LOCUS       sequence-201879-        5928 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 5928)
  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 5928)
  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-J6LE60
FEATURES             Location/Qualifiers
     source          1..5928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     83..102
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     296..318
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     311..327
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      364..373
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3559..3579
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3589..3605)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3589..3605)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3602..3624)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3613..3629
                     /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(3637..3643,3644..3661,3662..3667))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3682..3703
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4041..4629)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4121..4140)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4800..5591,5592..5660))
                     /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     5423..5442
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5661..5765)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5833..5851
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5889..5911)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
       61 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
      121 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
      181 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      241 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      301 tcacgacgtt gtaaaacgac ggccagtgtg ctgtctcatc attttggcaa agaattcgcg
      361 gccgccacca tgggaatccg cttcgccttt gatctcggca ccaacagcat cggctgggcc
      421 gtctggagga ctggccctgg cgtttttgga gaggacaccg cggctagcct ggacggttca
      481 ggggtcttga tctttaagga cggccgcaac cccaaggacg gccagtcact ggccactatg
      541 aggcgcgtcc cccggcagag ccgcaagcga agagatcggt ttgtgctccg gcgccgggac
      601 ctcctggccg ccctgcgcaa ggctggtctg ttcccagtgg acgtcgagga gggaaggcga
      661 ctcgcagcta cggacccgta tcaccttcgc gctaaagcgc tggacgagtc attgacacca
      721 cacgagatgg ggagagtgat ctttcacctg aaccagagac gcgggtttag gagtaataga
      781 aaagctgaca gacaagatag agagaagggc aaaatagctg agggctctaa acgcttggct
      841 gaaaccctcg ctgcaactaa ctgccggact ctgggggaat tcctgtggag cagacatcgc
      901 gggactccac ggacgcggag cccgacacgc attaggatgg agggagaggg agccaaagca
      961 ctctacgctt tctatccaac tcgggagatg gttagagctg aatttgagag attgtggaca
     1021 gctcaatctc gctttgcgcc tgatctgctc actccagagc ggcacgagga gattgcgggg
     1081 atcctgttta ggcagcggga tctggcccct cctaaaatcg ggtgctgcac ctttgagcct
     1141 agcgaaagga ggctcccacg cgcccttccc agcgtggagg ccaggggaat ctacgaacgg
     1201 ctggctcatc tgcggattac caccgggcct gtatccgacc ggggtttgac aaggcctgag
     1261 cgggatgtgc tcgccagcgc cctgctggca ggcaagtccc tgacatttaa ggcagtgagg
     1321 aagacactta agatcctccc ccacgctttg gtgaatttcg aggaagccgg ggagaaggga
     1381 ctcgacggcg ccctcactgc caaactgttg tccaagccgg atcattatgg agccgcttgg
     1441 cacggtctgt cattcgctga gaaggatacc ttcgttggga aactgcttga tgaagctgac
     1501 gaagagcggc tgattagacg cttggtcacc gaaaaccgcc tgagtgagga cgccgctaga
     1561 agatgtgcga gtatccccct ggcggatggg tacggccggt tgggtcggac agccaacact
     1621 gagattctgg cagctctggt tgaggagaca gatgagactg ggacagttgt aacatacgcc
     1681 gaagctgtac gcagggctgg cgagaggact ggccgcaact ggcatcactc tgatgaaagg
     1741 gacggagtca tcctggaccg gctgccttat tatggtgaaa ttcttcagcg gcacgttgtc
     1801 cccggcagtg gagagcccga agagaagaac gaggccgccc gctggggacg ccttgcaaac
     1861 cctactgtcc acatcggcct gaaccagctt cggaaagtcg tgaatagact gatcgccgct
     1921 catggaaggc ctgaccagat cgtggtagag ctggcccgcg agctgaaact gaaccgcgag
     1981 cagaaggagc gcttggatcg cgaaaataga aagaacaggg aggaaaacga acgcagaaca
     2041 gcaatcctgg ccgaacacgg acaacgcgat accgccgaaa ataagattag actgaggctg
     2101 ttcgaagaac aagcgcgggc caatgctggc atcgcactgt gcccctacac aggaagggcc
     2161 atcggaattg ccgagctgtt cacttccgag gtggagattg atcacattct gcccgtaagc
     2221 ctgaccctgg atgacagcct cgctaatcgc gttctttgtc gaagggaagc caatcgcgag
     2281 aagcgacggc agaccccatt ccaggcattc ggtgctaccc cagcatggaa cgacatcgtc
     2341 gcccgcgcag ctaagttgcc tcccaacaag cggtggagat tcgatccagc cgctctggag
     2401 aggttcgaaa gggaaggagg tttcctgggg cgccagctga acgaaacaaa gtatctgtcc
     2461 aggcttgcta aaatttattt gggaaagatc tgcgatcccg atcgcgtgta tgtgactccg
     2521 ggcactctca cgggcctcct cagggcccgg tggggattga attccatcct cagtgacagt
     2581 aactttaaaa accggtcaga tcatcgacat catgccgttg atgccgttgt gattggagtc
     2641 cttacgaggg gtatgatcca gagaattgca catgacgctg cccgcgccga ggatcaagat
     2701 ctggacagag tgttccgcga cgtaccagtg cccttcgaag actttagaga tcatgtgcga
     2761 gagagagtga gcaccatcac ggtggccgtg aagccagagc acggcaaagg cggcgctctc
     2821 catgaggata catcttacgg ccttgtacct gacaccgatc ccaacgcggc actgggcaat
     2881 ctggtcgtgc gcaaaccaat acggtccctg acagctggcg aagtcgacag ggttcgagat
     2941 cgggcgttga gggctcggct gggagccttg gctgccccat ttcgggacga gagtggaagg
     3001 gtgcgcgacg ctaaggggct cgcacaggcc ttggaagctt tcggggccga gaatggtatc
     3061 cggcgggtac ggattctgaa gcctgacgct agtgttgtga ctatcgccga tcgccgcacc
     3121 ggagtaccat atcgcgcagt cgctccaggg gagaatcatc acgtggacat tgtgcaaatg
     3181 cgggacggta gttggcgagg cttcgctgcc agtgtgtttg aggtaaatag gcccggatgg
     3241 cgcccagaat gggaggtgaa gaagctgggc ggcaaactcg tgatgcgcct gcacaagggg
     3301 gacatggttg agctgtccga caaagacggt cagcgcaggg tcaaggtcgt gcagcagatt
     3361 gaaatttcag ctaacagggt gagattgagc cctcataatg atggggggaa gctgcaagac
     3421 agacacgccg atgccgatga cccctttcgg tgggatctgg ccacaattcc actgcttaaa
     3481 gacagaggtt gtgtggccgt ccgcgtggat cccatcggtg tcgttaccct gaggagaagc
     3541 aacgtgagca gggctgaccc caagaagaag aggaaggtga ggtccatggt catagctgtt
     3601 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3661 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3721 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3781 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3841 ctcggtcgtt cggctgcgtc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3901 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     3961 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4021 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4081 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4141 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4201 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4261 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4321 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4381 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4441 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4501 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4561 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4621 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4681 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4741 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4801 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4861 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4921 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4981 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5041 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5101 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5161 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5221 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5281 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5341 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5401 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5461 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5521 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5581 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5641 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5701 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5761 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     5821 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     5881 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgt
//
