LOCUS       sequence-228007-        6390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, Sp-eCas9 1.1.
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: pEPOR0SP0018
FEATURES             Location/Qualifiers
     source          1..6390
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     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 acaagaagta ctccattggg ctcgatatcg
     2281 gcacaaacag cgtcggctgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat
     2341 tcaaagttct gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc
     2401 tgttcgactc cggggagacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat
     2461 atacccgcag aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta
     2521 aggtggatga ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa
     2581 agcacgagcg ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt
     2641 acccaaccat atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc
     2701 ggttgatcta tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg
     2761 gggacctgaa cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt
     2821 acaatcagct tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc
     2881 tgagcgctag gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg
     2941 agaagaagaa cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact
     3001 ttaaatctaa cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg
     3061 atgatgatct cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg
     3121 cggcaaagaa cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga
     3181 tcaccaaagc tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact
     3241 tgactttgct gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct
     3301 tcgatcagtc taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat
     3361 tttacaaatt tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa
     3421 agcttaacag agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc
     3481 accagattca cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct
     3541 ttttgaaaga taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg
     3601 taggccccct cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga
     3661 ctatcactcc ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca
     3721 tcgaaaggat gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact
     3781 ctctgctgta cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag
     3841 aagggatgag aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc
     3901 tcttcaagac gaaccggaaa gttaccgtga aacagctcaa agaagattat ttcaaaaaga
     3961 ttgaatgttt cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg
     4021 gaacgtatca cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga
     4081 acgaggacat tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga
     4141 ttgaagaacg cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca
     4201 agaggcgccg atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag
     4261 acaagcagag tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga
     4321 acttcatgca gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac
     4381 aagtttctgg ccagggggac agtctccacg agcacatcgc taatcttgca ggtagcccag
     4441 ctatcaaaaa gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg
     4501 gaaggcataa gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga
     4561 agggacagaa gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg
     4621 ggtcccaaat ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct
     4681 acctgtacta cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaatc
     4741 ggctctccga ctacgacgtg gatcatatcg tgccccagtc ttttctcgca gatgattcta
     4801 ttgataataa agtgttgaca agatccgata aaaatagagg gaagagtgat aacgtcccct
     4861 cagaagaagt tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga
     4921 tcacacaacg gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg
     4981 ataaagccgg cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg
     5041 cccaaattct cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag
     5101 aggtgaaagt tattactctg aagtctaagc tggtttcaga tttcagaaag gactttcagt
     5161 tttataaggt gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag
     5221 tggtaggcac tgcacttatc aaaaaatatc ccgcgcttga atctgaattt gtttacggag
     5281 actataaagt gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg
     5341 ccaccgctaa gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac
     5401 tggccaatgg agagattcgg aaggcaccac ttatcgaaac aaacggagaa acaggagaaa
     5461 tcgtgtggga caagggtagg gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg
     5521 tgaacatcgt taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc
     5581 cgaaaaggaa cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg
     5641 gcggattcga ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag
     5701 ggaagtctaa aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat
     5761 caagcttcga aaaaaacccc atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa
     5821 aagacctcat cattaagctt cccaagtact ctctctttga gcttgaaaac ggccggaaac
     5881 gaatgctcgc tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat
     5941 acgttaattt cttgtatctg gccagccact atgaaaagct caaaggatct cccgaagata
     6001 atgagcagaa gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc
     6061 aaataagcga attctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt
     6121 ctgcttacaa taagcacagg gataagccca tcagggagca ggcagaaaac attatccact
     6181 tgtttactct gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag
     6241 acagaaagcg gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa
     6301 ttacggggct ctatgaaaca agaatcgacc tctctcagct cggtggagac agcagggctg
     6361 accccaagaa gaagaggaag gtgtgagctt
//
