LOCUS       sequence-201872-        7245 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 7245)
  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 7245)
  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-H1DEI0
FEATURES             Location/Qualifiers
     source          1..7245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..108
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     302..324
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     316..333
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     317..333
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      370..379
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4882..4902
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4912..4928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4912..4928)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4925..4947)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4936..4952
                     /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(4960..4966,4967..4984,4985..4990))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5005..5026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5364..5952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5444..5463)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6123..6914,6915..6983))
                     /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     6746..6765
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6984..7088)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7156..7174
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7212..7234)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
       61 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
      121 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat
      181 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
      241 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
      301 tcccagtcac gacgttgtaa aacgacggcc agtgtgctgt ctcatcattt tggcaaagaa
      361 ttcgcggccg ccaccatgga aactacgctg ggtatcgacc ttgggaccaa tagtattgga
      421 ctggcactgg tcgaccaaga ggagcatcag attctgtact ccggagtaag aatcttcccc
      481 gaagggatca ataaagatac aatagggctg ggggagaagg aggagagccg gaacgcaaca
      541 aggcgcgcca aacggcaaat gaggcgacag tactttagaa agaagctgag aaaggccaag
      601 ttgctggaac tgctcatcgc ctacgatatg tgtcctctca agcccgagga cgtaaggaga
      661 tggaagaact gggataaaca gcagaaatcc actgtgagac agtttcctga cacacctgct
      721 tttagggaat ggttgaagca gaacccgtac gaactgagga agcaggccgt cacggaagat
      781 gtgaccagac cggagctcgg tcgcattctt taccagatga ttcaacgccg cggctttctg
      841 agttcacgca agggaaagga agaagggaaa atcttcacgg ggaaagatcg aatggtggga
      901 atcgatgaaa caaggaaaaa cctccagaag cagacgctgg gagcctacct ctatgatatt
      961 gctcccaaga atggagagaa atacagattc cgcactgaaa gggtcagagc acggtatacg
     1021 cttcgcgaca tgtacatccg cgagttcgaa atcatctggc aaagacaagc aggtcacctt
     1081 ggccttgccc acgagcaggc caccaggaag aagaatattt tcctggaggg gtctgccact
     1141 aatgttcgca actccaagtt gattacccat ctgcaggcaa aatacggtcg ggggcatgtc
     1201 ttgattgaag ataccagaat caccgtgacg tttcagctgc cccttaagga agttctcggg
     1261 ggcaaaattg aaatcgaaga agaacagctt aaattcaagt ctaatgaatc tgtgttgttc
     1321 tggcagcgtc cactccggag tcagaaatct cttctgtcta agtgtgtatt tgagggaagg
     1381 aatttttacg acccagttca tcagaagtgg attatcgccg gtccaacgcc cgcccctctg
     1441 agccacccag agtttgagga atttagggcg taccagttca tcaataacat tatctacgga
     1501 aagaacgagc atcttacagc aatccagcgc gaggctgtat tcgaattgat gtgtactgag
     1561 tccaaagact tcaattttga gaaaatcccg aagcacctga agctcttcga gaagtttaat
     1621 tttgacgaca ccacaaaggt gcctgcctgc accacaatct cacagcttcg gaaacttttc
     1681 ccacaccccg tgtgggagga gaagcgggag gagatctggc attgcttcta cttctacgat
     1741 gataacactc tcctctttga aaaactccag aaggactatg ctctccaaac caacgatctg
     1801 gagaaaatca agaagataag actgtccgaa tcctacggaa atgtcagcct gaaagccatt
     1861 cggaggatta atccatatct gaagaaagga tacgcttata gtaccgccgt cctgctcggg
     1921 ggtatccgga attcttttgg gaagagattt gaatatttca aagagtacga acctgagatt
     1981 gaaaaagctg tctgtaggat cctgaaggag aaaaatgcgg agggtgaagt gatccgcaag
     2041 atcaaagact atctggtgca caatagattt gggtttgcca aaaatgatcg ggctttccag
     2101 aaactgtacc accattccca agccatcacc actcaggcac agaaagagcg cttgcccgaa
     2161 accggcaatc tgcggaaccc catcgttcag cagggtctga acgagctgcg gaggaccgtt
     2221 aacaagctgc tggcgacctg tcgggagaag tacggcccaa gtttcaaatt tgaccacatc
     2281 cacgtggaaa tgggccggga gctgcggagt agcaaaaccg agcgagagaa gcagagcagg
     2341 cagattagag agaatgaaaa gaaaaacgag gccgccaaag tcaagctcgc tgagtacggt
     2401 ttgaaggcct accgagacaa catccagaaa tatcttctgt ataaagagat cgaggagaag
     2461 ggtggcaccg tctgttgccc ttacaccggt aaaactctga atatctccca cactttgggc
     2521 tcagataaca gtgtgcagat agaacatatt attccttata gcattagcct ggatgatagt
     2581 ctcgctaaca agaccctgtg cgacgccacc tttaatagag agaagggcga actcacccct
     2641 tacgattttt accagaagga ccctagtccc gagaaatggg gggcgtctag ctgggaggag
     2701 attgaagacc gggctttcag gctcctgcca tatgctaaag ctcagaggtt tattaggcgg
     2761 aagccccagg agtcaaacga atttatctcc cggcagctga atgacacccg gtacatctca
     2821 aaaaaggcag ttgagtatct tagtgccatc tgcagcgatg taaaggcttt tcctggccaa
     2881 ctgacagccg aactgagaca cctgtggggt ctgaacaaca tcctgcagag cgcacctgat
     2941 attaccttcc cactgcctgt gtctgccacc gagaaccacc gcgagtatta tgttatcacc
     3001 aacgagcaaa atgaggtgat tcgactgttc ccaaaacagg gagaaactcc aaggacggag
     3061 aaaggggagc ttctcctgac aggagaggtg gagcggaaag tgtttcggtg taaggggatg
     3121 caggagtttc agacagatgt gtccgatggc aagtattgga gacggatcaa gctctctagc
     3181 agcgtcacct ggtcccccct gttcgcccct aaaccaatca gcgctgatgg gcagattgta
     3241 ttgaagggtc gcattgagaa aggcgtcttt gtatgtaatc agctgaaaca gaagctcaaa
     3301 acaggcttgc ctgacggatc ctactggatt tccctccctg tgatcagcca aaccttcaag
     3361 gagggtgaga gcgtaaataa ttccaaactg actagccagc aggtgcagtt gtttggacgc
     3421 gtgagggaag gaattttccg atgtcacaac tatcagtgcc cggcaagtgg tgctgatggc
     3481 aacttctggt gtactttgga tactgacaca gcccaacctg ccttcacgcc aatcaaaaac
     3541 gcacctccag gggtcggcgg gggccagatc atccttaccg gcgatgttga cgataaggga
     3601 atatttcatg ccgacgatga cctgcattat gagctgcctg ccagcctccc caaagggaaa
     3661 tactacggga tcttcactgt cgagtcctgt gaccctacac ttatcccaat cgaactgtca
     3721 gccccaaaaa cttccaaagg cgagaatctc attgaaggca acatctgggt ggacgagcat
     3781 actggagagg tcaggtttga cccaaaaaaa aatcgagagg atcagcgcca ccatgccatc
     3841 gacgctattg ttatagctct ctctagtcag agccttttcc agagactgag cacttataat
     3901 gccaggagag agaacaaaaa aaggggactt gactccaccg agcattttcc aagcccgtgg
     3961 cctggattcg ctcaggacgt gagacagagt gttgtgcccc tcttggtttc ctataaacag
     4021 aatccgaaga ctctgtgcaa gatttcaaag accctctata aggatgggaa gaaaattcac
     4081 agctgcggta acgccgtgag gggccagctc cataaggaaa ctgtgtacgg tcagaggacc
     4141 gcgcccggcg caaccgagaa atcttaccat attcgaaaag atatcaggga actgaagact
     4201 agtaagcaca tcgggaaggt tgtcgacata accatccggc aaatgctgct gaagcatctg
     4261 caggagaatt atcatattga tatcactcaa gaatttaaca tcccaagcaa cgcctttttc
     4321 aaggagggag tttacaggat cttcctccct aataagcacg gcgaaccagt gcccatcaag
     4381 aaaatccgca tgaaagaaga gctgggaaac gctgagagac tgaaggataa tataaaccag
     4441 tacgtcaatc ccaggaataa tcaccatgtc atgatctacc aggacgccga tggcaacttg
     4501 aaagaagaaa ttgtcagctt ctggagcgtg atcgagcgcc agaaccaagg ccagcccatt
     4561 tatcagctcc cccgcgaagg gaggaatatc gtgagtattc tccagattaa cgacactttt
     4621 cttatcggac tgaaggagga ggagcctgag gtgtacagaa atgatctgtc aaccctgagc
     4681 aaacatctgt atagagtgca gaaactgtcc ggcatgtatt atacgttccg gcaccacctg
     4741 gcctctacac ttaataatga aagagaggaa ttcaggattc aaagtcttga ggcctggaag
     4801 agagccaacc ctgtcaaggt gcagattgac gagattggcc ggattacctt tctgaatggt
     4861 ccgttgtgta gcagggctga ccccaagaag aagaggaagg tgaggtccat ggtcatagct
     4921 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4981 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     5041 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     5101 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5161 gcgctcggtc gttcggctgc gtcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5221 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     5281 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     5341 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     5401 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     5461 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     5521 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     5581 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     5641 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     5701 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     5761 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     5821 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     5881 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5941 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6001 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6061 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6121 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6181 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6241 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     6301 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     6361 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     6421 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     6481 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     6541 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     6601 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     6661 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     6721 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     6781 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     6841 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     6901 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     6961 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7021 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7081 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     7141 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     7201 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcaca
//
