LOCUS       sequence-181768-        8095 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module A, Promoter: EC1.2, Gene: Csy4-P2A-AtCas9, 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_A0508
FEATURES             Location/Qualifiers
     source          1..8095
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1594..1650
                     /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             1651..5754
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1651..5754
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5767..5787
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      5800..6048
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     promoter        6125..6229
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7297..7885
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7786..7805
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8039..8056
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 actttgataa atgttcctcg ctgacgtaag aagacattag taatggttat aatatatagc
       61 tttctatgaa tgtatggtga gaaaatgtct gttcactgat tttgagtttg gaataaaagc
      121 atttgcgttt ggtttatcat tgcgtttata caaggacaga gatccactga gctggaatag
      181 cttaaaacca ttatcagaac aaaataaacc attttttgtt aagaatcaga gcatagtaaa
      241 caacagaaac aacctaagag aggtaacttg tccaagaaga tagctaatta tatctatttt
      301 ataaaagtta tcatagtttg taagtcacaa aagatgcaaa taacagagaa actaggagac
      361 ttgagaatat acattcttgt atatttgtat tcgagattgt gaaaatttga ccataagttt
      421 aaattcttaa aaagatatat ctgatctaga tgatggttat agactgtaat tttaccacat
      481 gtttaatgat ggatagtgac acacatgaca catcgacaac actatagcat cttatttaga
      541 ttacaacatg aaatttttct gtaatacatg tctttgtaca taatttaaaa gtaattccta
      601 agaaatatat ttatacaagg agtttaaaga aaacatagca taaagttcaa tgagtagtaa
      661 aaaccatata cagtatatag cataaagttc aatgagttta ttacaaaagc attggttcac
      721 tttctgtaac acgacgttaa accttcgtct ccaataggag cgctactgat tcaacatgcc
      781 aatatatact aaatacgttt ctacagtcaa atgctttaac gtttcatgat taagtgacta
      841 tttaccgtca atcctttccc attcctccca ctaatccaac tttttaatta ctcttaaatc
      901 accactaagc ttcgaatcca tccaaaacca caatataaaa acagaactct cgtaactcaa
      961 tcatcgcaaa acaaaacaaa acaaaacaaa aaccccaaaa agaaagaata acctgcagga
     1021 aaaatggatc attatcttga tattagactt agacctgatc cagaatttcc accagctcaa
     1081 cttatgtctg ttctttttgg aaaacttcat caagctcttg ttgctcaagg aggagataga
     1141 attggagttt cttttcctga tcttgatgaa tcaagatcaa gacttggaga aagacttaga
     1201 attcatgctt ctgctgatga tcttagagct ttgcttgcta gaccttggct tgaaggactt
     1261 agagatcatc ttcaatttgg agaaccagct gttgttccac atccaactcc ttatagacaa
     1321 gtttcaagag ttcaagctaa atctaatcca gaaagactta gaagaagact tatgagaaga
     1381 catgatcttt ctgaagaaga agctagaaaa agaattcctg atactgttgc tagagctttg
     1441 gatttgcctt ttgttacact tagatcacaa tctactggac aacattttag actttttatt
     1501 agacatggac cacttcaagt tactgctgaa gaaggaggat ttacttgtta tggactttct
     1561 aagggaggtt ttgttccttg gtttggatct ggagctacta atttttctct tcttaagcaa
     1621 gctggagatg ttgaagaaaa tcctggaccc atggataaga agtactctat cggactcgat
     1681 atcggaacta actctgtggg atgggctgtg atcaccgatg agtacaaggt gccatctaag
     1741 aagttcaagg ttctcggaaa caccgatagg cactctatca agaaaaacct tatcggtgct
     1801 ctcctcttcg attctggtga aactgctgag gctaccagac tcaagagaac cgctagaaga
     1861 aggtacacca gaagaaagaa caggatctgc tacctccaag agatcttctc taacgagatg
     1921 gctaaagtgg atgattcatt cttccacagg ctcgaagagt cattcctcgt ggaagaagat
     1981 aagaagcacg agaggcaccc tatcttcgga aacatcgttg atgaggtggc ataccacgag
     2041 aagtacccta ctatctacca cctcagaaag aagctcgttg attctactga taaggctgat
     2101 ctcaggctca tctacctcgc tctcgctcac atgatcaagt tcagaggaca cttcctcatc
     2161 gagggtgatc tcaaccctga taactctgat gtggataagt tgttcatcca gctcgtgcag
     2221 acctacaacc agcttttcga agagaaccct atcaacgctt caggtgtgga tgctaaggct
     2281 atcctctctg ctaggctctc taagtcaaga aggcttgaga acctcattgc tcagctccct
     2341 ggtgagaaga agaacggact tttcggaaac ttgatcgctc tctctctcgg actcacccct
     2401 aacttcaagt ctaacttcga tctcgctgag gatgcaaagc tccagctctc aaaggatacc
     2461 tacgatgatg atctcgataa cctcctcgct cagatcggag atcagtacgc tgatttgttc
     2521 ctcgctgcta agaacctctc tgatgctatc ctcctcagtg atatcctcag agtgaacacc
     2581 gagatcacca aggctccact ctcagcttct atgatcaaga gatacgatga gcaccaccag
     2641 gatctcacac ttctcaaggc tcttgttaga cagcagctcc cagagaagta caaagagatt
     2701 ttcttcgatc agtctaagaa cggatacgct ggttacatcg atggtggtgc atctcaagaa
     2761 gagttctaca agttcatcaa gcctatcctc gagaagatgg atggaaccga ggaactcctc
     2821 gtgaagctca atagagagga tcttctcaga aagcagagga ccttcgataa cggatctatc
     2881 cctcatcaga tccacctcgg agagttgcac gctatcctta gaaggcaaga ggatttctac
     2941 ccattcctca aggataacag ggaaaagatt gagaagattc tcaccttcag aatcccttac
     3001 tacgtgggac ctctcgctag aggaaactca agattcgctt ggatgaccag aaagtctgag
     3061 gaaaccatca ccccttggaa cttcgaagag gtggtggata agggtgctag tgctcagtct
     3121 ttcatcgaga ggatgaccaa cttcgataag aaccttccaa acgagaaggt gctccctaag
     3181 cactctttgc tctacgagta cttcaccgtg tacaacgagt tgaccaaggt taagtacgtg
     3241 accgagggaa tgaggaagcc tgcttttttg tcaggtgagc aaaagaaggc tatcgttgat
     3301 ctcttgttca agaccaacag aaaggtgacc gtgaagcagc tcaaagagga ttacttcaag
     3361 aaaatcgagt gcttcgattc agttgagatt tctggtgttg aggataggtt caacgcatct
     3421 ctcggaacct accacgatct cctcaagatc attaaggata aggatttctt ggataacgag
     3481 gaaaacgagg atatcttgga ggatatcgtt cttaccctca ccctctttga agatagagag
     3541 atgattgaag aaaggctcaa gacctacgct catctcttcg atgataaggt gatgaagcag
     3601 ttgaagagaa gaagatacac tggttgggga aggctctcaa gaaagctcat taacggaatc
     3661 agggataagc agtctggaaa gacaatcctt gatttcctca agtctgatgg attcgctaac
     3721 agaaacttca tgcagctcat ccacgatgat tctctcacct ttaaagagga tatccagaag
     3781 gctcaggttt caggacaggg tgatagtctc catgagcata tcgctaacct cgctggatct
     3841 cctgcaatca agaagggaat cctccagact gtgaaggttg tggatgagtt ggtgaaggtg
     3901 atgggaaggc ataagcctga gaacatcgtg atcgaaatgg ctagagagaa ccagaccact
     3961 cagaagggac agaagaactc tagggaaagg atgaagagga tcgaggaagg tatcaaagag
     4021 cttggatctc agatcctcaa agagcaccct gttgagaaca ctcagctcca gaatgagaag
     4081 ctctacctct actacctcca gaacggaagg gatatgtatg tggatcaaga gttggatatc
     4141 aacaggctct ctgattacga tgttgatcat atcgtgccac agtcattctt gaaggatgat
     4201 tctatcgata acaaggtgct caccaggtct gataagaaca ggggtaagag tgataacgtg
     4261 ccaagtgaag aggttgtgaa gaaaatgaag aactattgga ggcagctcct caacgctaag
     4321 ctcatcactc agagaaagtt cgataacttg actaaggctg agaggggagg actctctgaa
     4381 ttggataagg caggattcat caagaggcag cttgtggaaa ccaggcagat cactaagcac
     4441 gttgcacaga tcctcgattc taggatgaac accaagtacg atgagaacga taagttgatc
     4501 agggaagtga aggttatcac cctcaagtca aagctcgtgt ctgatttcag aaaggatttc
     4561 caattctaca aggtgaggga aatcaacaac taccaccacg ctcacgatgc ttaccttaac
     4621 gctgttgttg gaaccgctct catcaagaag tatcctaagc tcgagtcaga gttcgtgtac
     4681 ggtgattaca aggtgtacga tgtgaggaag atgatcgcta agtctgagca agagatcgga
     4741 aaggctaccg ctaagtattt cttctactct aacatcatga atttcttcaa gaccgagatt
     4801 accctcgcta acggtgagat cagaaagagg ccactcatcg agacaaacgg tgaaacaggt
     4861 gagatcgtgt gggataaggg aagggatttc gctaccgtta gaaaggtgct ctctatgcca
     4921 caggtgaaca tcgttaagaa aaccgaggtg cagaccggtg gattctctaa agagtctatc
     4981 ctccctaaga ggaactctga taagctcatt gctaggaaga aggattggga ccctaagaaa
     5041 tacggtggtt tcgattctcc taccgtggct tactctgttc tcgttgtggc taaggttgag
     5101 aagggaaaga gtaagaagct caagtctgtt aaggaacttc tcggaatcac tatcatggaa
     5161 aggtcatctt tcgagaagaa cccaatcgat ttcctcgagg ctaagggata caaagaggtt
     5221 aagaaggatc tcatcatcaa gctcccaaag tactcactct tcgaactcga gaacggtaga
     5281 aagaggatgc tcgcttctgc tggtgagctt caaaagggaa acgagcttgc tctcccatct
     5341 aagtacgtta actttcttta cctcgcttct cactacgaga agttgaaggg atctccagaa
     5401 gataacgagc agaagcaact tttcgttgag cagcacaagc actacttgga tgagatcatc
     5461 gagcagatct ctgagttctc taaaagggtg atcctcgctg atgcaaacct cgataaggtg
     5521 ttgtctgctt acaacaagca cagagataag cctatcaggg aacaggcaga gaacatcatc
     5581 catctcttca cccttaccaa cctcggtgct cctgctgctt tcaagtactt cgatacaacc
     5641 atcgatagga agagatacac ctctaccaaa gaagtgctcg atgctaccct catccatcag
     5701 tctatcactg gactctacga gactaggatc gatctctcac agctcggtgg tgattcaagg
     5761 gctgatccta agaagaagag gaaggtttga cgtcgacgat atgaagatga agatgaaata
     5821 tttggtgtgt caaataaaaa gcttgtgtgc ttaagtttgt gtttttttct tggcttgttg
     5881 tgttatgaat ttgtggcttt ttctaatatt aaatgaatgt aagatcacat tataatgaat
     5941 aaacaaatgt ttctataatc cattgtgaat gttttgttgg atctcttctg cagcatataa
     6001 ctactgtatg tgctatggta tggactatgg aatatgatta aagataagcc agagctctgg
     6061 tgacggacgt gggcaggtgt gatgaaaagc cttagggtca ggtggcactt ttcggggaaa
     6121 tgtgcgatga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctaat
     6181 gagacaataa ccctgataaa tgcttcaatt atattgaaaa aggaagagta tgagtatcca
     6241 acatttccgt gtcgccctta ttcccttttt tgcggccttc tgccttcctg tttttgctca
     6301 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgccc gagtgggtta
     6361 cattgaactg gatctcaaca gcggcaaaat cttagagagt tttcgccccg aagaacgctt
     6421 cccaatgatg agcactttca aagttctgct atgtggcgcg gtgttatccc gtattgatgc
     6481 tgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtattc
     6541 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gtagtgctgc
     6601 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgattg gaggaccgaa
     6661 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6721 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6781 ggcaacaact ctccgcaaac tattaactgg cgaactactt actcttgctt cacgccagca
     6841 actcattgac tggatggagg cggacaaagt tgcaggacca cttctgcgct cggcacttcc
     6901 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6961 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     7021 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     7081 gcattggtaa ctgtcagacg aagggaaata aatagtagcc cgcctgatgt gcgggctttt
     7141 ttttggtggt ggttaccagt gtcgcttagc taccgtaggt cctcttaggc ggtccgatta
     7201 gcgacatagt catcaatgcc ttgacggcaa aatcccttaa cgtgagtttt cgttccactg
     7261 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7321 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7381 agagctacta actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7441 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7501 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7561 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7621 gggttcgtgc tcacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7681 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7741 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7801 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7861 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7921 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7981 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     8041 cgagtcagtg agcgaggaag cggaagagcg cccacctgct catgatcggc gcgcc
//
