LOCUS       sequence-227984-        6389 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, SpCas9-p (no stop codon).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6389)
  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 6389)
  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: pEPOR0SP0009
FEATURES             Location/Qualifiers
     source          1..6389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(36..54)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        122..226
                     /gene="bla"
                     /label=AmpR promoter
     CDS             448..1239
                     /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      1410..1998
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1899..1918
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2152..2169
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2247..6350
                     /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             2247..6350
                     /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             6363..6383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 tgagaccacg aagtggctct tcagtggacg aaagggcctc gtgatacgcc tatttttata
       61 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
      121 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      181 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatgc gctcacgcaa
      241 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc
      301 ttgttatgac tgtttttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg
      361 cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc
      421 agtcgcccta aaacaaagtt aaacatcatg agggaagcgg tgatcgccga agtatcgact
      481 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta
      541 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagcgatat tgatttgctg
      601 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg
      661 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt
      721 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa
      781 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg
      841 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag
      901 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg
      961 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc
     1021 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg
     1081 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat
     1141 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccat
     1201 tacgtaaaag gcgagatcac caaggtagtc ggcaaataac tgtcagacca agtttactca
     1261 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     1321 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1381 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1441 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1501 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     1561 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1621 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1681 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1741 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1801 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1861 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     1921 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     1981 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2041 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2101 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2161 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2221 attcattaat cactctgtgg tctcaaatgg ataagaagta ctctatcgga ctcgatatcg
     2281 gaactaactc tgtgggatgg gctgtgatca ccgatgagta caaggtgcca tctaagaagt
     2341 tcaaggttct cggaaacacc gataggcact ctatcaagaa aaaccttatc ggtgctctcc
     2401 tcttcgattc tggtgaaact gctgaggcta ccagactcaa gagaaccgct agaagaaggt
     2461 acaccagaag aaagaacagg atctgctacc tccaagagat cttctctaac gagatggcta
     2521 aagtggatga ttcattcttc cacaggctcg aagagtcatt cctcgtggaa gaagataaga
     2581 agcacgagag gcaccctatc ttcggaaaca tcgttgatga ggtggcatac cacgagaagt
     2641 accctactat ctaccacctc agaaagaagc tcgttgattc tactgataag gctgatctca
     2701 ggctcatcta cctcgctctc gctcacatga tcaagttcag aggacacttc ctcatcgagg
     2761 gtgatctcaa ccctgataac tctgatgtgg ataagttgtt catccagctc gtgcagacct
     2821 acaaccagct tttcgaagag aaccctatca acgcttcagg tgtggatgct aaggctatcc
     2881 tctctgctag gctctctaag tcaagaaggc ttgagaacct cattgctcag ctccctggtg
     2941 agaagaagaa cggacttttc ggaaacttga tcgctctctc tctcggactc acccctaact
     3001 tcaagtctaa cttcgatctc gctgaggatg caaagctcca gctctcaaag gatacctacg
     3061 atgatgatct cgataacctc ctcgctcaga tcggagatca gtacgctgat ttgttcctcg
     3121 ctgctaagaa cctctctgat gctatcctcc tcagtgatat cctcagagtg aacaccgaga
     3181 tcaccaaggc tccactctca gcttctatga tcaagagata cgatgagcac caccaggatc
     3241 tcacacttct caaggctctt gttagacagc agctcccaga gaagtacaaa gagattttct
     3301 tcgatcagtc taagaacgga tacgctggtt acatcgatgg tggtgcatct caagaagagt
     3361 tctacaagtt catcaagcct atcctcgaga agatggatgg aaccgaggaa ctcctcgtga
     3421 agctcaatag agaggatctt ctcagaaagc agaggacctt cgataacgga tctatccctc
     3481 atcagatcca cctcggagag ttgcacgcta tccttagaag gcaagaggat ttctacccat
     3541 tcctcaagga taacagggaa aagattgaga agattctcac cttcagaatc ccttactacg
     3601 tgggacctct cgctagagga aactcaagat tcgcttggat gaccagaaag tctgaggaaa
     3661 ccatcacccc ttggaacttc gaagaggtgg tggataaggg tgctagtgct cagtctttca
     3721 tcgagaggat gaccaacttc gataagaacc ttccaaacga gaaggtgctc cctaagcact
     3781 ctttgctcta cgagtacttc accgtgtaca acgagttgac caaggttaag tacgtgaccg
     3841 agggaatgag gaagcctgct tttttgtcag gtgagcaaaa gaaggctatc gttgatctct
     3901 tgttcaagac caacagaaag gtgaccgtga agcagctcaa agaggattac ttcaagaaaa
     3961 tcgagtgctt cgattcagtt gagatttctg gtgttgagga taggttcaac gcatctctcg
     4021 gaacctacca cgatctcctc aagatcatta aggataagga tttcttggat aacgaggaaa
     4081 acgaggatat cttggaggat atcgttctta ccctcaccct ctttgaagat agagagatga
     4141 ttgaagaaag gctcaagacc tacgctcatc tcttcgatga taaggtgatg aagcagttga
     4201 agagaagaag atacactggt tggggaaggc tctcaagaaa gctcattaac ggaatcaggg
     4261 ataagcagtc tggaaagaca atccttgatt tcctcaagtc tgatggattc gctaacagaa
     4321 acttcatgca gctcatccac gatgattctc tcacctttaa agaggatatc cagaaggctc
     4381 aggtttcagg acagggtgat agtctccatg agcatatcgc taacctcgct ggatctcctg
     4441 caatcaagaa gggaatcctc cagactgtga aggttgtgga tgagttggtg aaggtgatgg
     4501 gaaggcataa gcctgagaac atcgtgatcg aaatggctag agagaaccag accactcaga
     4561 agggacagaa gaactctagg gaaaggatga agaggatcga ggaaggtatc aaagagcttg
     4621 gatctcagat cctcaaagag caccctgttg agaacactca gctccagaat gagaagctct
     4681 acctctacta cctccagaac ggaagggata tgtatgtgga tcaagagttg gatatcaaca
     4741 ggctctctga ttacgatgtt gatcatatcg tgccacagtc attcttgaag gatgattcta
     4801 tcgataacaa ggtgctcacc aggtctgata agaacagggg taagagtgat aacgtgccaa
     4861 gtgaagaggt tgtgaagaaa atgaagaact attggaggca gctcctcaac gctaagctca
     4921 tcactcagag aaagttcgat aacttgacta aggctgagag gggaggactc tctgaattgg
     4981 ataaggcagg attcatcaag aggcagcttg tggaaaccag gcagatcact aagcacgttg
     5041 cacagatcct cgattctagg atgaacacca agtacgatga gaacgataag ttgatcaggg
     5101 aagtgaaggt tatcaccctc aagtcaaagc tcgtgtctga tttcagaaag gatttccaat
     5161 tctacaaggt gagggaaatc aacaactacc accacgctca cgatgcttac cttaacgctg
     5221 ttgttggaac cgctctcatc aagaagtatc ctaagctcga gtcagagttc gtgtacggtg
     5281 attacaaggt gtacgatgtg aggaagatga tcgctaagtc tgagcaagag atcggaaagg
     5341 ctaccgctaa gtatttcttc tactctaaca tcatgaattt cttcaagacc gagattaccc
     5401 tcgctaacgg tgagatcaga aagaggccac tcatcgagac aaacggtgaa acaggtgaga
     5461 tcgtgtggga taagggaagg gatttcgcta ccgttagaaa ggtgctctct atgccacagg
     5521 tgaacatcgt taagaaaacc gaggtgcaga ccggtggatt ctctaaagag tctatcctcc
     5581 ctaagaggaa ctctgataag ctcattgcta ggaagaagga ttgggaccct aagaaatacg
     5641 gtggtttcga ttctcctacc gtggcttact ctgttctcgt tgtggctaag gttgagaagg
     5701 gaaagagtaa gaagctcaag tctgttaagg aacttctcgg aatcactatc atggaaaggt
     5761 catctttcga gaagaaccca atcgatttcc tcgaggctaa gggatacaaa gaggttaaga
     5821 aggatctcat catcaagctc ccaaagtact cactcttcga actcgagaac ggtagaaaga
     5881 ggatgctcgc ttctgctggt gagcttcaaa agggaaacga gcttgctctc ccatctaagt
     5941 acgttaactt tctttacctc gcttctcact acgagaagtt gaagggatct ccagaagata
     6001 acgagcagaa gcaacttttc gttgagcagc acaagcacta cttggatgag atcatcgagc
     6061 agatctctga gttctctaaa agggtgatcc tcgctgatgc aaacctcgat aaggtgttgt
     6121 ctgcttacaa caagcacaga gataagccta tcagggaaca ggcagagaac atcatccatc
     6181 tcttcaccct taccaacctc ggtgctcctg ctgctttcaa gtacttcgat acaaccatcg
     6241 ataggaagag atacacctct accaaagaag tgctcgatgc taccctcatc catcagtcta
     6301 tcactggact ctacgagact aggatcgatc tctcacagct cggtggtgat tcaagggctg
     6361 atcctaagaa gaagaggaag gttggttcg
//
