LOCUS       sequence-227986-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, SpCas9-KA.
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: pEPOR0SP0014
FEATURES             Location/Qualifiers
     source          1..6390
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..58)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        126..230
                     /gene="bla"
                     /label=AmpR promoter
     CDS             452..1243
                     /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      1414..2002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1903..1922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2156..2173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2251..6354
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNAVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             6367..6387
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gctttgagac cacgaagtgg ctcttcagtg gacgaaaggg cctcgtgata cgcctatttt
       61 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
      121 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
      181 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgcgctcac
      241 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
      301 tggcttgtta tgactgtttt tttttggggt acagtctatg cctcgggcat ccaagcagca
      361 agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg ttacgcagca
      421 gggcagtcgc cctaaaacaa agttaaacat catgagggaa gcggtgatcg ccgaagtatc
      481 gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc
      541 cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagcg atattgattt
      601 gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct
      661 tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat
      721 tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg
      781 agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga
      841 tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc
      901 ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt
      961 aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt
     1021 gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga
     1081 ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctagacaggc
     1141 ttatcttgga caagaagaag atcgcttggc ctcgcgcgca gatcagttgg aagaatttgt
     1201 ccattacgta aaaggcgaga tcaccaaggt agtcggcaaa taactgtcag accaagttta
     1261 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     1321 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     1381 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     1441 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     1501 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     1561 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     1621 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     1681 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     1741 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1801 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1861 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1921 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1981 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     2041 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     2101 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     2161 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     2221 gccgattcat taatcactct gtggtctcaa atggacaaga agtactccat tgggctcgat
     2281 atcggcacaa acagcgtcgg ctgggccgtc attacggacg agtacaaggt gccgagcaaa
     2341 aaattcaaag ttctgggcaa taccgatcgc cacagcataa agaagaacct cattggcgcc
     2401 ctcctgttcg actccgggga gacggccgaa gccacgcggc tcaaaagaac agcacggcgc
     2461 agatataccc gcagaaagaa tcggatctgc tacctgcagg agatctttag taatgagatg
     2521 gctaaggtgg atgactcttt cttccatagg ctggaggagt cctttttggt ggaggaggat
     2581 aaaaagcacg agcgccaccc aatctttggc aatatcgtgg acgaggtggc gtaccatgaa
     2641 aagtacccaa ccatatatca tctgaggaag aagcttgtag acagtactga taaggctgac
     2701 ttgcggttga tctatctcgc gctggcgcat atgatcaaat ttcggggaca cttcctcatc
     2761 gagggggacc tgaacccaga caacagcgat gtcgacaaac tctttatcca actggttcag
     2821 acttacaatc agcttttcga agagaacccg atcaacgcat ccggagttga cgccaaagca
     2881 atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa acctcatcgc acagctccct
     2941 ggggagaaga agaacggcct gtttggtaat cttatcgccc tgtcactcgg gctgaccccc
     3001 aactttaaat ctaacttcga cctggccgaa gatgccaagc ttcaactgag caaagacacc
     3061 tacgatgatg atctcgacaa tctgctggcc cagatcggcg accagtacgc agaccttttt
     3121 ttggcggcaa agaacctgtc agacgccatt ctgctgagtg atattctgcg agtgaacacg
     3181 gagatcacca aagctccgct gagcgctagt atgatcaagc gctatgatga gcaccaccaa
     3241 gacttgactt tgctgaaggc ccttgtcaga cagcaactgc ctgagaagta caaggaaatt
     3301 ttcttcgatc agtctaaaaa tggctacgcc ggatacattg acggcggagc aagccaggag
     3361 gaattttaca aatttattaa gcccatcttg gaaaaaatgg acggcaccga ggagctgctg
     3421 gtaaagctta acagagaaga tctgttgcgc aaacagcgca ctttcgacaa tggaagcatc
     3481 ccccaccaga ttcacctggg cgaactgcac gctatcctca ggcggcaaga ggatttctac
     3541 ccctttttga aagataacag ggaaaagatt gagaaaatcc tcacatttcg gataccctac
     3601 tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt ggatgactcg caaatcagaa
     3661 gagactatca ctccctggaa cttcgaggaa gtcgtggata agggggcctc tgcccagtcc
     3721 ttcatcgaaa ggatgactaa ctttgataaa aatctgccta acgaaaaggt gcttcctaaa
     3781 cactctctgc tgtacgagta cttcacagtt tataacgagc tcaccaaggt caaatacgtc
     3841 acagaaggga tgagaaagcc agcattcctg tctggagagc agaagaaagc tatcgtggac
     3901 ctcctcttca agacgaaccg gaaagttacc gtgaaacagc tcaaagaaga ttatttcaaa
     3961 aagattgaat gtttcgactc tgttgaaatc agcggagtgg aggatcgctt caacgcatcc
     4021 ctgggaacgt atcacgatct cctgaaaatc attaaagaca aggacttcct ggacaatgag
     4081 gagaacgagg acattcttga ggacattgtc ctcaccctta cgttgtttga agatagggag
     4141 atgattgaag aacgcttgaa aacttacgct catctcttcg acgacaaagt catgaaacag
     4201 ctcaagaggc gccgatatac aggatggggg cggctgtcaa gaaaactgat caatgggatc
     4261 cgagacaagc agagtggaaa gacaatcctg gattttctta agtccgatgg atttgccaac
     4321 cggaacttca tgcagttgat ccatgatgac tctctcacct ttaaggagga catccagaaa
     4381 gcacaagttt ctggccaggg ggacagtctc cacgagcaca tcgctaatct tgcaggtagc
     4441 ccagctatca aaaagggaat actgcagacc gttaaggtcg tggatgaact cgtcaaagta
     4501 atgggaaggc ataagcccga gaatatcgtt atcgagatgg cccgagagaa ccaaactacc
     4561 cagaagggac agaagaacag tagggaaagg atgaagagga ttgaagaggg tataaaagaa
     4621 ctggggtccc aaatccttaa ggaacaccca gttgaaaaca cccagcttca gaatgagaag
     4681 ctctacctgt actacctgca gaacggcagg gacatgtacg tggatcagga actggacatc
     4741 aatcggctct ccgactacga cgtggatcat atcgtgcccc agtcttttct caaagatgat
     4801 tctattgata atgcagtgtt gacaagatcc gataaaaata gagggaagag tgataacgtc
     4861 ccctcagaag aagttgtcaa gaaaatgaaa aattattggc ggcagctgct gaacgccaaa
     4921 ctgatcacac aacggaagtt cgataatctg actaaggctg aacgaggtgg cctgtctgag
     4981 ttggataaag ccggcttcat caaaaggcag cttgttgaga cacgccagat caccaagcac
     5041 gtggcccaaa ttctcgattc acgcatgaac accaagtacg atgaaaatga caaactgatt
     5101 cgagaggtga aagttattac tctgaagtct aagctggttt cagatttcag aaaggacttt
     5161 cagttttata aggtgagaga gatcaacaat taccaccatg cgcatgatgc ctacctgaat
     5221 gcagtggtag gcactgcact tatcaaaaaa tatcccaagc ttgaatctga atttgtttac
     5281 ggagactata aagtgtacga tgttaggaaa atgatcgcaa agtctgagca ggaaataggc
     5341 aaggccaccg ctaagtactt cttttacagc aatattatga attttttcaa gaccgagatt
     5401 acactggcca atggagagat tcggaagcga ccacttatcg aaacaaacgg agaaacagga
     5461 gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc ggaaggtcct gtccatgccg
     5521 caggtgaaca tcgttaaaaa gaccgaagta cagaccggag gcttctccaa ggaaagtatc
     5581 ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa aagattggga ccccaagaaa
     5641 tacggcggat tcgattctcc tacagtcgct tacagtgtac tggttgtggc caaagtggag
     5701 aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc tgggcatcac aatcatggag
     5761 cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg cgaaaggata taaagaggtc
     5821 aaaaaagacc tcatcattaa gcttcccaag tactctctct ttgagcttga aaacggccgg
     5881 aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta acgagctggc actgccctct
     5941 aaatacgtta atttcttgta tctggccagc cactatgaaa agctcaaagg atctcccgaa
     6001 gataatgagc agaagcagct gttcgtggaa caacacaaac actaccttga tgagatcatc
     6061 gagcaaataa gcgaattctc caaaagagtg atcctcgccg acgctaacct cgataaggtg
     6121 ctttctgctt acaataagca cagggataag cccatcaggg agcaggcaga aaacattatc
     6181 cacttgttta ctctgaccaa cttgggcgcg cctgcagcct tcaagtactt cgacaccacc
     6241 atagacagaa agcggtacac ctctacaaag gaggtcctgg acgccacact gattcatcag
     6301 tcaattacgg ggctctatga aacaagaatc gacctctctc agctcggtgg agacagcagg
     6361 gctgacccca agaagaagag gaaggtgtga
//
