LOCUS       sequence-201714-        5907 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 5907)
  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 5907)
  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-E3CY56
FEATURES             Location/Qualifiers
     source          1..5907
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     83..102
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     296..318
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     310..327
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     311..327
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             1985..1993
                     /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             3538..3558
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3568..3584)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3568..3584)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3581..3603)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3592..3608
                     /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(3616..3622,3623..3640,3641..3646))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3661..3682
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4020..4608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4100..4119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4779..5570,5571..5639))
                     /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     5402..5421
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5640..5744)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5812..5830
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5868..5890)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
       61 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
      121 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
      181 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
      241 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
      301 tcacgacgtt gtaaaacgac ggccagtgtg ctgtctcatc attttggcaa agaattcgcg
      361 gccgccacca tgattggaga gcacgtccgc ggcggctgcc tgtttgatga ccattggact
      421 cccaactggg gtgctttccg actgcccaat actgtaagaa cattcaccaa agccgagaac
      481 ccaaaagacg gctcctccct ggccgaaccg cgacggcagg cacgcggcct gagaagacgg
      541 ttgaggcgca agacccagag gctcgaagac ctcagacgcc ttcttgctaa ggagggcgtg
      601 ctgtcacttt cagaccttga gactttgttc cgggaaaccc ccgccaaaga tccatatcag
      661 ttgcgagctg agggtttgga caggcccctg agcttcccag agtgggtcag agttctgtat
      721 catattacca aacaccgggg atttcagagc aaccgaagga atccagtgga ggacggccag
      781 gaaagatcca gacaggaaga ggaggggaag ctcctgtccg gggtcgggga gaacgagcgg
      841 ctgctgcggg agggtgggta tcgcaccgca ggagagatgc tggcccggga tcctaagttc
      901 caggatcaca gaaggaatcg cgccggtgat tactctcaca ccctgtctcg ctccctgctg
      961 ctggaggaag caagaagact gttccagtca cagagaacgc tcggaaatcc ccacgcatca
     1021 tctaatctgg aggaagcatt cctgcatctg gtggcatttc agaatccatt cgcaagcggc
     1081 gaagacatcc ggaataaggc cggtcactgt tccctggagc cagatcaaat ccgcgcccct
     1141 cgaagaagcg cttccgcaga aacctttatg ctgctgcaga agaccggtaa cttgagactg
     1201 attcatcgca gaacagggga ggaaaggccc cttaccgata aagagcggga gcagattcat
     1261 ttgctggcat ggaaacagga gaaagttact cacaaaactc tgaggcggca tctcgagatt
     1321 cccgaggaat ggctgtttac cgggttgccc tatcaccggt caggagacaa ggccgaggag
     1381 aaattgtttg ttcacctggc tggaatccac gagattcgca aggctttgga caagggacct
     1441 gaccccgcag tttgggatac gcttcggagt cggagggacc tcctggatag cattgccgat
     1501 accttgacat tttataagaa tgaagacgag attctgcctc gactggaatc actgggactc
     1561 tcacccgaaa acgcacgggc cctggccccc ctgtcttttt ctggaaccgc acacctgtca
     1621 ttgtctgccc tggggaagct gctgccccac ctggaagaag gaaagagcta cacccaggct
     1681 cgagccgacg cgggatatgc ggccccacct ccggacagac accccaagtt gcctcccctg
     1741 gaagaagcgg actggcggaa ccccgtggtg tttcgcgccc ttacccagac caggaaagtg
     1801 gtcaatgcac ttgtgaggcg gtacggtccc ccttggtgca tacatctgga gactgcacgc
     1861 gaattgtccc agcctgcaaa agttaggcgg cgcattgaga ctgagcagca ggctaacgag
     1921 aagaagaagc agcaggctga gagggagttc ctggacattg tggggaccgc gccagggccc
     1981 ggggacctcc ttaaaatgcg attgtggcgg gagcaaggtg gcttctgtcc atactgtgag
     2041 gaatatctga accctacccg gctggctgag cccggctacg cggagatgga ccatatcctg
     2101 ccttattcca ggagtttgga taatggatgg cataacagag tgctcgtgca tgggaaggac
     2161 aatcgcgata aaggcaatcg aaccccattc gaagcgttcg gcggagatac tgcacggtgg
     2221 gacagactgg tggcctgggt ccaggcaagt cacctgtcag ccccaaagaa gagaaacctg
     2281 ctgagggagg actttgggga ggaagcggaa agggaactta aagatagaaa tttgaccgat
     2341 actcgcttta ttacaaagac cgcagccacc cttctgagag acaggctgac atttcaccca
     2401 gaagctccaa aagaccctgt catgacactt aacggaagac tcactgcctt tctcagaaag
     2461 caatgggggc tgcacaagaa ccggaagaac ggcgacctgc atcatgcact ggatgctgcg
     2521 gttctggctg tggcgtcccg aagctttgtg tatcgcctga gttcccacaa cgccgcgtgg
     2581 ggcgagttgc ctagggggcg agaagctgaa aatggcttca gcctcccata cccggctttc
     2641 aggtctgagg tgttggcgag actgtgcccg acccgggaag agattctctt gcggctggat
     2701 cagggcggag tgggttacga cgaagcattc cggaatggat tgcggccagt gtttgtgtct
     2761 cgagctcctt ctagacggct gcggggcaag gctcacatgg aaaccttgcg aagccctaag
     2821 tggaaggatc accctgaagg gccaagaact gcgtctagaa ttcctctcaa agacctgaat
     2881 cttgaaaaac tcgagagaat ggtgggcaag gatcgagata gaaaactcta tgaagccctt
     2941 cgagagagac tggcggcctt cggaggcaac ggaaaaaaag cttttgtagc tccttttcgc
     3001 aagccttgcc ggtccgggga gggccccctc gtgcgcagct tgagaatatt cgactcagga
     3061 tattccggtg tggaactgag agatggcggt gaagtatacg ccgtcgctga tcacgaatct
     3121 atggtgcgag tggatgtgta cgcaaaaaaa aataggttct acctcgtacc agtgtacgtg
     3181 gcggatgtgg cccggggcat tgttaagaac agagccattg tggcccataa aagtgaggag
     3241 gaatgggacc tggtggacgg cagtttcgac tttagattca gtctttttcc cggggatctt
     3301 gtagaaatcg agaagaagga cggcgcatat ctgggatatt ataagtcttg ccaccgggga
     3361 gatggacgcc tgctgttgga cagacacgat cgcatgcccc gagaaagcga ctgtggcacc
     3421 ttttatgtct ctacccgaaa ggacgtcttg tctatgagca agtatcaggt ggaccccctc
     3481 ggcgaaataa gactggtggg aagcgaaaaa ccacccttcg tgctgagcag ggctgacccc
     3541 aagaagaaga ggaaggtgag gtccatggtc atagctgttt cctgtgtgaa attgttatcc
     3601 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3661 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3721 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3781 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcgtct
     3841 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     3901 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     3961 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4021 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4081 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4141 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4201 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4261 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4321 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4381 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4441 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4501 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4561 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4621 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4681 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     4741 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     4801 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4861 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     4921 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     4981 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5041 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5101 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5161 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5221 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5281 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5341 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5401 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5461 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5521 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5581 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5641 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     5701 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5761 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     5821 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     5881 cagctcccgg agacggtcac agcttgt
//
