LOCUS       Exported                8214 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Human codon optimized Cas9 nuclease from Streptococcus pyogenes 
            (Cbh-3X-FLAG-NLS-SpCas9-NLS).
ACCESSION   .
VERSION     .
KEYWORDS    pSpCas9 (PX165)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8214)
  AUTHORS   Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F
  TITLE     Genome engineering using the CRISPR-Cas9 system.
  JOURNAL   Nat Protoc. 2013 Nov;8(11):2281-308. doi: 10.1038/nprot.2013.143. 
            Epub 2013 Oct 24.
  PUBMED    24157548
REFERENCE   2  (bases 1 to 8214)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8214
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     100..119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(136..591)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(273..294)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     485..504
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   666..806
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   677..806
                     /label=AAV2 ITR
     polyA_signal    815..1022
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1011..1028
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1056..1103)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(1104..5204)
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(5229..5249)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5256..5321)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     regulatory      5321..5330
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     intron          5342..5569
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     promoter        complement(5570..5847)
                     /label=chicken beta-actin promoter
     enhancer        5849..6134
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     repeat_region   6153..6282
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(6344..6932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6424..6443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7103..7963)
                     /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     7726..7745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7964..8068)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8136..8154
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8192..8214)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
       61 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
      121 tgagagtgca ccataaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt
      181 gttaaatcag ctcatttttt aaccaataga ccgaaatcgg caaaatccct tataaatcaa
      241 aagaatagcc cgagatagag ttgagtgttg ttccagtttg gaacaagagt ccactattaa
      301 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
      361 gtgaaccatc acccaaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
      421 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
      481 aggaagggaa gaaagcgaaa ggagcgggcg ctaaggcgct ggcaagtgta gcggtcacgc
      541 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tactatggtt
      601 gctttgacgt atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
      661 gcgcccctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg
      721 gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt
      781 ggccaactcc atcactaggg gttcctgcgg ccgctcccca gcatgcctgc tattctcttc
      841 ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact
      901 cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc
      961 cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac
     1021 agtcgaggct gatcagcgag ctctaggaat tcttactttt tcttttttgc ctggccggcc
     1081 tttttcgtgg ccgccggcct tttgtcgcct cccagctgag acaggtcgat ccgtgtctcg
     1141 tacaggccgg tgatgctctg gtggatcagg gtggcgtcca gcacctcttt ggtgctggtg
     1201 tacctcttcc ggtcgatggt ggtgtcaaag tacttgaagg cggcaggggc tcccagattg
     1261 gtcagggtaa acaggtggat gatattctcg gcctgctctc tgatgggctt atcccggtgc
     1321 ttgttgtagg cggacagcac tttgtccaga ttagcgtcgg ccaggatcac tctcttggag
     1381 aactcgctga tctgctcgat gatctcgtcc aggtagtgct tgtgctgttc cacaaacagc
     1441 tgtttctgct cattatcctc gggggagccc ttcagcttct catagtggct ggccaggtac
     1501 aggaagttca catatttgga gggcagggcc agttcgtttc ccttctgcag ttcgccggca
     1561 gaggccagca ttctcttccg gccgttttcc agctcgaaca gggagtactt aggcagcttg
     1621 atgatcaggt cctttttcac ttctttgtag cccttggctt ccagaaagtc gatgggattc
     1681 ttctcgaagc tgcttctttc catgatggtg atccccagca gctctttcac actcttcagt
     1741 ttcttggact tgcccttttc cactttggcc accaccagca cagaataggc cacggtgggg
     1801 ctgtcgaagc cgccgtactt cttagggtcc cagtccttct ttctggcgat cagcttatcg
     1861 ctgttcctct tgggcaggat agactctttg ctgaagccgc ctgtctgcac ctcggtcttt
     1921 ttcacgatat tcacttgggg catgctcagc actttccgca cggtggcaaa atcccggccc
     1981 ttatcccaca cgatctcccc ggtttcgccg tttgtctcga tcagaggccg cttccggatc
     2041 tcgccgttgg ccagggtaat ctcggtcttg aaaaagttca tgatgttgct gtagaagaag
     2101 tacttggcgg tagccttgcc gatttcctgc tcgctcttgg cgatcatctt ccgcacgtcg
     2161 tacaccttgt agtcgccgta cacgaactcg ctttccagct tagggtactt tttgatcagg
     2221 gcggttccca cgacggcgtt caggtaggcg tcgtgggcgt ggtggtagtt gttgatctcg
     2281 cgcactttgt aaaactggaa atccttccgg aaatcggaca ccagcttgga cttcagggtg
     2341 atcactttca cttcccggat cagcttgtca ttctcgtcgt acttagtgtt catccgggag
     2401 tccaggatct gtgccacgtg ctttgtgatc tgccgggttt ccaccagctg tctcttgatg
     2461 aagccggcct tatccagttc gctcaggccg cctctctcgg ccttggtcag attgtcgaac
     2521 tttctctggg taatcagctt ggcgttcagc agctgccgcc agtagttctt catcttcttc
     2581 acgacctctt cggagggcac gttgtcgctc ttgccccggt tcttgtcgct tctggtcagc
     2641 accttgttgt cgatggagtc gtccttcaga aagctctgag gcacgatatg gtccacatcg
     2701 tagtcggaca gccggttgat gtccagttcc tggtccacgt acatatcccg cccattctgc
     2761 aggtagtaca ggtacagctt ctcgttctgc agctgggtgt tttccacggg gtgttctttc
     2821 aggatctggc tgcccagctc tttgatgccc tcttcgatcc gcttcattct ctcgcggctg
     2881 ttcttctgtc ccttctgggt ggtctggttc tctctggcca tttcgatcac gatgttctcg
     2941 ggcttgtgcc ggcccatcac tttcacgagc tcgtccacca ccttcactgt ctgcaggatg
     3001 cccttcttaa tggcggggct gccggccaga ttggcaatgt gctcgtgcag gctatcgccc
     3061 tggccggaca cctgggcttt ctggatgtcc tctttaaagg tcaggctgtc gtcgtggatc
     3121 agctgcatga agtttctgtt ggcgaagccg tcggacttca ggaaatccag gattgtcttg
     3181 ccggactgct tgtcccggat gccgttgatc agcttccggc tcagcctgcc ccagccggtg
     3241 tatctccgcc gcttcagctg cttcatcact ttgtcgtcga acaggtgggc ataggttttc
     3301 agccgttcct cgatcatctc tctgtcctca aacagtgtca gggtcagcac gatatcttcc
     3361 agaatgtcct cgttttcctc attgtccagg aagtccttgt ccttgataat tttcagcaga
     3421 tcgtggtatg tgcccaggga ggcgttgaac cgatcttcca cgccggagat ttccacggag
     3481 tcgaagcact cgattttctt gaagtagtcc tctttcagct gcttcacggt cactttccgg
     3541 ttggtcttga acagcaggtc cacgatggcc tttttctgct cgccgctcag gaaggcgggc
     3601 tttctcattc cctcggtcac gtatttcact ttggtcagct cgttatacac ggtgaagtac
     3661 tcgtacagca ggctgtgctt gggcagcacc ttctcgttgg gcaggttctt atcgaagttg
     3721 gtcatccgct cgatgaagct ctgggcggaa gcgcccttgt ccaccacttc ctcgaagttc
     3781 cagggggtga tggtttcctc gctctttctg gtcatccagg cgaatctgct gtttcccctg
     3841 gccagagggc ccacgtagta ggggatgcgg aaggtcagga tcttctcgat cttttcccgg
     3901 ttgtccttca ggaatgggta aaaatcttcc tgccgccgca gaatggcgtg cagctctccc
     3961 aggtggatct ggtgggggat gctgccgttg tcgaaggtcc gctgcttccg cagcaggtcc
     4021 tctctgttca gcttcacgag cagttcctcg gtgccgtcca tcttttccag gatgggcttg
     4081 atgaacttgt agaactcttc ctggctggct ccgccgtcaa tgtagccggc gtagccgttc
     4141 ttgctctggt cgaagaaaat ctctttgtac ttctcaggca gctgctgccg cacgagagct
     4201 ttcagcaggg tcaggtcctg gtggtgctcg tcgtatctct tgatcataga ggcgctcagg
     4261 ggggccttgg tgatctcggt gttcactctc aggatgtcgc tcagcaggat ggcgtcggac
     4321 aggttcttgg cggccagaaa caggtcggcg tactggtcgc cgatctgggc cagcaggttg
     4381 tccaggtcgt cgtcgtaggt gtccttgctc agctgcagtt tggcatcctc ggccaggtcg
     4441 aagttgctct tgaagttggg ggtcaggccc aggctcaggg caatcaggtt tccgaacagg
     4501 ccattcttct tctcgccggg cagctgggcg atcagatttt ccagccgtct gctcttgctc
     4561 agtctggcag acaggatggc cttggcgtcc acgccgctgg cgttgatggg gttttcctcg
     4621 aacagctggt tgtaggtctg caccagctgg atgaacagct tgtccacgtc gctgttgtcg
     4681 gggttcaggt cgccctcgat caggaagtgg ccccggaact tgatcatgtg ggccagggcc
     4741 agatagatca gccgcaggtc ggccttgtcg gtgctgtcca ccagtttctt tctcaggtgg
     4801 tagatggtgg ggtacttctc gtggtaggcc acctcgtcca cgatgttgcc gaagatgggg
     4861 tgccgctcgt gcttcttatc ctcttccacc aggaaggact cttccagtct gtggaagaag
     4921 ctgtcgtcca ccttggccat ctcgttgctg aagatctctt gcagatagca gatccggttc
     4981 ttccgtctgg tgtatcttct tctggcggtt ctcttcagcc gggtggcctc ggctgtttcg
     5041 ccgctgtcga acagcagggc tccgatcagg ttcttcttga tgctgtgccg gtcggtgttg
     5101 cccagcacct tgaatttctt gctgggcacc ttgtactcgt cggtgatcac ggcccagccc
     5161 acagagttgg tgccgatgtc caggccgatg ctgtacttct tgtcggctgc tgggactccg
     5221 tggataccga ccttccgctt cttctttggg gccatcttat cgtcatcgtc tttgtaatca
     5281 atatcatgat ccttgtagtc tccgtcgtgg tccttatagt ccatggtggc accggtccaa
     5341 cctgaaaaaa agtgatttca ggcaggtgct ccaggtaatt aaacattaat accccaccaa
     5401 ccaaccatcc cttaaaccct tacctcttgc tcagctaatt acagcccgga ggagaagggc
     5461 cgtcccgccc gctcacctgt gggagtaacg cggtcagtca gagccggggc gggcggcgcg
     5521 aggcggcggc ggagcggggc acggggcgaa ggcagcgcgc agcgactccc gcccgccgcg
     5581 cgcttcgctt tttatagggc cgccgccgcc gccgcctcgc cataaaagga aactttcgga
     5641 gcgcgccgct ctgattggct gccgccgcac ctctccgcct cgccccgccc cgcccctcgc
     5701 cccgccccgc cccgcctggc gcgcgccccc cccccccccc cgcccccatc gctgcacaaa
     5761 ataattaaaa aataaataaa tacaaaattg ggggtgggga ggggggggag atggggagag
     5821 tgaagcagaa cgtggggctc acctcgacca tggtaatagc gatgactaat acgtagatgt
     5881 actgccaagt aggaaagtcc cataaggtca tgtactgggc acaatgccag gcgggccatt
     5941 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     6001 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     6061 ttactattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg
     6121 taagttatgt aacgggtacc tctagaggcc gcaggaaccc ctagtgatgg agttggccac
     6181 tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc
     6241 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggacatgtga gcaaaaggcc
     6301 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6361 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     6421 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     6481 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     6541 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     6601 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     6661 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     6721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     6781 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     6841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     6901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     6961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     7021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     7081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     7141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     7201 gggagggctt accatctggc cccagtgctg caatgatacc gcggcttcca cgctcaccgg
     7261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     7321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     7381 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     7441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     7501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     7561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     7621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     7681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     7741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     7801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     7861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     7921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     7981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     8041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     8101 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     8161 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccgg
//
