LOCUS       sequence-227456-        6389 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level0 Golden Gate part: CDS, Sp-xCas9 3.7 (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: pEPOR0SP0011
FEATURES             Location/Qualifiers
     source          1..6389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      592..1180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1081..1100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1334..1351
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             1429..5532
                     /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
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5545..5565
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5607..5625)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5693..5797
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6019..6389,1..421)
                     /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"
ORIGIN
        1 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
       61 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
      121 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
      181 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
      241 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
      301 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
      361 tcagttggaa gaatttgtcc attacgtaaa aggcgagatc accaaggtag tcggcaaata
      421 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
      481 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      541 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      601 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      661 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      721 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      781 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      841 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      901 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      961 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1021 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1081 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1141 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1201 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1261 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     1321 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     1381 gcctctcccc gcgcgttggc cgattcatta atcactctgt ggtctcaaat ggacaagaag
     1441 tactccattg ggctcgatat cggcacaaac agcgtcggct gggccgtcat tacggacgag
     1501 tacaaggtgc cgagcaaaaa attcaaagtt ctgggcaata ccgatcgcca cagcataaag
     1561 aagaacctca ttggcgccct cctgttcgac tccggggaga cggccgaagc cacgcggctc
     1621 aaaagaacag cacggcgcag atatacccgc agaaagaatc ggatctgcta cctgcaggag
     1681 atctttagta atgagatggc taaggtggat gactctttct tccataggct ggaggagtcc
     1741 tttttggtgg aggaggataa aaagcacgag cgccacccaa tctttggcaa tatcgtggac
     1801 gaggtggcgt accatgaaaa gtacccaacc atatatcatc tgaggaagaa gcttgtagac
     1861 agtactgata aggctgactt gcggttgatc tatctcgcgc tggcgcatat gatcaaattt
     1921 cggggacact tcctcatcga gggggacctg aacccagaca acagcgatgt cgacaaactc
     1981 tttatccaac tggttcagac ttacaatcag cttttcgaag agaacccgat caacgcatcc
     2041 ggagttgacg ccaaagcaat cctgagcgct aggctgtcca aatcccggcg gctcgaaaac
     2101 ctcatcgcac agctccctgg ggagaagaag aacggcctgt ttggtaatct tatcgccctg
     2161 tcactcgggc tgacccccaa ctttaaatct aacttcgacc tggccgaaga tactaagctt
     2221 caactgagca aagacaccta cgatgatgat ctcgacaatc tgctggccca gatcggcgac
     2281 cagtacgcag accttttttt ggcggcaaag aacctgtcag acgccattct gctgagtgat
     2341 attctgcgag tgaacacgga gatcaccaaa gctccgctga gcgctagtat gatcaagctg
     2401 tatgatgagc accaccaaga cttgactttg ctgaaggccc ttgtcagaca gcaactgcct
     2461 gagaagtaca aggaaatttt cttcgatcag tctaaaaatg gctacgccgg atacattgac
     2521 ggcggagcaa gccaggagga attttacaaa tttattaagc ccatcttgga aaaaatggac
     2581 ggcaccgagg agctgctggt aaagcttaac agagaagatc tgttgcgcaa acagcgcact
     2641 ttcgacaatg gaatcatccc ccaccagatt cacctgggcg aactgcacgc tatcctcagg
     2701 cggcaagagg atttctaccc ctttttgaaa gataacaggg aaaagattga gaaaatcctc
     2761 acatttcgga taccctacta tgtaggcccc ctcgcccggg gaaattccag attcgcgtgg
     2821 atgactcgca aatcagaaga gactatcact ccctggaact tcgagaaggt cgtggataag
     2881 ggggcctctg cccagtcctt catcgaaagg atgactaact ttgataaaaa tctgcctaac
     2941 gaaaaggtgc ttcctaaaca ctctctgctg tacgagtact tcacagttta taacgagctc
     3001 accaaggtca aatacgtcac agaagggatg agaaagccag cattcctgtc tggagatcag
     3061 aagaaagcta tcgtggacct cctcttcaag acgaaccgga aagttaccgt gaaacagctc
     3121 aaagaagatt atttcaaaaa gattgaatgt ttcgactctg ttgaaatcag cggagtggag
     3181 gatcgcttca acgcatccct gggaacgtat cacgatctcc tgaaaatcat taaagacaag
     3241 gacttcctgg acaatgagga gaacgaggac attcttgagg acattgtcct cacccttacg
     3301 ttgtttgaag atagggagat gattgaagaa cgcttgaaaa cttacgctca tctcttcgac
     3361 gacaaagtca tgaaacagct caagaggcgc cgatatacag gatgggggcg gctgtcaaga
     3421 aaactgatca atgggatccg agacaagcag agtggaaaga caatcctgga ttttcttaag
     3481 tccgatggat ttgccaaccg gaacttcatc cagttgatcc atgatgactc tctcaccttt
     3541 aaggaggaca tccagaaagc acaagtttct ggccaggggg acagtctcca cgagcacatc
     3601 gctaatcttg caggtagccc agctatcaaa aagggaatac tgcagaccgt taaggtcgtg
     3661 gatgaactcg tcaaagtaat gggaaggcat aagcccgaga atatcgttat cgagatggcc
     3721 cgagagaacc aaactaccca gaagggacag aagaacagta gggaaaggat gaagaggatt
     3781 gaagagggta taaaagaact ggggtcccaa atccttaagg aacacccagt tgaaaacacc
     3841 cagcttcaga atgagaagct ctacctgtac tacctgcaga acggcaggga catgtacgtg
     3901 gatcaggaac tggacatcaa tcggctctcc gactacgacg tggatcatat cgtgccccag
     3961 tcttttctca aagatgattc tattgataat aaagtgttga caagatccga taaaaataga
     4021 gggaagagtg ataacgtccc ctcagaagaa gttgtcaaga aaatgaaaaa ttattggcgg
     4081 cagctgctga acgccaaact gatcacacaa cggaagttcg ataatctgac taaggctgaa
     4141 cgaggtggcc tgtctgagtt ggataaagcc ggcttcatca aaaggcagct tgttgagaca
     4201 cgccagatca ccaagcacgt ggcccaaatt ctcgattcac gcatgaacac caagtacgat
     4261 gaaaatgaca aactgattcg agaggtgaaa gttattactc tgaagtctaa gctggtttca
     4321 gatttcagaa aggactttca gttttataag gtgagagaga tcaacaatta ccaccatgcg
     4381 catgatgcct acctgaatgc agtggtaggc actgcactta tcaaaaaata tcccaagctt
     4441 gaatctgaat ttgtttacgg agactataaa gtgtacgatg ttaggaaaat gatcgcaaag
     4501 tctgagcagg aaataggcaa ggccaccgct aagtacttct tttacagcaa tattatgaat
     4561 tttttcaaga ccgagattac actggccaat ggagagattc ggaagcgacc acttatcgaa
     4621 acaaacggag aaacaggaga aatcgtgtgg gacaagggta gggatttcgc gacagtccgg
     4681 aaggtcctgt ccatgccgca ggtgaacatc gttaaaaaga ccgaagtaca gaccggaggc
     4741 ttctccaagg aaagtatcct cccgaaaagg aacagcgaca agctgatcgc acgcaaaaaa
     4801 gattgggacc ccaagaaata cggcggattc gattctccta cagtcgctta cagtgtactg
     4861 gttgtggcca aagtggagaa agggaagtct aaaaaactca aaagcgtcaa ggaactgctg
     4921 ggcatcacaa tcatggagcg atcaagcttc gaaaaaaacc ccatcgactt tctcgaggcg
     4981 aaaggatata aagaggtcaa aaaagacctc atcattaagc ttcccaagta ctctctcttt
     5041 gagcttgaaa acggccggaa acgaatgctc gctagtgcgg gcgtactgca gaaaggtaac
     5101 gagctggcac tgccctctaa atacgttaat ttcttgtatc tggccagcca ctatgaaaag
     5161 ctcaaaggat ctcccgaaga taatgagcag aagcagctgt tcgtggaaca acacaaacac
     5221 taccttgatg agatcatcga gcaaataagc gaattctcca aaagagtgat cctcgccgac
     5281 gctaacctcg ataaggtgct ttctgcttac aataagcaca gggataagcc catcagggag
     5341 caggcagaaa acattatcca cttgtttact ctgaccaact tgggcgcgcc tgcagccttc
     5401 aagtacttcg acaccaccat agacagaaag cggtacacct ctacaaagga ggtcctggac
     5461 gccacactga ttcatcagtc aattacgggg ctctatgaaa caagaatcga cctctctcag
     5521 ctcggtggag acagcagggc tgaccccaag aagaagagga aggtgggttc gtgagaccac
     5581 gaagtggctc ttcagtggac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5641 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5701 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5761 ctgataaatg cttcaataat attgaaaaag gaagagtatg cgctcacgca actggtccag
     5821 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     5881 ctgttttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc gcgttacgcc
     5941 gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg cagtcgccct
     6001 aaaacaaagt taaacatcat gagggaagcg gtgatcgccg aagtatcgac tcaactatca
     6061 gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt acatttgtac
     6121 ggctccgcag tggatggcgg cctgaagcca cacagcgata ttgatttgct ggttacggtg
     6181 accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt ggaaacttcg
     6241 gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt tgtgcacgac
     6301 gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga atggcagcgc
     6361 aatgacattc ttgcaggtat cttcgagcc
//
