LOCUS       sequence-170554-        7604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of wild type Nme Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7604)
  AUTHORS   Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, 
            Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR
  TITLE     Naturally Occurring Off-Switches for CRISPR-Cas9.
  JOURNAL   Cell. 2016 Dec 15;167(7):1829-1838.e9. doi: 
            10.1016/j.cell.2016.11.017. Epub 2016 Dec 8.
  PUBMED    27984730
REFERENCE   2  (bases 1 to 7604)
  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"; doi: 
            "10.1016/j.cell.2016.11.017"; journalName: "Cell"; date: 
            "2016-12-15- 15"; volume: "167"; issue: "7"; pages: "1829-1838.e9"
COMMENT     Sequence Label: pCSDest2-NmeCas9-NLS-3XHA-NLS
FEATURES             Location/Qualifiers
     source          1..7604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        37..55
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     37..54
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    110..134
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      138..147
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             144..3389
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             3402..3422
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3423..3512
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3525..3551
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     protein_bind    complement(3589..3613)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3658..3792
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3682..3701)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3736..3755
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3905..3925)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3905..3923)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3944..3960)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3944..3960)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3957..3979)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3968..3984
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3992..3998,3999..4016,4017..4022))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4037..4058
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4175..4192)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4346..4934)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4426..4445)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5105..5896,5897..5965))
                     /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     5728..5747
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5966..6070)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6096..6551)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6233..6254)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6445..6464
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(6622..7604,1..2)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 accgccattc tgcctgggga cgtcggagca agcttgattt aggtgacact atagaataca
       61 agctacttgt tctttttgca ggatcccatc gattcgaatt caaggatcaa caagtttgta
      121 caaaaaagca ggctggcgcc accatggctg ccttcaaacc taattcaatc aactacatcc
      181 tcggcctcga tatcggcatc gcatccgtcg gctgggcgat ggtagaaatt gacgaagaag
      241 aaaaccccat ccgcctgatt gatttgggcg tgcgcgtatt tgagcgtgcc gaagtaccga
      301 aaacaggcga ctcccttgcc atggcaaggc gtttggcgcg cagtgttcgc cgcctgaccc
      361 gccgtcgcgc ccaccgcctg cttcggaccc gccgcctatt gaaacgcgaa ggcgtattac
      421 aagccgccaa ttttgacgaa aacggcttga ttaaatcctt accgaataca ccatggcaac
      481 ttcgcgcagc cgcattagac cgcaaactga cgcctttaga gtggtcggca gtcttgttgc
      541 atttaatcaa acatcgcggc tatttatcgc aacggaaaaa cgagggcgaa actgccgata
      601 aggagcttgg cgctttgctt aaaggcgtag ccggcaatgc ccatgcctta cagacaggcg
      661 atttccgcac accggccgaa ttggctttaa ataaatttga gaaagaaagc ggccatatcc
      721 gcaatcagcg cagcgattat tcgcatacgt tcagccgcaa agatttacag gcggagctga
      781 ttttgctgtt tgaaaaacaa aaagaatttg gcaatccgca tgtttcaggc ggccttaaag
      841 aaggtattga aaccctactg atgacgcaac gccctgccct gtccggcgat gccgttcaaa
      901 aaatgttggg gcattgcacc ttcgaaccgg cagagccgaa agccgctaaa aacacctaca
      961 cagccgaacg tttcatctgg ctgaccaagc tgaacaacct gcgtatttta gagcaaggca
     1021 gcgagcggcc attgaccgat accgaacgcg ccacgcttat ggacgagcca tacagaaaat
     1081 ccaaactgac ttacgcacaa gcccgtaagc tgctgggttt agaagatacc gcctttttca
     1141 aaggcttgcg ctatggtaaa gacaatgccg aagcctcaac attgatggaa atgaaggcct
     1201 accatgccat cagccgtgca ctggaaaaag aaggattgaa agacaaaaaa tccccattaa
     1261 acctttctcc cgaattacaa gacgaaatcg gcacggcatt ctccctgttc aaaaccgatg
     1321 aagacattac aggccgtctg aaagaccgta tacagcccga aatcttagaa gcgctgttga
     1381 aacacatcag cttcgataag ttcgtccaaa tttccttgaa agcattgcgc cgaattgtgc
     1441 ctctaatgga acaaggcaaa cgttacgatg aagcctgcgc cgaaatctac ggagaccatt
     1501 acggcaagaa gaatacggaa gaaaagattt atctgccgcc gattcccgcc gacgaaatcc
     1561 gcaaccccgt cgtcttgcgc gccttatctc aagcacgtaa ggtcattaac ggcgtggtac
     1621 gccgttacgg ctccccagct cgtatccata ttgaaactgc aagggaagta ggtaaatcgt
     1681 ttaaagaccg caaagaaatt gagaaacgcc aagaagaaaa ccgcaaagac cgggaaaaag
     1741 ccgccgccaa attccgagag tatttcccca attttgtcgg agaacccaaa tccaaagata
     1801 ttctgaaact gcgcctgtac gagcaacaac acggcaaatg cctgtattcg ggcaaagaaa
     1861 tcaacttagg ccgtctgaac gaaaaaggct atgtcgaaat cgaccatgcc ctgccgttct
     1921 cgcgcacatg ggacgacagt ttcaacaata aagtactggt attgggcagc gaaaaccaaa
     1981 acaaaggcaa tcaaacccct tacgaatact tcaacggcaa agacaacagc cgcgaatggc
     2041 aggaatttaa agcgcgtgtc gaaaccagcc gtttcccgcg cagtaaaaaa caacggattc
     2101 tgctgcaaaa attcgatgaa gacggcttta aagaacgcaa tctgaacgac acgcgctacg
     2161 tcaaccgttt cctgtgtcaa tttgttgccg accgtatgcg gctgacaggt aaaggcaaga
     2221 aacgtgtctt tgcatccaac ggacaaatta ccaatctgtt gcgcggcttt tggggattgc
     2281 gcaaagtgcg tgcggaaaac gaccgccatc acgccttgga cgccgtcgtc gttgcctgct
     2341 cgaccgttgc catgcagcag aaaattaccc gttttgtacg ctataaagag atgaacgcgt
     2401 ttgacggtaa aaccatagac aaagaaacag gagaagtgct gcatcaaaaa acacacttcc
     2461 cacaaccttg ggaatttttc gcacaagaag tcatgattcg cgtcttcggc aaaccggacg
     2521 gcaaacccga attcgaagaa gccgataccc tagaaaaact gcgcacgttg cttgccgaaa
     2581 aattatcatc tcgccccgaa gccgtacacg aatacgttac gccactgttt gtttcacgcg
     2641 cgcccaatcg gaagatgagc gggcaagggc atatggagac cgtcaaatcc gccaaacgac
     2701 tggacgaagg cgtcagcgtg ttgcgcgtac cgctgacaca gttaaaactg aaagacttgg
     2761 aaaaaatggt caatcgggag cgcgaaccta agctatacga agcactgaaa gcacggctgg
     2821 aagcacataa agacgatcct gccaaagcct ttgccgagcc gttttacaaa tacgataaag
     2881 caggcaaccg cacccaacag gtaaaagccg tacgcgtaga gcaagtacag aaaaccggcg
     2941 tatgggtgcg caaccataac ggtattgccg acaacgcaac catggtgcgc gtagatgtgt
     3001 ttgagaaagg cgacaagtat tatctggtac cgatttacag ttggcaggta gcgaaaggga
     3061 ttttgccgga tagggctgtt gtacaaggaa aagatgaaga agattggcaa cttattgatg
     3121 atagtttcaa ctttaaattc tcattacacc ctaatgattt agtcgaggtt ataacaaaaa
     3181 aagctagaat gtttggttac tttgccagct gccatcgagg cacaggtaat atcaatatac
     3241 gcattcatga tcttgatcat aaaattggca aaaatggaat actggaaggt atcggcgtca
     3301 aaaccgccct ttcattccaa aaataccaaa ttgacgaact gggcaaagaa atcagaccat
     3361 gccgtctgaa aaaacgcccg cctgtccgtg gcaccggcgg gcccaagaag aagaggaagg
     3421 tatacccata cgatgttcct gactatgcgg gctatcccta tgacgtcccg gactatgcag
     3481 gatcgtatcc ttatgacgtt ccagattacg ctggatccgc cgctccggca gctaagaaaa
     3541 agaaactgga tttcgaatcc ggataagcgg ccgcctcgag atatctagac ccagctttct
     3601 tgtacaaagt ggttgatcct ctcgagcctc tagaactata gtgagtcgta ttacgtagat
     3661 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     3721 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     3781 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     3841 tgggaggttt tttaattcgc ggccgcggcg ccaatgcatt gggcccggta cccagctttt
     3901 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     3961 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4021 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4081 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4141 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4201 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4261 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4321 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4381 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4441 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4501 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4561 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4621 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4681 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4741 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     4801 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4861 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4921 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4981 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5041 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5101 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5161 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5221 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5281 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5341 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5401 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5461 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5521 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5581 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5641 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5701 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5761 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5821 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5881 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5941 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6001 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6061 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     6121 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     6181 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     6241 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     6301 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     6361 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     6421 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     6481 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     6541 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     6601 ggcctcttcg ctattacgcc agtcgaccgc caattcaata tggcgtatat ggactcatgc
     6661 caattcaata tggtggatct ggacctgtgc caattcaata tggcgtatat ggactcgtgc
     6721 caattcaata tggtggatct ggaccccagc caattcaata tggcggactt ggcaccatgc
     6781 caattcaata tggcggactt ggcactgtgc caactgggga ggggtctact tggcacggtg
     6841 ccaagtttga ggaggggtct tggccctgtg ccaagtccgc catattgaat tggcatggtg
     6901 ccaataatgg cggccatatt ggctatatgc caggatcaat atataggcaa tatccaatat
     6961 ggccctatgc caatatggct attggccagg ttcaatacta tgtattggcc ctatgccata
     7021 tagtattcca tatatgggtt ttcctattga cgtagatagc ccctcccaat gggcggtccc
     7081 atataccata tatggggctt cctaataccg cccatagcca ctcccccatt gacgtcaatg
     7141 gtctctatat atggtctttc ctattgacgt catatgggcg gtcctattga cgtatatggc
     7201 gcctccccca ttgacgtcaa ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt
     7261 caataggacc acccaccatt gacgtcaatg ggatggctca ttgcccattc atatccgttc
     7321 tcacgccccc tattgacgtc aatgacggta aatggcccac ttggcagtac atcaatatct
     7381 attaatagta acttggcaag tacattacta ttggaagtac gccagggtac attggcagta
     7441 ctcccattga cgtcaatggc ggtaaatggc ccgcgatggc tgccaagtac atccccattg
     7501 acgtcaatgg ggaggggcaa tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag
     7561 gcgtgcctaa tgggaggtct atataagcaa tgctcgttta ggga
//
