LOCUS       sequence-201733-        5946 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 5946)
  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 5946)
  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-B1UZL4
FEATURES             Location/Qualifiers
     source          1..5946
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..93
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     287..309
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     301..318
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     302..318
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     RBS             645..653
                     /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)"
     RBS             1481..1489
                     /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             3568..3588
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3598..3614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3598..3614)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3611..3633)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3622..3638
                     /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(3646..3652,3653..3670,3671..3676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3691..3712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4050..4638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4130..4149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4809..5600,5601..5669))
                     /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     5432..5451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5670..5774)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5842..5860
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5898..5920)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
       61 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
      121 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg
      181 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa
      241 gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt
      301 tgtaaaacga cggccagtgt gctgtctcat cattttggca aagaattcgc ggccgccacc
      361 atgaacaaag acatcaacta cgccctgggg ctggatatcg gcatcacctc cgtcggctgg
      421 gcagttataa acctggattt gaaccgaatc gaagatctgg gcgtgagaat ttttaacgca
      481 gccgagaatc tcaaagatgg ttcatcattg gctctcccga ggcggcttgc acgcggacga
      541 agaaggctcc tgcgcaggaa agcataccga gtcgaaagag tgcggaagct gattcttaaa
      601 aacaacatac tcaccaagga ggaattggag ggtttgttca aaaataagga ggtgatcgac
      661 gtctgggaag cccgcgtgaa aggtctggat tacaaactgt ctagagaaga gtgggccaag
      721 attctcatca atttttgcaa gaggcgcggt tttaagagca acagaaaaaa tgaagccaag
      781 gataaggaag caggacaaat attgtccagc atatcttaca acatcgaaaa gatgaaggaa
      841 accaacagta ggacgatcgg cgagtatatc tataacgaag tgaagaattc cgaggattca
      901 tacgccccac ttcgcaataa gttcggcgaa tataaaatgt gtgtgagccg agatgtgatc
      961 cgggaagaga ttcataccct ttttgagaaa cagcgcgagc tgggcaataa ttttgccagc
     1021 gatgaaatcg aggataagta tttcgagatt ttcaattctc agcggccttt tagcaacttc
     1081 gaggacctgg aaaagctcgt gggtttttgc actttcgagc gaaagaaaca taaacgagca
     1141 ccaaaacact gcatcagcag cgaagagttc acactttatg aaggcatcaa caagctgtca
     1201 attattaagg acggtgagaa gagaaagctc tcagaggagg aacgaactct tgtcgttaac
     1261 gaggcattca ataaaaagga aattaaatat actactctgc ggaagctcct tggcctctcc
     1321 gaggaggagc tgttttctac actgacatat tccatcgaca aagacatttc caaaactgaa
     1381 aacaccaagt ttgtctcatt gaaaggatac catgagatta aaagggccat tgaaaagggc
     1441 gtgagtaaaa aggcgtggca ggatatcgag aataatagaa acctccttaa tgatatcgcc
     1501 tacgtgttga cactggggaa aacagatgat gagattgaga agcagttgag gctgaggaac
     1561 gtcccagagg agcttttcga atcacttctt gatatgtctt ttaataagtt taacaatctt
     1621 tctatctacg ccttggagaa aatactccct tttatgaaag aggggtatca gtacaatgaa
     1681 gcatgcgaaa aggccggcta caacttcaag gcgatctatg agggcgtcag aaccaagaag
     1741 ctccccgtaa tcgaaataga cgaaatagtt aatcccgtgg ttaacagggc tctggctcag
     1801 acaagaaaag tgatcaactc agtgatcgac aagtacggat ctcctgtgcg gattaacatc
     1861 gagctggcca gggatctggc aaagaatttc aaggacagaa aactcatcga gaaagaacag
     1921 aaagaaaata gggcaaacct ggacaagatt cgcgagaacc tccgggaatt gatggacaag
     1981 gagccaacag tcgtcgaggt gctgaagtat aggctctggg agcagcaaag gggggaatgc
     2041 gcctacaccc aatctcaaat acctctggaa agactcttta gtccaggata ctgcgagatc
     2101 gatcatatta ttccctttag ccgctccttc gatgattcac tgagcaacaa aatcctggtg
     2161 ttgggctcag aaaaccagag gaaaggtaac cgcactcctt acgaatattt tggtgaagac
     2221 tctgagagat ggaattcctt cgagatctgg gtgaaagggt cctatctgaa ctataagaaa
     2281 aaggccaatc ttttgaaaaa aaaattttcc gaagaagaac agcgcgactg gaaagcccgc
     2341 aaccttcagg acactaagta catctgcaga tacatcagca actttgtgaa taaccggctc
     2401 gaattcaaag aaagcgagaa taaacagaag gtgatcaccg tgaatggtcg agccactgct
     2461 tatctgagga gtcgctgggg cctcaacaag gtgagagagg atggcgataa gcatcacgcc
     2521 ctcgatgctg ccgtggtcgg ggtgacaacg caggggatgg tgcagaaaat cagcaagtat
     2581 agtaaagcac acgaattgta tacagtgaga aaaaatgatg aattcgtgga tgtcgagaca
     2641 ggcgagtgtg tagatgtcga cgaatataaa gaaataagaa aggacattct gcctcgacca
     2701 tggagaggat ttagtgagga actgaaactt aggctcagtg acaaccccct ggaggagctg
     2761 aaaaagagcc ccatagagac gtacaatgag gagttcatta aggagaacgt gaaacccatt
     2821 tttgtatccc gcgtaccatt ccgaaaaatc tcaggtaaac tgtttaaaga aaccatttat
     2881 tccgaaagag ctttcaaaga tggatacttc gtgagcaaaa aatctctcgt cgacctcaag
     2941 cggggcgacc tggacaattt ctacaattat gaatgtgaca aaaaacttta cgatgctatt
     3001 tcagaacgga tgaaagagtt caactacaat ggcaagaagg cctttgagag tccattccgg
     3061 aagcccacca agtctggcaa ggagggacct gtcgtgagat ccgtgaaaat taaaagtgcc
     3121 gtgccgttta aagacggcat tcccatgaat gagggtctgg tcgctaaaga ggggatggtg
     3181 agaatagata tttacgagaa ggagggcaag tacttcgtgg tgcccgttta ccggtaccag
     3241 ctggccaagg gaatcgtccc taaacgcgcg gcgatagcaa agaaagatga gctgaagtgg
     3301 cctatcatgg accagactta taacttcaag ttctcaattt acaaaaacga ccttattgag
     3361 gtgaagtatg ataagaaagg tgtctatttc ggctactatg acgggttcga ccgcaactca
     3421 gcatccttga caattgaaaa gcacgataat tctgagcgct accgaagcat cgggctgaag
     3481 gttggcgttc tggaaataaa taaatatgag gtcgatgtac tcggcaacta ccataaagta
     3541 aagcttggcg gtaatagcag ggctgacccc aagaagaaga ggaaggtgag gtccatggtc
     3601 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3661 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3721 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3781 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     3841 ctcgctgcgc tcggtcgttc ggctgcgtct tccgcttcct cgctcactga ctcgctgcgc
     3901 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3961 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4021 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4081 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4141 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4201 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4261 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4321 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4381 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4441 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4501 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4561 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4621 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4681 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4741 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4801 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4861 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4921 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4981 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5041 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5101 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5161 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5221 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5281 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5341 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5401 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5461 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5521 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5581 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5641 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5701 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5761 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     5821 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     5881 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     5941 taagcg
//
