LOCUS       sequence-201814-        6732 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 6732)
  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 6732)
  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-E7MR72
FEATURES             Location/Qualifiers
     source          1..6732
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(4..26)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     126..145
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     339..361
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     353..370
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     354..370
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      407..416
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4406..4426
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4436..4452)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4436..4452)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4449..4471)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4460..4476
                     /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(4484..4490,4491..4508,4509..4514))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4529..4550
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4888..5476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4968..4987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5647..6438,6439..6507))
                     /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     6270..6289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6508..6612)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6680..6698
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
       61 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
      121 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
      181 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
      241 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
      301 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
      361 gacggccagt gtgctgtctc atcattttgg caaagaattc gcggccgcca ccatggaagg
      421 acaaatgaaa aacaacggca acaacttgca acagggcaat tattatcttg gcctggacgt
      481 gggcacttca tccgtaggct gggccgtgac ggacaccgac tacaatgttc tgaaattccg
      541 aggcaaaagt atgtggggtg cccgactgtt cgatgaggct agcactgcgg aggaacgccg
      601 gacacatagg ggcaacagac gcagactggc tagacggaag taccgcctgc tgttgctgga
      661 gcagctgttt gagaaggaga tccggaagat tgacgacaac ttttttgtgc ggctgcacga
      721 gtctaatctg tgggctgatg acaagagtaa gccatccaag ttcctcctct tcaacgatac
      781 aaactttact gataaggatt atcttaagaa gtatcctact atttatcatc tcagatccga
      841 cctgatccat aatagcacag agcatgatat tagactggtc tttctcgcac ttcaccacct
      901 tatcaaatac aggggccatt tcatctacga caactctgcc aatggggacg tgaagacgct
      961 tgacgaggcc gtgtccgact tcgaagagta cctgaatgaa aacgacatcg aattcaatat
     1021 cgagaataag aaggagttca ttaacgtgct tagcgataag catctgacca aaaaagaaaa
     1081 aaagatcagc cttaagaaac tttatggaga tattaccgac agtgagaaca ttaatatttc
     1141 agtactcata gagatgctgt ccggcagcag tatcagcctc agcaatctgt ttaaagatat
     1201 tgaatttgac ggcaagcaga acctgagctt ggattcagac attgaagaga cgcttaacga
     1261 cgtggtggac atcttggggg ataacattga ccttttgatc cacgctaaag aggtttatga
     1321 catcgcagtc ctcacctcaa gtctgggaaa acacaagtac ctgtgtgacg caaaggtgga
     1381 actcttcgaa aaaaataaga aagacctgat gatccttaaa aaatacatca aaaagaacca
     1441 cccggaggac tacaaaaaga tcttttcctc tccaaccgag aagaaaaact acgccgccta
     1501 ctcacagacc aattcaaaga acgtgtgttc tcaggaagaa ttctgcctct tcataaaacc
     1561 ctacatccgg gatatggtga agtccgagaa tgaagatgag gtaaggatcg cgaaggaggt
     1621 tgaagacaag agcttcttga ctaagcttaa gggcaccaac aattctgtgg taccctatca
     1681 gatccatgag cgcgaactga atcaaattct taaaaatatc gtcgcatacc ttccttttat
     1741 gaacgacgag caggaagaca tctcagttgt agacaaaatc aaattgattt tcaagttcaa
     1801 gatcccttac tacgtggggc ctctgaatac caaaagtact cggtcctggg tatacagatc
     1861 tgacgagaag atttacccct ggaactttag caacgtcatc gatctggata aaaccgcaca
     1921 tgagtttatg aaccgattga taggtcgatg tacttacacc aacgacccgg tgctgcctat
     1981 ggactccctc ttgtattcca aatacaatgt cttgaacgag atcaatccta tcaaggtcaa
     2041 cgggaaagcc attccagtag aggttaagca ggctatttat acagatttgt ttgaaaactc
     2101 taaaaaaaag gtgacacgaa aatcaattta catctacctg cttaagaatg gatatatcga
     2161 aaaggaagat attgtaagtg gcattgatat agaaattaaa tctaagctga agagccacca
     2221 cgactttacc caaattgtcc aggagaacaa gtgtacaccc gaggaaatcg aacgcataat
     2281 caagggtatt ctcgtgtatt ccgacgacaa gtctatgctg cgcagatggt tgaagaacaa
     2341 tattaagggg ctgtctgaga acgacgtcaa gtacttggcc aagctcaatt ataaggagtg
     2401 gggccggctt agtaagaccc tgctgacaga catctacacg atcaaccccg aggacgggga
     2461 ggcgtgttca atcctcgaca ttatgtggaa taccaatgcc acactcatgg aaatactcag
     2521 taacgagaaa taccagttta aacagaacat cgaaaactac aaagctgaga actacgacga
     2581 aaagcagaat ctgcacgagg aattggatga catgtacatc tcacccgccg cccggcggtc
     2641 tatctggcag gcgcttcgca ttgtcgatga aatcgtcgat atcaagaaga gtgcccccaa
     2701 gaagatcttt atcgagatgg caagggaaaa gaagagcgct atgaaaaaga aacgcactga
     2761 aagcaggaaa gatactctcc tcgagctcta caagtcttgt aagagtcagg ccgacggttt
     2821 ctacgatgag gagttgtttg aaaaacttag caacgagtca aatagccggc tgagacgcga
     2881 ccagctttac ctctactaca cccaaatggg tcgatcaatg tacacaggca aaagaataga
     2941 cttcgacaaa ctgatcaacg acaagaacac ttacgacata gatcatatat atcctcgaag
     3001 caaaataaaa gatgatagca tcactaaccg ggtgctcgtt gagaaggaca taaatgggga
     3061 gaaaaccgac atttatccca tttcagaaga cataaggcag aagatgcaac ctttttggaa
     3121 aattctcaaa gagaaagggt tgatcaacga agaaaagtac aaacggctga ccaggaacta
     3181 cgagcttact gacgaggagc tgagtagctt cgtcgcccga caacttgtgg aaacccagca
     3241 gagcacaaaa gcactggcca cattgctgaa gaaggaatac cccagtgcaa agatcgtcta
     3301 cagcaaggcc gggaatgtgt ctgaattcag gaacagaaaa gacaaggaac tgccaaagtt
     3361 ccgcgaaatc aacgacctcc accatgctaa ggacgcatac ttgaacatcg tggtgggcaa
     3421 tgtgtacgat actaagttta ccgagaaatt ttttaacaac atccggaacg aaaattatag
     3481 cctcaagagg gtgttcgatt tctctgtgcc aggggcctgg gacgcaaaag ggtccacatt
     3541 taatacgata aagaaatata tggctaagaa caatcccatt atcgcgtttg caccttatga
     3601 ggtcaaaggc gagctgttcg accagcaaat cgttcctaag ggaaagggcc aatttcccat
     3661 taagcaggga aaagacatcg agaagtacgg cgggtacaat aagctctcaa gcgctttcct
     3721 gttcgcagtg gagtataagg gaaagaaagc cagggagcgc tccctcgaaa cagtgtacat
     3781 caaagacgtg gagttgtacc ttcaggatcc cattaagtat tgtgaatccg tgctgggcct
     3841 taaggaacca cagatcatta agccaaaaat cctgatgggg tccctgttct ccatcaataa
     3901 caagaagctg gtggtcaccg ggcggagcgg caaacagtat gtgtgccacc acatctacca
     3961 gctcagcatt aatgacgaag actcacaata ccttaagaac atcgccaagt accttcagga
     4021 agagcccgac gggaatattg aacggcagaa catcctgaat attaccagcg tcaataacat
     4081 taagcttttc gatgttctgt gcactaaatt caacagcaac acatacgaaa ttattttgaa
     4141 ttcactcaaa aacgatgtca atgaggggcg cgagaagttc agcgagctcg acattctcga
     4201 acagtgtaac attctgctcc aactgctgaa agctttcaaa tgtaacaggg agtcctctaa
     4261 tctggagaag ctcaacaaca agaagcaggc aggggttatc gtaatcccac atctttttac
     4321 caagtgctcc gtatttaaag taatacacca gtccataaca ggcctgttcg aaaaggagat
     4381 ggatctgctg aaaagcaggg ctgaccccaa gaagaagagg aaggtgaggt ccatggtcat
     4441 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4501 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4561 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4621 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4681 cgctgcgctc ggtcgttcgg ctgcgtcttc cgcttcctcg ctcactgact cgctgcgctc
     4741 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4801 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4861 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4921 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4981 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5041 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5101 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5161 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5221 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5281 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5341 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5401 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5461 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5521 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5581 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5641 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5701 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5761 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5821 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaacct tatccgcctc
     5881 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5941 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6001 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6061 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6121 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6181 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6241 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6301 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6361 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6421 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6481 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6541 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6601 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     6661 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     6721 tgatgacggt ga
//
