LOCUS       sequence-201695-        6144 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 6144)
  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 6144)
  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-B9M9X8
FEATURES             Location/Qualifiers
     source          1..6144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     214..236
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     228..245
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     229..245
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      282..291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3693..3713
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3723..3739)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3723..3739)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3736..3758)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3747..3763
                     /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(3771..3777,3778..3795,3796..3801))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3816..3837
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4175..4763)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4255..4274)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4934..5725,5726..5794))
                     /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     5557..5576
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5795..5899)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5967..5985
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6023..6045)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
       61 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg
      121 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
      181 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg
      241 ccagtgtgct gtctcatcat tttggcaaag aattcgcggc cgccaccatg gcccaacatg
      301 tattcggcct cgacatcggg attgcttccg tgggatgggc tatcctgggg gaacagagga
      361 taatcgatct cggcgtgcga tgttttgaca aggcagagac ggccaaggaa ggcgatcctt
      421 tgaacttgac caggaggcag gcccgccttc tgcgacggcg cctgtacagg cgcgcttgga
      481 gattgacaca gctcagcagg ctcctgaaac gaaaagggct gattgccgat gccaagctct
      541 tcgccaaagc tcctagctat ggtgacagtg cgtgggagct gcgccgacag gggctggatc
      601 ggcttctgac accactggaa tgggcccggg tcatctatca tcagtgtaaa catcgagggt
      661 tccattggac gtctaaagcc gaggaagcaa aggccgatag tgatgccgag ggaggtcgag
      721 ttaagcaagg tctggcccat accaaagccc tcatgcaggc aaagaattac agaagcgccg
      781 ctgagatggt gctggcggaa ttcccagacg cccagcgaaa taagcgagga cagtatgaca
      841 aagctttgag ccgggttctg ctgggggaag agctggcact cttgttcgca acccaaaggc
      901 gcctggggaa tccacacgcc agtgattttt tcgagaaact gattctcggc gacggagata
      961 ggaaatctgg gctgttttgg caacagaagc ccgcactctc cggggccgac cttctgaaaa
     1021 tgttggggaa atgtaccttc gagaaaggcg agtaccgcgc ccccaaagcc tctttttctg
     1081 tcgagagaca tgtttggctg acaaggctta acaatttgcg gatagttgtg gatggacgga
     1141 gccggcctct gaatgaagca gaacggcagg ccgccctcct gctgccctat cagactgaga
     1201 cgagtaaata caagacgctc aaaaacgcct ttatcaaggc aggcttgtgg ggtgatggcg
     1261 tgcgatttgg agggctggcc tacccaagcc aggcgcaaat agacgcggaa aaaactaagg
     1321 acccagagga tcaattcctg gtcaagttgc cagcttggca tgaactgaga aaggctttca
     1381 aggcagcagg tcacgaggcc ctgtggcagc agatcagcac gcccgcgctg gacggagatc
     1441 caacactcct ggaccaaata gccactgtgc tgagcgttta caaggacggt gctgaagtag
     1501 tgcagcagct cagacaactg gccctgccag aacctgctgc ttctatcgcc gttctcgaaa
     1561 agatttcatt cgataagttt tccagcttga gcctgaaagc tctgcggaga atagtcccgc
     1621 tgatgcagag tgggcttcga tatgacgagg ccgtcgcaca aattccagag tatggccatc
     1681 attctcaacg gattgaacct ggagcggcca aacaccttta cctgccacca ttctacgaag
     1741 ctcagcggaa gtatgcaggc aaaggcgacc atatcggcag tatgcagttt agggatgatg
     1801 cagatatccc ccgaaatcct gttgtcttgc gcgctctcaa tcaagcaaga aaggtggtca
     1861 atgcccttat tcgggagtac ggttctccta tcgctgttaa catagagatg gcacgggatc
     1921 tgtcccgccc tctggatgag cgaaacaagg tcaagcgcgc ccaggaggaa tttagggata
     1981 ggaatgacag ggctcggagc gaatttgaga gggattttgg atacaaacct aaggctgccg
     2041 cgttcgaaaa gtggatgctg tatagggagc agctgggcca gtgcgcgtac tcccagcagc
     2101 cgctggatat ccagagggtc ttggacgatc acaattatgc ccaggtcgac cacgctttgc
     2161 cttacagtag atcatatgat gattcaaaaa ataacaaagt tctggtcctg actcacgaaa
     2221 atcagaacaa gggaaaccgg acggcctttg aatacctcac atcatttccc gacggcgagg
     2281 atggcgagag gtggaggacc tttgtggcat gggtgcaggg gaacaaggct taccgcatgg
     2341 ccaaaagaaa tcgccttctg cgcaagaact acggcgtgga cgagtccaaa gggttcattg
     2401 acaggaatct caacgatacc agatacatct gtaaattttt taagaactac gtggaggaac
     2461 acctgcagct cgcagcaaga gcagacgggg atacagcccg ccgatgcgta gtcgttaacg
     2521 ggcagctgac agcgttcctg agagcccgat ggggacttac aaaagtgcgc ggtgattccg
     2581 atcgccatca tgccttggat gcagccgtgg tggccgcctg cactcatgga atggtcaaag
     2641 ccctggccga ctacagtcgg agaaaagaga tcagcttcct gcaggagggc ttccccgacc
     2701 cagagactgg agagattttg aaccctgcag cctttgaccg cgcccgccag cacttcccag
     2761 aaccctggac acacttcgca catgaattga aagctcggct gttcaccgat gatttggcag
     2821 ccctgcgaga ggatatgcag cgactgggaa gttacaccac ggaagatctg ggtcgcctgc
     2881 gaaccttgtt cgtcagcagg gcccctcaga ggcgcagcgg gggcgcagtg cacaaagaaa
     2941 caatatacgc tcagcctgag agcctgaagc aacagggcgg cgtgattgag aaaatcctgc
     3001 ttacatctct caagcttcaa gatttcgaca aactgctgaa cccagaatct aacgatcact
     3061 tcgtggagcc tcacagaaac gagaggctct atgccgcaat caggcagagg ctcgaacagt
     3121 ttggtggaag agcagataag gcttttggac cagacaactt gtttcataaa cctgataaaa
     3181 ataatcagcc caccggaccc gtggtaagga gtataaagct ggttaggggc aaacagacgg
     3241 gaatccctat tcgggggggc ttggctaaga acgactccat gctgcgagtt gatatcttta
     3301 ccaaagctgg aaaatttcat ctggtgccag tgtacgttca ccatcgggtg acaggcctgc
     3361 ccaaccgggc aatcgtagcc tttaaggacg aggatgagtg gacactgatc gacgaaagct
     3421 tcgcttttct ctttagcgta tatcccaacg attatgtgaa ggtgacactg aaaaaggaac
     3481 aacagtcagg atattactca ggagctgata ggagcactgg cgccatgaat ctctgggccc
     3541 acgaccgggc cgcaagtgtt gggaaagatg ggttgatccg cggtataggg gtgaagacgg
     3601 ccctgtccgt ggaaaaattt aacgtcgatg tgctcggccg catttatttg gcgcccccgg
     3661 agacacggag cggtttggcc agcagggctg accccaagaa gaagaggaag gtgaggtcca
     3721 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3781 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3841 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3901 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3961 actgactcgc tgcgctcggt cgttcggctg cgtcttccgc ttcctcgctc actgactcgc
     4021 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4081 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4141 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4201 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4261 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4321 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4381 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4441 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4501 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4561 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4621 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4681 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4741 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4801 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4861 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4921 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4981 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5041 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5101 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5161 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5221 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5281 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5341 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5401 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5461 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5521 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5581 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5641 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5701 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5761 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5821 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5881 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     5941 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     6001 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     6061 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     6121 ggtgtcgggg ctggcttaac tatg
//
