LOCUS       sequence-201806-        6765 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 6765)
  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 6765)
  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-D4J3S7
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..111
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     305..327
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     319..336
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     320..336
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4405..4425
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4435..4451)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4435..4451)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4448..4470)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4459..4475
                     /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(4483..4489,4490..4507,4508..4513))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4528..4549
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4887..5475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4967..4986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5646..6437,6438..6506))
                     /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     6269..6288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6507..6611)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6679..6697
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6735..6757)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
       61 gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg
      121 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
      181 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
      241 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg
      301 ttttcccagt cacgacgttg taaaacgacg gccagtgtgc tgtctcatca ttttggcaaa
      361 gaattcgcgg ccgccaccat gaagcaagaa tactttttgg gactggacat gggcaccggc
      421 agcctcggct gggcagtaac agattccact taccaagtga tgagaaaaca cggaaaggcc
      481 ctttggggca cccgactctt tgagagtgca agtacagccg aagagaggcg aatgttccgg
      541 acagcaagga gacggttgga tcgacgaaac tggcgaatac aggtacttca ggaaatcttc
      601 agcgaggaaa taagtaaggt ggatcctggg ttcttcctga gaatgaagga gagtaagtac
      661 tacccagagg ataaaaggga cgcagaaggg aattgcccag agctgccata tgctctcttt
      721 gtggatgata attatactga taagaattat cataaagact acccaaccat ctaccatctg
      781 cgcaaaatgc ttatggaaac cactgagatt ccagatatca ggctcgtcta tttggttctt
      841 catcatatga tgaagcaccg gggacacttc ctgctgtctg gggatatctc tcagatcaag
      901 gagtttaagt ctacattcga acagctgatc cagaacatcc aggatgagga gcttgagtgg
      961 catattagct tggacgatgc cgccattcag tttgtggagc atgtactgaa agacaggaat
     1021 ctcacgagat ctaccaagaa gagccggttg ataaagcagc ttaacgcaaa gagcgcatgc
     1081 gaaaaagcta tccttaatct cctgtccggg ggcactgtca aactgtcaga catatttaat
     1141 aataaggagc tggacgagtc cgaaagacca aaagtcagct tcgccgactc tggttatgac
     1201 gattacatcg gcatagttga ggctgaactg gccgagcagt attatattat cgccagcgca
     1261 aaagccgtgt atgattggag cgtgctggtg gaaattctcg gcaactccgt gagtatttct
     1321 gaggcgaaga tcaaggtgta ccagaaacat caggctgact tgaagactct gaaaaagatt
     1381 gttaggcaat acatgactaa ggaagattac aaaagggtgt tcgtcgacac cgaagaaaaa
     1441 ctgaacaatt attcagccta tatcggaatg actaaaaaga atggaaagaa ggttgatctt
     1501 aagagtaaac agtgtaccca ggccgacttt tatgattttt tgaaaaagaa cgttatcaag
     1561 gtgatcgatc ataaagaaat cacacaggag atagaatccg aaatcgagaa ggaaaacttc
     1621 ctccccaagc aggtgaccaa ggataatggg gttatcccat accaggttca cgattatgag
     1681 ctcaagaaga tactggacaa tttgggtaca cgcatgccat ttatcaaaga aaacgctgaa
     1741 aagatccagc agctgttcga gtttcggatc ccctattacg ttggacctct gaaccgagtt
     1801 gacgacggca aggacggaaa atttacatgg tccgtgagga aatctgacgc caggatctat
     1861 ccctggaact tcactgaggt cattgatgtt gaggcctccg cggagaagtt tatacggagg
     1921 atgacaaaca agtgcacata tctcgtcggc gaagatgttc ttcccaaaga ttcacttgtg
     1981 tacagtaagt tcatggtgct caacgaactt aacaacttga gactgaacgg agagaagata
     2041 agcgtggagc tcaagcaaag gatctacgag gaactgtttt gcaagtatcg caaagtgacc
     2101 aggaaaaagc tggagcggta tctcgtgatc gaggggattg caaagaaggg cgtggagatc
     2161 actgggatcg atggggattt taaggcaagc cttaccgctt atcacgattt caaggagcgc
     2221 ctgacagacg tgcagctgtc ccagagagcc aaggaggcta tagtgcttaa cgtggtgctg
     2281 tttggggacg acaagaaact cctcaaacag cggttgtcaa agatgtaccc caatctcact
     2341 accggacagc tgaaaggcat ttgcagcctg tcctatcagg gatggggacg actttccaaa
     2401 acgtttctgg aggaaattac cgtaccagcc cccggtacgg gcgaagtttg gaacataatg
     2461 accgcacttt ggcagaccaa tgacaacctc atgcaactcc tgagtcgcaa ttacggtttt
     2521 acaaacgagg tggaggagtt caacactctg aagaaagaaa ccgacttgag ctacaaaaca
     2581 gtggatgagc tttacgttag ccctgccgtc aaacggcaaa tctggcagac cctcaaagta
     2641 gtgaaagaga tacagaaggt tatgggaaat gcaccgaagc gggtttttgt ggaaatggct
     2701 cgagagaaac aggagggcaa gaggtccgat tctagaaaaa agcagcttgt tgaactgtac
     2761 cgcgcttgta aaaacgagga gagagactgg atcactgaac tgaacgctca gagcgatcaa
     2821 cagcttagga gtgacaagct gttcctgtac tacattcaga agggtagatg catgtactcc
     2881 ggggaaacca ttcagctgga cgaattgtgg gataatacca aatatgatat tgaccatatc
     2941 tatcctcagt caaagacaat ggacgattcc ctgaataacc gggtcctcgt aaagaaaaat
     3001 tacaacgcca tcaagagcga tacctatcct ctgagccttg atatccagaa gaaaatgatg
     3061 tccttctgga aaatgcttca acaacagggc ttcattacga aggaaaagta tgtgcggctt
     3121 gtgcggtctg acgagctgag cgctgatgaa ttggccggct tcatagagag acaaattgtg
     3181 gaaaccagac agagtaccaa ggcggttgcg acgatcctta aggaggctct gcccgacaca
     3241 gagattgtgt acgtaaaggc tgggaatgtt tctaacttcc ggcagaccta tgagctgctg
     3301 aaggtgcgcg aaatgaacga tctccaccac gctaaggacg catacttgaa tatagtggtg
     3361 gggaacgcgt acttcgtgaa gtttacgaaa aacgctgcgt ggtttatccg caacaatcca
     3421 ggccgctctt ataacctgaa acggatgttc gagttcgata tcgagcggtc tggtgaaata
     3481 gcttggaaag ccggtaataa gggcagtatt gtcactgtta agaaggtgat gcaaaagaat
     3541 aatatccttg tgacaaggaa agcctatgag gtaaaaggcg ggctgttcga tcagcagatt
     3601 atgaaaaaag gcaagggaca ggtcccgatc aagggtaatg atgagcgact ggctgacatc
     3661 gagaagtacg ggggctataa taaggcggcc gggacttatt tcatgctggt gaagtctctg
     3721 gataagaaag gaaaggaaat tcgaactatc gagttcgtac cactgtatct gaagaaccaa
     3781 atcgaaatca atcatgagtc cgccattcag tacctggctc aggaaagagg actgaatagc
     3841 ccagaaattc tcctttccaa gatcaagatc gacactctgt ttaaggtcga cggattcaag
     3901 atgtggctct caggacggac agggaatcag ctgatcttta agggcgcaaa ccagctgatt
     3961 ctttcacacc aggaagctgc gatcctgaaa ggtgtggtga agtacgtgaa taggaagaac
     4021 gagaacaaag atgccaagct gtctgaacga gatgggatga ccgaggagaa actgcttcag
     4081 ctgtacgaca ccttcctgga caagctgtct aacacggttt atagtattcg actctccgca
     4141 cagatcaaga ctctgacaga gaagagggca aaattcatcg ggctgtccaa tgaagaccag
     4201 tgcatagttc tcaacgagat cttgcatatg tttcagtgtc agtccggcag cgccaatctt
     4261 aagctcatag ggggccccgg cagcgctggt atcctggtta tgaataacaa tatcaccgct
     4321 tgcaaacaga tttcagtgat caatcagtca cccaccggca tatatgaaaa ggaaatcgac
     4381 ctgatcaaac tgagcagggc tgaccccaag aagaagagga aggtgaggtc catggtcata
     4441 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4501 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4561 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4621 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4681 gctgcgctcg gtcgttcggc tgcgtcttcc gcttcctcgc tcactgactc gctgcgctcg
     4741 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4801 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5521 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5581 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6481 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6541 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6601 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6661 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     6721 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtc
//
