LOCUS       sequence-227983-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, SpCas9-p .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6390)
  AUTHORS   Raitskin O, Schudoma C, West A, Patron NJ
  TITLE     Comparison of efficiency and specificity of CRISPR-associated (Cas) 
            nucleases in plants: An expanded toolkit for precision genome 
            engineering.
  JOURNAL   PLoS One. 2019 Feb 27;14(2):e0211598. doi: 
            10.1371/journal.pone.0211598. eCollection 2019.
  PUBMED    30811422
REFERENCE   2  (bases 1 to 6390)
  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: "PLoS One.";
            date: "2019-02-27"; volume: "14(2):e0211598. doi"; pages: " 
            10.1371/journal.pone.0211598. eCollection 2019"
COMMENT     Sequence Label: pEPOR0SP0013
FEATURES             Location/Qualifiers
     source          1..6390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     139..156
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             234..4337
                     /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             234..4337
                     /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             4350..4370
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4413..4431)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4499..4603
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4825..5616
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5787..6375
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6276..6295
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
       61 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
      121 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc
      181 aaaccgcctc tccccgcgcg ttggccgatt cattaatcac tctgtggtct caaatggata
      241 agaagtactc tatcggactc gatatcggaa ctaactctgt gggatgggct gtgatcaccg
      301 atgagtacaa ggtgccatct aagaagttca aggttctcgg aaacaccgat aggcactcta
      361 tcaagaaaaa ccttatcggt gctctcctct tcgattctgg tgaaactgct gaggctacca
      421 gactcaagag aaccgctaga agaaggtaca ccagaagaaa gaacaggatc tgctacctcc
      481 aagagatctt ctctaacgag atggctaaag tggatgattc attcttccac aggctcgaag
      541 agtcattcct cgtggaagaa gataagaagc acgagaggca ccctatcttc ggaaacatcg
      601 ttgatgaggt ggcataccac gagaagtacc ctactatcta ccacctcaga aagaagctcg
      661 ttgattctac tgataaggct gatctcaggc tcatctacct cgctctcgct cacatgatca
      721 agttcagagg acacttcctc atcgagggtg atctcaaccc tgataactct gatgtggata
      781 agttgttcat ccagctcgtg cagacctaca accagctttt cgaagagaac cctatcaacg
      841 cttcaggtgt ggatgctaag gctatcctct ctgctaggct ctctaagtca agaaggcttg
      901 agaacctcat tgctcagctc cctggtgaga agaagaacgg acttttcgga aacttgatcg
      961 ctctctctct cggactcacc cctaacttca agtctaactt cgatctcgct gaggatgcaa
     1021 agctccagct ctcaaaggat acctacgatg atgatctcga taacctcctc gctcagatcg
     1081 gagatcagta cgctgatttg ttcctcgctg ctaagaacct ctctgatgct atcctcctca
     1141 gtgatatcct cagagtgaac accgagatca ccaaggctcc actctcagct tctatgatca
     1201 agagatacga tgagcaccac caggatctca cacttctcaa ggctcttgtt agacagcagc
     1261 tcccagagaa gtacaaagag attttcttcg atcagtctaa gaacggatac gctggttaca
     1321 tcgatggtgg tgcatctcaa gaagagttct acaagttcat caagcctatc ctcgagaaga
     1381 tggatggaac cgaggaactc ctcgtgaagc tcaatagaga ggatcttctc agaaagcaga
     1441 ggaccttcga taacggatct atccctcatc agatccacct cggagagttg cacgctatcc
     1501 ttagaaggca agaggatttc tacccattcc tcaaggataa cagggaaaag attgagaaga
     1561 ttctcacctt cagaatccct tactacgtgg gacctctcgc tagaggaaac tcaagattcg
     1621 cttggatgac cagaaagtct gaggaaacca tcaccccttg gaacttcgaa gaggtggtgg
     1681 ataagggtgc tagtgctcag tctttcatcg agaggatgac caacttcgat aagaaccttc
     1741 caaacgagaa ggtgctccct aagcactctt tgctctacga gtacttcacc gtgtacaacg
     1801 agttgaccaa ggttaagtac gtgaccgagg gaatgaggaa gcctgctttt ttgtcaggtg
     1861 agcaaaagaa ggctatcgtt gatctcttgt tcaagaccaa cagaaaggtg accgtgaagc
     1921 agctcaaaga ggattacttc aagaaaatcg agtgcttcga ttcagttgag atttctggtg
     1981 ttgaggatag gttcaacgca tctctcggaa cctaccacga tctcctcaag atcattaagg
     2041 ataaggattt cttggataac gaggaaaacg aggatatctt ggaggatatc gttcttaccc
     2101 tcaccctctt tgaagataga gagatgattg aagaaaggct caagacctac gctcatctct
     2161 tcgatgataa ggtgatgaag cagttgaaga gaagaagata cactggttgg ggaaggctct
     2221 caagaaagct cattaacgga atcagggata agcagtctgg aaagacaatc cttgatttcc
     2281 tcaagtctga tggattcgct aacagaaact tcatgcagct catccacgat gattctctca
     2341 cctttaaaga ggatatccag aaggctcagg tttcaggaca gggtgatagt ctccatgagc
     2401 atatcgctaa cctcgctgga tctcctgcaa tcaagaaggg aatcctccag actgtgaagg
     2461 ttgtggatga gttggtgaag gtgatgggaa ggcataagcc tgagaacatc gtgatcgaaa
     2521 tggctagaga gaaccagacc actcagaagg gacagaagaa ctctagggaa aggatgaaga
     2581 ggatcgagga aggtatcaaa gagcttggat ctcagatcct caaagagcac cctgttgaga
     2641 acactcagct ccagaatgag aagctctacc tctactacct ccagaacgga agggatatgt
     2701 atgtggatca agagttggat atcaacaggc tctctgatta cgatgttgat catatcgtgc
     2761 cacagtcatt cttgaaggat gattctatcg ataacaaggt gctcaccagg tctgataaga
     2821 acaggggtaa gagtgataac gtgccaagtg aagaggttgt gaagaaaatg aagaactatt
     2881 ggaggcagct cctcaacgct aagctcatca ctcagagaaa gttcgataac ttgactaagg
     2941 ctgagagggg aggactctct gaattggata aggcaggatt catcaagagg cagcttgtgg
     3001 aaaccaggca gatcactaag cacgttgcac agatcctcga ttctaggatg aacaccaagt
     3061 acgatgagaa cgataagttg atcagggaag tgaaggttat caccctcaag tcaaagctcg
     3121 tgtctgattt cagaaaggat ttccaattct acaaggtgag ggaaatcaac aactaccacc
     3181 acgctcacga tgcttacctt aacgctgttg ttggaaccgc tctcatcaag aagtatccta
     3241 agctcgagtc agagttcgtg tacggtgatt acaaggtgta cgatgtgagg aagatgatcg
     3301 ctaagtctga gcaagagatc ggaaaggcta ccgctaagta tttcttctac tctaacatca
     3361 tgaatttctt caagaccgag attaccctcg ctaacggtga gatcagaaag aggccactca
     3421 tcgagacaaa cggtgaaaca ggtgagatcg tgtgggataa gggaagggat ttcgctaccg
     3481 ttagaaaggt gctctctatg ccacaggtga acatcgttaa gaaaaccgag gtgcagaccg
     3541 gtggattctc taaagagtct atcctcccta agaggaactc tgataagctc attgctagga
     3601 agaaggattg ggaccctaag aaatacggtg gtttcgattc tcctaccgtg gcttactctg
     3661 ttctcgttgt ggctaaggtt gagaagggaa agagtaagaa gctcaagtct gttaaggaac
     3721 ttctcggaat cactatcatg gaaaggtcat ctttcgagaa gaacccaatc gatttcctcg
     3781 aggctaaggg atacaaagag gttaagaagg atctcatcat caagctccca aagtactcac
     3841 tcttcgaact cgagaacggt agaaagagga tgctcgcttc tgctggtgag cttcaaaagg
     3901 gaaacgagct tgctctccca tctaagtacg ttaactttct ttacctcgct tctcactacg
     3961 agaagttgaa gggatctcca gaagataacg agcagaagca acttttcgtt gagcagcaca
     4021 agcactactt ggatgagatc atcgagcaga tctctgagtt ctctaaaagg gtgatcctcg
     4081 ctgatgcaaa cctcgataag gtgttgtctg cttacaacaa gcacagagat aagcctatca
     4141 gggaacaggc agagaacatc atccatctct tcacccttac caacctcggt gctcctgctg
     4201 ctttcaagta cttcgataca accatcgata ggaagagata cacctctacc aaagaagtgc
     4261 tcgatgctac cctcatccat cagtctatca ctggactcta cgagactagg atcgatctct
     4321 cacagctcgg tggtgattca agggctgatc ctaagaagaa gaggaaggtt tgagctttga
     4381 gaccacgaag tggctcttca gtggacgaaa gggcctcgtg atacgcctat ttttataggt
     4441 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     4501 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     4561 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgcgct cacgcaactg
     4621 gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg
     4681 ttatgactgt ttttttttgg ggtacagtct atgcctcggg catccaagca gcaagcgcgt
     4741 tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca gcagggcagt
     4801 cgccctaaaa caaagttaaa catcatgagg gaagcggtga tcgccgaagt atcgactcaa
     4861 ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct ggccgtacat
     4921 ttgtacggct ccgcagtgga tggcggcctg aagccacaca gcgatattga tttgctggtt
     4981 acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga ccttttggaa
     5041 acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac cattgttgtg
     5101 cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt tggagaatgg
     5161 cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat tgatctggct
     5221 atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc ggcggaggaa
     5281 ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac cttaacgcta
     5341 tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc
     5401 atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca
     5461 atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca ggcttatctt
     5521 ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgtccattac
     5581 gtaaaaggcg agatcaccaa ggtagtcggc aaataactgt cagaccaagt ttactcatat
     5641 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5701 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5761 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5821 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5881 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5941 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6001 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6061 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6121 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6181 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     6241 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6301 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6361 cggagcctat ggaaaaacgc cagcaacgcg
//
