LOCUS       sequence-201815-        6219 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 6219)
  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 6219)
  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-E9S7M7
FEATURES             Location/Qualifiers
     source          1..6219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     113..132
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     326..348
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     340..357
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     341..357
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      394..403
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3880..3900
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3910..3926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3910..3926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3923..3945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3934..3950
                     /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(3958..3964,3965..3982,3983..3988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4003..4024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4362..4950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4442..4461)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5121..5912,5913..5981))
                     /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     5744..5763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5982..6086)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6154..6172
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(6210..6219,1..13))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
       61 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
      121 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
      181 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
      241 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
      301 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgtg
      361 ctgtctcatc attttggcaa agaattcgcg gccgccacca tggggaacta ctacctgggc
      421 ttggacgtcg gaataggctc aatcgggtgg gccgtcatca acatcgagaa gaagcgcatt
      481 gaggatttta atgtccggat ttttaagtct ggcgagatac aggagaagaa ccggaatagt
      541 cgggcgagtc aacagtgtag aagatcccgc ggtctgcgac gactgtatcg ccggaagtca
      601 catcgcaagc tgaggctcaa gaattatctg tcaatcattg ggctgaccac tagcgagaag
      661 atcgattact attatgagac tgctgataat aacgtgatac aacttcgaaa taaaggactc
      721 tctgagaagc tcactccgga agagatcgcc gcctgtctga ttcacatctg caataatagg
      781 gggtacaagg acttctacga ggtgaatgtt gaggatattg aggatccaga cgagcgcaac
      841 gagtacaaag aggaacacga cagtatcgta ctgattagca acctcatgaa tgaaggcggg
      901 tactgtaccc ctgctgaaat gatctgcaac tgcagggagt ttgatgaacc aaacagtgtg
      961 tacagaaagt tccataactc cgccgctagt aagaatcatt atttgatcac gcgacatatg
     1021 ctcgtgaaag aggtcgacct tatcctggag aatcagagca agtattacgg tattctcgac
     1081 gacaagacca ttgctaaaat caaagatatt atctttgccc agcgggattt tgagatcggg
     1141 cccggcaaga acgagcggtt ccggcggttc acaggttatc tggatagtat tggtaaatgc
     1201 cagtttttca aggaccaaga acgcggttct cggttcacag tgattgcaga catttacgca
     1261 tttgtgaatg ttctgagcca gtacacgtac actaacaata ggggagagtc tgtattcgat
     1321 accagctttg ccaacgacct gatcaacagt gcactcaaaa acggatcaat ggataaacgc
     1381 gagctgaagg ctattgccaa gtcctaccat atagatatct ctgataagaa ttccgacaca
     1441 agcctgacta agtgcttcaa atatatcaaa gtcgtgaagc ctcttttcga aaagtatgga
     1501 tatgactggg ataaactgat tgagaattat accgacaccg acaataatgt gctcaacagg
     1561 atcgggattg tcctttctca ggcacagacc ccaaagcggc gacgcgagaa actcaaggct
     1621 ctgaatatag ggttggacga tggcctgatc aatgaactga ccaaactgaa actcagcggc
     1681 actgcgaacg tgtcctacaa gtatatgcag gggagcattg aggccttctg cgagggcgat
     1741 ctctatggaa aatatcaggc caaatttaac aaggagattc cggatattga tgaaaacgcc
     1801 aaacctcaaa aactgcctcc cttcaagaac gaggacgact gtgagttctt caaaaaccca
     1861 gtggtgttcc ggtctatcaa cgaaaccagg aagctcatca atgcgataat agacaagtac
     1921 ggatatcccg cggccgtgaa cattgagacg gcagacgagc tcaacaagac attcgaggac
     1981 cgggctattg acaccaagcg aaacaatgat aaccagaagg aaaatgatcg catcgttaaa
     2041 gaaatcatag aatgcataaa atgcgacgaa gttcacgcaa gacacctgat tgagaagtac
     2101 aagctgtggg aggctcagga aggcaaatgt ttgtactctg gggaaacgat caccaaggag
     2161 gatatgctta gggacaagga caaactcttc gaagtggacc acattgtccc ttatagtctc
     2221 atccttgaca acaccattaa taataaagct ctcgtttatg ccgaggaaaa tcaaaaaaaa
     2281 ggccaaagaa cacctctgat gtatatgaat gaggctcagg ccgccgacta ccgagttcga
     2341 gtgaacacca tgttcaaaag caaaaaatgc tctaaaaaga agtaccagta cctgatgctc
     2401 ccggacctga acgatcagga actcctcgga ggatggagat ctcggaactt gaacgacacg
     2461 cggtatattt gcaagtacct tgtgaactac ctgcgcaaaa acttgcgctt tgatcggagc
     2521 tacgaatcaa gtgacgaaga cgacctcaag atccgcgacc actatcgcgt gttccccgtg
     2581 aaatctcgct ttacgtctat gtttcggcgg tggtggctta acgagaaaac atggggccgc
     2641 tacgataaag ctgagctgaa gaagttgacc tacctcgacc acgccgcaga cgctatcatt
     2701 atcgcgaatt gtaggcccga atatgtagtg ctcgccggag agaagctcaa actcaataag
     2761 atgtatcacc aggcgggaaa aaggatcacc cccgagtacg aacaatccaa gaaggcctgc
     2821 attgacaatc tgtacaaact tttccgcatg gatagaagaa ctgctgaaaa gttgctcagc
     2881 ggacacggcc gcttgactcc tatcattcct aacctgtccg aggaggtcga caagcgcctg
     2941 tgggacaaga atatctatga acagttctgg aaagacgaca aggataagaa gagctgcgag
     3001 gaactttacc gggagaacgt cgcatcactg tacaaagggg atcccaagtt tgccagcagt
     3061 ttgagtatgc ccgtcattag cctgaagcct gaccataaat acagagggac tataacagga
     3121 gaggaagcca tccgagtcaa agaaattgat ggcaagctga taaagctgaa acgcaagagt
     3181 atcagcgaaa taaccgccga gtccatcaat agcatctata cagacgacaa aatcttgatt
     3241 gattcactga aaaccatatt cgaacaggct gattataaag atgttgggga ctatttgaag
     3301 aagaccaatc aacacttttt cactacctca agtggtaaga gagtcaataa agtaaccgtc
     3361 atcgagaagg ttccaagcag gtggctgaga aaggagattg atgacaataa tttctctctg
     3421 ttgaacgaca gctcttacta ctgcattgag ctgtacaagg attcaaaggg cgacaacaat
     3481 ctgcagggga tcgcaatgtc cgacattgtg catgaccgga agacaaagaa actttatctt
     3541 aagccagact ttaattaccc cgatgactac tacactcatg tcatgtatat tttccccggt
     3601 gactatctgc ggattaaatc tacgtcaaaa aaatccggag agcaactgaa gtttgagggg
     3661 tatttcatta gcgttaaaaa cgtcaatgag aactcattcc gatttatttc cgacaataag
     3721 ccgtgtgcaa aggataaacg cgtctcaatt acaaagaagg atatcgtgat caagctggct
     3781 gtggacctta tgggcaaggt acagggcgag aacaacggta agggaatttc ttgtggggag
     3841 ccacttagcc tgcttaagga gaagaatagc agggctgacc ccaagaagaa gaggaaggtg
     3901 aggtccatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3961 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4021 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4081 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4141 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgt cttccgcttc ctcgctcact
     4201 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4261 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4321 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4381 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4441 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4501 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4561 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4621 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4681 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4741 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4801 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4861 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4921 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4981 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5041 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5101 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5161 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5221 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5281 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5341 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5401 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5461 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5521 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5581 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5641 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5701 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5761 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5821 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5881 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5941 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6001 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6061 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6121 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     6181 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacac
//
