LOCUS       sequence-201739-        5814 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 5814)
  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 5814)
  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-B8I085
FEATURES             Location/Qualifiers
     source          1..5814
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..60
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     254..276
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     268..285
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     269..285
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1531..1542)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3403..3423
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3433..3449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3433..3449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3446..3468)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3457..3473
                     /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(3481..3487,3488..3505,3506..3511))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3526..3547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3885..4473)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3965..3984)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4644..5435,5436..5504))
                     /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     5267..5286
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5505..5609)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5677..5695
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5733..5755)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
       61 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
      121 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      181 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      241 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgtgct gtctcatcat
      301 tttggcaaag aattcgcggc cgccaccatg aagtataccc tcggacttga cgtgggaatc
      361 gcttctgttg gatgggcagt aattgacaag gataacaata agatcatcga cttgggcgta
      421 aggtgcttcg ataaagcaga ggaatctaag accggggagt ctctcgctac cgctcgcaga
      481 atagcccgcg gcatgagaag acggatttca agacgctccc aacggcttcg gcttgtgaaa
      541 aaactctttg tgcagtacga gattattaaa gatagcagtg agtttaacag aatattcgac
      601 actagcagag atggatggaa agacccatgg gagctgagat ataacgccct cagccgcata
      661 ctcaagccct atgagcttgt ccaggttctt acccacatta cgaaaagacg aggattcaag
      721 tccaacagaa aggaggatct gtcaacgacg aaggagggag tcgtaataac ttctataaag
      781 aacaactcag aaatgctgag aactaaaaac tatcgaacaa taggagagat gatcttcatg
      841 gaaacccctg aaaattcaaa caagagaaat aaggtggatg agtacattca taccattgcg
      901 agggaagacc tgctgaacga aatcaaatac attttctcta tacagagaaa attggggtcc
      961 cccttcgtga ccgagaagtt ggagcatgat tttctgaaca tctgggaatt ccaacgcccg
     1021 ttcgccagtg gcgacagcat actgtctaag gtgggcaaat gcactttgct caaggaggag
     1081 ctgagagcac ccacctcctg ttatactagt gaatatttcg ggttgctgca gagcattaat
     1141 aatctcgtcc tcgttgagga taataacaca ctgactctga acaacgacca gcgcgccaag
     1201 atcatcgagt acgcgcactt taagaatgaa ataaaatact ctgagatccg caaacttttg
     1261 gatattgagc cagagatact ctttaaagct cataacctga cacacaagaa tccttccgga
     1321 aataacgaga gcaagaagtt ctatgagatg aagtcctatc acaaactgaa atctactctg
     1381 ccaactgata tatggggaaa gttgcacagc aacaaggaat ctcttgacaa cctcttctac
     1441 tgtctgacgg tctataaaaa tgacaacgag ataaaggatt acttgcaagc aaataacctt
     1501 gattacctga tcgagtacat tgcaaaactc cctaccttca ataaatttaa gcacctttcc
     1561 ctggtagcga tgaagaggat aattcccttc atggaaaagg gttataagta ctctgatgcc
     1621 tgtaatatgg cagagctcga tttcacagga tcatctaagc tcgagaagtg caacaaactg
     1681 actgtcgagc caattatcga aaatgtgact aaccctgtcg tgatccgagc cctgactcaa
     1741 gctagaaaag ttattaatgc tattatacag aagtatggtc tgccatatat ggtcaacatt
     1801 gaactggccc gcgaggctgg catgacgcgg caagacagag ataacttgaa gaaagagcac
     1861 gaaaataacc ggaaggcacg cgagaagatc agcgacctta ttaggcaaaa tggtagagta
     1921 gcctcagggt tggacatcct gaaatggcgg ctgtgggagg accagggggg acggtgtgcc
     1981 tactccggga agcctattcc tgtgtgtgat ctcctgaatg attccctgac tcaaattgac
     2041 cacatttacc cctactcacg gagcatggat gactcatata tgaacaaagt gttggttctc
     2101 accgacgaaa atcagaacaa gcgcagttac acaccatatg aggtgtgggg gtctacagaa
     2161 aagtgggagg actttgaagc aagaatttac tccatgcact tgcctcagag taaggaaaag
     2221 cggctcctga accggaactt catcacgaag gatctggaca gctttattag cagaaatctg
     2281 aacgataccc gatatatcag tcgcttcctg aagaattata tcgagagtta tctgcaattc
     2341 tccaacgact cccccaagag ctgtgtggtc tgcgtgaacg gccagtgtac agcccaactg
     2401 cggtccagat ggggactcaa taagaaccga gaagagagcg atttgcatca cgctctcgat
     2461 gccgcagtca tagcctgcgc agatcggaag attatcaaag agattaccaa ttactacaac
     2521 gaaagggaga accacaatta taaagtaaag tatcccctcc cttggcacag ttttaggcag
     2581 gacctgatgg agacactcgc aggtgttttc attagtcgcg cgccccgcag gaagattaca
     2641 ggacccgctc atgacgaaac catacgctct cccaagcatt tcaacaaggg cctgacaagc
     2701 gtaaagattc cactgaccac agtcacactg gagaagctcg aaaccatggt aaagaatact
     2761 aaaggtggga tatccgacaa ggcagtctac aacgtactca agaaccgctt gatcgaacac
     2821 aacaataaac cccttaaagc tttcgcggag aaaatctata agccattgaa aaatggcacc
     2881 aacggcgcca taataagaag cattagggtg gagactccga gctacaccgg agtgttcaga
     2941 aatgagggaa aaggcatctc tgataacagc ctcatggtcc gcgttgacgt gttcaagaag
     3001 aaagacaaat actatttggt ccccatatac gttgctcaca tgatcaaaaa ggaactgcct
     3061 agcaaagcca tcgtgcctct caaacccgaa agccaatggg agcttattga ctccacccac
     3121 gaattcctct tctccctgta ccagaatgac tacctggtaa taaaaaccaa gaaggggatc
     3181 accgagggtt attatcggtc ctgtcataga ggcacaggga gcctgtcttt gatgcctcat
     3241 ttcgccaata acaaaaatgt gaagattgac atcggcgtaa ggacggctat tagcattgaa
     3301 aagtataatg ttgacattct cgggaacaaa agtattgtga agggggaacc ccggaggggc
     3361 atggaaaaat acaattcctt caagtctaat agcagggctg accccaagaa gaagaggaag
     3421 gtgaggtcca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     3481 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     3541 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     3601 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     3661 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cgtcttccgc ttcctcgctc
     3721 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3781 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3841 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3901 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3961 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4021 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4081 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4141 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4201 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4261 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4321 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4381 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4441 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4501 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4561 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4621 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4681 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4741 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4801 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4861 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4921 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4981 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5041 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5101 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5161 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     5221 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     5281 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     5341 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     5401 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     5461 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     5521 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5581 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     5641 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     5701 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     5761 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcg
//
