LOCUS       40924_365        8100 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Human expression vector for S .aureus Cas9: 
            CAG-humanSaCas9-NLS-3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8100)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 
            Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
  JOURNAL   Nature. 2015 Jun 22. doi: 10.1038/nature14592.
  PUBMED    26098369
REFERENCE   2  (bases 1 to 8100)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8100)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2015 Jun 22. doi: 10.1038/nature14592."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8100
                     /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..1673
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     primer_bind     1681..1700
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     regulatory      1766..1775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1778..4933
                     /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
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4940..4960
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4967..5032
                     /codon_start=1
                     /label=3xFLAG
                     /note="three tandem FLAG(R) epitope tags, followed by an 
                     enterokinase cleavage site"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5122..5141)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5187..5242
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5241..5260)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also
                     called rb-glob-pA-term-R"
     primer_bind     complement(5603..5619)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5627..5643)
                     /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(5651..5681)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5696..5717)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5775..5971
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     polyA_signal    5977..6111
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6179..6196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6350..6938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7112..7969)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7970..8074)
                     /label=AmpR promoter
     promoter        8100
                     /label=CAG
                     /note="CMV early enhancer fused to modified chicken 
                     beta-actin 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 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct
     1441 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
     1501 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg
     1561 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1621 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1681 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattctgc agtcgacggt
     1741 accgcgggcc cgggatccac cggtcgccac catggggaaa cggaactaca tcctggggct
     1801 tgacattggg ataaccagcg ttggctacgg aattattgat tatgagacac gcgatgtgat
     1861 tgacgccggg gttaggctgt tcaaagaggc caacgttgaa aacaacgagg gaagacggag
     1921 taagcgcgga gcaagaagac tcaagcgcag acggagacat cggattcaga gggtgaaaaa
     1981 gctgctcttc gattacaatc tcctgaccga tcatagtgag ctgagcggaa tcaaccccta
     2041 cgaggcgcga gtgaaagggc tttcccagaa gctgtccgaa gaggagttct ccgccgcgtt
     2101 gctgcacctg gccaaacgga ggggggttca caatgtaaac gaagtggagg aggacacggg
     2161 caatgaactt agtacgaaag aacagatcag taggaactct aaggctctcg aagagaaata
     2221 cgtcgctgag ttgcagcttg agagactgaa aaaagacggc gaagtacgcg gatctattaa
     2281 taggttcaag acttcagatt acgtaaagga agccaagcag ctcctgaaag tacagaaagc
     2341 gtaccatcag ctcgatcaga gcttcatcga tacctacata gatttgctgg agacacggag
     2401 gacatactac gagggcccag gggaaggatc tccttttggg tggaaggaca tcaaggaatg
     2461 gtacgagatg cttatgggac attgtacata ttttccggag gagctcagga gcgtcaagta
     2521 cgcctacaat gccgacctgt acaatgccct caatgacctc aataacctcg tgattaccag
     2581 ggacgagaac gagaagctgg agtactatga aaagttccag attatcgaga atgtgtttaa
     2641 gcagaagaag aagccgacac ttaagcagat tgcaaaggaa atcctcgtga atgaggaaga
     2701 tatcaaggga tacagagtga caagtacagg caagcccgag ttcacaaatc tgaaggtgta
     2761 ccacgatatt aaggacataa ccgcacgaaa ggagataatc gaaaacgctg agctcctcga
     2821 tcagatcgca aaaattctta ccatctacca gtctagtgag gacattcagg aggaactgac
     2881 taatctgaac agtgagctca cccaagagga aattgagcag atttcaaacc tgaaaggcta
     2941 caccgggacg cacaatctga gcctcaaagc aatcaacctc attctggatg aactttggca
     3001 cacaaatgac aaccaaattg ccatattcaa ccgcctgaaa ctggtgccaa aaaaagtgga
     3061 tctgtcacag caaaaggaaa tccctacaac cttggttgac gattttattc tgtcccccgt
     3121 tgtcaagcgg agcttcatcc agtcaatcaa ggtgatcaat gccatcatta aaaaatacgg
     3181 attgccaaac gatataatta tcgagcttgc acgagagaag aactcaaagg acgcccagaa
     3241 gatgattaac gaaatgcaga agcgcaaccg ccagacaaac gaacgcatag aggaaattat
     3301 aagaacaacc ggcaaagaga atgccaagta tctgatcgag aaaatcaagc tgcacgacat
     3361 gcaagaaggc aagtgcctgt actctctgga agctatccca ctcgaagatc tgctgaataa
     3421 tccattcaat tacgaggtgg accacatcat ccctagatcc gtaagctttg acaattcctt
     3481 caataacaaa gttctggtta aacaggagga aaattctaaa aaagggaacc ggaccccgtt
     3541 ccagtacctg agctccagtg acagcaagat tagctacgag acttttaaga aacatattct
     3601 gaatctggcc aaaggcaaag gcaggatcag caagaccaag aaggagtacc tcctcgaaga
     3661 acgcgacatt aacagattta gtgtgcagaa agatttcatc aaccgaaacc ttgtcgatac
     3721 tcggtacgcc acgagaggcc tgatgaatct cctcaggagc tacttccgcg tcaataatct
     3781 ggacgttaaa gtcaagagca taaatggggg attcaccagc tttctgagga gaaagtggaa
     3841 gtttaagaag gaacgaaaca aaggatacaa gcaccatgct gaggatgctt tgatcatcgc
     3901 taacgcggac tttatcttta aggaatggaa aaagctggat aaggcaaaga aagtgatgga
     3961 aaaccagatg ttcgaggaga agcaggcaga gtcaatgcct gagatcgaga cagagcagga
     4021 atacaaggaa attttcatca cccctcatca gattaaacac ataaaggact tcaaagacta
     4081 taaatactct catagggtgg acaaaaaacc caatcgcgag ctcattaatg acaccctgta
     4141 ctcaacacgg aaggatgata aaggtaatac cttgattgtg aataatctta atggattgta
     4201 tgacaaagat aacgacaagc tcaagaagct gatcaacaag tctccagaga agctccttat
     4261 gtatcaccac gacccacaga cttatcagaa attgaaactg atcatggagc aatacgggga
     4321 tgagaagaac ccactctaca aatattatga ggaaacaggt aattacctga ccaagtactc
     4381 caagaaggat aacggaccag tgatcaaaaa gataaagtac tatggcaaca aacttaatgc
     4441 gcatttggac ataactgacg attaccccaa ttctcgaaac aaggttgtga agctctccct
     4501 gaagccttat agatttgacg tgtacctgga taatggggtt tataaattcg tcaccgtgaa
     4561 aaatctggac gtgatcaaaa aggagaacta ttatgaagta aactcaaagt gctatgagga
     4621 ggcgaagaag ctgaagaaga tctccaatca ggccgagttc atcgcttcct tctataataa
     4681 cgatctcatc aagatcaatg gagagcttta tcgcgtcatt ggtgtgaaca atgacttgct
     4741 gaacaggatc gaagtcaata tgatagacat tacctaccgg gagtatctcg aaaacatgaa
     4801 tgataaacgg ccgcctcgca tcatcaagac aatcgcatct aaaactcagt caataaaaaa
     4861 gtactctacc gatatcctgg ggaatctcta tgaagtgaag tcaaagaagc acccacaaat
     4921 cattaaaaaa ggtggatccc ccaagaagaa gaggaaagtc tcgagcgact acaaagacca
     4981 tgacggtgat tataaagatc atgacatcga ttacaaggat gacgatgaca agtaaagcgg
     5041 ccgcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct
     5101 ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa
     5161 gccccttgag catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg
     5221 ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca
     5281 tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa
     5341 caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt
     5401 attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt
     5461 tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct
     5521 ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca
     5581 gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5641 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5701 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5761 cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     5821 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     5881 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     5941 aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat
     6001 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     6061 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt
     6121 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     6181 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     6241 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     6301 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     6361 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     6421 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     6481 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     6541 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     6601 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     6661 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6721 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6781 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6841 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6901 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6961 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     7021 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     7081 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     7141 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     7201 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     7261 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     7321 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     7381 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     7441 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     7501 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     7561 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     7621 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     7681 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7741 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7801 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7861 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7921 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7981 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     8041 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
