LOCUS       sequence-201819-        5937 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 5937)
  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 5937)
  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-G2ZYP2
FEATURES             Location/Qualifiers
     source          1..5937
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..44)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     144..163
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     357..379
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     372..388
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      425..434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             503..514
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3629..3649
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3659..3675)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3659..3675)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3672..3694)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3683..3699
                     /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(3707..3713,3714..3731,3732..3737))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3752..3773
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4111..4699)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4191..4210)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4870..5661,5662..5730))
                     /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     5493..5512
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5731..5835)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5903..5921
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
       61 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
      121 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
      181 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc
      241 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
      301 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
      361 gtcacgacgt tgtaaaacga cggccagtgt gctgtctcat cattttggca aagaattcgc
      421 ggccgccacc atggcagaga aacaacatag atggggcctt gatataggaa ccaactctat
      481 cggatgggct gtcatcgcac tgatcgaggg gcggcccgca ggactcgtgg cgacaggcag
      541 ccggattttc tccgacggac gaaacccgaa agatgggagt tcactggccg tcgagaggcg
      601 cggacctaga cagatgcggc gccgacggga cagatatctt aggaggagag atcggttcat
      661 gcaggctctg atcaacgtgg ggctgatgcc aggtgatgct gcagctcgga aggcactggt
      721 tacggagaat ccctatgtac tccgccagcg cggcttggat caggctctga ccctccccga
      781 gttcggcaga gccctgttcc atttgaatca gagacgaggc ttccagagca acaggaaaac
      841 agatcgcgcc acggcaaagg agagcggaaa ggttaagaat gccatcgccg cctttagagc
      901 tgggatgggc aacgcaagaa ccgtgggcga ggccctcgcg cgccgccttg aggacgggag
      961 gcctgtccgc gcgcgaatgg tggggcaggg gaaagatgaa cattacgaac tttacatcgc
     1021 cagagagtgg atcgcccagg aattcgacgc cctgtgggct agccagcaac gatttcatgc
     1081 cgaagtgctg gccgacgccg cacgcgaccg gctccgcgcg attctgttgt ttcagcgaaa
     1141 gttgctgcct gtgcctgtgg gcaaatgttt cttggagccc aatcaacccc gcgttgccgc
     1201 cgcacttcct tcagcccaga gattccgcct gatgcaggag ctcaatcacc tcagggtcat
     1261 gaccctcgcg gataaacgcg agaggccttt gtctttccag gagagaaacg acttgctggc
     1321 gcaactcgtt gctcggccaa agtgtggatt tgatatgctc cggaaaattg tcttcggagc
     1381 caataaagaa gcctatcgct tcaccatcga atcagagaga cggaaggagc tgaaaggttg
     1441 cgacactgcc gcaaagctgg ccaaggtgaa cgctttgggt acgcggtggc aggccctcag
     1501 tctggatgaa caagatagac tggtctgttt gctgctggac ggagagaacg acgccgtttt
     1561 ggctgatgca cttcgggaac actatggcct cactgacgca caaattgata ctctgcttgg
     1621 cctgagtttc gaagatggac atatgagact tgggcgaagc gctcttctgc gggtgctgga
     1681 cgccctggag agcgggcgag acgagcaggg gttgcctctg tcttatgaca aggccgtcgt
     1741 cgctgccggg tatccagccc acaccgctga tctggagaat ggtgagagag atgcgctccc
     1801 atactatgga gaactcctgt ggcgctacac acaggacgca cctactgcca agaacgacgc
     1861 tgagagaaaa tttgggaaaa ttgccaatcc aaccgtgcat attgggctta accagttgcg
     1921 caaactcgtc aatgccctga ttcagagata cggaaagcca gcgcagattg tggtcgaact
     1981 tgcgagaaac ttgaaggcag gcctggagga gaaagagcga attaagaagc agcagacagc
     2041 taacctcgag agaaacgaaa gaattcgcca gaagctgcag gacgctgggg tacctgataa
     2101 tcgggaaaat cgactgagga tgaggctgtt tgaagagctg gggcaaggaa atggactggg
     2161 taccccatgc atttattcag ggcgccagat ctccctgcag aggctcttta gcaatgacgt
     2221 gcaggtcgat cacatcctgc ccttctcaaa aacacttgac gactcattcg caaataaggt
     2281 cctcgcacag catgacgcta atcggtataa aggaaataga ggccccttcg aagcctttgg
     2341 agccaatcgc gacggttacg catgggacga catcagagca agagcggccg ttcttccaag
     2401 gaataagcgg aatagatttg ccgaaaccgc catgcaggat tggctccata acgaaaccga
     2461 tttccttgcc aggcagctta cggacaccgc atatctgtct cgagtggcac gccagtacct
     2521 cactgctatc tgcagtaagg acgatgtcta cgttagtcca ggtaggctta cagccatgct
     2581 gcgagcaaaa tgggggctca atcgggtgtt ggatggtgtt atggaggaac aaggccgccc
     2641 tgcagtcaag aaccgggatg accaccgaca ccacgccatc gacgccgttg tgattggcgc
     2701 gaccgacaga gctatgcttc aacaggtagc caccctggcc gcccgggcta gagaacagga
     2761 cgcagaacgg ctgatcggag atatgcctac cccttggcct aatttccttg aagacgttag
     2821 agcagccgtg gcacgctgtg ttgtgtctca caaaccggac catggacccg agggcgggct
     2881 ccacaacgat acagcatacg gcatcgtggc cggtccattc gaagacggac ggtaccgggt
     2941 gagacacaga gtgtccctct tcgacctgaa acccggggat ctcagcaacg tgcggtgcga
     3001 cgcgcctctg caggccgagt tggagcctat cttcgaacag gatgacgcca gagcacgcga
     3061 ggtggctctg acagcactgg ctgagcgata tcgacagcgg aaggtctggc tggaagagct
     3121 gatgagtgtg ctgcccatcc gacctcgagg ggaggacggt aaaacactgc ccgatagcgc
     3181 accatacaaa gcctacaaag gggattccaa ctactgctac gagttgttca ttaacgagcg
     3241 gggccgctgg gacggcgagc tgatctctac tttcagagcc aatcaagccg cttacagacg
     3301 attcaggaac gatcccgcga gatttcggcg atatactgcc ggagggcggc ccctcttgat
     3361 gagactttgc ataaatgact atatcgccgt tgggacagca gcagagcgga ctatcttccg
     3421 agttgtgaag atgtcagaga acaagattac attggccgaa cactttgagg gaggtacgct
     3481 gaaacaacgg gatgctgaca aggatgatcc attcaaatac ctgactaaaa gtccaggagc
     3541 tctgagagat ttgggagccc ggcggatctt cgtggatctg attggccgcg tgctggatcc
     3601 cgggattaag ggcgacagca gggctgaccc caagaagaag aggaaggtga ggtccatggt
     3661 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3721 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3781 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3841 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     3901 actcgctgcg ctcggtcgtt cggctgcgtc ttccgcttcc tcgctcactg actcgctgcg
     3961 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4021 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4081 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4141 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4201 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4261 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4321 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4381 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4441 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4501 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4561 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4621 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4681 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4741 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4801 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4861 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4921 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4981 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5041 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5101 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5161 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5221 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5281 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5341 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5401 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5461 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5521 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5581 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5641 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5701 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5761 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5821 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     5881 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcg
//
