LOCUS       sequence-201877-        6450 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 6450)
  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 6450)
  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-I0ZZR5
FEATURES             Location/Qualifiers
     source          1..6450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     271..293
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     285..302
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     286..302
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      339..348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4056..4076
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4086..4102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4086..4102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4099..4121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4110..4126
                     /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(4134..4140,4141..4158,4159..4164))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4179..4200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4538..5126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4618..4637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5297..6088,6089..6157))
                     /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     5920..5939
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6158..6262)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6330..6348
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6386..6408)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
       61 catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
      121 taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag
      181 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
      241 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
      301 gtgtgctgtc tcatcatttt ggcaaagaat tcgcggccgc caccatggaa aagaaaagaa
      361 aagtgacgtt gggattcgac ctgggcatag catctgtggg atgggccatt gtggattctg
      421 aaaccaacca ggtttacaaa ctgggttcca gactgttcga cgcccccgat accaatctgg
      481 agaggcggac ccagaggggc acccgcaggc tgctccgaag gaggaaatac agaaatcaga
      541 agttctataa tttggtcaaa aggacggagg tcttcggatt gtcatcccgg gaggccattg
      601 agaaccggtt tagagagttg tctataaagt atcctaatat catagagctg aaaacgaagg
      661 ccctctccca ggaggtgtgt cctgacgaaa ttgcatggat ccttcacgat tatctgaaaa
      721 atcgcgggta cttctacgac gagaaggaga ctaaggaaga cttcgatcag cagaccgtgg
      781 agagcatgcc atcttacaag ctgaatgagt tttacaagaa gtacggctac tttaaaggcg
      841 ccctgtccca acccaccgag tccgaaatga aagataacaa ggacctgaaa gaagcatttt
      901 tcttcgactt cagtaacaaa gagtggctca aagaaatcaa ctatttcttc aacgtccaga
      961 aaaacatcct gagcgagaca tttatcgagg agtttaagaa aatattttcc tttactcggg
     1021 atatctcaaa gggccctggg agcgacaaca tgccctctcc ctacggaatt ttcggcgaat
     1081 tcggagataa tggtcaagga gggcggtatg agcatatctg ggacaagaac atcggcaaat
     1141 gctccatttt caccaatgaa cagagagccc caaagtatct gccctccgca ctcattttca
     1201 actttctgaa tgagctggcc aatatccgcc tgtactccac tgacaagaag aatatccagc
     1261 cattgtggaa gctgtcctca gtagacaagt tgaatattct tctcaatttg tttaacctgc
     1321 ctattagtga gaagaaaaag aaacttacct ctacaaatat taatgacata gtgaagaaag
     1381 aatctattaa atctattatg atctccgtag aggacatcga tatgattaag gacgaatggg
     1441 ctgggaagga acccaatgtg tacggagtgg gtctgtctgg attgaacatc gaagagtccg
     1501 ccaaggaaaa caagttcaag ttccaggatc ttaagattct gaatgttttg atcaatctgc
     1561 tggacaacgt ggggatcaag ttcgaattta aagacagaaa cgatattatc aagaatctgg
     1621 agctgctcga taatctgtac ctcttcctga tctaccaaaa ggaatctaac aacaaagact
     1681 catctattga tctctttatt gctaaaaacg aatctcttaa catcgagaac ctgaaactga
     1741 aactgaagga gttcttgctg ggtgctggga acgagttcga aaatcataat tccaagacac
     1801 acagtttgag taaaaaagct atcgacgaga tcttgccaaa actactcgac aacaatgagg
     1861 ggtggaacct ggaagctatc aagaattacg atgaggaaat caaatcacag atagaggaca
     1921 attcttctct gatggccaag caggataaaa agtatctcaa cgacaatttt ttgaaagatg
     1981 ctatcctgcc cccaaatgta aaggtaactt ttcagcaggc catcctcatc ttcaataaaa
     2041 tcatacagaa gttttctaaa gacttcgaga ttgataaagt ggtgatagag cttgctcggg
     2101 agatgaccca ggaccaggaa aacgatgcgc ttaaaggcat agccaaggcg cagaaatcca
     2161 aaaagagcct ggtggaggag cggctggagg caaacaacat tgacaaatca gtcttcaacg
     2221 acaagtatga gaagctcatc tacaagatct ttctttggat tagccaggat ttcaaagacc
     2281 catatacagg cgctcagata tctgtgaacg agattgtcaa caacaaggtg gagattgatc
     2341 acatcatccc atattctttg tgcttcgacg actctagtgc aaataaggta ctcgtccata
     2401 agcagtccaa ccaggagaaa tctaattctc tcccttatga atacataaag caaggacata
     2461 gtggctggaa ctgggatgag ttcactaagt acgtaaagcg ggtttttgtt aacaacgtgg
     2521 acagtattct tagtaaaaaa gagaggttga aaaaatccga gaatctgctg actgcgtcct
     2581 acgacgggta tgacaagctg ggcttcctcg cacgcaactt gaacgacaca cgctacgcaa
     2641 cgattttgtt tcgagatcag ctgaacaact acgccgagca ccatttgatt gacaacaaga
     2701 agatgttcaa agtgatcgca atgaatgggg ctgtgacaag tttcatacga aagaatatgt
     2761 catacgacaa caaactgcgc ctgaaggaca ggtccgactt cagccaccac gcttacgacg
     2821 ccgcaatcat tgcactcttt agtaataaga ccaagacact gtacaacctg attgatccct
     2881 ccctgaatgg tatcatatcc aaaaggagcg aaggatactg ggtcattgaa gaccggtaca
     2941 ccggggaaat taaagaactg aaaaaggaag actggactag catcaaaaat aacgtccagg
     3001 ccaggaagat cgcaaaggag atcgaagagt accttattga cctggacgac gaagtgttct
     3061 tttctagaaa aacaaaaagg aagaccaatc gacagctgta taacgagaca atttacggaa
     3121 tcgcaaccaa gaccgacgaa gacggtatta caaactatta caaaaaggag aaattctcca
     3181 ttctggatga caaagatatc tatcttagac tcctccgcga aagggagaaa tttgtgataa
     3241 atcaaagcaa ccccgaagtg atcgaccaga taattgaaat cattgagagc tacggcaagg
     3301 agaataacat ccccagtagg gatgaggcca tcaacattaa gtataccaaa aacaagataa
     3361 actacaacct gtatctgaaa caatacatgc gatctctcac caagtctctg gaccagttca
     3421 gtgaggagtt tatcaaccag atgatcgcta acaagacatt tgttctctac aaccctacaa
     3481 agaacactac aagaaagata aagttcctga gattggtgaa tgacgtgaag atcaatgaca
     3541 ttaggaagaa ccaagtaatt aacaagttta atggcaagaa taatgaacca aaagcctttt
     3601 atgaaaatat caactctctt ggcgcaatcg tctttaaaaa ttctgcgaat aattttaaga
     3661 cgctctctat caatacacag atagctatct ttggagacaa gaattgggac atagaggatt
     3721 tcaagacata taatatggag aagatcgaaa agtataagga gatatatggc atcgataaaa
     3781 cgtacaactt ccacagtttc atttttcccg ggaccattct gttggataaa caaaataaag
     3841 aattttatta tatatcatcc attcagaccg tgcgagacat tattgagatc aagtttctca
     3901 acaagattga gtttaaagat gagaacaaga atcaggatac atccaaaacc ccaaagagat
     3961 tgatgtttgg tatcaaatct attatgaaca actatgagca agttgacatc agcccgttcg
     4021 gcattaataa gaaaattttt gaaagcaggg ctgaccccaa gaagaagagg aaggtgaggt
     4081 ccatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4141 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4201 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4261 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4321 ctcactgact cgctgcgctc ggtcgttcgg ctgcgtcttc cgcttcctcg ctcactgact
     4381 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4441 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4501 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4561 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4621 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4681 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4741 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4801 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4861 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4921 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4981 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5041 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5101 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5161 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5221 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5281 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5341 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5401 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5461 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5521 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5581 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5641 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5701 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5761 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5821 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5881 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5941 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6001 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6061 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6121 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6181 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6241 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     6301 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     6361 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     6421 cttgtctgta agcggatgcc gggagcagac
//
