LOCUS       sequence-201882-        5702 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 5702)
  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 5702)
  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-Q0P897
FEATURES             Location/Qualifiers
     source          1..5702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     67..86
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     280..302
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     294..311
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     295..311
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      347..356
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             353..3304
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjCas9
                     /translation="MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLA
                     LPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYEL
                     RFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVG
                     EYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKF
                     EEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNT
                     EGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIK
                     ALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFK
                     ALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKE
                     YRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGL
                     KINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVL
                     VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDR
                     NLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTW
                     GFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKR
                     KFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKAL
                     ELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGE
                     IKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFET
                     LSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     CDS             3317..3337
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3347..3363)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3347..3363)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3360..3382)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3371..3387
                     /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(3395..3401,3402..3419,3420..3425))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3440..3461
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3799..4387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3879..3898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4558..5349,5350..5418))
                     /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     5181..5200
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5419..5523)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5591..5609
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5647..5669)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
       61 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
      121 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact
      181 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
      241 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      301 cgacggccag tgtgctgtct catcattttg gcaaagaatc gcggccgcca ccatggcaag
      361 aatcttggct tttgacattg gaatcagctc tatcggttgg gcgttttccg aaaatgatga
      421 gctgaaagat tgcggcgttc ggattttcac caaggtcgaa aaccccaaga ccggggagtc
      481 tctcgctctt ccacgaagac tcgccagatc tgcccggaag cgcctcgcta ggagaaaagc
      541 tagacttaac cacctgaagc atctgattgc gaatgaattc aaactgaact acgaggacta
      601 ccagtcattc gatgaaagcc ttgccaaggc ttataagggg agcttgatct ccccctacga
      661 gctgcgattt cgggcgctga atgagttgct gagcaaacag gacttcgcac gcgtcatact
      721 gcacatcgca aagagaagag ggtacgatga catcaagaac agcgacgata aggagaaagg
      781 agccatcctt aaagcgataa aacaaaacga agaaaagctt gcaaactacc agagcgtggg
      841 agagtatctc tacaaagagt attttcagaa atttaaggaa aacagcaagg aatttaccaa
      901 cgttcggaat aaaaaggaga gctatgagag atgcattgct cagagttttc tgaaagacga
      961 gttgaagctt atatttaaaa agcagcggga atttggcttc agctttagta aaaaattcga
     1021 ggaggaggtc ctgtccgtgg ccttttataa gcgggcgctc aaagatttct cacatttggt
     1081 ggggaactgc tcatttttca ccgacgaaaa gagagctcct aagaactccc ccctggcttt
     1141 catgttcgtc gcactcacga ggatcattaa cttgctgaat aacttgaaga acactgaggg
     1201 catactttac acaaaagatg atctgaacgc cttgctcaat gaagtcctta agaacgggac
     1261 actgacttat aagcaaacaa aaaaactcct gggtctgtcc gatgactatg agtttaaagg
     1321 agaaaaggga acctacttca tcgagttcaa gaaatataag gaatttataa aagcattggg
     1381 ggagcataac ttgtcccagg atgatttgaa tgagattgca aaagacatca ctctcattaa
     1441 ggatgaaatc aagctgaaaa aggctctggc gaaatacgac ctcaaccaga accagatcga
     1501 ttcactgagt aaactggagt ttaaggatca tctgaatatc tccttcaaag cactcaaatt
     1561 ggtcaccccc ctgatgctgg agggcaagaa atacgacgaa gcttgtaacg agctgaatct
     1621 taaggtagca attaacgagg ataaaaagga ctttctgccc gcctttaatg agacgtatta
     1681 caaagacgag gtcacgaacc ctgtggtgct gagggcaatc aaagagtata gaaaggtact
     1741 taatgccctc cttaaaaagt atggaaaagt gcataaaatt aatattgagc tggcccgcga
     1801 ggtcggcaag aaccactctc agagggcaaa gatcgagaaa gagcagaacg agaactataa
     1861 agccaagaaa gatgcggagc tcgagtgtga gaagctgggc ttgaaaatta actctaagaa
     1921 tatcctgaaa ctgagactgt tcaaagaaca gaaggagttt tgtgcttata gcggcgagaa
     1981 gatcaaaatt tccgatctcc aggatgagaa gatgctggag attgatcata tataccctta
     2041 ttccaggagt ttcgacgaca gctatatgaa taaagtgttg gtgtttacga agcagaacca
     2101 ggagaagttg aatcaaacac cttttgaagc ctttggtaac gattccgcaa agtggcaaaa
     2161 aattgaggtg ctcgcaaaga acctgcctac gaaaaaacag aagcgcatcc tcgataagaa
     2221 ttacaaagac aaggaacaga agaatttcaa agaccggaat ctgaacgaca ccaggtacat
     2281 cgctagactg gtgcttaatt acactaagga ctacctcgac tttctccctc tgtctgacga
     2341 cgagaacacc aaacttaacg acactcaaaa aggatccaag gtgcatgttg aggccaagag
     2401 tggcatgctt acaagtgctc tgcgacacac atggggtttc agcgccaagg acaggaataa
     2461 tcatctccac cacgccatcg acgcggtcat tatcgcttac gcaaataaca gtatcgtcaa
     2521 ggccttttca gattttaaga aagagcagga atcaaattca gccgagctgt acgccaagaa
     2581 aatatctgag cttgactaca aaaacaagcg caagtttttt gagccgtttt ccggattcag
     2641 gcagaaagtg ctcgacaaaa ttgacgagat tttcgtctca aaacctgaaa gaaaaaagcc
     2701 atcaggcgcc ctgcacgagg agacgttcag gaaggaggag gaattctacc agtcttacgg
     2761 aggcaaggaa ggggtgctta aggcactgga gctgggcaaa atccgcaagg ttaacggaaa
     2821 aatagtgaaa aacggtgata tgttccgcgt ggatattttt aaacacaaaa agaccaacaa
     2881 gttttatgcc gtcccaattt acacaatgga cttcgctctc aaggtgctcc cgaacaaagc
     2941 tgttgcgaga tctaagaaag gtgaaatcaa ggactggatc ttgatggacg aaaattatga
     3001 gttctgtttc agcctgtata aagactcact gatcttgatc caaaccaagg atatgcagga
     3061 gcccgaattc gtctactaca atgccttcac ttctagtacc gtttccctca tagtgtccaa
     3121 gcatgacaac aaatttgaaa ctctgtccaa aaatcagaaa atactgttca agaatgccaa
     3181 tgagaaagag gttattgcaa agtccatcgg catacaaaac ttgaaggtgt tcgagaagta
     3241 tatcgtgagc gcacttggcg aagtcacgaa agccgaattt agacagaggg aggacttcaa
     3301 gaaaagcagg gctgacccca agaagaagag gaaggtgagg tccatggtca tagctgtttc
     3361 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3421 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3481 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3541 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3601 cggtcgttcg gctgcgtctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3661 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3721 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3781 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3841 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3901 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3961 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4021 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4081 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4141 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4201 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4261 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4321 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4381 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4441 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4501 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4561 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4621 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4681 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4741 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4801 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4861 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4921 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4981 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5041 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5101 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5161 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5221 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5281 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5341 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5401 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5461 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5521 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     5581 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     5641 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     5701 cg
//
