LOCUS       sequence-275195-        6198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Puchta Arabidopsis codon-optimised Cas9 for CRISPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6198)
  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 6198)
  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: CDS_Cas9-NLS
FEATURES             Location/Qualifiers
     source          1..6198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(117..705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(197..216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(888..982)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1003..1662)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1577..1596
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     1824..1842
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1846..1889)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(1912..1932)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(1945..6048)
                     /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             complement(1945..6048)
                     /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"
     terminator      6063..6120
                     /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."
     primer_bind     complement(6144..6161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
       61 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      121 cacaggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      181 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      241 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      301 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      361 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      421 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      481 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      541 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      601 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      661 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
      721 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
      781 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
      841 atctaaagta tatatgagta aacttggtct gacagctcga ggcttggatt ctcaccaata
      901 aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt
      961 actggatcta tcaacaggag tccaagcgag ctcgatatca aattacgccc cgccctgcca
     1021 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacaaac
     1081 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     1141 gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact
     1201 ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg
     1261 gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt gtagaaactg
     1321 ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa
     1381 aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat
     1441 acgaaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa
     1501 cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg
     1561 gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg
     1621 ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc
     1681 tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat tatggtgaaa
     1741 gttggaacct cttacgtgcc cgatcaactc gagtgccacc tgacgtctaa gaaaccatta
     1801 ttatcatgac attaacctat aaaaataggc gtatcacgag gcagaatttc agataaaaaa
     1861 aatccttagc tttcgctaag gatgatttct ggaattcggt ctcaaagctc aaaccttcct
     1921 cttcttctta ggatcagccc ttgaatcacc accgagctgt gagagatcga tcctagtctc
     1981 gtagagtcca gtgatagact gatggatgag ggtagcatcg agcacttctt tggtagaggt
     2041 gtatctcttc ctatcgatgg ttgtatcgaa gtacttgaaa gcagcaggag caccgaggtt
     2101 ggtaagggtg aagagatgga tgatgttctc tgcctgttcc ctgataggct tatctctgtg
     2161 cttgttgtaa gcagacaaca ccttatcgag gtttgcatca gcgaggatca cccttttaga
     2221 gaactcagag atctgctcga tgatctcatc caagtagtgc ttgtgctgct caacgaaaag
     2281 ttgcttctgc tcgttatctt ctggagatcc cttcaacttc tcgtagtgag aagcgaggta
     2341 aagaaagtta acgtacttag atgggagagc aagctcgttt cccttttgaa gctcaccagc
     2401 agaagcgagc atcctctttc taccgttctc gagttcgaag agtgagtact ttgggagctt
     2461 gatgatgaga tccttcttaa cctctttgta tcccttagcc tcgaggaaat cgattgggtt
     2521 cttctcgaaa gatgaccttt ccatgatagt gattccgaga agttccttaa cagacttgag
     2581 cttcttactc tttcccttct caaccttagc cacaacgaga acagagtaag ccacggtagg
     2641 agaatcgaaa ccaccgtatt tcttagggtc ccaatccttc ttcctagcaa tgagcttatc
     2701 agagttcctc ttagggagga tagactcttt agagaatcca ccggtctgca cctcggtttt
     2761 cttaacgatg ttcacctgtg gcatagagag cacctttcta acggtagcga aatcccttcc
     2821 cttatcccac acgatctcac ctgtttcacc gtttgtctcg atgagtggcc tctttctgat
     2881 ctcaccgtta gcgagggtaa tctcggtctt gaagaaattc atgatgttag agtagaagaa
     2941 atacttagcg gtagcctttc cgatctcttg ctcagactta gcgatcatct tcctcacatc
     3001 gtacaccttg taatcaccgt acacgaactc tgactcgagc ttaggatact tcttgatgag
     3061 agcggttcca acaacagcgt taaggtaagc atcgtgagcg tggtggtagt tgttgatttc
     3121 cctcaccttg tagaattgga aatcctttct gaaatcagac acgagctttg acttgagggt
     3181 gataaccttc acttccctga tcaacttatc gttctcatcg tacttggtgt tcatcctaga
     3241 atcgaggatc tgtgcaacgt gcttagtgat ctgcctggtt tccacaagct gcctcttgat
     3301 gaatcctgcc ttatccaatt cagagagtcc tcccctctca gccttagtca agttatcgaa
     3361 ctttctctga gtgatgagct tagcgttgag gagctgcctc caatagttct tcattttctt
     3421 cacaacctct tcacttggca cgttatcact cttacccctg ttcttatcag acctggtgag
     3481 caccttgtta tcgatagaat catccttcaa gaatgactgt ggcacgatat gatcaacatc
     3541 gtaatcagag agcctgttga tatccaactc ttgatccaca tacatatccc ttccgttctg
     3601 gaggtagtag aggtagagct tctcattctg gagctgagtg ttctcaacag ggtgctcttt
     3661 gaggatctga gatccaagct ctttgatacc ttcctcgatc ctcttcatcc tttccctaga
     3721 gttcttctgt cccttctgag tggtctggtt ctctctagcc atttcgatca cgatgttctc
     3781 aggcttatgc cttcccatca ccttcaccaa ctcatccaca accttcacag tctggaggat
     3841 tcccttcttg attgcaggag atccagcgag gttagcgata tgctcatgga gactatcacc
     3901 ctgtcctgaa acctgagcct tctggatatc ctctttaaag gtgagagaat catcgtggat
     3961 gagctgcatg aagtttctgt tagcgaatcc atcagacttg aggaaatcaa ggattgtctt
     4021 tccagactgc ttatccctga ttccgttaat gagctttctt gagagccttc cccaaccagt
     4081 gtatcttctt ctcttcaact gcttcatcac cttatcatcg aagagatgag cgtaggtctt
     4141 gagcctttct tcaatcatct ctctatcttc aaagagggtg agggtaagaa cgatatcctc
     4201 caagatatcc tcgttttcct cgttatccaa gaaatcctta tccttaatga tcttgaggag
     4261 atcgtggtag gttccgagag atgcgttgaa cctatcctca acaccagaaa tctcaactga
     4321 atcgaagcac tcgattttct tgaagtaatc ctctttgagc tgcttcacgg tcacctttct
     4381 gttggtcttg aacaagagat caacgatagc cttcttttgc tcacctgaca aaaaagcagg
     4441 cttcctcatt ccctcggtca cgtacttaac cttggtcaac tcgttgtaca cggtgaagta
     4501 ctcgtagagc aaagagtgct tagggagcac cttctcgttt ggaaggttct tatcgaagtt
     4561 ggtcatcctc tcgatgaaag actgagcact agcaccctta tccaccacct cttcgaagtt
     4621 ccaaggggtg atggtttcct cagactttct ggtcatccaa gcgaatcttg agtttcctct
     4681 agcgagaggt cccacgtagt aagggattct gaaggtgaga atcttctcaa tcttttccct
     4741 gttatccttg aggaatgggt agaaatcctc ttgccttcta aggatagcgt gcaactctcc
     4801 gaggtggatc tgatgaggga tagatccgtt atcgaaggtc ctctgctttc tgagaagatc
     4861 ctctctattg agcttcacga ggagttcctc ggttccatcc atcttctcga ggataggctt
     4921 gatgaacttg tagaactctt cttgagatgc accaccatcg atgtaaccag cgtatccgtt
     4981 cttagactga tcgaagaaaa tctctttgta cttctctggg agctgctgtc taacaagagc
     5041 cttgagaagt gtgagatcct ggtggtgctc atcgtatctc ttgatcatag aagctgagag
     5101 tggagccttg gtgatctcgg tgttcactct gaggatatca ctgaggagga tagcatcaga
     5161 gaggttctta gcagcgagga acaaatcagc gtactgatct ccgatctgag cgaggaggtt
     5221 atcgagatca tcatcgtagg tatcctttga gagctggagc tttgcatcct cagcgagatc
     5281 gaagttagac ttgaagttag gggtgagtcc gagagagaga gcgatcaagt ttccgaaaag
     5341 tccgttcttc ttctcaccag ggagctgagc aatgaggttc tcaagccttc ttgacttaga
     5401 gagcctagca gagaggatag ccttagcatc cacacctgaa gcgttgatag ggttctcttc
     5461 gaaaagctgg ttgtaggtct gcacgagctg gatgaacaac ttatccacat cagagttatc
     5521 agggttgaga tcaccctcga tgaggaagtg tcctctgaac ttgatcatgt gagcgagagc
     5581 gaggtagatg agcctgagat cagccttatc agtagaatca acgagcttct ttctgaggtg
     5641 gtagatagta gggtacttct cgtggtatgc cacctcatca acgatgtttc cgaagatagg
     5701 gtgcctctcg tgcttcttat cttcttccac gaggaatgac tcttcgagcc tgtggaagaa
     5761 tgaatcatcc actttagcca tctcgttaga gaagatctct tggaggtagc agatcctgtt
     5821 ctttcttctg gtgtaccttc ttctagcggt tctcttgagt ctggtagcct cagcagtttc
     5881 accagaatcg aagaggagag caccgataag gtttttcttg atagagtgcc tatcggtgtt
     5941 tccgagaacc ttgaacttct tagatggcac cttgtactca tcggtgatca cagcccatcc
     6001 cacagagtta gttccgatat cgagtccgat agagtacttc ttatccattc gagaccctgc
     6061 agtccggcaa aaaagggcaa ggtgtcacca ccctgccctt tttctttaaa accgaaaaga
     6121 ttacttcgcg ttatgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     6181 cggcgagcgg tatcagct
//
