LOCUS       sequence-202487-        7586 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 7586)
  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 7586)
  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..7586
                     /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             159..179
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             204..3359
                     /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             3381..3401
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3402..3491
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             3504..3530
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     protein_bind    complement(3568..3592)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3637..3771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3661..3680)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3715..3734
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3884..3904)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3884..3902)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3923..3939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3923..3939)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3936..3958)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3947..3963
                     /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(3971..3977,3978..3995,3996..4001))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4016..4037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4154..4171)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4325..4913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4405..4424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5084..5875,5876..5944))
                     /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     5707..5726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5945..6049)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6075..6530)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6212..6233)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6424..6443
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(6604..7586,1..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 accatgggac gtacggcccc aaagaagaag cggaaggtcg
      181 gtatccacgg agtcccagca gccaagcgga actacatcct gggcctggac atcggcatca
      241 ccagcgtggg ctacggcatc atcgactacg agacacggga cgtgatcgat gccggcgtgc
      301 ggctgttcaa agaggccaac gtggaaaaca acgagggcag gcggagcaag agaggcgcca
      361 gaaggctgaa gcggcggagg cggcatagaa tccagagagt gaagaagctg ctgttcgact
      421 acaacctgct gaccgaccac agcgagctga gcggcatcaa cccctacgag gccagagtga
      481 agggcctgag ccagaagctg agcgaggaag agttctctgc cgccctgctg cacctggcca
      541 agagaagagg cgtgcacaac gtgaacgagg tggaagagga caccggcaac gagctgtcca
      601 ccaaagagca gatcagccgg aacagcaagg ccctggaaga gaaatacgtg gccgaactgc
      661 agctggaacg gctgaagaaa gacggcgaag tgcggggcag catcaacaga ttcaagacca
      721 gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca gaaggcctac caccagctgg
      781 accagagctt catcgacacc tacatcgacc tgctggaaac ccggcggacc tactatgagg
      841 gacctggcga gggcagcccc ttcggctgga aggacatcaa agaatggtac gagatgctga
      901 tgggccactg cacctacttc cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg
      961 acctgtacaa cgccctgaac gacctgaaca atctcgtgat caccagggac gagaacgaga
     1021 agctggaata ttacgagaag ttccagatca tcgagaacgt gttcaagcag aagaagaagc
     1081 ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga agaggatatt aagggctaca
     1141 gagtgaccag caccggcaag cccgagttca ccaacctgaa ggtgtaccac gacatcaagg
     1201 acattaccgc ccggaaagag attattgaga acgccgagct gctggatcag attgccaaga
     1261 tcctgaccat ctaccagagc agcgaggaca tccaggaaga actgaccaat ctgaactccg
     1321 agctgaccca ggaagagatc gagcagatct ctaatctgaa gggctatacc ggcacccaca
     1381 acctgagcct gaaggccatc aacctgatcc tggacgagct gtggcacacc aacgacaacc
     1441 agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga
     1501 aagagatccc caccaccctg gtggacgact tcatcctgag ccccgtcgtg aagagaagct
     1561 tcatccagag catcaaagtg atcaacgcca tcatcaagaa gtacggcctg cccaacgaca
     1621 tcattatcga gctggcccgc gagaagaact ccaaggacgc ccagaaaatg atcaacgaga
     1681 tgcagaagcg gaaccggcag accaacgagc ggatcgagga aatcatccgg accaccggca
     1741 aagagaacgc caagtacctg atcgagaaga tcaagctgca cgacatgcag gaaggcaagt
     1801 gcctgtacag cctggaagcc atccctctgg aagatctgct gaacaacccc ttcaactatg
     1861 aggtggacca catcatcccc agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc
     1921 tcgtgaagca ggaagaaaac agcaagaagg gcaaccggac cccattccag tacctgagca
     1981 gcagcgacag caagatcagc tacgaaacct tcaagaagca catcctgaat ctggccaagg
     2041 gcaagggcag aatcagcaag accaagaaag agtatctgct ggaagaacgg gacatcaaca
     2101 ggttctccgt gcagaaagac ttcatcaacc ggaacctggt ggataccaga tacgccacca
     2161 gaggcctgat gaacctgctg cggagctact tcagagtgaa caacctggac gtgaaagtga
     2221 agtccatcaa tggcggcttc accagctttc tgcggcggaa gtggaagttt aagaaagagc
     2281 ggaacaaggg gtacaagcac cacgccgagg acgccctgat cattgccaac gccgatttca
     2341 tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg
     2401 aggaaaagca ggccgagagc atgcccgaga tcgaaaccga gcaggagtac aaagagatct
     2461 tcatcacccc ccaccagatc aagcacatta aggacttcaa ggactacaag tacagccacc
     2521 gggtggacaa gaagcctaat agagagctga ttaacgacac cctgtactcc acccggaagg
     2581 acgacaaggg caacaccctg atcgtgaaca atctgaacgg cctgtacgac aaggacaatg
     2641 acaagctgaa aaagctgatc aacaagagcc ccgaaaagct gctgatgtac caccacgacc
     2701 cccagaccta ccagaaactg aagctgatta tggaacagta cggcgacgag aagaatcccc
     2761 tgtacaagta ctacgaggaa accgggaact acctgaccaa gtactccaaa aaggacaacg
     2821 gccccgtgat caagaagatt aagtattacg gcaacaaact gaacgcccat ctggacatca
     2881 ccgacgacta ccccaacagc agaaacaagg tcgtgaagct gtccctgaag ccctacagat
     2941 tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga
     3001 tcaaaaaaga aaactactac gaagtgaata gcaagtgcta tgaggaagct aagaagctga
     3061 agaagatcag caaccaggcc gagtttatcg cctccttcta caacaacgat ctgatcaaga
     3121 tcaacggcga gctgtataga gtgatcggcg tgaacaacga cctgctgaac cggatcgaag
     3181 tgaacatgat cgacatcacc taccgcgagt acctggaaaa catgaacgac aagaggcccc
     3241 ccaggatcat taagacaatc gcctccaaga cccagagcat taagaagtac agcacagaca
     3301 ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc tcagatcatc aaaaagggca
     3361 aaaggccggg caccggcggg cccaagaaga agaggaaggt atacccatac gatgttcctg
     3421 actatgcggg ctatccctat gacgtcccgg actatgcagg atcgtatcct tatgacgttc
     3481 cagattacgc tggatccgcc gctccggcag ctaagaaaaa gaaactggat ttcgaatccg
     3541 gataagcggc cgcctcgaga tatctagacc cagctttctt gtacaaagtg gttgatcctc
     3601 tcgagcctct agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt
     3661 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt
     3721 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac
     3781 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg
     3841 gccgcggcgc caatgcattg ggcccggtac ccagcttttg ttccctttag tgagggttaa
     3901 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     3961 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4021 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4081 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4141 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4201 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4261 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4321 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4381 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4441 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4501 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4561 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4621 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4681 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4741 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     4801 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4861 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4921 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4981 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5041 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5101 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5161 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5221 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5281 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5341 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5401 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5461 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5521 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5581 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5641 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5701 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5761 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5821 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5881 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5941 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6001 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6061 gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     6121 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     6181 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     6241 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     6301 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     6361 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     6421 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     6481 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc
     6541 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
     6601 gtcgaccata gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat
     6661 ctggacctgt gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat
     6721 ctggacccca gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac
     6781 ttggcactgt gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt
     6841 cttggccctg tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata
     6901 ttggctatat gccaggatca atatataggc aatatccaat atggccctat gccaatatgg
     6961 ctattggcca ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg
     7021 ttttcctatt gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc
     7081 ttcctaatac cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt
     7141 tcctattgac gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc
     7201 aattacggta aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca
     7261 ttgacgtcaa tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg
     7321 tcaatgacgg taaatggccc acttggcagt acatcaatat ctattaatag taacttggca
     7381 agtacattac tattggaagg acgccagggt acattggcag tactcccatt gacgtcaatg
     7441 gcggtaaatg gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc
     7501 aatgacgcaa atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt
     7561 ctatataagc aatgctcgtt taggga
//
