LOCUS       sequence-275199-        6197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puchta Arabidopsis codon-optimised Cas9 for CRISPR. CDS12 for N-term
            fusion with a CTAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6197)
  AUTHORS   Sauret-Gueto S, Frangedakis E, Silvestri L, Rebmann M, Tomaselli M, 
            Markel K, Delmans M, West A, Patron NJ, Haseloff J
  TITLE     Systematic tools for reprogramming plant gene expression in a simple
            model, Marchantia polymorpha.
  JOURNAL   ACS Synth Biol. 2020 Mar 12. doi: 10.1021/acssynbio.9b00511.
  PUBMED    32163700
REFERENCE   2  (bases 1 to 6197)
  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: "ACS Synth 
            Biol. 2020 Mar 12. doi: 10.1021/acssynbio.9b00511."
COMMENT     Sequence Label: CDS12_Cas9-NLS
FEATURES             Location/Qualifiers
     source          1..6197
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..128
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(152..209)
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             224..4327
                     /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             224..4327
                     /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             4340..4360
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      4382..4425
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     primer_bind     complement(4429..4447)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             4609..5268
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4675..4694)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5289..5383
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      5566..6154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6055..6074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
       61 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
      121 tgagcgagga agcctgcata acgcgaagta atcttttcgg ttttaaagaa aaagggcagg
      181 gtggtgacac cttgcccttt tttgccggac tgcagggtct cgaatggata agaagtactc
      241 tatcggactc gatatcggaa ctaactctgt gggatgggct gtgatcaccg atgagtacaa
      301 ggtgccatct aagaagttca aggttctcgg aaacaccgat aggcactcta tcaagaaaaa
      361 ccttatcggt gctctcctct tcgattctgg tgaaactgct gaggctacca gactcaagag
      421 aaccgctaga agaaggtaca ccagaagaaa gaacaggatc tgctacctcc aagagatctt
      481 ctctaacgag atggctaaag tggatgattc attcttccac aggctcgaag agtcattcct
      541 cgtggaagaa gataagaagc acgagaggca ccctatcttc ggaaacatcg ttgatgaggt
      601 ggcataccac gagaagtacc ctactatcta ccacctcaga aagaagctcg ttgattctac
      661 tgataaggct gatctcaggc tcatctacct cgctctcgct cacatgatca agttcagagg
      721 acacttcctc atcgagggtg atctcaaccc tgataactct gatgtggata agttgttcat
      781 ccagctcgtg cagacctaca accagctttt cgaagagaac cctatcaacg cttcaggtgt
      841 ggatgctaag gctatcctct ctgctaggct ctctaagtca agaaggcttg agaacctcat
      901 tgctcagctc cctggtgaga agaagaacgg acttttcgga aacttgatcg ctctctctct
      961 cggactcacc cctaacttca agtctaactt cgatctcgct gaggatgcaa agctccagct
     1021 ctcaaaggat acctacgatg atgatctcga taacctcctc gctcagatcg gagatcagta
     1081 cgctgatttg ttcctcgctg ctaagaacct ctctgatgct atcctcctca gtgatatcct
     1141 cagagtgaac accgagatca ccaaggctcc actctcagct tctatgatca agagatacga
     1201 tgagcaccac caggatctca cacttctcaa ggctcttgtt agacagcagc tcccagagaa
     1261 gtacaaagag attttcttcg atcagtctaa gaacggatac gctggttaca tcgatggtgg
     1321 tgcatctcaa gaagagttct acaagttcat caagcctatc ctcgagaaga tggatggaac
     1381 cgaggaactc ctcgtgaagc tcaatagaga ggatcttctc agaaagcaga ggaccttcga
     1441 taacggatct atccctcatc agatccacct cggagagttg cacgctatcc ttagaaggca
     1501 agaggatttc tacccattcc tcaaggataa cagggaaaag attgagaaga ttctcacctt
     1561 cagaatccct tactacgtgg gacctctcgc tagaggaaac tcaagattcg cttggatgac
     1621 cagaaagtct gaggaaacca tcaccccttg gaacttcgaa gaggtggtgg ataagggtgc
     1681 tagtgctcag tctttcatcg agaggatgac caacttcgat aagaaccttc caaacgagaa
     1741 ggtgctccct aagcactctt tgctctacga gtacttcacc gtgtacaacg agttgaccaa
     1801 ggttaagtac gtgaccgagg gaatgaggaa gcctgctttt ttgtcaggtg agcaaaagaa
     1861 ggctatcgtt gatctcttgt tcaagaccaa cagaaaggtg accgtgaagc agctcaaaga
     1921 ggattacttc aagaaaatcg agtgcttcga ttcagttgag atttctggtg ttgaggatag
     1981 gttcaacgca tctctcggaa cctaccacga tctcctcaag atcattaagg ataaggattt
     2041 cttggataac gaggaaaacg aggatatctt ggaggatatc gttcttaccc tcaccctctt
     2101 tgaagataga gagatgattg aagaaaggct caagacctac gctcatctct tcgatgataa
     2161 ggtgatgaag cagttgaaga gaagaagata cactggttgg ggaaggctct caagaaagct
     2221 cattaacgga atcagggata agcagtctgg aaagacaatc cttgatttcc tcaagtctga
     2281 tggattcgct aacagaaact tcatgcagct catccacgat gattctctca cctttaaaga
     2341 ggatatccag aaggctcagg tttcaggaca gggtgatagt ctccatgagc atatcgctaa
     2401 cctcgctgga tctcctgcaa tcaagaaggg aatcctccag actgtgaagg ttgtggatga
     2461 gttggtgaag gtgatgggaa ggcataagcc tgagaacatc gtgatcgaaa tggctagaga
     2521 gaaccagacc actcagaagg gacagaagaa ctctagggaa aggatgaaga ggatcgagga
     2581 aggtatcaaa gagcttggat ctcagatcct caaagagcac cctgttgaga acactcagct
     2641 ccagaatgag aagctctacc tctactacct ccagaacgga agggatatgt atgtggatca
     2701 agagttggat atcaacaggc tctctgatta cgatgttgat catatcgtgc cacagtcatt
     2761 cttgaaggat gattctatcg ataacaaggt gctcaccagg tctgataaga acaggggtaa
     2821 gagtgataac gtgccaagtg aagaggttgt gaagaaaatg aagaactatt ggaggcagct
     2881 cctcaacgct aagctcatca ctcagagaaa gttcgataac ttgactaagg ctgagagggg
     2941 aggactctct gaattggata aggcaggatt catcaagagg cagcttgtgg aaaccaggca
     3001 gatcactaag cacgttgcac agatcctcga ttctaggatg aacaccaagt acgatgagaa
     3061 cgataagttg atcagggaag tgaaggttat caccctcaag tcaaagctcg tgtctgattt
     3121 cagaaaggat ttccaattct acaaggtgag ggaaatcaac aactaccacc acgctcacga
     3181 tgcttacctt aacgctgttg ttggaaccgc tctcatcaag aagtatccta agctcgagtc
     3241 agagttcgtg tacggtgatt acaaggtgta cgatgtgagg aagatgatcg ctaagtctga
     3301 gcaagagatc ggaaaggcta ccgctaagta tttcttctac tctaacatca tgaatttctt
     3361 caagaccgag attaccctcg ctaacggtga gatcagaaag aggccactca tcgagacaaa
     3421 cggtgaaaca ggtgagatcg tgtgggataa gggaagggat ttcgctaccg ttagaaaggt
     3481 gctctctatg ccacaggtga acatcgttaa gaaaaccgag gtgcagaccg gtggattctc
     3541 taaagagtct atcctcccta agaggaactc tgataagctc attgctagga agaaggattg
     3601 ggaccctaag aaatacggtg gtttcgattc tcctaccgtg gcttactctg ttctcgttgt
     3661 ggctaaggtt gagaagggaa agagtaagaa gctcaagtct gttaaggaac ttctcggaat
     3721 cactatcatg gaaaggtcat ctttcgagaa gaacccaatc gatttcctcg aggctaaggg
     3781 atacaaagag gttaagaagg atctcatcat caagctccca aagtactcac tcttcgaact
     3841 cgagaacggt agaaagagga tgctcgcttc tgctggtgag cttcaaaagg gaaacgagct
     3901 tgctctccca tctaagtacg ttaactttct ttacctcgct tctcactacg agaagttgaa
     3961 gggatctcca gaagataacg agcagaagca acttttcgtt gagcagcaca agcactactt
     4021 ggatgagatc atcgagcaga tctctgagtt ctctaaaagg gtgatcctcg ctgatgcaaa
     4081 cctcgataag gtgttgtctg cttacaacaa gcacagagat aagcctatca gggaacaggc
     4141 agagaacatc atccatctct tcacccttac caacctcggt gctcctgctg ctttcaagta
     4201 cttcgataca accatcgata ggaagagata cacctctacc aaagaagtgc tcgatgctac
     4261 cctcatccat cagtctatca ctggactcta cgagactagg atcgatctct cacagctcgg
     4321 tggtgattca agggctgatc ctaagaagaa gaggaaggtt gcttcgtgag accgaattcc
     4381 agaaatcatc cttagcgaaa gctaaggatt ttttttatct gaaattctgc ctcgtgatac
     4441 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactc
     4501 gagttgatcg ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg
     4561 ggcgtatttt ttgagttatc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
     4621 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
     4681 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
     4741 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
     4801 cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata
     4861 tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg
     4921 ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg
     4981 gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
     5041 gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac
     5101 aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg
     5161 atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg
     5221 cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tgatatcgag
     5281 ctcgcttgga ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt
     5341 cagaacgctc ggttgccgcc gggcgttttt tattggtgag aatccaagcc tcgagctgtc
     5401 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5461 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5521 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5581 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5641 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5701 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5761 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5821 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5881 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5941 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6001 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6061 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6121 gtgatgctcg tcaggggggc ggagcctgtg gaaaaacgcc agcaacgcgg cctttttacg
     6181 gttcctggcc ttttgct
//
