LOCUS       sequence-174053-        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"
     regulatory      22..31
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             28..3273
                     /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             3286..3306
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3307..3396
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3409..3435
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     protein_bind    complement(3473..3497)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3542..3676
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3566..3585)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3620..3639
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3789..3809)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3789..3807)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3828..3844)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3828..3844)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3841..3863)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3852..3868
                     /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(3876..3882,3883..3900,3901..3906))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3921..3942
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4059..4076)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4230..4818)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4310..4329)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4989..5780,5781..5849))
                     /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     5612..5631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5850..5954)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5980..6435)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6117..6138)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6329..6348
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6506..7490
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        7525..7543
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     7525..7542
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    join(7598..7604,1..18)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tgtacaaaaa agcaggctgg cgccaccatg gctgccttca aacctaattc aatcaactac
       61 atcctcggcc tcgatatcgg catcgcatcc gtcggctggg cgatggtaga aattgacgaa
      121 gaagaaaacc ccatccgcct gattgatttg ggcgtgcgcg tatttgagcg tgccgaagta
      181 ccgaaaacag gcgactccct tgccatggca aggcgtttgg cgcgcagtgt tcgccgcctg
      241 acccgccgtc gcgcccaccg cctgcttcgg acccgccgcc tattgaaacg cgaaggcgta
      301 ttacaagccg ccaattttga cgaaaacggc ttgattaaat ccttaccgaa tacaccatgg
      361 caacttcgcg cagccgcatt agaccgcaaa ctgacgcctt tagagtggtc ggcagtcttg
      421 ttgcatttaa tcaaacatcg cggctattta tcgcaacgga aaaacgaggg cgaaactgcc
      481 gataaggagc ttggcgcttt gcttaaaggc gtagccggca atgcccatgc cttacagaca
      541 ggcgatttcc gcacaccggc cgaattggct ttaaataaat ttgagaaaga aagcggccat
      601 atccgcaatc agcgcagcga ttattcgcat acgttcagcc gcaaagattt acaggcggag
      661 ctgattttgc tgtttgaaaa acaaaaagaa tttggcaatc cgcatgtttc aggcggcctt
      721 aaagaaggta ttgaaaccct actgatgacg caacgccctg ccctgtccgg cgatgccgtt
      781 caaaaaatgt tggggcattg caccttcgaa ccggcagagc cgaaagccgc taaaaacacc
      841 tacacagccg aacgtttcat ctggctgacc aagctgaaca acctgcgtat tttagagcaa
      901 ggcagcgagc ggccattgac cgataccgaa cgcgccacgc ttatggacga gccatacaga
      961 aaatccaaac tgacttacgc acaagcccgt aagctgctgg gtttagaaga taccgccttt
     1021 ttcaaaggct tgcgctatgg taaagacaat gccgaagcct caacattgat ggaaatgaag
     1081 gcctaccatg ccatcagccg tgcactggaa aaagaaggat tgaaagacaa aaaatcccca
     1141 ttaaaccttt ctcccgaatt acaagacgaa atcggcacgg cattctccct gttcaaaacc
     1201 gatgaagaca ttacaggccg tctgaaagac cgtatacagc ccgaaatctt agaagcgctg
     1261 ttgaaacaca tcagcttcga taagttcgtc caaatttcct tgaaagcatt gcgccgaatt
     1321 gtgcctctaa tggaacaagg caaacgttac gatgaagcct gcgccgaaat ctacggagac
     1381 cattacggca agaagaatac ggaagaaaag atttatctgc cgccgattcc cgccgacgaa
     1441 atccgcaacc ccgtcgtctt gcgcgcctta tctcaagcac gtaaggtcat taacggcgtg
     1501 gtacgccgtt acggctcccc agctcgtatc catattgaaa ctgcaaggga agtaggtaaa
     1561 tcgtttaaag accgcaaaga aattgagaaa cgccaagaag aaaaccgcaa agaccgggaa
     1621 aaagccgccg ccaaattccg agagtatttc cccaattttg tcggagaacc caaatccaaa
     1681 gatattctga aactgcgcct gtacgagcaa caacacggca aatgcctgta ttcgggcaaa
     1741 gaaatcaact taggccgtct gaacgaaaaa ggctatgtcg aaatcgacca tgccctgccg
     1801 ttctcgcgca catgggacga cagtttcaac aataaagtac tggtattggg cagcgaaaac
     1861 caaaacaaag gcaatcaaac cccttacgaa tacttcaacg gcaaagacaa cagccgcgaa
     1921 tggcaggaat ttaaagcgcg tgtcgaaacc agccgtttcc cgcgcagtaa aaaacaacgg
     1981 attctgctgc aaaaattcga tgaagacggc tttaaagaac gcaatctgaa cgacacgcgc
     2041 tacgtcaacc gtttcctgtg tcaatttgtt gccgaccgta tgcggctgac aggtaaaggc
     2101 aagaaacgtg tctttgcatc caacggacaa attaccaatc tgttgcgcgg cttttgggga
     2161 ttgcgcaaag tgcgtgcgga aaacgaccgc catcacgcct tggacgccgt cgtcgttgcc
     2221 tgctcgaccg ttgccatgca gcagaaaatt acccgttttg tacgctataa agagatgaac
     2281 gcgtttgacg gtaaaaccat agacaaagaa acaggagaag tgctgcatca aaaaacacac
     2341 ttcccacaac cttgggaatt tttcgcacaa gaagtcatga ttcgcgtctt cggcaaaccg
     2401 gacggcaaac ccgaattcga agaagccgat accctagaaa aactgcgcac gttgcttgcc
     2461 gaaaaattat catctcgccc cgaagccgta cacgaatacg ttacgccact gtttgtttca
     2521 cgcgcgccca atcggaagat gagcgggcaa gggcatatgg agaccgtcaa atccgccaaa
     2581 cgactggacg aaggcgtcag cgtgttgcgc gtaccgctga cacagttaaa actgaaagac
     2641 ttggaaaaaa tggtcaatcg ggagcgcgaa cctaagctat acgaagcact gaaagcacgg
     2701 ctggaagcac ataaagacga tcctgccaaa gcctttgccg agccgtttta caaatacgat
     2761 aaagcaggca accgcaccca acaggtaaaa gccgtacgcg tagagcaagt acagaaaacc
     2821 ggcgtatggg tgcgcaacca taacggtatt gccgacaacg caaccatggt gcgcgtagat
     2881 gtgtttgaga aaggcgacaa gtattatctg gtaccgattt acagttggca ggtagcgaaa
     2941 gggattttgc cggatagggc tgttgtacaa ggaaaagatg aagaagattg gcaacttatt
     3001 gatgatagtt tcaactttaa attctcatta caccctaatg atttagtcga ggttataaca
     3061 aaaaaagcta gaatgtttgg ttactttgcc agctgccatc gaggcacagg taatatcaat
     3121 atacgcattc atgatcttga tcataaaatt ggcaaaaatg gaatactgga aggtatcggc
     3181 gtcaaaaccg ccctttcatt ccaaaaatac caaattgacg aactgggcaa agaaatcaga
     3241 ccatgccgtc tgaaaaaacg cccgcctgtc cgtggcaccg gcgggcccaa gaagaagagg
     3301 aaggtatacc catacgatgt tcctgactat gcgggctatc cctatgacgt cccggactat
     3361 gcaggatcgt atccttatga cgttccagat tacgctggat ccgccgctcc ggcagctaag
     3421 aaaaagaaac tggatttcga atccggataa gcggccgcct cgagatatct agacccagct
     3481 ttcttgtaca aagtggttga tcctctcgag cctctagaac tatagtgagt cgtattacgt
     3541 agatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg
     3601 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     3661 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     3721 ggtgtgggag gttttttaat tcgcggccgc ggcgccaatg cattgggccc ggtacccagc
     3781 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     3841 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg gagcataaag
     3901 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3961 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4021 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4081 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4141 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4201 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4261 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4321 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4381 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4441 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4501 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4561 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4621 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4681 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4741 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4801 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4861 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4921 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4981 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5041 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5101 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5161 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5221 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5281 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5341 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5401 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5461 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5521 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5581 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5641 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5701 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5761 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5821 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5881 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5941 ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc gttaatattt
     6001 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     6061 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     6121 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     6181 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     6241 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     6301 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     6361 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     6421 cgctacaggg cgcgtcccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     6481 tgcgggcctc ttcgctatta cgccagtcga ccgccaattc aatatggcgt atatggactc
     6541 atgccaattc aatatggtgg atctggacct gtgccaattc aatatggcgt atatggactc
     6601 gtgccaattc aatatggtgg atctggaccc cagccaattc aatatggcgg acttggcacc
     6661 atgccaattc aatatggcgg acctggcact gtgccaactg gggaggggtc tacttggcac
     6721 ggtgccaagt ttgaggaggg gtcttggccc tgtgccaagt ccgccatatt gaattggcat
     6781 ggtgccaata atggcggcca tattggctat atgccaggat caatatatag gcaatatcca
     6841 atatggccct atgccaatat ggctattggc caggttcaat actatgtatt ggccctatgc
     6901 catatagtat tccatatatg ggttttccta ttgacgtaga tagcccctcc caatgggcgg
     6961 tcccatatac catatatggg gcttcctaat accgcccata gccactcccc cattgacgtc
     7021 aatggtctct atatatggtc tttcctattg acgtcatatg ggcggtccta ttgacgtata
     7081 tggcgcctcc cccattgacg tcaattacgg taaatggccc gcctggctca atgcccattg
     7141 acgtcaatag gaccacccac cattgacgtc aatgggatgg ctcattgccc attcatatcc
     7201 gttctcacgc cccctattga cgtcaatgac ggtaaatggc ccacttggca gtacatcaat
     7261 atctattaat agtaacttgg caagtacatt actattggaa gtacgccagg gtacattggc
     7321 agtactccca ttgacgtcaa tggcggtaaa tggcccgcga tggctgccaa gtacatcccc
     7381 attgacgtca atggggaggg gcaatgacgc aaatgggcgt tccattgacg taaatgggcg
     7441 gtaggcgtgc ctaatgggag gtctatataa gcaatgctcg tttagggaac cgccattctg
     7501 cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag ctacttgttc
     7561 tttttgcagg atcccatcga ttcgaattca aggatcaaca agtt
//
