LOCUS       sequence-478607-        4413 bp    DNA     linear   UNA 14-JUL-2025
DEFINITION  Expresses Cas9 nuclease with fused p2c targeting ligand for 
            expression in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4413)
  AUTHORS   Chaverra-Rodriguez D, Macias VM, Hughes GL, Pujhari S, Suzuki Y, 
            Peterson DR, Kim D, McKeand S, Rasgon JL
  TITLE     Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod 
            ovaries for heritable germline gene editing.
  JOURNAL   Nat Commun. 2018 Aug 1;9(1):3008. doi: 10.1038/s41467-018-05425-9.
  PUBMED    30068905
REFERENCE   2  (bases 1 to 4413)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-08-1"; volume: "9(1):3008. doi"; pages: " 
            10.1038/s41467-018-05425-9"
COMMENT     Sequence Label: pET28(a)-p2c-Cas9
FEATURES             Location/Qualifiers
     source          1..4413
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             28..45
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             49..81
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             241..4344
                     /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             241..4344
                     /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             4360..4380
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4381..4407
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             4381..4407
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
ORIGIN
        1 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat ggctagcatg
       61 actggtggac agcaaatggg tcgcggatcc aatctgcagc agcagcgcca gcacggcaag
      121 aacggcaacc aggactacca ggatcagagc aacgaacaga ggaagaacca gaggaccagc
      181 agcgaggagg actacagcga ggaggttaag aacgagctcc gtcgacaagc ttgcggccgc
      241 atggacaaga agtacagcat cggcctggac atcggtacca acagcgtggg ctgggccgtg
      301 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgaccgc
      361 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag
      421 gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc
      481 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccaccgc
      541 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agcgccaccc catcttcggc
      601 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgcgcaag
      661 aagctggtgg acagcaccga caaggccgac ctgcgcctga tctacctggc cctggcccac
      721 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
      781 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc
      841 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg cccgcctgag caagagccgc
      901 cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac
      961 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     1021 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     1081 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc
     1141 ctgctgagcg acatcctgcg cgtgaacacc gagatcacca aggcccccct gagcgccagc
     1201 atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgcgc
     1261 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc
     1321 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg
     1381 gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga cctgctgcgc
     1441 aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac
     1501 gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc
     1561 gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg cggcaacagc
     1621 cgcttcgcct ggatgacccg caagagcgag gagaccatca ccccctggaa cttcgaggag
     1681 gtggtggaca agggcgccag cgcccagagc ttcatcgagc gcatgaccaa cttcgacaag
     1741 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     1801 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc cgccttcctg
     1861 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg caaggtgacc
     1921 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc
     1981 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc
     2041 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg
     2101 ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa gacctacgcc
     2161 cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac cggctggggc
     2221 cgcctgagcc gcaagcttat caacggcatc cgcgacaagc agagcggcaa gaccatcctg
     2281 gacttcctga agagcgacgg cttcgccaac cgcaacttca tgcagctgat ccacgacgac
     2341 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg
     2401 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc
     2461 gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga gaacatcgtg
     2521 atcgagatgg cccgcgagaa ccagaccacc cagaagggcc agaagaacag ccgcgagcgc
     2581 atgaagcgca tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc
     2641 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc
     2701 gacatgtacg tggaccagga gctggacatc aaccgcctga gcgactacga cgtggaccac
     2761 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gacccgcagc
     2821 gacaagaacc gcggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag
     2881 aactactggc gccagctgct gaacgccaag ctgatcaccc agcgcaagtt cgacaacctg
     2941 accaaggccg agcgcggcgg cctgagcgag ctggacaagg ccggcttcat caagcgccag
     3001 ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggacag ccgcatgaac
     3061 accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac cctgaagagc
     3121 aagctggtga gcgacttccg caaggacttc cagttctaca aggtgcgcga gatcaacaac
     3181 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag
     3241 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcgcaag
     3301 atgatcgcca agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc
     3361 aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat ccgcaagcgc
     3421 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg ccgcgacttc
     3481 gccaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg
     3541 cagaccggcg gcttcagcaa ggagagcatc ctgcccaagc gcaacagcga caagctgatc
     3601 gcccgcaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc
     3661 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg
     3721 aaggagctgc tgggcatcac catcatggag cgcagcagct tcgagaagaa ccccatcgac
     3781 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag
     3841 tacagcctgt tcgagctgga gaacggccgc aagcgcatgc tggccagcgc cggcgagctg
     3901 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc
     3961 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag
     4021 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagcgcgtg
     4081 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca ccgcgacaag
     4141 cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc
     4201 cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac cagcaccaag
     4261 gaggtgctgg acgccaccct gatccaccag agcatcaccg gtctgtacga gacccgcatc
     4321 gacctgagcc agctgggcgg cgacggcggc tccggacctc caaagaaaaa gagaaaagta
     4381 tacccctacg acgtgcccga ctacgcctgt taa
//
