LOCUS       sequence-201867-        6015 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 6015)
  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 6015)
  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-G9QLF2
FEATURES             Location/Qualifiers
     source          1..6015
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     42..61
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     255..277
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     269..286
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     270..286
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(988..999)
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             3605..3625
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3635..3651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3635..3651)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3648..3670)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3659..3675
                     /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(3683..3689,3690..3707,3708..3713))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3728..3749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4087..4675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4167..4186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4846..5637,5638..5706))
                     /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     5469..5488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5707..5811)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5879..5897
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5935..5957)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
       61 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
      121 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
      181 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
      241 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgtgc tgtctcatca
      301 ttttggcaaa gaattcgcgg ccgccaccat gaattacaag atgggccttg acataggtat
      361 cgcgtccgtg gggtgggccg tcatcaacct tgacctgaaa agaatcgagg atcttggagt
      421 tagaattttc gacaaggcgg agcaccctca gaacggtgaa tctttggccc ttccccggag
      481 aattgcgcgc agtgctagga ggaggctgag acgacggaaa catcgccttg aacggattag
      541 acggctgctg gtgagcgaga atgtgcttac gaaggaagag atgaacttgc tgtttaagca
      601 aaaaaagcag atagacgtgt ggcagctgcg cgtggatgcc ctggaacgga agctgaacaa
      661 cgacgaactg gcacgcgtgc ttttgcacct ggctaagaga agggggttca agtctaacag
      721 aaagtcagaa agaaacagca aagaaagctc agaattcctg aagaacattg aggaaaacca
      781 gtctattctt gctcaatata ggagcgttgg tgaaatgatt gtaaaggatt ccaaatttgc
      841 ttaccacaag cgcaataagc tggacagcta ctcaaacatg attgcccggg acgacttgga
      901 gagggaaatt aaacttatct tcgagaaaca gagagaattt aacaaccccg tatgcacgga
      961 gcgactcgag gaaaagtatt tgaacatctg gagctcccag cgaccgttcg ccagcaagga
     1021 ggacatcgag aagaaggttg gattttgcac tttcgagcct aaggagaaac gggccccaaa
     1081 ggccacatac acattccagt cctttattgt ttgggaacac atcaacaaac ttaggctcgt
     1141 cagcccagac gaaaccaggg cgctgacaga aatcgagaga aacctgcttt acaagcaagc
     1201 attttctaag aataaaatga cgtattacga cattcgcaag ctcctcaatc tcagcgatga
     1261 tatccatttt aaggggttgc tgtacgaccc taaaagctca ctgaaacaga ttgagaacat
     1321 caggtttctg gagctggact cctatcacaa gattagaaaa tgcatcgaaa atgtgtacgg
     1381 gaaggacggc atcaggatgt ttaacgaaac cgacatcgat acgttcggat acgcacttac
     1441 catcttcaag gacgacgaag acatcgtggc ttatctgcag aatgagtaca tcaccaagaa
     1501 tggcaaacgc gtaagcaacc ttgctaacaa agtgtatgat aagagcctga tcgacgaact
     1561 gctgaatctc tcttttagta aatttgctca tctctctatg aaggcgatca ggaatatcct
     1621 gccttatatg gaacagggtg aaatctattc caaggcctgc gagttggccg gatataattt
     1681 cacaggccca aagaagaaag aaaaggcact gctgcttcca gtgataccaa acattgcgaa
     1741 tccggttgtg atgcgagctc tcacacagtc ccgcaaggtg gtcaatgcaa tcatcaagaa
     1801 gtatggttcc cccgtgtcca tccacatcga gcttgctcgg gacctgtctc actccttcga
     1861 tgagagaaaa aagattcaga aggatcagac cgagaaccgg aaaaaaaatg aaacagctat
     1921 caaacagctg atagaatatg aactgactaa gaacccaaca ggacttgata tcgttaagtt
     1981 taaactctgg tctgagcagc agggccgatg catgtacagc ctgaagccga tcgagttgga
     2041 gcgactgttg gagcctggat acgtcgaagt ggatcacatc ttgccgtact cacggtcact
     2101 cgatgatagc tacgctaata aggtccttgt actcactaaa gaaaacaggg agaaaggcaa
     2161 tcataccccg gtcgagtacc tgggactggg gtctgagcgg tggaaaaagt tcgagaagtt
     2221 cgtgcttgct aataagcagt tttctaaaaa gaaaaagcag aatctgttgc gcctgcgata
     2281 tgaagagaca gaggaaaaag aattcaagga gcggaacctc aacgacaccc gctacatttc
     2341 caagttcttc gccaacttta ttaaagaaca tctgaagttt gcggatggcg atgggggcca
     2401 aaaagtctac acaatcaacg gaaagatcac agctcatctg agatcccgct gggacttcaa
     2461 caaaaaccgc gaggagtctg atctgcatca cgcagtcgat gccgtcatcg tcgcatgcgc
     2521 cacgcagggc atgatcaaga agataacgga attttacaaa gcaagagagc agaacaaaga
     2581 gagcgctaag aagaaagaac ccatctttcc tcagccctgg ccccactttg ccgatgaact
     2641 caaagccaga ctgagcaagt tccctcagga gtctatcgag gctttcgcct tgggaaatta
     2701 tgaccggaaa aagctggaat ctctgcgtcc agtctttgtt agtaggatgc ctaagaggtc
     2761 agtgacgggt gcagcgcatc aggaaaccct gcgccggtgc gtcggtattg atgaacaatc
     2821 aggcaagatt cagacagccg taaaaaccaa gctcagcgat attaaactgg acaaagatgg
     2881 gcattttccc atgtaccaga aagagtctga ccctcggacc tacgaggcta tcaggcagag
     2941 gctcctggag cacaataatg accccaaaaa ggcctttcag gaacccttgt acaagccgaa
     3001 gaaaaatggg gaaccggggc ccgtcattcg gaccgtcaag atcatagaca ccaagaacaa
     3061 agtcgtgcat ctcgacggct caaaaactgt cgcctacaat agtaacatag tccgcacgga
     3121 tgtcttcgag aaggacggca agtactactg cgtcccagtt tacaccatgg atatcatgaa
     3181 gggcactctg cctaacaaag ctatcgaggc caacaagccg tactctgaat ggaaggaaat
     3241 gaccgaggag tacacctttc aattcagcct gttcccaaat gacctggtgc gcatcgtcct
     3301 gccccgcgaa aagacaatca agacctccac aaatgaggag atcatcatca aggatatttt
     3361 tgcctattat aaaaccatag actcagcaac cggaggcctc gagctgattt ctcacgacag
     3421 gaactttagc ctgcgcggcg tcgggagcaa aacgctcaaa agattcgaaa agtatcaagt
     3481 tgacgtgttg ggaaacatcc ataaggtgaa aggcgagaag cgggtcggtc tggccgctcc
     3541 caccaaccag aagaagggga aaaccgtcga ctcccttcag tcagtgagcg acagcagggc
     3601 tgaccccaag aagaagagga aggtgaggtc catggtcata gctgtttcct gtgtgaaatt
     3661 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     3721 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     3781 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     3841 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     3901 tgcgtcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3961 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4021 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4081 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4141 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4201 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4261 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4321 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4381 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4441 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4501 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4561 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4621 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4681 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4741 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4801 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4861 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4921 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4981 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5041 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5101 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5161 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5221 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5281 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5341 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5401 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5461 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5521 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5581 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5641 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5701 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5761 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5821 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     5881 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     5941 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     6001 agcccgtcag ggcgc
//
