LOCUS       sequence-201888-        6663 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 6663)
  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 6663)
  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-Q4A5I2
FEATURES             Location/Qualifiers
     source          1..6663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     102..121
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     315..337
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     329..346
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     330..346
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4313..4333
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4343..4359)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4343..4359)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4356..4378)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4367..4383
                     /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(4391..4397,4398..4415,4416..4421))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4436..4457
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4795..5383)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4875..4894)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5554..6345,6346..6414))
                     /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
                     LIRHW"
     primer_bind     6177..6196
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6415..6519)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6587..6605
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(6643..6663,1..2))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
       61 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
      121 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
      181 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
      241 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
      301 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgtgc tgtctcatca
      361 ttttggcaaa gaattcgcgg ccgccaccat gctgaacaac gtgcaaaacc tgtggaagta
      421 tcttctcttg cttatctttg acaagaaaat tatttttctg ttcaagatca aggtaatctt
      481 gattcgcagg tacatggaga acaacaataa agaaaaaatt gtgatagggt ttgatctggg
      541 tgtggcctca gttggttgga gtatcgtgaa tgctgaaaca aaagaggtca tcgatctggg
      601 cgtgcgactg ttcagtgagc ctgagaaggc agattacagg cgggctaagc gaaccacacg
      661 cagactgctg cgaaggaaaa agtttaaaag agagaaattt cacaaactga tactgaaaaa
      721 cgcagagatt ttcggacttc agagtaggaa cgaaattctg aacgtctata aggatcagag
      781 ctctaagtat cgcaacatcc ttaaattgaa aatcaacgcc ttgaaagaag agatcaaacc
      841 cagtgaactg gtttggattc tccgggacta tctgcaaaat cggggatact tctacaaaaa
      901 tgaaaagctg acagacgaat tcgtttccaa tagttttcct agcaagaagc ttcacgagca
      961 ctatgaaaaa tacggattct ttagagggag cgtgaagttg gacaataagc ttgacaataa
     1021 aaaggacaag gccaaggaga aggacgagga agaggagtct gatgccaaaa aagagagtga
     1081 agagctgatc ttcagtaaca aacagtggat taacgagatc gtcaaagtgt tcgagaacca
     1141 gtcttacctc acagagtcct tcaaagaaga gtacttgaaa ctgtttaatt atgtgcgacc
     1201 cttcaataaa ggccccggga gcaagaatag ccgcacagcg tacggagtct ttagcaccga
     1261 tatcgacccc gaaaccaata agtttaaaga ctacagcaat atatgggata aaacaatcgg
     1321 caagtgcagt ttgttcgaag aggagattcg agcccctaag aacctcccgt ccgccctgat
     1381 ctttaacctt cagaacgaga tttgtactat caaaaatgag ttcactgaat tcaaaaactg
     1441 gtggttgaat gcagaacaga aaagcgaaat cctgaaattc gtctttaccg agcttttcaa
     1501 ctggaaggac aagaagtata gcgacaaaaa atttaataag aatctgcaag ataaaattaa
     1561 gaagtacctc ctcaacttcg ctctggagaa ctttaatctg aatgaggaga tacttaagaa
     1621 tcgcgatctg gagaacgata ccgtgctggg cctgaaaggt gtaaagtact acgagaagtc
     1681 aaacgccacc gccgatgccg ccctggagtt ttcctccttg aaacctctct acgtatttat
     1741 aaagtttctc aaggaaaaaa aactggactt gaactatctt cttggcctcg aaaataccga
     1801 gattctttac tttctggact ccatatacct ggctatctct tactccagtg acctcaagga
     1861 acgcaatgag tggtttaaga agctgttgaa ggaactctac ccaaagatca aaaacaacaa
     1921 cctggaaatt atcgagaacg tggaggacat tttcgaaatc acagaccagg agaagtttga
     1981 aagcttttcc aagactcaca gtctgagccg agaggcattt aatcacatca taccactcct
     2041 cctcagcaac aatgagggaa agaattacga gtcactcaag cattccaacg aagagctgaa
     2101 gaagagaact gagaaagcag agttgaaagc ccagcagaat cagaagtatc tgaaagataa
     2161 cttcctgaag gaagcattgg tgcccctgtc cgtcaagact tctgtgctgc aggccattaa
     2221 aatattcaac cagattatca agaattttgg aaagaaatat gagatttccc aagtggtgat
     2281 cgagatggct cgggagctta ccaagcctaa tctcgaaaag ctcctcaaca acgccacgaa
     2341 ttccaacatt aagattctga aggagaagct ggaccagaca gagaagtttg acgactttac
     2401 caagaagaag ttcatagaca aaatcgagaa ctcagtcgtt ttccggaata aactgttttt
     2461 gtggttcgag caggatcgca aagaccccta cacacagctt gacatcaaaa tcaatgagat
     2521 tgaagatgaa accgagatag atcatgtgat cccctactca aaatccgccg acgattcctg
     2581 gttcaacaaa ctgctggtga aaaaaagtac taatcagctt aaaaaaaaca aaacagtgtg
     2641 ggaatactat cagaatgagt ccgacccaga ggcgaaatgg aataagtttg ttgcatgggc
     2701 taaacgaatt tacctggtgc agaaatctga taaggagtcc aaggacaatt ccgaaaagaa
     2761 tagcatattt aagaataaga aaccaaacct gaagtttaag aatatcacca aaaaactttt
     2821 cgatccatac aaagacttgg gcttcctggc cagaaacctg aatgatacac gctacgccac
     2881 taaggttttc agagatcaac tgaacaacta ctctaagcat cattctaagg atgatgagaa
     2941 taagctgttt aaagtggtat gtatgaatgg tagtattacc tcctttctgc gcaaatccat
     3001 gtggaggaaa aacgaagaac aggtgtacag gttcaacttt tggaagaaag accgggacca
     3061 atttttccac catgccgtag acgcctctat tattgccatc ttctcactgc tgaccaagac
     3121 cctttacaat aagttgcgcg tgtacgagag ctatgatgtt cagaggcggg aagacggggt
     3181 ttatttgata aacaaggaaa ctggggaggt taagaaggcc gacaaagact actggaagga
     3241 ccagcacaac tttttgaaaa tcagggaaaa tgccattgaa atcaagaacg tgctgaacaa
     3301 tgtggatttt caaaatcaag tgagatacag tcggaaggcg aacaccaagt tgaacacaca
     3361 gctctttaac gagacgctgt atggcgtaaa agagttcgag aacaatttct acaagctgga
     3421 aaaagtgaac ttgttctcaa gaaaggatct gaggaaattt atactggagg acctcaacga
     3481 agagagtgaa aaaaataaga aaaacgaaaa tggttccaga aaacggatcc tgactgagaa
     3541 gtatatcgtt gacgaaatcc tgcagatact ggaaaatgaa gaatttaaag actctaaatc
     3601 tgacataaat gccctcaata aatacatgga cagccttccc agcaagtttt ccgagttttt
     3661 ttcacaggac ttcattaata aatgcaaaaa ggagaattcc ctgattctga cgttcgacgc
     3721 tataaaacat aacgacccca agaaagtgat caaaatcaag aatcttaagt tcttccgcga
     3781 ggatgccacc cttaagaata aacaagctgt tcacaaggac tcaaagaatc agatcaagtc
     3841 cttctacgag tcctacaagt gcgtggggtt catttggctg aagaataaaa acgatctgga
     3901 ggagagcata ttcgtcccaa ttaactcacg agtgatccac tttggtgaca aggacaagga
     3961 catttttgac tttgactctt ataacaagga aaagttgctc aacgaaatca acctcaagcg
     4021 gcccgaaaat aagaagttta actcaattaa cgaaattgag ttcgttaaat ttgttaaacc
     4081 gggtgcactt cttcttaact ttgagaacca gcaaatttac tatatcagca ccttggaaag
     4141 cagctccctc cgcgcgaaaa tcaagctgct caacaagatg gataaaggga aggctgtgag
     4201 tatgaaaaaa attactaacc ccgatgagta taaaataatt gaacacgtga acccactggg
     4261 aattaatctg aattggacaa agaaattgga gaacaacaat agcagggctg accccaagaa
     4321 gaagaggaag gtgaggtcca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4381 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4441 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4501 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4561 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cgtcttccgc
     4621 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4681 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4741 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4801 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4861 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     4921 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     4981 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5041 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5101 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5161 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5221 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5281 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5341 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5401 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5461 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5521 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcctaatca gtgaggcacc
     5581 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5641 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5701 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5761 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5821 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     5881 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     5941 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6001 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6061 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6121 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6181 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6241 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6301 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6361 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6421 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagtg gatacatatt
     6481 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6541 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     6601 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     6661 ccc
//
