LOCUS       sequence-209054-        5913 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 5913)
  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 5913)
  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-E6MBW6
FEATURES             Location/Qualifiers
     source          1..5913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(183..771)
                     /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(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2031..2053)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2153..2172
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2366..2388
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2380..2397
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2381..2397
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5614..5634
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5644..5660)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5644..5660)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5657..5679)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5668..5684
                     /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(5692..5698,5699..5716,5717..5722))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5737..5758
                     /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 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2041 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2101 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     2161 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     2221 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     2281 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     2341 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgtg
     2401 ctgtctcatc attttggcaa agaattcgcg gccgccacca tgaaccagaa gtttatactg
     2461 gggctcgaca ttgggattac aagcgtgggt tacggcctta ttgactacga aaccaaaaat
     2521 attatagatg ccggtgtcag gctcttccct gaggcaaatg tagagaacaa cgaaggacgg
     2581 agaagcaaac gcggttcacg gcggctgaaa cgcaggagga tacataggct ggagcgggtc
     2641 aagaagctgc tggaggatta caatttgctg gatcagtctc aaattcctca gagcaccaat
     2701 ccatatgcta tacgggtgaa gggcctgtct gaggctctga gcaaagatga acttgtcata
     2761 gcacttttgc acattgcaaa gcggcggggt atccacaaaa tcgacgtgat cgattctaac
     2821 gacgatgtgg ggaatgaact ctccactaaa gaacaactga acaaaaactc caagctgctc
     2881 aaagataaat ttgtgtgtca aattcagctg gagcgcatga atgaggggca ggtcagaggt
     2941 gagaaaaatc ggtttaaaac agccgacata attaaggaga ttatccaatt gctcaatgtg
     3001 caaaaaaatt tccatcaact cgatgagaat ttcatcaata aatacattga gcttgtagag
     3061 atgcgcagag agtacttcga gggaccagga aaaggcagtc cctatggttg ggaaggcgac
     3121 cctaaagcgt ggtacgaaac attgatgggg cactgcacgt actttcccga cgaactgcgc
     3181 tccgtcaagt acgcctacag cgcagacctg ttcaacgctc tgaacgatct taacaacctg
     3241 gttattcaaa gagatggcct gagcaagctg gagtaccatg agaaatatca catcatcgaa
     3301 aacgttttta aacaaaagaa gaaaccaaca ctgaaacaga tcgctaatga gattaatgta
     3361 aaccccgaag atatcaaggg ttaccgaatc accaaatccg gaaagcccca gtttacggag
     3421 tttaaactct accacgatct caagtcagtc ctttttgacc aaagtatctt ggagaatgag
     3481 gacgtactgg atcagattgc agagattctg accatttatc aagacaagga ctctatcaag
     3541 tcaaaactta ctgaacttga catccttctg aacgaggaag ataaggagaa cattgcccag
     3601 ctgaccggtt acaccggaac tcaccgcttg tcactgaagt gtatccgcct ggtccttgag
     3661 gagcagtggt actcctcacg caaccaaatg gaaatcttca ctcacctgaa catcaaaccc
     3721 aagaagatca acctgaccgc ggctaacaaa atccctaaag ctatgatcga cgaatttatc
     3781 cttagccctg tggttaaaag aactttcggc caggcaatta atttgataaa caagataatc
     3841 gagaaatacg gggtcccgga ggatatcata attgagctgg ctcgggaaaa caactctaaa
     3901 gacaagcaga aattcattaa cgagatgcag aagaagaacg aaaacaccag gaaacggatt
     3961 aacgaaatca tcgggaaata tgggaatcag aatgcgaaga gactggtgga gaagattcgc
     4021 ctccacgacg agcaggaggg gaaatgtctg tatagcctgg aatcaattcc actggaggat
     4081 ctgctgaaca atccgaatca ctacgaagtg gaccacataa ttcctcgcag tgtgtccttc
     4141 gacaacagct atcacaacaa agttttggtg aaacaaagcg aaaacagcaa gaaaagcaat
     4201 ctcacaccat atcagtactt taattcagga aaaagcaaac ttagctacaa ccagttcaag
     4261 cagcatatac ttaacctgag taagtcccag gatcggatca gtaagaaaaa gaaagaatat
     4321 ttgctggaag agcgggatat taacaaattc gaagtgcaaa aagaattcat aaaccgaaat
     4381 ctggtggata cccgctatgc cacccgagaa ttgacaaatt acctgaaagc atactttagc
     4441 gccaataaca tgaacgtgaa ggtgaagact atcaacggat cattcaccga ctaccttcgc
     4501 aaagtgtgga agttcaagaa agagagaaac catggataca agcaccacgc tgaggatgca
     4561 cttatcattg ccaacgctga ttttctcttc aaggaaaata agaagctgaa ggctgtaaat
     4621 agcgttttgg agaagcccga aatagaatcc aaacagcttg acattcaggt cgatagtgaa
     4681 gacaattact ctgagatgtt cattattccc aagcaagttc aggacatcaa ggatttcaga
     4741 aattttaaat attcacatag ggtagataag aaaccgaaca gacagctgat taatgacaca
     4801 ctgtactcta cacggaagaa agataattcc acatacattg tgcaaactat caaggacata
     4861 tatgccaagg acaacaccac actcaagaaa caatttgata agtcacccga gaaatttctg
     4921 atgtaccagc acgacccccg cacattcgaa aagctggaag tgattatgaa acaatatgca
     4981 aacgagaaaa atccactggc taagtatcac gaagagactg gcgaatatct cacaaagtac
     5041 tcaaagaaaa acaacggccc catagtgaag agcctgaagt atatcgggaa taagctgggg
     5101 agccaccttg acgttactca tcagtttaag tcctctacta aaaaattggt caagctcagc
     5161 attaagccct acaggttcga cgtatacctc acggacaagg gatataaatt cattactatt
     5221 agctatctgg atgttctcaa aaaggacaat tattactaca tccctgaaca gaagtacgat
     5281 aaacttaaac tgggtaaggc cattgataag aacgccaaat tcatcgcctc cttttacaaa
     5341 aatgacttga tcaagctcga cggcgagatc tacaagatta tcggcgtgaa cagcgataca
     5401 cggaatatga tcgaactgga cctgccagac atacggtaca aggagtactg cgagctgaat
     5461 aatatcaagg gcgagcctcg gatcaagaaa accatcggaa aaaaagtcaa ttccatcgaa
     5521 aagctgacaa cagatgtcct gggtaacgtg ttcacaaaca cccagtatac caaaccccaa
     5581 ctgctcttta agaggggtaa cagcagggct gaccccaaga agaagaggaa ggtgaggtcc
     5641 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     5701 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5761 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5821 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5881 cactgactcg ctgcgctcgg tcgttcggct gcg
//
