LOCUS       sequence-201738-        6855 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 6855)
  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 6855)
  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-B5XLC1
FEATURES             Location/Qualifiers
     source          1..6855
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     75..94
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     288..310
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     302..319
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     303..319
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      356..365
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             4378..4386
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             4478..4498
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4508..4524)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4508..4524)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4521..4543)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4532..4548
                     /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(4556..4562,4563..4580,4581..4586))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4601..4622
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4960..5548)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5040..5059)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5719..6510,6511..6579))
                     /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     6342..6361
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6580..6684)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6752..6770
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6808..6830)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
       61 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
      121 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct
      181 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
      241 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
      301 ttgtaaaacg acggccagtg tgctgtctca tcattttggc aaagaattcg cggccgccac
      361 catggataag aaatattcta taggacttga catagggaca aatagtgtgg gctgggccgt
      421 gatcaccgac gattataaag tgccgtcaaa gaagttcaaa gtgctgggca ataccgaccg
      481 gcacagtata aagaaaaacc tgatcggggc cctgctgttt gactccgggg aaacagccga
      541 ggccactcgc cttaaaagaa cagctaggcg gcgatacacc aggcgcaaaa atagaatctg
      601 ctatctgcag gagatttttt caaatgagat cgccaaggtg gatgatagct tctttcaccg
      661 gttggaggag tccttcttgg tcgaggagga taaaaaacac gagcgacacc ctatctttgg
      721 caatatcgta gacgaagtcg catatcacga gaaatatcct acaatctacc acctgcggaa
      781 aaagttggcc gacagcaccg acaaagccga cctgaggctg atctatctgg cccttgccca
      841 tatgataaaa ttccgaggac atttcctgat cgaaggagat ctcaaccccg ataatagcga
      901 tgtggataag ctgttcattc agctcgtgca gacctacaat cagcttttcg aggaaaatcc
      961 catcaacgcc tccagggtcg acgccaaggc tatcctgagc gctaggcttt ctaagagtag
     1021 gcgactggag aaccttatcg ctcagctgcc cggagagaag cggaacggtc tgttcggcaa
     1081 cctgattgcc ctgtcactcg gcctgacacc caattttaag tccaacttcg acctggctga
     1141 ggatgccaaa ctccagctga gcaaggacac ttatgatgat gatctggaca acctgctggc
     1201 tcagatcggt gaccagtacg cagacctctt tctggccgcc aaaaacctca gcgacgctat
     1261 actcctgtca gacattctta gggtgaatac agaaataacc aaagcccctt tgtccgcatc
     1321 catgatcaaa agatatgatg agcaccacca ggatctgact ctccttaaag cgctggtccg
     1381 gcaacagctg ccagaaaagt ataaggaaat cttctttgat cagagcaaaa acgggtatgc
     1441 gggttacatc gacggggggg cctcacagga agagttttac aaattcatta agcctatcct
     1501 cgagaagatg gatggcactg aagagctcct ggcgaaactg aatcgcgaag accttctgcg
     1561 gaaacaacgg acatttgata atgggagcat ccctcaccag attcacctgg gggaactgca
     1621 tgccatactt aggcggcagg aggacttcta ccccttcctg aaggacaacc gggaaaaaat
     1681 cgaaaaaatt ctgacgttca gaataccgta ttacgtggga ccgctggcca ggggaaacag
     1741 caggtttgcc tggatgactc ggaagtccga ggaaacgatc accccctgga atttcgagga
     1801 ggtcgttgac aagggagcat ccgcgcagtc ttttatcgaa agaatgacga attttgataa
     1861 aaacctgcct aatgaaaaag ttctccctaa acattctctg ctctatgaat acttcaccgt
     1921 gtataatgag ctcaccaagg ttaaatatgt tactgagggc atgcgcaaac ccgcgttctt
     1981 gagcggcgag cagaaaaagg ccattgtcga ccttctgttt aagactaaca ggaaagtcac
     2041 agtcaagcag ctcaaagaag actatttcaa gaagattgag tgtttcgata gtgtggagat
     2101 ctcaggcgtc gaggacagat ttaacgcctc actgggaacc taccacgatc tcttgaagat
     2161 catcaaagac aaggatttcc tggacaatga ggagaacgaa gatatcctcg aggatattgt
     2221 gttgactctc accctttttg aggatcgcga aatgattgaa gagaggctta agacttatgc
     2281 ccacctcttt gatgataaag tgatgaaaca gctgaagagg cggagataca ctggttgggg
     2341 tcggctctcc cgcaaactca tcaacggaat cagggacaag caaagtggta aaaccatatt
     2401 ggattttctt aagagtgacg gctttgctaa ccgcaacttc atgcagctga ttcatgacga
     2461 cagtttgaca tttaaggagg acttgcagaa agctcaagtg agcgggcagg gggacagtct
     2521 gcatgaacat attgctaatt tggctggctc tccagcgatt aagaagggca ttctccagac
     2581 agtgaaggtg gttgacgaac tggtcaaggt catgggtcgg cacaagcctg agaacatcgt
     2641 tatagaaatg gctcgcgaaa accaaacaac ccagaaaggt cagaagaata gtagggagcg
     2701 catgaagcgc atcgaagagg gcatcaagga actcgggagc gatattctca aagagtaccc
     2761 cgtagaaacg acacagctgc agaatgaaaa actgtatctg tattacctcc agaacgggag
     2821 ggacatgtat gtcgaccaag agctggacat taaccgactg agcgactacg atgtcgacca
     2881 tattgtaccc cagtcattcc tcaaagacga cagtatcgat aacaaagtgc tgacaagatc
     2941 cgacaaaaat aggggaaaaa gtgacaatgt cccttccgaa gaggttgtca aaaagatgaa
     3001 aaattactgg aagcagcttc tcaacgcaaa gctgattaca cagcgcaagt tcgataacct
     3061 gaccaaagct gagcgaggtg gtttgtcaga acttgacaag gctggcttta tcaagagaca
     3121 gctcgtcgaa accagacaaa tcaccaagca cgtagctcag atcctggact cccggatgaa
     3181 tacaaagtac gacgagaacg acaaactgat tagagaggtg cgagtgataa ctctcaaatc
     3241 caaacttgtg agcgatttca ggaaggattt ccagttctac aaagtgcgag agataaataa
     3301 ttatcaccac gcgcacgatg cttacctcaa tgctgtggtg ggcacggccc ttattaagaa
     3361 gtatcccaag ctggaatccg aattcgttta tggtgattat aaggtgtacg acattagaaa
     3421 gatgattgca aagagcgagc aagaaatcgg gaaggccaca gctaagtatt tcttctattc
     3481 taacatcatg aattttttca aaacagaaat cacactcgct aatggtgaga ttcggaaaag
     3541 accacttatc gaaaccaatg gtgagacagg agagatcgtg tgggataagg gacgagactt
     3601 cgcaaccgtc aggaaggtac tctccatgcc ccaggtgaat atcgtcaaga aaactgaggt
     3661 gcagacaggt gggttctcaa aagaaagtat cctgccaaaa cggaatagcg acaaactcat
     3721 tgcccgcaag aaagactggg atccgaagaa gtacggaggc ttcgattccc caacagtggc
     3781 ctacagtgtc ctggttgtcg caaaagttga aaagggtaaa agtaagaaac tgaagtccgt
     3841 caaagaactt gtcggtatca cgattatgga acgcagctca ttcgaaaagg acccagtgga
     3901 tttcctggag gctaaggggt acaaagaggt acggaaagac ctgattatta agttgccaaa
     3961 atacagcctg ttcgagctgg aaaacgggag gaagcggatg ctcgcctctg ccggggagct
     4021 gcagaaggga aacgagctgg ccttgcccag caagtatgtg aacttcctgt acctggcttc
     4081 acactacgaa aagcttaagg gatcacccga ggataacgaa cagaagcagc tgtttgtcga
     4141 gcagcacaaa cactatctgg atgagatcat agaacagatc agcgagtttt ccaagcgagt
     4201 gatactcgcc gatgcaaacc tggacaaagt gctcagcgcg tataataaac accgggacaa
     4261 gccaatcaga gagcaagccg agaacattat tcacttgttt acactgacca acttgggtgc
     4321 tcctgccgct ttcaaatgct tcgatactac catcgggagg aacagatata aatcaattaa
     4381 ggaggtgttg gatgccaccc tcattcacca gagtatcacc ggattgtacg aaacccggat
     4441 tgacttgagc caactggggg gcgatagcag ggctgacccc aagaagaaga ggaaggtgag
     4501 gtccatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4561 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4621 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4681 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4741 cgctcactga ctcgctgcgc tcggtcgttc ggctgcgtct tccgcttcct cgctcactga
     4801 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     4861 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     4921 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     4981 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5041 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5101 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5161 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5221 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5281 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5341 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     5401 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     5461 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     5521 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     5581 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     5641 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     5701 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     5761 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     5821 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     5881 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     5941 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6001 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6061 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6121 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6181 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6241 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6301 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6361 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     6421 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     6481 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     6541 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     6601 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     6661 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     6721 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     6781 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     6841 agcttgtctg taagc
//
