LOCUS       sequence-181840-        8901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module A, Promoter: 35S, Gene: dAtCas9-VPR, Terminator: HSP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8901)
  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 8901)
  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_A3501
FEATURES             Location/Qualifiers
     source          1..8901
                     /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             join(1745..1771,1772..1774,1775..4261,4262..4264,
                     4265..5848)
                     /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             5861..5881
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5897..6046
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             6071..6091
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6521..6877
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             6896..7465
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     terminator      7484..7732
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     promoter        7809..7913
                     /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 cctgcaggta gatcgctcgt
     1741 cgacatggat aagaagtact ctatcggact cgctatcgga actaactctg tgggatgggc
     1801 tgtgatcacc gatgagtaca aggtgccatc taagaagttc aaggttctcg gaaacaccga
     1861 taggcactct atcaagaaaa accttatcgg tgctctcctc ttcgattctg gtgaaactgc
     1921 tgaggctacc agactcaaga gaaccgctag aagaaggtac accagaagaa agaacaggat
     1981 ctgctacctc caagagatct tctctaacga gatggctaaa gtggatgatt cattcttcca
     2041 caggctcgaa gagtcattcc tcgtggaaga agataagaag cacgagaggc accctatctt
     2101 cggaaacatc gttgatgagg tggcatacca cgagaagtac cctactatct accacctcag
     2161 aaagaagctc gttgattcta ctgataaggc tgatctcagg ctcatctacc tcgctctcgc
     2221 tcacatgatc aagttcagag gacacttcct catcgagggt gatctcaacc ctgataactc
     2281 tgatgtggat aagttgttca tccagctcgt gcagacctac aaccagcttt tcgaagagaa
     2341 ccctatcaac gcttcaggtg tggatgctaa ggctatcctc tctgctaggc tctctaagtc
     2401 aagaaggctt gagaacctca ttgctcagct ccctggtgag aagaagaacg gacttttcgg
     2461 aaacttgatc gctctctctc tcggactcac ccctaacttc aagtctaact tcgatctcgc
     2521 tgaggatgca aagctccagc tctcaaagga tacctacgat gatgatctcg ataacctcct
     2581 cgctcagatc ggagatcagt acgctgattt gttcctcgct gctaagaacc tctctgatgc
     2641 tatcctcctc agtgatatcc tcagagtgaa caccgagatc accaaggctc cactctcagc
     2701 ttctatgatc aagagatacg atgagcacca ccaggatctc acacttctca aggctcttgt
     2761 tagacagcag ctcccagaga agtacaaaga gattttcttc gatcagtcta agaacggata
     2821 cgctggttac atcgatggtg gtgcatctca agaagagttc tacaagttca tcaagcctat
     2881 cctcgagaag atggatggaa ccgaggaact cctcgtgaag ctcaatagag aggatcttct
     2941 cagaaagcag aggaccttcg ataacggatc tatccctcat cagatccacc tcggagagtt
     3001 gcacgctatc cttagaaggc aagaggattt ctacccattc ctcaaggata acagggaaaa
     3061 gattgagaag attctcacct tcagaatccc ttactacgtg ggacctctcg ctagaggaaa
     3121 ctcaagattc gcttggatga ccagaaagtc tgaggaaacc atcacccctt ggaacttcga
     3181 agaggtggtg gataagggtg ctagtgctca gtctttcatc gagaggatga ccaacttcga
     3241 taagaacctt ccaaacgaga aggtgctccc taagcactct ttgctctacg agtacttcac
     3301 cgtgtacaac gagttgacca aggttaagta cgtgaccgag ggaatgagga agcctgcttt
     3361 tttgtcaggt gagcaaaaga aggctatcgt tgatctcttg ttcaagacca acagaaaggt
     3421 gaccgtgaag cagctcaaag aggattactt caagaaaatc gagtgcttcg attcagttga
     3481 gatttctggt gttgaggata ggttcaacgc atctctcgga acctaccacg atctcctcaa
     3541 gatcattaag gataaggatt tcttggataa cgaggaaaac gaggatatct tggaggatat
     3601 cgttcttacc ctcaccctct ttgaagatag agagatgatt gaagaaaggc tcaagaccta
     3661 cgctcatctc ttcgatgata aggtgatgaa gcagttgaag agaagaagat acactggttg
     3721 gggaaggctc tcaagaaagc tcattaacgg aatcagggat aagcagtctg gaaagacaat
     3781 ccttgatttc ctcaagtctg atggattcgc taacagaaac ttcatgcagc tcatccacga
     3841 tgattctctc acctttaaag aggatatcca gaaggctcag gtttcaggac agggtgatag
     3901 tctccatgag catatcgcta acctcgctgg atctcctgca atcaagaagg gaatcctcca
     3961 gactgtgaag gttgtggatg agttggtgaa ggtgatggga aggcataagc ctgagaacat
     4021 cgtgatcgaa atggctagag agaaccagac cactcagaag ggacagaaga actctaggga
     4081 aaggatgaag aggatcgagg aaggtatcaa agagcttgga tctcagatcc tcaaagagca
     4141 ccctgttgag aacactcagc tccagaatga gaagctctac ctctactacc tccagaacgg
     4201 aagggatatg tatgtggatc aagagttgga tatcaacagg ctctctgatt acgatgttga
     4261 tgctatcgtg ccacagtcat tcttgaagga tgattctatc gataacaagg tgctcaccag
     4321 gtctgataag aacaggggta agagtgataa cgtgccaagt gaagaggttg tgaagaaaat
     4381 gaagaactat tggaggcagc tcctcaacgc taagctcatc actcagagaa agttcgataa
     4441 cttgactaag gctgagaggg gaggactctc tgaattggat aaggcaggat tcatcaagag
     4501 gcagcttgtg gaaaccaggc agatcactaa gcacgttgca cagatcctcg attctaggat
     4561 gaacaccaag tacgatgaga acgataagtt gatcagggaa gtgaaggtta tcaccctcaa
     4621 gtcaaagctc gtgtctgatt tcagaaagga tttccaattc tacaaggtga gggaaatcaa
     4681 caactaccac cacgctcacg atgcttacct taacgctgtt gttggaaccg ctctcatcaa
     4741 gaagtatcct aagctcgagt cagagttcgt gtacggtgat tacaaggtgt acgatgtgag
     4801 gaagatgatc gctaagtctg agcaagagat cggaaaggct accgctaagt atttcttcta
     4861 ctctaacatc atgaatttct tcaagaccga gattaccctc gctaacggtg agatcagaaa
     4921 gaggccactc atcgagacaa acggtgaaac aggtgagatc gtgtgggata agggaaggga
     4981 tttcgctacc gttagaaagg tgctctctat gccacaggtg aacatcgtta agaaaaccga
     5041 ggtgcagacc ggtggattct ctaaagagtc tatcctccct aagaggaact ctgataagct
     5101 cattgctagg aagaaggatt gggaccctaa gaaatacggt ggtttcgatt ctcctaccgt
     5161 ggcttactct gttctcgttg tggctaaggt tgagaaggga aagagtaaga agctcaagtc
     5221 tgttaaggaa cttctcggaa tcactatcat ggaaaggtca tctttcgaga agaacccaat
     5281 cgatttcctc gaggctaagg gatacaaaga ggttaagaag gatctcatca tcaagctccc
     5341 aaagtactca ctcttcgaac tcgagaacgg tagaaagagg atgctcgctt ctgctggtga
     5401 gcttcaaaag ggaaacgagc ttgctctccc atctaagtac gttaactttc tttacctcgc
     5461 ttctcactac gagaagttga agggatctcc agaagataac gagcagaagc aacttttcgt
     5521 tgagcagcac aagcactact tggatgagat catcgagcag atctctgagt tctctaaaag
     5581 ggtgatcctc gctgatgcaa acctcgataa ggtgttgtct gcttacaaca agcacagaga
     5641 taagcctatc agggaacagg cagagaacat catccatctc ttcaccctta ccaacctcgg
     5701 tgctcctgct gctttcaagt acttcgatac aaccatcgat aggaagagat acacctctac
     5761 caaagaagtg ctcgatgcta ccctcatcca tcagtctatc actggactct acgagactag
     5821 gatcgatctc tcacagctcg gtggtgattc aagggctgat cctaagaaga agaggaaggt
     5881 tggatccgga cgggctgacg cattggacga ttttgatctg gatatgctgg gaagtgacgc
     5941 cctcgatgat tttgaccttg acatgcttgg ttcggatgcc cttgatgact ttgacctcga
     6001 catgctcggc agtgacgccc ttgatgattt cgacctggac atgctgatta actctagaag
     6061 ttccggatct ccgaaaaaga aacgcaaagt tggtagccag tacctgcccg acaccgacga
     6121 ccggcaccgg atcgaggaaa agcggaagcg gacctacgag acattcaaga gcatcatgaa
     6181 gaagtccccc ttcagcggcc ccaccgaccc tagacctcca cctagaagaa tcgccgtgcc
     6241 cagcagatcc agcgccagcg tgccaaaacc tgccccccag ccttacccct tcaccagcag
     6301 cctgagcacc atcaactacg acgagttccc taccatggtg ttccccagcg gccagatctc
     6361 tcaggcctct gctctggctc cagcccctcc tcaggtgctg cctcaggctc ctgctcctgc
     6421 accagctcca gccatggtgt ctgcactggc tcaggcacca gcacccgtgc ctgtgctggc
     6481 tcctggacct ccacaggctg tggctccacc agcccctaaa cctacacagg ccggcgaggg
     6541 cacactgtct gaagctctgc tgcagctgca gttcgacgac gaggatctgg gagccctgct
     6601 gggaaacagc accgatcctg ccgtgttcac cgacctggcc agcgtggaca acagcgagtt
     6661 ccagcagctg ctgaaccagg gcatccctgt ggcccctcac accaccgagc ccatgctgat
     6721 ggaatacccc gaggccatca cccggctcgt gacaggcgct cagaggcctc ctgatccagc
     6781 tcctgcccct ctgggagcac caggcctgcc taatggactg ctgtctggcg acgaggactt
     6841 cagctctatc gccgacatgg acttctccgc actgctgggt agcggatcgg gatctcggga
     6901 ttccagggaa gggatgtttt tgccgaagcc tgaggccggc tccgctatta gtgacgtgtt
     6961 tgagggccgc gaggtgtgcc agccaaaacg aatccggcca tttcatcctc caggaagtcc
     7021 atgggccaac cgcccactcc ccgccagcct cgcaccaaca ccaaccggtc cagtacatga
     7081 gccagtcggg tcactgaccc cggcaccagt ccctcagcca ctggatccag cgcccgcagt
     7141 gactcccgag gccagtcacc tgttggagga tcccgatgaa gagactagcc aggctgtcaa
     7201 agcccttcgg gagatggccg atactgtgat tccccagaag gaagaggctg caatctgtgg
     7261 ccaaatggac ctttcccatc cgcccccaag gggccatctg gatgagctga caaccacact
     7321 tgagtccatg accgaggatc tgaacctgga ctcacccctg accccggaat tgaacgagat
     7381 tctggatacc ttcctgaacg acgagtgcct cttgcatgcc atgcatatca gcacaggact
     7441 gtccatcttc gacacatctc tgttctagat gacgcgtcga cgatatgaag atgaagatga
     7501 aatatttggt gtgtcaaata aaaagcttgt gtgcttaagt ttgtgttttt ttcttggctt
     7561 gttgtgttat gaatttgtgg ctttttctaa tattaaatga atgtaagatc acattataat
     7621 gaataaacaa atgtttctat aatccattgt gaatgttttg ttggatctct tctgcagcat
     7681 ataactactg tatgtgctat ggtatggact atggaatatg attaaagata agccagagct
     7741 ctggtgacgg acgtgggcag gtgtgatgaa aagccttagg gtcaggtggc acttttcggg
     7801 gaaatgtgcg atgaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     7861 taatgagaca ataaccctga taaatgcttc aattatattg aaaaaggaag agtatgagta
     7921 tccaacattt ccgtgtcgcc cttattccct tttttgcggc cttctgcctt cctgtttttg
     7981 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcccgagtgg
     8041 gttacattga actggatctc aacagcggca aaatcttaga gagttttcgc cccgaagaac
     8101 gcttcccaat gatgagcact ttcaaagttc tgctatgtgg cgcggtgtta tcccgtattg
     8161 atgctgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     8221 attcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgtagtg
     8281 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg attggaggac
     8341 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     8401 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     8461 caatggcaac aactctccgc aaactattaa ctggcgaact acttactctt gcttcacgcc
     8521 agcaactcat tgactggatg gaggcggaca aagttgcagg accacttctg cgctcggcac
     8581 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     8641 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     8701 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     8761 ttaagcattg gtaactgtca gacgaaggga aataaatagt agcccgcctg atgtgcgggc
     8821 ttttttttgg tggtggttac cagtgtcgct tagctaccgt aggtcctctt aggcggtccg
     8881 attagcgaca tagtcatcaa t
//
