LOCUS       40924_2104        8097 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Human expression plasmid for SaCas9 KKH variant: 
            CAG-humanSaCas9(E782K/N968K/R1015H)-NLS-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8097)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Nguyen NT, Topkar VV, Zheng Z, 
            Joung JK
  TITLE     Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 
            by modifying PAM recognition.
  JOURNAL   Nat Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404.
  PUBMED    26524662
REFERENCE   2  (bases 1 to 8097)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8097)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2015 Nov 2. doi: 10.1038/nbt.3404."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8097
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     regulatory      1763..1772
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1775..4930
                     /codon_start=1
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4937..4957
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4964..5029
                     /codon_start=1
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5119..5138)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5184..5239
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5238..5257)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     primer_bind     complement(5600..5616)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5624..5640)
                     /label=lac operator
                     /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(5648..5678)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5693..5714)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5772..5968
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     polyA_signal    5974..6108
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6176..6193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6347..6935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7109..7966)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7967..8071)
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attctgcagt cgacggtacc
     1741 gcgggcccgg gatccaccgg tcgccaccat ggggaaacgg aactacatcc tggggcttga
     1801 cattgggata accagcgttg gctacggaat tattgattat gagacacgcg atgtgattga
     1861 cgccggggtt aggctgttca aagaggccaa cgttgaaaac aacgagggaa gacggagtaa
     1921 gcgcggagca agaagactca agcgcagacg gagacatcgg attcagaggg tgaaaaagct
     1981 gctcttcgat tacaatctcc tgaccgatca tagtgagctg agcggaatca acccctacga
     2041 ggcgcgagtg aaagggcttt cccagaagct gtccgaagag gagttctccg ccgcgttgct
     2101 gcacctggcc aaacggaggg gggttcacaa tgtaaacgaa gtggaggagg acacgggcaa
     2161 tgaacttagt acgaaagaac agatcagtag gaactctaag gctctcgaag agaaatacgt
     2221 cgctgagttg cagcttgaga gactgaaaaa agacggcgaa gtacgcggat ctattaatag
     2281 gttcaagact tcagattacg taaaggaagc caagcagctc ctgaaagtac agaaagcgta
     2341 ccatcagctc gatcagagct tcatcgatac ctacatagat ttgctggaga cacggaggac
     2401 atactacgag ggcccagggg aaggatctcc ttttgggtgg aaggacatca aggaatggta
     2461 cgagatgctt atgggacatt gtacatattt tccggaggag ctcaggagcg tcaagtacgc
     2521 ctacaatgcc gacctgtaca atgccctcaa tgacctcaat aacctcgtga ttaccaggga
     2581 cgagaacgag aagctggagt actatgaaaa gttccagatt atcgagaatg tgtttaagca
     2641 gaagaagaag ccgacactta agcagattgc aaaggaaatc ctcgtgaatg aggaagatat
     2701 caagggatac agagtgacaa gtacaggcaa gcccgagttc acaaatctga aggtgtacca
     2761 cgatattaag gacataaccg cacgaaagga gataatcgaa aacgctgagc tcctcgatca
     2821 gatcgcaaaa attcttacca tctaccagtc tagtgaggac attcaggagg aactgactaa
     2881 tctgaacagt gagctcaccc aagaggaaat tgagcagatt tcaaacctga aaggctacac
     2941 cgggacgcac aatctgagcc tcaaagcaat caacctcatt ctggatgaac tttggcacac
     3001 aaatgacaac caaattgcca tattcaaccg cctgaaactg gtgccaaaaa aagtggatct
     3061 gtcacagcaa aaggaaatcc ctacaacctt ggttgacgat tttattctgt cccccgttgt
     3121 caagcggagc ttcatccagt caatcaaggt gatcaatgcc atcattaaaa aatacggatt
     3181 gccaaacgat ataattatcg agcttgcacg agagaagaac tcaaaggacg cccagaagat
     3241 gattaacgaa atgcagaagc gcaaccgcca gacaaacgaa cgcatagagg aaattataag
     3301 aacaaccggc aaagagaatg ccaagtatct gatcgagaaa atcaagctgc acgacatgca
     3361 agaaggcaag tgcctgtact ctctggaagc tatcccactc gaagatctgc tgaataatcc
     3421 attcaattac gaggtggacc acatcatccc tagatccgta agctttgaca attccttcaa
     3481 taacaaagtt ctggttaaac aggaggaaaa ttctaaaaaa gggaaccgga ccccgttcca
     3541 gtacctgagc tccagtgaca gcaagattag ctacgagact tttaagaaac atattctgaa
     3601 tctggccaaa ggcaaaggca ggatcagcaa gaccaagaag gagtacctcc tcgaagaacg
     3661 cgacattaac agatttagtg tgcagaaaga tttcatcaac cgaaaccttg tcgatactcg
     3721 gtacgccacg agaggcctga tgaatctcct caggagctac ttccgcgtca ataatctgga
     3781 cgttaaagtc aagagcataa atgggggatt caccagcttt ctgaggagaa agtggaagtt
     3841 taagaaggaa cgaaacaaag gatacaagca ccatgctgag gatgctttga tcatcgctaa
     3901 cgcggacttt atctttaagg aatggaaaaa gctggataag gcaaagaaag tgatggaaaa
     3961 ccagatgttc gaggagaagc aggcagagtc aatgcctgag atcgagacag agcaggaata
     4021 caaggaaatt ttcatcaccc ctcatcagat taaacacata aaggacttca aagactataa
     4081 atactctcat agggtggaca aaaaacccaa tcgcaagctc attaatgaca ccctgtactc
     4141 aacacggaag gatgataaag gtaatacctt gattgtgaat aatcttaatg gattgtatga
     4201 caaagataac gacaagctca agaagctgat caacaagtct ccagagaagc tccttatgta
     4261 tcaccacgac ccacagactt atcagaaatt gaaactgatc atggagcaat acggggatga
     4321 gaagaaccca ctctacaaat attatgagga aacaggtaat tacctgacca agtactccaa
     4381 gaaggataac ggaccagtga tcaaaaagat aaagtactat ggcaacaaac ttaatgcgca
     4441 tttggacata actgacgatt accccaattc tcgaaacaag gttgtgaagc tctccctgaa
     4501 gccttataga tttgacgtgt acctggataa tggggtttat aaattcgtca ccgtgaaaaa
     4561 tctggacgtg atcaaaaagg agaactatta tgaagtaaac tcaaagtgct atgaggaggc
     4621 gaagaagctg aagaagatct ccaatcaggc cgagttcatc gcttccttct ataagaacga
     4681 tctcatcaag atcaatggag agctttatcg cgtcattggt gtgaacaatg acttgctgaa
     4741 caggatcgaa gtcaatatga tagacattac ctaccgggag tatctcgaaa acatgaatga
     4801 taaacggccg cctcacatca tcaagacaat cgcatctaaa actcagtcaa taaaaaagta
     4861 ctctaccgat atcctgggga atctctatga agtgaagtca aagaagcacc cacaaatcat
     4921 taaaaaaggt ggatccccca agaagaagag gaaagtctcg agcgactaca aagaccatga
     4981 cggtgattat aaagatcatg acatcgatta caaggatgac gatgacaagt aaagcggccg
     5041 cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc
     5101 tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc
     5161 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg
     5221 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca
     5281 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa
     5341 aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt
     5401 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt
     5461 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc
     5521 ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc
     5581 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     5641 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     5701 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     5761 gccagcggat ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     5821 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     5881 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     5941 cttttttgga ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt
     6001 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     6061 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat
     6121 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     6181 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6241 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6301 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6361 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6421 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6481 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6541 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6601 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6661 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6721 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6781 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6841 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6901 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6961 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     7021 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     7081 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     7141 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     7201 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7261 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7321 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7381 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7441 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7501 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7561 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7621 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7681 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7741 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7801 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7861 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7921 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7981 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     8041 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
