LOCUS       sequence-181767-        7549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Module A, Promoter: CmYLCV, Gene: Csy4-P2A-AtCas9, Terminator: HSP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7549)
  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 7549)
  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_A0503
FEATURES             Location/Qualifiers
     source          1..7549
                     /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             1926..1982
                     /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             1983..6086
                     /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             1983..6086
                     /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             6099..6119
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      6132..6380
                     /label=HSP terminator
                     /note="efficient transcription terminator from the 
                     Arabidopsis thaliana heat shock protein 18.2 gene (Nagaya 
                     et al., 2010)"
     promoter        6457..6561
                     /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 ggcgcgcctg gcagacatac tgtcccacaa
      901 atgaagatgg aatctgtaaa agaaaacgcg tgaaataatg cgtctgacaa aggttaggtc
      961 ggctgccttt aatcaatacc aaagtggtcc ctaccacgat ggaaaaactg tgcagtcggt
     1021 ttggcttttt ctgacgaaca aataagattc gtggccgaca ggtgggggtc caccatgtga
     1081 aggcatcttc agactccaat aatggagcaa tgacgtaagg gcttacgaaa taagtaaggg
     1141 tagtttggga aatgtccact cacccgtcag tctataaata cttagcccct ccctcattgt
     1201 taagggagca aaatctcaga gagatagtcc tagagagaga aagagagcaa gtagcctaga
     1261 agtagtcaag gcggcgaagt attcaggcac gtggccagga agaagaaaag ccaagacgac
     1321 gaaaacaggt aagagctaag cttcctgcag gaaaaatgga tcattatctt gatattagac
     1381 ttagacctga tccagaattt ccaccagctc aacttatgtc tgttcttttt ggaaaacttc
     1441 atcaagctct tgttgctcaa ggaggagata gaattggagt ttcttttcct gatcttgatg
     1501 aatcaagatc aagacttgga gaaagactta gaattcatgc ttctgctgat gatcttagag
     1561 ctttgcttgc tagaccttgg cttgaaggac ttagagatca tcttcaattt ggagaaccag
     1621 ctgttgttcc acatccaact ccttatagac aagtttcaag agttcaagct aaatctaatc
     1681 cagaaagact tagaagaaga cttatgagaa gacatgatct ttctgaagaa gaagctagaa
     1741 aaagaattcc tgatactgtt gctagagctt tggatttgcc ttttgttaca cttagatcac
     1801 aatctactgg acaacatttt agacttttta ttagacatgg accacttcaa gttactgctg
     1861 aagaaggagg atttacttgt tatggacttt ctaagggagg ttttgttcct tggtttggat
     1921 ctggagctac taatttttct cttcttaagc aagctggaga tgttgaagaa aatcctggac
     1981 ccatggataa gaagtactct atcggactcg atatcggaac taactctgtg ggatgggctg
     2041 tgatcaccga tgagtacaag gtgccatcta agaagttcaa ggttctcgga aacaccgata
     2101 ggcactctat caagaaaaac cttatcggtg ctctcctctt cgattctggt gaaactgctg
     2161 aggctaccag actcaagaga accgctagaa gaaggtacac cagaagaaag aacaggatct
     2221 gctacctcca agagatcttc tctaacgaga tggctaaagt ggatgattca ttcttccaca
     2281 ggctcgaaga gtcattcctc gtggaagaag ataagaagca cgagaggcac cctatcttcg
     2341 gaaacatcgt tgatgaggtg gcataccacg agaagtaccc tactatctac cacctcagaa
     2401 agaagctcgt tgattctact gataaggctg atctcaggct catctacctc gctctcgctc
     2461 acatgatcaa gttcagagga cacttcctca tcgagggtga tctcaaccct gataactctg
     2521 atgtggataa gttgttcatc cagctcgtgc agacctacaa ccagcttttc gaagagaacc
     2581 ctatcaacgc ttcaggtgtg gatgctaagg ctatcctctc tgctaggctc tctaagtcaa
     2641 gaaggcttga gaacctcatt gctcagctcc ctggtgagaa gaagaacgga cttttcggaa
     2701 acttgatcgc tctctctctc ggactcaccc ctaacttcaa gtctaacttc gatctcgctg
     2761 aggatgcaaa gctccagctc tcaaaggata cctacgatga tgatctcgat aacctcctcg
     2821 ctcagatcgg agatcagtac gctgatttgt tcctcgctgc taagaacctc tctgatgcta
     2881 tcctcctcag tgatatcctc agagtgaaca ccgagatcac caaggctcca ctctcagctt
     2941 ctatgatcaa gagatacgat gagcaccacc aggatctcac acttctcaag gctcttgtta
     3001 gacagcagct cccagagaag tacaaagaga ttttcttcga tcagtctaag aacggatacg
     3061 ctggttacat cgatggtggt gcatctcaag aagagttcta caagttcatc aagcctatcc
     3121 tcgagaagat ggatggaacc gaggaactcc tcgtgaagct caatagagag gatcttctca
     3181 gaaagcagag gaccttcgat aacggatcta tccctcatca gatccacctc ggagagttgc
     3241 acgctatcct tagaaggcaa gaggatttct acccattcct caaggataac agggaaaaga
     3301 ttgagaagat tctcaccttc agaatccctt actacgtggg acctctcgct agaggaaact
     3361 caagattcgc ttggatgacc agaaagtctg aggaaaccat caccccttgg aacttcgaag
     3421 aggtggtgga taagggtgct agtgctcagt ctttcatcga gaggatgacc aacttcgata
     3481 agaaccttcc aaacgagaag gtgctcccta agcactcttt gctctacgag tacttcaccg
     3541 tgtacaacga gttgaccaag gttaagtacg tgaccgaggg aatgaggaag cctgcttttt
     3601 tgtcaggtga gcaaaagaag gctatcgttg atctcttgtt caagaccaac agaaaggtga
     3661 ccgtgaagca gctcaaagag gattacttca agaaaatcga gtgcttcgat tcagttgaga
     3721 tttctggtgt tgaggatagg ttcaacgcat ctctcggaac ctaccacgat ctcctcaaga
     3781 tcattaagga taaggatttc ttggataacg aggaaaacga ggatatcttg gaggatatcg
     3841 ttcttaccct caccctcttt gaagatagag agatgattga agaaaggctc aagacctacg
     3901 ctcatctctt cgatgataag gtgatgaagc agttgaagag aagaagatac actggttggg
     3961 gaaggctctc aagaaagctc attaacggaa tcagggataa gcagtctgga aagacaatcc
     4021 ttgatttcct caagtctgat ggattcgcta acagaaactt catgcagctc atccacgatg
     4081 attctctcac ctttaaagag gatatccaga aggctcaggt ttcaggacag ggtgatagtc
     4141 tccatgagca tatcgctaac ctcgctggat ctcctgcaat caagaaggga atcctccaga
     4201 ctgtgaaggt tgtggatgag ttggtgaagg tgatgggaag gcataagcct gagaacatcg
     4261 tgatcgaaat ggctagagag aaccagacca ctcagaaggg acagaagaac tctagggaaa
     4321 ggatgaagag gatcgaggaa ggtatcaaag agcttggatc tcagatcctc aaagagcacc
     4381 ctgttgagaa cactcagctc cagaatgaga agctctacct ctactacctc cagaacggaa
     4441 gggatatgta tgtggatcaa gagttggata tcaacaggct ctctgattac gatgttgatc
     4501 atatcgtgcc acagtcattc ttgaaggatg attctatcga taacaaggtg ctcaccaggt
     4561 ctgataagaa caggggtaag agtgataacg tgccaagtga agaggttgtg aagaaaatga
     4621 agaactattg gaggcagctc ctcaacgcta agctcatcac tcagagaaag ttcgataact
     4681 tgactaaggc tgagagggga ggactctctg aattggataa ggcaggattc atcaagaggc
     4741 agcttgtgga aaccaggcag atcactaagc acgttgcaca gatcctcgat tctaggatga
     4801 acaccaagta cgatgagaac gataagttga tcagggaagt gaaggttatc accctcaagt
     4861 caaagctcgt gtctgatttc agaaaggatt tccaattcta caaggtgagg gaaatcaaca
     4921 actaccacca cgctcacgat gcttacctta acgctgttgt tggaaccgct ctcatcaaga
     4981 agtatcctaa gctcgagtca gagttcgtgt acggtgatta caaggtgtac gatgtgagga
     5041 agatgatcgc taagtctgag caagagatcg gaaaggctac cgctaagtat ttcttctact
     5101 ctaacatcat gaatttcttc aagaccgaga ttaccctcgc taacggtgag atcagaaaga
     5161 ggccactcat cgagacaaac ggtgaaacag gtgagatcgt gtgggataag ggaagggatt
     5221 tcgctaccgt tagaaaggtg ctctctatgc cacaggtgaa catcgttaag aaaaccgagg
     5281 tgcagaccgg tggattctct aaagagtcta tcctccctaa gaggaactct gataagctca
     5341 ttgctaggaa gaaggattgg gaccctaaga aatacggtgg tttcgattct cctaccgtgg
     5401 cttactctgt tctcgttgtg gctaaggttg agaagggaaa gagtaagaag ctcaagtctg
     5461 ttaaggaact tctcggaatc actatcatgg aaaggtcatc tttcgagaag aacccaatcg
     5521 atttcctcga ggctaaggga tacaaagagg ttaagaagga tctcatcatc aagctcccaa
     5581 agtactcact cttcgaactc gagaacggta gaaagaggat gctcgcttct gctggtgagc
     5641 ttcaaaaggg aaacgagctt gctctcccat ctaagtacgt taactttctt tacctcgctt
     5701 ctcactacga gaagttgaag ggatctccag aagataacga gcagaagcaa cttttcgttg
     5761 agcagcacaa gcactacttg gatgagatca tcgagcagat ctctgagttc tctaaaaggg
     5821 tgatcctcgc tgatgcaaac ctcgataagg tgttgtctgc ttacaacaag cacagagata
     5881 agcctatcag ggaacaggca gagaacatca tccatctctt cacccttacc aacctcggtg
     5941 ctcctgctgc tttcaagtac ttcgatacaa ccatcgatag gaagagatac acctctacca
     6001 aagaagtgct cgatgctacc ctcatccatc agtctatcac tggactctac gagactagga
     6061 tcgatctctc acagctcggt ggtgattcaa gggctgatcc taagaagaag aggaaggttt
     6121 gacgtcgacg atatgaagat gaagatgaaa tatttggtgt gtcaaataaa aagcttgtgt
     6181 gcttaagttt gtgttttttt cttggcttgt tgtgttatga atttgtggct ttttctaata
     6241 ttaaatgaat gtaagatcac attataatga ataaacaaat gtttctataa tccattgtga
     6301 atgttttgtt ggatctcttc tgcagcatat aactactgta tgtgctatgg tatggactat
     6361 ggaatatgat taaagataag ccagagctct ggtgacggac gtgggcaggt gtgatgaaaa
     6421 gccttagggt caggtggcac ttttcgggga aatgtgcgat gaacccctat ttgtttattt
     6481 ttctaaatac attcaaatat gtatccgcta atgagacaat aaccctgata aatgcttcaa
     6541 ttatattgaa aaaggaagag tatgagtatc caacatttcc gtgtcgccct tattcccttt
     6601 tttgcggcct tctgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6661 gctgaagatc agttgggtgc ccgagtgggt tacattgaac tggatctcaa cagcggcaaa
     6721 atcttagaga gttttcgccc cgaagaacgc ttcccaatga tgagcacttt caaagttctg
     6781 ctatgtggcg cggtgttatc ccgtattgat gctgggcaag agcaactcgg tcgccgcata
     6841 cactattctc agaatgactt ggttgagtat tcaccagtca cagaaaagca tcttacggat
     6901 ggcatgacag taagagaatt atgtagtgct gccataacca tgagtgataa cactgcggcc
     6961 aacttacttc tgacaacgat tggaggaccg aaggagctaa ccgctttttt gcacaacatg
     7021 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     7081 gacgagcgtg acaccacgat gcctgtagca atggcaacaa ctctccgcaa actattaact
     7141 ggcgaactac ttactcttgc ttcacgccag caactcattg actggatgga ggcggacaaa
     7201 gttgcaggac cacttctgcg ctcggcactt ccggctggct ggtttattgc tgataaatct
     7261 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     7321 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7381 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga cgaagggaaa
     7441 taaatagtag cccgcctgat gtgcgggctt ttttttggtg gtggttacca gtgtcgctta
     7501 gctaccgtag gtcctcttag gcggtccgat tagcgacata gtcatcaat
//
