LOCUS       sequence-201720-        5997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 coding gene template with optimized sequence for human codon 
            usage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5997)
  AUTHORS   Cho N, Seo HN, Ryu T, Kwon E, Huh S, Noh J, Yeom H, Hwang B, Ha H, 
            Lee JH, Kwon S, Bang D
  TITLE     High-throughput construction of multiple cas9 gene variants via 
            assembly of high-depth tiled and sequence-verified oligonucleotides.
  JOURNAL   Nucleic Acids Res. 2018 May 18;46(9):e55. doi: 10.1093/nar/gky112.
  PUBMED    29529247
REFERENCE   2  (bases 1 to 5997)
  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: "Nucleic 
            Acids Res."; date: "2018-05-18"; volume: "46(9):e55. doi"; pages: " 
            10.1093/nar/gky112"
COMMENT     Sequence Label: pUC19-A1IQ68
FEATURES             Location/Qualifiers
     source          1..5997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     51..70
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     264..286
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     278..295
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     279..295
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      332..341
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             338..3583
                     /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="MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     DNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             3596..3616
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3626..3642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3626..3642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3639..3661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3650..3666
                     /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(3674..3680,3681..3698,3699..3704))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3719..3740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4078..4666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4158..4177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4837..5628,5629..5697))
                     /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     5460..5479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5698..5802)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5870..5888
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5926..5948)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
       61 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
      121 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
      181 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      241 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgtgct
      301 gtctcatcat tttggcaaag aattcgcggc cgccaccatg gctgctttca agccaaaccc
      361 cattaactac attctgggac tggacattgg tattgcatca gtaggatggg ctatggtcga
      421 gatcgatgaa gatgagaatc ctatatgtct gatcgacctg ggggtgcgag tgttcgagcg
      481 ggcagaggtg cccaagacag gtgatagtct ggcgatggca cgcagactgg ccaggtccgt
      541 cagacgcttg acccgccgac gggcccatag gctgttgagg gccaggcgac tgctgaaaag
      601 ggaaggagtg cttcaagcag ccgatttcga cgagaacggg cttataaaga gccttccaaa
      661 tactccatgg cagttgcgcg ccgcagcact tgaccgcaaa ctgacaccgc tggagtggag
      721 cgccgttctg ttgcatctca ttaaacacag aggttatctc tcccagagaa aaaacgaggg
      781 agagacagct gacaaggaat tgggtgccct gcttaaggga gttgccgata acgctcatgc
      841 cctgcaaacc ggagacttca gaactcccgc cgagctggct ctgaacaagt tcgagaagga
      901 atcagggcac atacgcaatc agagggggga ctacagtcac acatttagca gaaaggacct
      961 gcaggctgaa cttatccttc tgtttgagaa acagaaggag ttcggtaatc ctcatgtgag
     1021 cgggggcctg aaggagggca ttgaaacatt gctgatgacc cagcgccccg ctctgtccgg
     1081 agacgctgtg caaaagatgc tgggccactg tactttcgag ccagccgaac caaaagcggc
     1141 caagaacact tacactgctg agcgcttcat ctggctgact aaacttaata acctcaggat
     1201 tcttgagcag gggtccgagc gaccactgac tgatacagaa cgggctacac tgatggatga
     1261 gccataccgc aaaagcaaac tgacttacgc tcaggcacgc aaactgctgg gcctggagga
     1321 taccgccttc ttcaaggggc ttaggtatgg taaggataat gccgaggcaa gcaccctcat
     1381 ggaaatgaag gcataccatg cgatatcccg ggcactcgaa aaggaggggc tcaaagacaa
     1441 gaaatcaccg ctcaaccttt ccccagaact tcaagacgaa atcggaacag ccttttcact
     1501 gttcaagaca gatgaagaca ttaccggaag actcaaagat cgcatccagc ctgagatcct
     1561 ggaagcactg ctcaaacaca tctcctttga caaattcgtg caaatatctc tgaaggctct
     1621 gcggcggatt gtgcctctga tggagcaggg caaaaggtat gacgaagctt gcgcggagat
     1681 ttacggcgac cattacggga agaagaacac ggaggaaaag atttacctcc ctccgatccc
     1741 ggccgatgaa attagaaatc cagtggttct gagggcactc agtcaagccc gcaaggtgat
     1801 caatggagta gtgaggcggt atggcagtcc tgcacgcata cacattgaaa ccgctagaga
     1861 ggtcggcaag tcattcaagg acagaaagga aatcgagaaa cgacaggagg agaaccggaa
     1921 agacagggaa aaagctgccg caaagttcag agaatacttt cccaattttg tgggagaacc
     1981 taaatctaaa gatattctca aactcagact ctacgagcag cagcatggta agtgcctcta
     2041 ttcagggaaa gaaatcaatc tgggacggct caacgaaaag ggttatgtag aaatcgatca
     2101 cgctctcccc ttcagtagga catgggacga cagtttcaac aataaggttc ttgtactggg
     2161 ctctgagaac cagaataaag gcaaccagac tccatatgag tatttcaacg gaaaagacaa
     2221 ttcaagagag tggcaggaat ttaaggcgcg cgttgaaacc tcccggtttc caaggagcaa
     2281 aaagcagcga attctgctgc agaagtttga tgaagacgga ttcaaggaaa ggaacttgaa
     2341 cgacacgagg tatgtaaatc gattcctgtg ccagtttgta gcggatagaa tgcgactcac
     2401 tggcaaaggg aaaaaaaggg tgtttgcctc caatggacag ataactaacc tgctccgagg
     2461 gttctggggt ctgcgcaaag tgcgcgctga gaacgatcga caccacgcac tggacgccgt
     2521 ggtggtggcc tgtagtaccg tcgctatgca acagaagatc acccggtttg tgagatacaa
     2581 agagatgaac gcattcgatg gcaagactat tgacaaggaa acgggtgagg tcctccacca
     2641 aaaaacgcat ttcccccaac cttgggaatt tttcgctcag gaggtaatga tccgcgtgtt
     2701 cggcaaaccg gatggaaaac cagaattcga agaggcggac acccccgaaa agctcaggac
     2761 actcctcgcc gagaaacttt cctcacgccc cgaggcagta catgagtacg tcacccccct
     2821 cttcgtgtcc agagcaccca acagaaagat gtctggacag ggacacatgg aaaccgtgaa
     2881 atcagctaaa cgccttgatg aaggtgtgag tgtgcttcgc gtacccctga ctcagcttaa
     2941 gctgaaagat ttggagaaga tggtcaacag ggagcgcgaa cctaagctgt acgaagccct
     3001 gaaggcaaga ctcgaagcac acaaggacga tcccgccaag gcctttgctg agcctttcta
     3061 caaatacgat aaggcgggga atagaactca acaggtgaag gcggtgcggg tggaacaggt
     3121 ccagaaaacc ggtgtctggg taagaaacca taatggcatc gctgacaacg caactatggt
     3181 gcgggtcgac gtgttcgaga aaggcgataa gtattatttg gtgcctatct attcatggca
     3241 ggtggccaaa ggtatattgc cagaccgggc tgtcgtacag ggcaaagacg aggaagactg
     3301 gcagctgata gacgacagct tcaatttcaa gttttctctc cacccaaacg atcttgtgga
     3361 agtgatcact aaaaaagccc gaatgttcgg gtacttcgct agttgtcacc gcggcacagg
     3421 gaacataaat atccgcatcc acgatttgga tcacaaaatc ggcaagaacg ggatcctgga
     3481 gggcatcggc gtaaaaacag cactgagctt tcagaagtac cagatagatg aactggggaa
     3541 ggaaattaga ccctgccgct tgaagaaacg gcctcccgtt cgcagcaggg ctgaccccaa
     3601 gaagaagagg aaggtgaggt ccatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3661 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     3721 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     3781 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3841 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcgtcttc
     3901 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3961 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4021 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4081 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4141 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4201 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4261 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4321 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4381 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4441 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4501 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4561 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4621 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4681 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4741 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4801 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4861 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4921 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4981 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5041 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5101 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5161 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5221 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5281 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5341 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5401 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5461 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5521 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5581 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5641 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5701 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5761 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5821 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     5881 cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     5941 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccg
//
