LOCUS       sequence-201724-        5986 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 5986)
  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 5986)
  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-A8LN05
FEATURES             Location/Qualifiers
     source          1..5986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(183..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1974..1992
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2030..2052)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2152..2171
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2365..2387
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2379..2396
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2380..2396
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5687..5707
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5717..5733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5717..5733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5730..5752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5741..5757
                     /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(5765..5771,5772..5789,5790..5795))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5810..5831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     1981 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     2041 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     2101 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     2161 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     2221 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     2281 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     2341 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgtgc
     2401 tgtctcatca ttttggcaaa gaatcgcggc cgccaccatg cgcctggggc tggatattgg
     2461 cacatcatct attgggtggt ggctctatga aaccgacggg gctggaagtg acgctagaat
     2521 cactggcgtc gtcgatgggg gagtgagaat ctttagcgac ggccgggacc ccaagtccgg
     2581 tgccagcctc gctgtcgacc ggagagcagc cagagcaatg cgccgcagac gagatcggta
     2641 cctccgccgc agggctactc tcatgaaagt gctcgcagag actggcctca tgccagcgga
     2701 ccccgccgaa gccaaagccc tggaggcatt ggatccattt gccttgcgag cagcagggct
     2761 ggatgaacct ctccctcttc cccacctcgg gagagctctt ttccacctca atcagaggcg
     2821 aggttttaag tctaatcgaa agacggaccg gggcgacaat gaatcaggca agatcaaaga
     2881 cgccactgct aggctcgaca tggaaatgat ggctaacggc gccagaacgt atggggaatt
     2941 tctgcataaa agaagacaga aagccacaga tccgcgacac gtcccttccg tccggacgcg
     3001 gttgagcata gccaaccggg gaggtcctga cggtaaagaa gaggcggggt acgactttta
     3061 tcccgaccgc cgccatctcg aagaggaatt ccataagctt tgggccgcac aaggagcaca
     3121 ccatcccgaa ctgacggaaa ccctgcgcga cctcctgttt gaaaagatat tctttcagcg
     3181 ccctctgaag gaacccgaag tgggcctgtg ccttttctcc ggacaccacg gagtgccgcc
     3241 taaagatccc aggcttccaa aagcccatcc actcactcag cgacgagtgc tgtacgaaac
     3301 tgtcaaccag ttgagagtaa cagcagatgg gagggaggca agaccactga ctagagagga
     3361 gcgcgaccaa gtcattcacg ctctggataa taagaagcct acgaaaagtc tgtccagtat
     3421 ggtacttaaa ttgcctgcgc ttgccaaagt gttgaaactg cgggacggtg aaaggtttac
     3481 tcttgaaacc ggcgttcggg atgccatcgc ctgcgatcct ctccgcgcga gccctgcgca
     3541 tccagaccgg ttcggcccgc gctggtctat tctcgatgcc gacgcacagt gggaagtgat
     3601 ttcccgcatc agaagggtgc agtctgatgc tgaacacgca gccctggttg attggctgac
     3661 agaggctcac ggtcttgaca gggcacacgc cgaagccaca gcccacgccc ctcttcctga
     3721 tgggtatgga cggctgggcc tgaccgctac tacccgaata ctttaccaac tcactgccga
     3781 tgtggttacg tatgccgatg ctgtaaaggc ctgcgggtgg caccactccg acggacgaac
     3841 gggagaatgc ttcgatcgac tgccttatta cggagaggtc cttgagagac atgtgattcc
     3901 tggctcatat catccagacg acgacgacat cactcggttc ggaaggatta cgaatccaac
     3961 tgtgcacatt gggctgaatc agctgcgcag actcgtcaac cgcataatcg aaacccacgg
     4021 caaaccacac cagatcgtgg tggaactcgc ccgggacctc aaaaaatcag aagagcagaa
     4081 gcgggctgat atcaaaagaa tacgggatac cacagaagcg gccaaaaagc ggtccgagaa
     4141 gctcgaggag ctggagattg aggataacgg gcggaatcgg atgctcttgc gactttggga
     4201 agaccttaat cccgacgacg ccatgaggcg cttctgcccc tataccggga cgaggatctc
     4261 cgccgcgatg atcttcgacg gctcctgcga tgtcgaccac atcctgccat atagtcggac
     4321 gctcgacgat tctttcccca accgaactct gtgccttagg gaagcaaata ggcagaaaag
     4381 aaatcagaca ccttggcaag catggggtga cacccctcac tggcatgcaa tcgcggctaa
     4441 cctgaaaaat ctgcccgaga acaaacgctg gaggttcgcg ccggatgcta tgacccggtt
     4501 cgaaggcgaa aatggattct tggatagggc cctgaaagat acgcagtact tggcccgcat
     4561 cagccgcagc tacctggaca ctctttttac aaaggggggc cacgtgtggg tagtccccgg
     4621 acggttcacc gagatgttgc gcaggcattg ggggcttaac tctctgctct ctgatgcggg
     4681 ccggggggcc gtcaaggcta agaacaggac cgatcaccgc catcacgcta tagacgccgc
     4741 cgtgattgcc gccactgacc ccggacttct taatagaatc tctcgggccg ctgggcaggg
     4801 agaagccgcc ggacagtcag cggagctgat agcccgggat actcctccgc cttgggaagg
     4861 ctttcgggac gaccttcggg tccggctgga tagaattatt gtcagccata gggcagacca
     4921 tgggcggatt gaccacgccg caagaaagca ggggcgcgac agcaccgccg ggcaactgca
     4981 ccaggaaact gcctactcca tcgtggatga tatccacgtt gccagtcgca ccgacttgct
     5041 gagccttaag ccagctcagc tgctggatga acccggcagg tctgggcagg tgcgcgaccc
     5101 acagcttcgc aaagctctgc gcgtggccac cggaggtaaa accggtaagg actttgaaaa
     5161 tgccctgcgg tattttgcct ctaagcctgg tccctatcaa gccataagac gggtgcggat
     5221 tatcaagcct ctccaagcac aagccagggt gcccgtgccc gcgcaggatc caattaaggc
     5281 ctaccaggga ggaagcaacc acctcttcga gatctggcgg cttcccgatg gagagattga
     5341 agctcaagtg atcacctctt tcgaggccca cacccttgag ggcgagaagc ggccacatcc
     5401 tgccgccaag agactgctta gagttcacaa aggggatatg gtggccctgg aacgggatgg
     5461 aaggcgcgtg gtgggccatg tccagaaaat ggatatcgcc aatgggcttt tcatcgtgcc
     5521 ccataatgag gcgaatgccg atacaaggaa taatgacaaa tccgatcctt tcaaatggat
     5581 acaaatcggt gcaaggcccg caatcgccag cggaatccga cgcgtgtcag tcgacgagat
     5641 tggcagactg agggacggtg gcacccgccc aattagcagg gctgacccca agaagaagag
     5701 gaaggtgagg tccatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     5761 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5821 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5881 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5941 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcg
//
