LOCUS       sequence-201722-        5862 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 5862)
  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 5862)
  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-A7HP89
FEATURES             Location/Qualifiers
     source          1..5862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     94..113
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     307..329
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     321..338
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     322..338
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      375..384
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3504..3524
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3534..3550)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3534..3550)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3547..3569)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3558..3574
                     /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(3582..3588,3589..3606,3607..3612))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3627..3648
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3986..4574)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4066..4085)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4745..5536,5537..5605))
                     /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     5368..5387
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5606..5710)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5778..5796
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5834..5856)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
       61 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
      121 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
      181 gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
      241 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
      301 ggttttccca gtcacgacgt tgtaaaacga cggccagtgt gctgtctcat cattttggca
      361 aagaattcgc ggccgccacc atggaaagaa tctttggttt tgacattggt accacttcaa
      421 tcggctttag cgttatagac tatagctcaa ctcagtctgc tggcaatatc cagcggttgg
      481 gggtcaggat attccctgag gctcgagatc ctgatgggac gccactcaat cagcagcgcc
      541 ggcagaagag gatgatgagg aggcagctgc gacgacggcg gatccgacgc aaggctctca
      601 atgagactct ccatgaggca ggctttctcc cagcctacgg tagcgctgat tggcctgtgg
      661 tcatggctga tgagccctac gagctgcgga gacgcgggtt ggaagagggc ctctctgctt
      721 acgaattcgg tagagccatt taccatctgg cccagcacag gcacttcaag ggccgagagt
      781 tggaggaaag tgatacccca gacccagacg tggacgacga gaaagaggct gcgaatgaaa
      841 gagcagcgac tctcaaggcc ctgaaaaatg agcaaacaac tctcggggct tggttggccc
      901 gccgcccgcc cagcgatcgg aagcgcggga tccatgcaca taggaacgtc gtggccgaag
      961 aatttgagag gctgtgggag gtccaatcaa agtttcatcc cgccctgaag tcagaggaga
     1021 tgcgcgctcg aatcagcgac actattttcg ctcagcggcc agttttttgg agaaaaaaca
     1081 cccttggcga atgtagattt atgcccggag aacccctgtg tcctaagggc tcatggctca
     1141 gccagcagag gagaatgctc gagaaactga ataacctggc gatcgcaggc ggaaacgcta
     1201 gacctctgga tgccgaagag cgagacgcta tcctgagcaa acttcaacaa caggcaagca
     1261 tgtcttggcc cggggtgaga tctgctctga aggctctcta caaacaacga ggcgaaccag
     1321 gcgcagagaa aagtctgaag ttcaatttgg aactgggggg ggagtccaag ctcctgggta
     1381 acgcgctgga agctaaactg gcagacatgt tcgggccgga ttggccggcc catccccgca
     1441 agcaggagat acgccatgcc gttcacgaga ggctttgggc cgctgactac ggcgagactc
     1501 cagataagaa acgggttatt attctgagtg agaaggaccg aaaagcgcat agagaggcgg
     1561 ccgctaactc attcgtcgcc gactttggaa tcaccgggga gcaggcagcg cagctccagg
     1621 cgctgaagct gcccacaggt tgggagccat acagcatccc tgccttgaat ctgtttctgg
     1681 cggagctcga gaagggcgag agatttggcg ccctggttaa tggacccgac tgggagggtt
     1741 ggagaaggac caactttccc caccggaatc agccaacagg ggaaatcctg gataaacttc
     1801 ccagccccgc tagtaaggaa gaaagagagc ggatcagtca gctgagaaat ccaacagtgg
     1861 tccgaactca gaatgagctg cgaaaggttg tgaataattt gataggcctg tatggaaagc
     1921 ctgacaggat acgcattgag gtcggccggg acgtgggaaa atctaaaaga gagcgcgagg
     1981 aaatacagag tgggatccgg cggaacgaga aacagcgcaa aaaggctacc gaggacctca
     2041 taaagaatgg gattgcaaac ccaagcagag atgacgtgga aaaatggatt ttgtggaagg
     2101 aaggacaaga gagatgcccc tatacgggcg accagatcgg ctttaatgcc ctttttcggg
     2161 agggtcgata tgaggttgag cacatttggc ctagaagccg gtcctttgat aacagcccaa
     2221 gaaacaagac actgtgtaga aaagacgtga acatagagaa gggcaatagg atgccttttg
     2281 aagcctttgg acatgatgag gaccgctggt ctgccattca gattagactg cagggcatgg
     2341 tatctgctaa aggcggcacc ggaatgtcac ctgggaaagt caagaggttc ctggcgaaga
     2401 ctatgcccga ggattttgct gcacgccagt tgaatgacac cagatatgcc gcaaaacaaa
     2461 tccttgctca gttgaaacgc ctgtggcctg atatgggtcc agaggcacct gtcaaggtcg
     2521 aagccgtgac cggccaggtc accgcacagc tgcgcaagct ctggactctc aacaacattc
     2581 tcgctgatga tggcgagaag acaagggctg accatcggca ccacgccatc gatgccctga
     2641 ctgtggcctg tacacatcca ggcatgacta ataaactttc ccgatattgg cagctccggg
     2701 atgaccctcg agcagagaaa cctgccctga cacccccttg ggatacgata agggctgacg
     2761 ctgagaaggc tgtcagcgaa atcgtggtat cccaccgggt gagaaagaaa gtcagcggac
     2821 cacttcacaa ggagacgacc tatggcgaca ccggaaccga cataaaaacc aagagcggca
     2881 cctatcggca gtttgtcacc cgaaagaaaa tagaatctct ttccaagggg gagctggacg
     2941 agatcaggga cccgcggatc aaggagatag ttgccgccca cgtggctggc aggggcggcg
     3001 acccaaagaa ggcattccct ccatatccct gcgtgtcccc cggggggcct gaaatccgga
     3061 aagtccggct gacttccaag cagcagctca acctcatggc ccagacaggt aacggatacg
     3121 cagacctcgg cagcaaccac cacatcgcta tatatcggct gcctgatgga aaagccgatt
     3181 ttgaaattgt ctccctcttt gacgcctctc gcagacttgc ccagcgcaac cctatagttc
     3241 aaagaacacg cgctgacggc gcgagctttg tgatgagtct ggctgctggc gaggcaatta
     3301 tgatcccaga gggtagtaag aaggggatct ggattgtgca gggcgtgtgg gcatctggtc
     3361 aggtggtgct tgagcgcgac accgacgctg accactccac cactacccgg ccgatgccca
     3421 accctatctt gaaggacgac gccaagaagg tgtcaatcga tcccatcgga agagtccggc
     3481 cttctaatga tagcagggct gaccccaaga agaagaggaa ggtgaggtcc atggtcatag
     3541 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3601 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3661 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3721 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3781 ctgcgctcgg tcgttcggct gcgtcttccg cttcctcgct cactgactcg ctgcgctcgg
     3841 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     3901 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     3961 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4021 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4081 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4141 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4201 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4261 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4321 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4381 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4441 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4501 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4561 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     4621 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     4681 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     4741 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4801 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4861 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     4921 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4981 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5041 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5101 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5161 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5221 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5281 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5341 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5401 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5461 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5521 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     5581 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     5641 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5701 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     5761 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     5821 atgacggtga aaacctctga cacatgcagc tcccggagac gg
//
