LOCUS       sequence-201737-        6942 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 6942)
  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 6942)
  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-B5JU35
FEATURES             Location/Qualifiers
     source          1..6942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     252..274
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     266..283
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     267..283
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4529..4549
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4559..4575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4559..4575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4572..4594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4583..4599
                     /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(4607..4613,4614..4631,4632..4637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4652..4673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5011..5599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5091..5110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5770..6561,6562..6630))
                     /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     6393..6412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6631..6735)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6803..6821
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6859..6881)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
       61 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
      121 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
      181 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
      241 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgtgctgt ctcatcattt
      301 tggcaaagaa ttcgcggccg ccaccatgac caagaactat atcagcccaa ttgccatcga
      361 cctgggggcg aaattcacag gcgtcgctct ctatcaatat ctggaaggcg cagattgcac
      421 ccaggaagtc gccaagggcc tcctcgtgga tgaccggggt aacgtcacct ggtcccaaga
      481 gggaagacgc ggaaagcggc accaggtccg aggatataag agacggaaga tggccaagcg
      541 cctgctgtgg ctcatattgg acagcgaata cggcataaag agagaggaag tgaccgagcc
      601 cctcctcaaa tttatcaatg gcctcttgaa ccggcgcggg tacacttata tatccgagga
      661 agtggacgaa gaatctatga atgtctcccc cctccccttc tccgagatga tgccagatta
      721 ctttaattct tctgccccct tgctggagca gcttgccaaa ctcctgagcg acaagaacaa
      781 acttgtccgg tttcgggccg aaggcaagat ccctagtaat aagaacgagt tcaagaaact
      841 gctcgacacc gccctggacg ggaagtacaa agatgagaaa aaagagctgt ccgaggcctg
      901 gggcaacata ctgatcgctt ccgaaaatgt cctcaaatct actgtcgatg gccacaagtc
      961 tagaagcgag tatctggcaa acatcaaaga ggacatcaag agcaatgaag aacttgagaa
     1021 gcagataagt agtaaggaga tcgacggatt ttataatctg gtcggacacc tgtcaaactt
     1081 ccaacttagg ttgctccgga agtatttcaa tgaccccaat atgagtggcg tgtcttattg
     1141 ggatgagaag cggctggaaa aatacttcta ccagtgggta cagggatggc acactaaagg
     1201 aggtaccgac gaggctgaaa agaaaaatat aatcctgaag accaaagggg ctcctctgct
     1261 gaaaaccctc aagagtctgt ctgccgacct gactataccc ccctacgagg atcaaaataa
     1321 ccggcgaccg ccgaaatgcc agagcgtgct gctgagtgat gagaaactca ccatgcatta
     1381 ccccaagtgg aaagaatggg tgggacagtt ggtgaagcaa aatgacaatg cctacctgaa
     1441 tgagaacgtc accttggcca acgccctgca caggatcgtc gagagatctc gaagtataga
     1501 cccatatcag ctgagactct tgatctccat taccgacgct gagaagcgca atgaccttgc
     1561 tggatacaag aggctcaagc tcagtttggg tagcgaggtg gacgagtttc tgcttttggt
     1621 gaaaaatatc gtggatgaga caaaggaggc acgcgagggt ctgtggttcg agacagagaa
     1681 caaacttttc tttaaatgcg gtaagactcc cccacgcaaa gaaaagctca agtctactct
     1741 gctgagcgct gtcctcggga agaacctctc cgacgatgaa cagagctctt ttattgagga
     1801 gttttggaaa agcgggaccc caaaaattga acgaaggaac gtgagaggct ggtgccgctt
     1861 ggcctcccaa gtgcagaaga cctacggggt gtacttgaag gagtacggcc tccaacagct
     1921 ccacaaattg gaggctggca aaaaactgga tgacaagcca ctggctctgc tgtataagaa
     1981 tagtggcctg attgcaagca agatcggtga agccttgaac atcgaacccg atgaagtctc
     2041 ccgatttgca tctccacata gtctggctca gatctttaat atcattgagg gcgatgtagc
     2101 cggatttaac aaaacttgtc gcgcatgcac ctacgaaaac atttggagaa tgcaggagga
     2161 aaaggtagag agcctcctga cgaatcagct gctctctgaa attcacgggg aaaggaaagt
     2221 gccgttgaaa tccgcaatgt gcacaagact ttccgccgat agcacccgac cttttgatgg
     2281 ccagatggcg agcattattg aacacatagc ccgcaagata gcccaacaca aaattgcaca
     2341 gatcaatgac gttcccaagg aatttagtat cgatattcct atcataatcg aaagcaatca
     2401 gttttccttc actgctgaac tcgaggagat caaacggggc agaggctctg caaaagctaa
     2461 aaaagccaag gagctcggcg agaagtctaa agcaggctgg gtcagcaaga ctgagaggat
     2521 caagaccagc agcgaaggta tctgcccata cactggcgcc ccactcgggg gatctggtga
     2581 aattgaccac attatacccc gcagtctcac cggccggacc aaaaagacag tcttcaacag
     2641 cgaggcaaat ctcatatatt gctcatccaa agggaatcac gataaaggaa atcgggttta
     2701 tgtgattgag cagctcaatg acaagtattt gaaaaagcag ttcagtacct cagacgtcaa
     2761 cctgatcaaa aagaaaatca agaccactat ccagaggttt actgaagggg gcgagaagtt
     2821 gcgcagcttt agcgaactgt ctcgcgaaga tcagaaggcc tttagacatg cgctgttcgt
     2881 gcctgaactt aagtccgaag tgacctccct gttggcagtg aagaatatta ctcgggtgaa
     2941 cggaacgcag gcctggctcg caaagaagat agccagcctg ctggccgagc atttggataa
     3001 gcaggggaga gactatacac tgagcgcgca tcagatcgac ccctggagtg taagtaagca
     3061 gcgaaaaatg cttgcatctg ccgagcctat ttgggctaaa aaggacccgc aaccagcggc
     3121 ctctcatgtg gtggacgctg tgtgtacatt cctcgaggcc ttggaacagc cgcacaccgc
     3181 atcccggctt aaaactatta gctcaaccag tttcgagaag acaggctggc gcagcgcact
     3241 gatcccagat ctgatcaaag ttgacgcact ggaccggcgg cctaagtaca gacgctataa
     3301 tataggctca acttctcttt ttaaagacgg gatctatgcc gagcggttcc tccccatcct
     3361 cattgatgaa aacggcctga tggccgggta tgacatcgat aatagtctga aagccaaggg
     3421 cgccgacgtg gtctttgagt ccctgagtcc tttccttctg ttcaaaggag aggaggtggg
     3481 cgctcaaagc ctctcagact ggcaggagcg catagatggc cgatacctct acatgtctat
     3541 cgacaaagta aaggccttcg attacttgca agaaaaagta ggcgagaaag acatcgctgc
     3601 cgagctgctg aacagcatcc atttcaccca gcggaagacc gagctgagag ccaagttcag
     3661 cgacgactca ggcaagaaga tgaaaaccct ggacgcaatc aggaagtccc tcaagcttac
     3721 cgtgacagtg aacgagattg ggaaacgcaa ggagaagtgc ggtttcagcg gaacaatagg
     3781 gatcccagcc aagtccgctt gggagaatct gctggatgag cccttgcttg aaacctattg
     3841 gggcacaaaa atgcctcctc aagagatctg ggagaaagta tataggaagc acttccctag
     3901 gaacatacca aaccaggcac ataggaaggt gcgaaaggac ttttccctgc cagtcgtgga
     3961 ttctgtctct ggtgggttca gggtcaaacg caaaacccct aacggctaca attaccagtt
     4021 gctggcaatt gatgggtact cagcggttgg attcaagaag gagggtgata atgtcgactt
     4081 taagagccct gccctggtcc cccagatagc ggaaagcaag agcgtgaccc ccatcagtag
     4141 cgagctcgtt catctcgaca agaatgaaat tgtgtacttc gacgagtggc gcaagataga
     4201 tataagcgac tctgatctta agcagttcgt gtccagcctt gaactggcac ccggctctca
     4261 aaaccggttc tacatccgat tcactgtgga cgaggaccag ttcgaaaggc actttaaatc
     4321 tgccctgcgg gtcaacggca ttcaggacct ggacactgtg aataagacct tcgattggaa
     4381 ccgcgaaatc ccctcccttt tgattcctcc taggtccaac ctgtttctcc tggaaaccgg
     4441 gcagaagatc actttcgagt atattgcaaa tggagctaac gccgaggtga agaaggccta
     4501 ctctctgcgc cgggcaagca gggctgaccc caagaagaag aggaaggtga ggtccatggt
     4561 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4621 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4681 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4741 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4801 actcgctgcg ctcggtcgtt cggctgcgtc ttccgcttcc tcgctcactg actcgctgcg
     4861 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4921 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4981 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5041 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5101 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5161 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5221 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5281 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5341 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5401 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5461 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5521 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5581 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5641 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5701 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5761 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5821 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5881 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5941 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6001 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6061 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6121 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6181 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6241 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6301 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6361 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6421 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6481 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6541 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6601 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6721 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     6781 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     6841 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     6901 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tc
//
