LOCUS       sequence-201715-        6294 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 6294)
  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 6294)
  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-E6KTN8
FEATURES             Location/Qualifiers
     source          1..6294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     99..118
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     312..334
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     326..343
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     327..343
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3941..3961
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3971..3987)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3971..3987)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3984..4006)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3995..4011
                     /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(4019..4025,4026..4043,4044..4049))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4064..4085
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4423..5011)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4503..4522)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5182..5973,5974..6042))
                     /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     5805..5824
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6043..6147)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6215..6233
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6271..6293)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
       61 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
      121 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
      181 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
      241 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
      301 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgtgctgt ctcatcattt
      361 tggcaaagaa ttcgcggccg ccaccatgct gcactgcatt gcagtcattc gggtacctcc
      421 tagtgaagag cccggcttct tcgaaaccca tgcagatagt tgcgccttgt gtcaccacgg
      481 atgcatgacc tatgcggcca atgacaaagc tatcagatac agagtaggga tagacgtcgg
      541 gcttcggtct atcgggttct gcgcagtaga ggtggacgac gaggaccatc ctatcagaat
      601 actgaattca gtcgtccacg ttcacgacgc cgggactggg ggcccaggcg aaactgaaag
      661 ccttcggaag cggtctggcg ttgctgcacg cgccaggcgg cgcggaagag ccgagaagca
      721 acgcctcaag aagttggacg tgcttttgga agagctggga tggggtgtgt ctagtaatga
      781 actgttggac tcacacgctc catggcacat cagaaagcgg ttggtttccg agtatatcga
      841 ggacgaaacc gaacgaaggc aatgtctttc cgttgccatg gcccacatcg ctaggcaccg
      901 cggctggcgg aatagctttt ctaaggttga caccctgctc ctcgagcagg cccctagtga
      961 tcggatgcaa ggtctgaaag aaagggttga ggacaggacc gggctccagt ttagtgaaga
     1021 ggtgacacag ggagagctgg tggctaccct gctggaacat gacggagacg tcacgattcg
     1081 cggatttgtc cgcaagggag gtaaagccac caaagtgcac ggtgtcctcg aaggaaagta
     1141 catgcagagc gatcttgttg ctgagctcag acagatctgc cgcacccagc gcgtctccga
     1201 gactactttt gaaaaactgg ttctttctat tttccattcc aaagagccag cgccctctgc
     1261 cgcacgacag agagagaggg taggacttga cgagctgcaa ctcgcgctcg atccagctgc
     1321 aaaacaacca cgagccgaaa gggcccatcc agcctttcag aaattcaagg tagtggccac
     1381 cctggcaaac atgaggatta gggagcagag cgccggggaa cgaagcctga cttcagaaga
     1441 gctgaatcgg gtggctcgct acctcctcaa tcacaccgaa tcagagagcc ccacgtggga
     1501 tgatgttgcc cggaaactgg aggtgccacg gcaccgcctg cgaggttcat ccagagccag
     1561 cctggagaca ggtgggggac tgacctatcc gcccgtggac gacactactg tgagggtgat
     1621 gtccgcagag gtggactggc tggccgactg gtgggattgt gctaatgatg aatcaagggg
     1681 ccacatgatc gacgctatct ctaacgggtg cggatcagag cctgacgacg tcgaagatga
     1741 agaggtgaac gagttgatca gctcagcaac tgctgaggac atgcttaaac tggagctgct
     1801 cgccaaaaag ctgccgtctg gtcgggtggc gtacagtctg aagaccttgc gggaggtcac
     1861 cgcagccatt ctggaaacag gcgacgatct ttcccaagca attaccagac tgtatggcgt
     1921 tgatcccggg tgggttccta cacccgcccc aatcgaagcg ccggtcggga acccatccgt
     1981 cgacagagtt ttgaagcagg ttgctagatg gctgaagttt gcttccaaga gatggggtgt
     2041 cccccagaca gttaatatcg agcataccag agaagggctc aagtcagcgt ccctcttgga
     2101 ggaggagagg gagagatggg agcgcttcga ggcccggcgc gaaatcagac aaaaggaaat
     2161 gtacaagaga ctgggcatta gcggcccatt tagacggtcc gaccaagtga ggtatgagat
     2221 actggacctg caagactgcg catgtctgta ttgcggcaac gaaatcaact ttcaaacctt
     2281 tgaggtggat catataatcc cgcgagtgga cgcctctagc gattcccgcc gcaccaacct
     2341 ggcagcggtg tgtcacagtt gtaacagtgc caagggtggg ttggcatttg ggcagtgggt
     2401 gaagaggggc gactgtccaa gcggcgtctc actggaaaac gctattaaac gcgtcagatc
     2461 ttggtccaaa gacagactgg gcctgactga gaaggctatg ggcaaaagga aatccgaggt
     2521 aatcagcagg ctgaagaccg aaatgccata tgaagagttc gacggacgca gtatggagag
     2581 cgtcgcttgg atggctattg aactcaagaa acggatcgag ggttatttca actccgatcg
     2641 gcccgaaggc tgtgccgccg ttcaggtgaa cgcttatagc ggtagattga cagcctgtgc
     2701 tcggagggct gcccatgtgg ataagcgcgt acgcctgatt cggctgaagg gcgacgacgg
     2761 ccatcataaa aacagattcg accggagaaa ccacgcaatg gatgcactcg ttattgcgtt
     2821 gatgacgccc gccatcgcgc ggaccattgc cgtgcgggaa gaccgccggg aggctcagca
     2881 gctgacacgg gcatttgaat cctggaagaa tttcctgggc tctgaagaac gcatgcagga
     2941 taggtgggaa tcctggatcg gagatgttga gtacgcgtgt gaccgactga atgagctgat
     3001 cgatgccgat aagatcccag tgacagagaa cctgagactt aggaacagcg ggaaactcca
     3061 tgctgaccag ccagaaagcc tgaagaaggc tcgcagagga agtaagcgac ctcgccccca
     3121 gcggtatgtg ctcggcgacg ccttgcctgc tgacgtgatc aatcgcgtga cagaccccgg
     3181 tctgtggacc gcgttggtgc gggccccagg gttcgatagc cagctggggc ttcctgcgga
     3241 tctgaatagg gggctgaagt tgagaggtaa gcggatatct gctgatttcc caatagacta
     3301 cttcccaaca gactcccccg cattggcagt ccagggcggg tacgtaggcc tggagttcca
     3361 ccacgcaagg ctgtaccgga tcataggccc taaagaaaaa gtcaagtacg ctctccttcg
     3421 ggtctgtgcc atcgacctgt gtggcataga ctgcgacgac ctcttcgagg ttgagctgaa
     3481 accaagtagc atcagcatgc gaactgctga tgccaaactg aaagaggcta tgggtaacgg
     3541 ctctgccaag cagattggat ggctggtcct gggtgacgag atacagatcg atcccacaaa
     3601 attccctaaa cagagcatcg gcaaattcct taaggagtgc ggaccagtgt cctcatggcg
     3661 cgtgtcagcc ctggacacac cctctaaaat aactctgaag cctagattgt tgtctaacga
     3721 gcccctgctg aagacctccc gcgtgggcgg gcacgagtca gacctcgtag tggccgaatg
     3781 tgtggagaag attatgaaga aaacagggtg ggtcgtcgaa atcaacgccc tgtgccaatc
     3841 tggactcatc cgggtgatca ggcggaatgc actgggggaa gtgaggacat cccctaagtc
     3901 aggcctgcct atctccctga atctgcggag cagggctgac cccaagaaga agaggaaggt
     3961 gaggtccatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     4021 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     4081 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     4141 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4201 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg tcttccgctt cctcgctcac
     4261 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4321 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4381 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4441 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4501 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4561 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4621 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4681 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4741 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4801 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4861 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4921 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4981 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5041 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5101 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5161 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5221 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5281 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5341 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5401 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5461 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5521 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5581 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5641 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5701 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5761 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5821 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5881 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5941 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6001 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6061 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6121 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6181 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6241 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cgga
//
