LOCUS       sequence-204651-        7583 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7583)
  AUTHORS   Bolukbasi MF, Liu P, Luk K, Kwok SF, Gupta A, Amrani N, Sontheimer 
            EJ, Zhu LJ, Wolfe SA
  TITLE     Orthogonal Cas9-Cas9 chimeras provide a versatile platform for 
            genome editing.
  JOURNAL   Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.
  PUBMED    30451839
REFERENCE   2  (bases 1 to 7583)
  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: "Nat 
            Commun."; date: "2018-11-19"; volume: "9(1):4856. doi"; pages: " 
            10.1038/s41467-018-07310-x"
COMMENT     Sequence Label: pCSD_1xNLS-SaCas9-1xNLS-3xHA-1xNLS
FEATURES             Location/Qualifiers
     source          1..7583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        38..56
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     38..55
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    111..135
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      139..148
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             160..180
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             205..3360
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             3382..3402
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3403..3492
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3505..3531
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     protein_bind    complement(3569..3593)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3638..3772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3662..3681)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3716..3735
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3885..3905)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3885..3903)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3924..3940)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3924..3940)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3937..3959)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3948..3964
                     /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(3972..3978,3979..3996,3997..4002))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4017..4038
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4155..4172)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4326..4914)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4406..4425)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5085..5876,5877..5945))
                     /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     5708..5727
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5946..6050)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6076..6531)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6213..6234)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6425..6444
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(6602..7583,1..3)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 aaccgccatt ctgcctgggg acgtcggagc aagcttgatt taggtgacac tatagaatac
       61 aagctacttg ttctttttgc aggatcccat cgattcgaat tcaaggatca acaagtttgt
      121 acaaaaaagc aggctggcgc caccatggga cgtacggccc caaagaagaa gcggaaggtc
      181 ggtatccacg gagtcccagc agccaagcgg aactacatcc tgggcctgga catcggcatc
      241 accagcgtgg gctacggcat catcgactac gagacacggg acgtgatcga tgccggcgtg
      301 cggctgttca aagaggccaa cgtggaaaac aacgagggca ggcggagcaa gagaggcgcc
      361 agaaggctga agcggcggag gcggcataga atccagagag tgaagaagct gctgttcgac
      421 tacaacctgc tgaccgacca cagcgagctg agcggcatca acccctacga ggccagagtg
      481 aagggcctga gccagaagct gagcgaggaa gagttctctg ccgccctgct gcacctggcc
      541 aagagaagag gcgtgcacaa cgtgaacgag gtggaagagg acaccggcaa cgagctgtcc
      601 accaaagagc agatcagccg gaacagcaag gccctggaag agaaatacgt ggccgaactg
      661 cagctggaac ggctgaagaa agacggcgaa gtgcggggca gcatcaacag attcaagacc
      721 agcgactacg tgaaagaagc caaacagctg ctgaaggtgc agaaggccta ccaccagctg
      781 gaccagagct tcatcgacac ctacatcgac ctgctggaaa cccggcggac ctactatgag
      841 ggacctggcg agggcagccc cttcggctgg aaggacatca aagaatggta cgagatgctg
      901 atgggccact gcacctactt ccccgaggaa ctgcggagcg tgaagtacgc ctacaacgcc
      961 gacctgtaca acgccctgaa cgacctgaac aatctcgtga tcaccaggga cgagaacgag
     1021 aagctggaat attacgagaa gttccagatc atcgagaacg tgttcaagca gaagaagaag
     1081 cccaccctga agcagatcgc caaagaaatc ctcgtgaacg aagaggatat taagggctac
     1141 agagtgacca gcaccggcaa gcccgagttc accaacctga aggtgtacca cgacatcaag
     1201 gacattaccg cccggaaaga gattattgag aacgccgagc tgctggatca gattgccaag
     1261 atcctgacca tctaccagag cagcgaggac atccaggaag aactgaccaa tctgaactcc
     1321 gagctgaccc aggaagagat cgagcagatc tctaatctga agggctatac cggcacccac
     1381 aacctgagcc tgaaggccat caacctgatc ctggacgagc tgtggcacac caacgacaac
     1441 cagatcgcta tcttcaaccg gctgaagctg gtgcccaaga aggtggacct gtcccagcag
     1501 aaagagatcc ccaccaccct ggtggacgac ttcatcctga gccccgtcgt gaagagaagc
     1561 ttcatccaga gcatcaaagt gatcaacgcc atcatcaaga agtacggcct gcccaacgac
     1621 atcattatcg agctggcccg cgagaagaac tccaaggacg cccagaaaat gatcaacgag
     1681 atgcagaagc ggaaccggca gaccaacgag cggatcgagg aaatcatccg gaccaccggc
     1741 aaagagaacg ccaagtacct gatcgagaag atcaagctgc acgacatgca ggaaggcaag
     1801 tgcctgtaca gcctggaagc catccctctg gaagatctgc tgaacaaccc cttcaactat
     1861 gaggtggacc acatcatccc cagaagcgtg tccttcgaca acagcttcaa caacaaggtg
     1921 ctcgtgaagc aggaagaaaa cagcaagaag ggcaaccgga ccccattcca gtacctgagc
     1981 agcagcgaca gcaagatcag ctacgaaacc ttcaagaagc acatcctgaa tctggccaag
     2041 ggcaagggca gaatcagcaa gaccaagaaa gagtatctgc tggaagaacg ggacatcaac
     2101 aggttctccg tgcagaaaga cttcatcaac cggaacctgg tggataccag atacgccacc
     2161 agaggcctga tgaacctgct gcggagctac ttcagagtga acaacctgga cgtgaaagtg
     2221 aagtccatca atggcggctt caccagcttt ctgcggcgga agtggaagtt taagaaagag
     2281 cggaacaagg ggtacaagca ccacgccgag gacgccctga tcattgccaa cgccgatttc
     2341 atcttcaaag agtggaagaa actggacaag gccaaaaaag tgatggaaaa ccagatgttc
     2401 gaggaaaagc aggccgagag catgcccgag atcgaaaccg agcaggagta caaagagatc
     2461 ttcatcaccc cccaccagat caagcacatt aaggacttca aggactacaa gtacagccac
     2521 cgggtggaca agaagcctaa tagagagctg attaacgaca ccctgtactc cacccggaag
     2581 gacgacaagg gcaacaccct gatcgtgaac aatctgaacg gcctgtacga caaggacaat
     2641 gacaagctga aaaagctgat caacaagagc cccgaaaagc tgctgatgta ccaccacgac
     2701 ccccagacct accagaaact gaagctgatt atggaacagt acggcgacga gaagaatccc
     2761 ctgtacaagt actacgagga aaccgggaac tacctgacca agtactccaa aaaggacaac
     2821 ggccccgtga tcaagaagat taagtattac ggcaacaaac tgaacgccca tctggacatc
     2881 accgacgact accccaacag cagaaacaag gtcgtgaagc tgtccctgaa gccctacaga
     2941 ttcgacgtgt acctggacaa tggcgtgtac aagttcgtga ccgtgaagaa tctggatgtg
     3001 atcaaaaaag aaaactacta cgaagtgaat agcaagtgct atgaggaagc taagaagctg
     3061 aagaagatca gcaaccaggc cgagtttatc gcctccttct acaacaacga tctgatcaag
     3121 atcaacggcg agctgtatag agtgatcggc gtgaacaacg acctgctgaa ccggatcgaa
     3181 gtgaacatga tcgacatcac ctaccgcgag tacctggaaa acatgaacga caagaggccc
     3241 cccaggatca ttaagacaat cgcctccaag acccagagca ttaagaagta cagcacagac
     3301 attctgggca acctgtatga agtgaaatct aagaagcacc ctcagatcat caaaaagggc
     3361 aaaaggccgg gcaccggcgg gcccaagaag aagaggaagg tatacccata cgatgttcct
     3421 gactatgcgg gctatcccta tgacgtcccg gactatgcag gatcgtatcc ttatgacgtt
     3481 ccagattacg ctggatccgc cgctccggca gctaagaaaa agaaactgga tttcgaatcc
     3541 ggataagcgg ccgcctcgag atatctagac ccagctttct tgtacaaagt ggttgatcct
     3601 ctcgagcctc tagaactata gtgagtcgta ttacgtagat ccagacatga taagatacat
     3661 tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat
     3721 ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa
     3781 caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaattcgc
     3841 ggccgcggcg ccaatgcatt gggcccggta cccagctttt gttcccttta gtgagggtta
     3901 attgcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3961 acaattccac acaacatacg agccgggagc ataaagtgta aagcctgggg tgcctaatga
     4021 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4081 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4141 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4201 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4261 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4321 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4381 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4441 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4501 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4561 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4621 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4681 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4741 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     4801 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4861 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4921 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4981 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5041 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5101 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5161 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5221 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5281 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5341 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5401 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5461 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5521 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5581 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5641 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5701 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5761 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5821 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5881 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5941 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6001 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6061 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     6121 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     6181 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     6241 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     6301 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     6361 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     6421 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     6481 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     6541 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     6601 agtcgaccgc caattcaata tggcgtatat ggactcatgc caattcaata tggtggatct
     6661 ggacctgtgc caattcaata tggcgtatat ggactcgtgc caattcaata tggtggatct
     6721 ggaccccagc caattcaata tggcggactt ggcaccatgc caattcaata tggcggacct
     6781 ggcactgtgc caactgggga ggggtctact tggcacggtg ccaagtttga ggaggggtct
     6841 tggccctgtg ccaagtccgc catattgaat tggcatggtg ccaataatgg cggccatatt
     6901 ggctatatgc caggatcaat atataggcaa tatccaatat ggccctatgc caatatggct
     6961 attggccagg ttcaatacta tgtattggcc ctatgccata tagtattcca tatatgggtt
     7021 ttcctattga cgtagatagc ccctcccaat gggcggtccc atataccata tatggggctt
     7081 cctaataccg cccatagcca ctcccccatt gacgtcaatg gtctctatat atggtctttc
     7141 ctattgacgt catatgggcg gtcctattga cgtatatggc gcctccccca ttgacgtcaa
     7201 ttacggtaaa tggcccgcct ggctcaatgc ccattgacgt caataggacc acccaccatt
     7261 gacgtcaatg ggatggctca ttgcccattc atatccgttc tcacgccccc tattgacgtc
     7321 aatgacggta aatggcccac ttggcagtac atcaatatct attaatagta acttggcaag
     7381 tacattacta ttggaagtac gccagggtac attggcagta ctcccattga cgtcaatggc
     7441 ggtaaatggc ccgcgatggc tgccaagtac atccccattg acgtcaatgg ggaggggcaa
     7501 tgacgcaaat gggcgttcca ttgacgtaaa tgggcggtag gcgtgcctaa tgggaggtct
     7561 atataagcaa tgctcgttta ggg
//
