcacgaca cacttgtcta
     1321 ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga cttttcaaca
     1381 aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt
     1441 gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc
     1501 catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag
     1561 catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat
     1621 ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat
     1681 ataaggaagt tcatttcatt tggagagaac acgggggact cctgcaggaa aaatggatca
     1741 ttatcttgat attagactta gacctgatcc agaatttcca ccagctcaac ttatgtctgt
     1801 tctttttgga aaacttcatc aagctcttgt tgctcaagga ggagatagaa ttggagtttc
     1861 ttttcctgat cttgatgaat caagatcaag acttggagaa agacttagaa ttcatgcttc
     1921 tgctgatgat cttagagctt tgcttgctag accttggctt gaaggactta gagatcatct
     1981 tcaatttgga gaaccagctg ttgttccaca tccaactcct tatagacaag tttcaagagt
     2041 tcaagctaaa tctaatccag aaagacttag aagaagactt atgagaagac atgatctttc
     2101 tgaagaagaa gctagaaaaa gaattcctga tactgttgct agagctttgg atttgccttt
     2161 tgttacactt agatcacaat ctactggaca acattttaga ctttttatta gacatggacc
     2221 acttcaagtt actgctgaag aaggaggatt tacttgttat ggactttcta agggaggttt
     2281 tgttccttgg tttggatctg gagctactaa tttttctctt cttaagcaag ctggagatgt
     2341 tgaagaaaat cctggaccca tggataagaa gtactctatc ggactcgcta tcggaactaa
     2401 ctctgtggga tgggctgtga tcaccgatga gtacaaggtg ccatctaaga agttcaaggt
     2461 tctcggaaac accgataggc actctatcaa gaaaaacctt atcggtgctc tcctcttcga
     2521 ttctggtgaa actgctgagg ctaccagact caagagaacc gctagaagaa ggtacaccag
     2581 aagaaagaac aggatctgct acctccaaga gatcttctct aacgagatgg ctaaagtgga
     2641 tgattcattc ttccacaggc tcgaagagtc attcctcgtg gaagaagata agaagcacga
     2701 gaggcaccct atcttcggaa acatcgttga tgaggtggca taccacgaga agtaccctac
     2761 tatctaccac ctcagaaaga agctcgttga ttctactgat aaggctgatc tcaggctcat
     2821 ctacctcgct ctcgctcaca tgatcaagtt cagaggacac ttcctcatcg agggtgatct
     2881 caaccctgat aactctgatg tggataagtt gttcatccag ctcgtgcaga cctacaacca
     2941 gcttttcgaa gagaacccta tcaacgcttc aggtgtggat gctaaggcta tcctctctgc
     3001 taggctctct aagtcaagaa ggcttgagaa cctcattgct cagctccctg gtgagaagaa
     3061 gaacggactt ttcggaaact tgatcgctct ctctctcgga ctcaccccta acttcaagtc
     3121 taacttcgat ctcgctgagg atgcaaagct ccagctctca aaggatacct acgatgatga
     3181 tctcgataac ctcctcgctc agatcggaga tcagtacgct gatttgttcc tcgctgctaa
     3241 gaacctctct gatgctatcc tcctcagtga tatcctcaga gtgaacaccg agatcaccaa
     3301 ggctccactc tcagcttcta tgatcaagag atacgatgag caccaccagg atctcacact
     3361 tctcaaggct cttgttagac agcagctccc agagaagtac aaagagattt tcttcgatca
     3421 gtctaagaac ggatacgctg gttacatcga tggtggtgca tctcaagaag agttctacaa
     3481 gttcatcaag cctatcctcg agaagatgga tggaaccgag gaactcctcg tgaagctcaa
     3541 tagagaggat cttctcagaa agcagaggac cttcgataac ggatctatcc ctcatcagat
     3601 ccacctcgga gagttgcacg ctatccttag aaggcaagag gatttctacc cattcctcaa
     3661 ggataacagg gaaaagattg agaagattct caccttcaga atcccttact acgtgggacc
     3721 tctcgctaga ggaaactcaa gattcgcttg gatgaccaga aagtctgagg aaaccatcac
     3781 cccttggaac ttcgaagagg tggtggataa gggtgctagt gctcagtctt tcatcgagag
     3841 gatgaccaac ttcgataaga accttccaaa cgagaaggtg ctccctaagc actctttgct
     3901 ctacgagtac ttcaccgtgt acaacgagtt gaccaaggtt aagtacgtga ccgagggaat
     3961 gaggaagcct gcttttttgt caggtgagca aaagaaggct atcgttgatc tcttgttcaa
     4021 gaccaacaga aaggtgaccg tgaagcagct caaagaggat tacttcaaga aaatcgagtg
     4081 cttcgattca gttgagattt ctggtgttga ggataggttc aacgcatctc tcggaaccta
     4141 ccacgatctc ctcaagatca ttaaggataa ggatttcttg gataacgagg aaaacgagga
     4201 tatcttggag gatatcgttc ttaccctcac cctctttgaa gatagagaga tgattgaaga
     4261 aaggctcaag acctacgctc atctcttcga tgataaggtg atgaagcagt tgaagagaag
     4321 aagatacact ggttggggaa ggctctcaag aaagctcatt aacggaatca gggataagca
     4381 gtctggaaag acaatccttg atttcctcaa gtctgatgga ttcgctaaca gaaacttcat
     4441 gcagctcatc cacgatgatt ctctcacctt taaagaggat atccagaagg ctcaggtttc
     4501 aggacagggt gatagtctcc atgagcatat cgctaacctc gctggatctc ctgcaatcaa
     4561 gaagggaatc ctccagactg tgaaggttgt ggatgagttg gtgaaggtga tgggaaggca
     4621 taagcctgag aacatcgtga tcgaaatggc tagagagaac cagaccactc agaagggaca
     4681 gaagaactct agggaaagga tgaagaggat cgaggaaggt atcaaagagc ttggatctca
     4741 gatcctcaaa gagcaccctg ttgagaacac tcagctccag aatgagaagc tctacctcta
     4801 ctacctccag aacggaaggg atatgtatgt ggatcaagag ttggatatca acaggctctc
     4861 tgattacgat gttgatgcta tcgtgccaca gtcattcttg aaggatgatt ctatcgataa
     4921 caaggtgctc accaggtctg ataagaacag gggtaagagt gataacgtgc caagtgaaga
     4981 ggttgtgaag aaaatgaaga actattggag gcagctcctc aacgctaagc tcatcactca
     5041 gagaaagttc gataacttga ctaaggctga gaggggagga ctctctgaat tggataaggc
     5101 aggattcatc aagaggcagc ttgtggaaac caggcagatc actaagcacg ttgcacagat
     5161 cctcgattct aggatgaaca ccaagtacga tgagaacgat aagttgatca gggaagtgaa
     5221 ggttatcacc ctcaagtcaa agctcgtgtc tgatttcaga aaggatttcc aattctacaa
     5281 ggtgagggaa atcaacaact accaccacgc tcacgatgct taccttaacg ctgttgttgg
     5341 aaccgctctc atcaagaagt atcctaagct cgagtcagag ttcgtgtacg gtgattacaa
     5401 ggtgtacgat gtgaggaaga tgatcgctaa gtctgagcaa gagatcggaa aggctaccgc
     5461 taagtatttc ttctactcta acatcatgaa tttcttcaag accgagatta ccctcgctaa
     5521 cggtgagatc agaaagaggc cactcatcga gacaaacggt gaaacaggtg agatcgtgtg
     5581 ggataaggga agggatttcg ctaccgttag aaaggtgctc tctatgccac aggtgaacat
     5641 cgttaagaaa accgaggtgc agaccggtgg attctctaaa gagtctatcc tccctaagag
     5701 gaactctgat aagctcattg ctaggaagaa ggattgggac cctaagaaat acggtggttt
     5761 cgattctcct accgtggctt actctgttct cgttgtggct aaggttgaga agggaaagag
     5821 taagaagctc aagtctgtta aggaacttct cggaatcact atcatggaaa ggtcatcttt
     5881 cgagaagaac ccaatcgatt tcctcgaggc taagggatac aaagaggtta agaaggatct
     5941 catcatcaag ctcccaaagt actcactctt cgaactcgag aacggtagaa agaggatgct
     6001 cgcttctgct ggtgagcttc aaaagggaaa cgagcttgct ctcccatcta agtacgttaa
     6061 ctttctttac ctcgcttctc actacgagaa gttgaaggga tctccagaag ataacgagca
     6121 gaagcaactt ttcgttgagc agcacaagca ctacttggat gagatcatcg agcagatctc
     6181 tgagttctct aaaagggtga tcctcgctga tgcaaacctc gataaggtgt tgtctgctta
     6241 caacaagcac agagataagc ctatcaggga acaggcagag aacatcatcc atctcttcac
     6301 ccttaccaac ctcggtgctc ctgctgcttt caagtacttc gatacaacca tcgataggaa
     6361 gagatacacc tctaccaaag aagtgctcga tgctaccctc atccatcagt ctatcactgg
     6421 actctacgag actaggatcg atctctcaca gctcggtggt gattcaaggg ctgatcctaa
     6481 gaagaagagg aaggtttgac gtcgacgata tgaagatgaa gatgaaatat ttggtgtgtc
     6541 aaataaaaag cttgtgtgct taagtttgtg tttttttctt ggcttgttgt gttatgaatt
     6601 tgtggctttt tctaatatta aatgaatgta agatcacatt ataatgaata aacaaatgtt
     6661 tctataatcc attgtgaatg ttttgttgga tctcttctgc agcatataac tactgtatgt
     6721 gctatggtat ggactatgga atatgattaa agataagcca gagctctggt gacggacgtg
     6781 ggcaggtgtg atgaaaagcc ttagggtcag gtggcacttt tcggggaaat gtgcgatgaa
     6841 cccctatttg tttatttttc taaatacatt caaatatgta tccgctaatg agacaataac
     6901 cctgataaat gcttcaatta tattgaaaaa ggaagagtat gagtatccaa catttccgtg
     6961 tcgcccttat tccctttttt gcggccttct gccttcctgt ttttgctcac ccagaaacgc
     7021 tggtgaaagt aaaagatgct gaagatcagt tgggtgcccg agtgggttac attgaactgg
     7081 atctcaacag cggcaaaatc ttagagagtt ttcgccccga agaacgcttc ccaatgatga
     7141 gcactttcaa agttctgcta tgtggcgcgg tgttatcccg tattgatgct gggcaagagc
     7201 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag
     7261 aaaagcatct tacggatggc atgacagtaa gagaattatg tagtgctgcc ataaccatga
     7321 gtgataacac tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg
     7381 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     7441 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaactc
     7501 tccgcaaact attaactggc gaactactta ctcttgcttc acgccagcaa ctcattgact
     7561 ggatggaggc ggacaaagtt gcaggaccac ttctgcgctc ggcacttccg gctggctggt
     7621 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     7681 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7741 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7801 tgtcagacga agggaaataa atagtagccc gcctgatgtg cgggcttttt tttggtggtg
     7861 gttaccagtg tcgcttagct accgtaggtc ctcttaggcg gtccgattag cgacatagtc
     7921 atcaat
//
