LOCUS       sequence-296472-        8214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eSpCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8214)
  AUTHORS   Schmid-Burgk JL, Gao L, Li D, Gardner Z, Strecker J, Lash B, Zhang F
  TITLE     Highly Parallel Profiling of Cas9 Variant Specificity.
  JOURNAL   Mol Cell. 2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023.
  PUBMED    32187529
REFERENCE   2  (bases 1 to 8214)
  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: "Mol Cell. 
            2020 Mar 17. pii: S1097-2765(20)30143-X. doi: 
            10.1016/j.molcel.2020.02.023."
COMMENT     Sequence Label: pX165-eSpCas9
FEATURES             Location/Qualifiers
     source          1..8214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..23
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(61..79)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        147..251
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(252..320,321..1112)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(470..489)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1283..1871
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1772..1791
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1933..2062
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2081..2366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          2646..2873
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2885..2894
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2894..2959
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2966..2986
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(3011..5548,5549..5551,5552..6013,6014..6016,6017..6184,
                     6185..6187,6188..7111)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Slaymaker et al., 2016)"
                     /label=eSpCas9(1.1)
                     /note="carries the mutations K848A, K1003A, and R1060A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLADDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKAPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             7112..7159
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(7187..7204)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7193..7400
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7409..7549
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7409..7538
                     /label=AAV2 ITR
     rep_origin      7624..8079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7711..7730)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7921..7942
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8096..8115)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
       61 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
      121 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
      181 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
      241 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
      301 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
      361 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
      421 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
      481 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
      541 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
      601 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
      661 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
      721 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
      781 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
      841 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
      901 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
      961 agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1021 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     1081 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1141 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     1201 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     1261 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     1321 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     1381 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     1441 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     1501 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     1561 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     1621 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     1681 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     1741 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     1801 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     1861 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     1921 ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct cactgaggcc gcccgggcgt
     1981 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     2041 aactccatca ctaggggttc ctgcggcctc tagaggtacc cgttacataa cttacggtaa
     2101 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata gtaacgccaa
     2161 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     2221 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     2281 ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     2341 acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc ttcactctcc
     2401 ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa ttattttgtg
     2461 cagcgatggg ggcggggggg gggggggggc gsgcgcsggg gggggggggg gggggsgrgg
     2521 ggsggggsgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
     2581 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
     2641 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
     2701 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
     2761 ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg ttggttggtg
     2821 gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt caggttggac
     2881 cggtgccacc atggactata aggaccacga cggagactac aaggatcatg atattgatta
     2941 caaagacgat gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt
     3001 cccagcagcc gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
     3061 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     3121 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     3181 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     3241 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     3301 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     3361 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     3421 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     3481 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     3541 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     3601 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     3661 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     3721 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     3781 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     3841 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     3901 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     3961 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     4021 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     4081 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     4141 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     4201 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     4261 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     4321 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     4381 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     4441 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
     4501 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     4561 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     4621 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
     4681 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     4741 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     4801 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     4861 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     4921 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     4981 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     5041 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     5101 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     5161 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     5221 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     5281 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     5341 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     5401 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     5461 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     5521 ggaccatatc gtgcctcaga gctttctggc cgacgactcc atcgacaaca aggtgctgac
     5581 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     5641 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
     5701 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     5761 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     5821 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
     5881 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     5941 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     6001 caaaaagtac cctgcgctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     6061 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     6121 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     6181 gaaggcgcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     6241 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     6301 cgaggtgcag acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa
     6361 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac
     6421 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     6481 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     6541 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     6601 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg
     6661 cgaactgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     6721 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     6781 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     6841 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     6901 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     6961 gggagcccct gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag
     7021 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     7081 acggatcgac ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg
     7141 ccaggcaaaa aagaaaaagt aagaattcct agagctcgct gatcagcctc gactgtgcct
     7201 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     7261 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     7321 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     7381 aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt ggccactccc
     7441 tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc
     7501 tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg
     7561 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca aagcaaccat
     7621 agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     7681 ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc tttcttccct tcctttctcg
     7741 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     7801 ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt tcacgtagtg
     7861 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     7921 gtggactctt gttccaaact ggaacaacac tcaactctat ctcgggctat tcttttgatt
     7981 tataagggat tttgccgatt tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat
     8041 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact ctcagtacaa
     8101 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     8161 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtct
//
