LOCUS       sequence-201721-        5943 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 5943)
  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 5943)
  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-A5EIM8
FEATURES             Location/Qualifiers
     source          1..5943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     243..265
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     257..274
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     258..274
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1451..1462)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3521..3541
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3551..3567)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3551..3567)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3564..3586)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3575..3591
                     /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(3599..3605,3606..3623,3624..3629))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3644..3665
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4003..4591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4083..4102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4762..5553,5554..5622))
                     /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     5385..5404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5623..5727)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5795..5813
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5851..5873)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
       61 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
      121 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
      181 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
      241 ttcccagtca cgacgttgta aaacgacggc cagtgtgctg tctcatcatt ttggcaaaga
      301 attcgcggcc gccaccatga agcggaccag tctgcgcgcg tacaggctgg gagttgatct
      361 cggggctaac tcattggggt ggttcgtagt ctggctggac gatcatggac agcctgaggg
      421 cctgggcccc gggggcgtga ggatcttccc tgatggccgg aatcctcaga gtaagcagag
      481 taacgccgcc ggacgacggc tcgcccgcag cgcccggagg cgccgggacc gctacctgca
      541 gcggaggggc aaactcatgg gcctccttgt aaagcacggc ctgatgcctg cagatgaacc
      601 agccaggaag agactggagt gcctggatcc ttacgggctg cgagcaaagg ctctggacga
      661 agtgctccct ttgcatcacg ttgggcgggc cctgtttcac ctgaaccagc gccggggact
      721 tttcgccaat agagccatcg aacagggcga caaggacgcg tccgccatca aagcagcggc
      781 ggggagactc cagacaagca tgcaagcttg tggggcaaga accctcggag aattcctgaa
      841 ccgacggcat cagctgagag ccaccgttcg ggcccggtca cctgtcggcg gcgacgtgca
      901 ggcccggtat gaattttacc ctacgagagc aatggtggac gccgagttcg aggctatatg
      961 ggccgctcag gccccacatc acccaacaat gactgcggaa gcacacgaca caatcaggga
     1021 agcaattttt agccagcggg ccatgaagcg tccaagtata ggaaaatgca gcctggatcc
     1081 agccacctca caggatgacg tggacggctt ccggtgtgca tggtcccacc ctctcgccca
     1141 gaggtttcgc atctggcaag atgttagaaa tctggcagtg gttgaaactg gtcccacgag
     1201 ttccaggctt gggaaagagg atcaagacaa ggtggctcgg gctttgctgc agaccgacca
     1261 actttccttc gacgaaataa gaggcttgct cggcctcccc tctgacgccc ggtttaatct
     1321 ggaatcagat agaagggatc atctgaaggg cgatgccaca ggtgcaatcc tgagtgcaag
     1381 acggcacttc gggccagcat ggcacgacag gagtctggat aggcagatcg acattgtggc
     1441 cctgctcgaa tctgccctcg atgaagctgc catcattgcc tctttgggta ctacacacag
     1501 cctggatgaa gccgccgctc aaagggcatt gtcagctctg cttcctgatg gttactgtcg
     1561 gctggggctg agggccataa agagggtttt gcctctcatg gaagctggcc ggacatacgc
     1621 cgaggctgcg tccgccgcag gctacgacca cgctctgctg cccggcggca aactgagtcc
     1681 gactggctat ctgccctact acgggcagtg gctgcagaat gacgtcgtgg gaagcgacga
     1741 tgagcgggac accaatgagc gacgctgggg ccggctcccc aaccctacag tgcacattgg
     1801 cattggccag cttcggcgcg tggttaatga gctgatacga tggcatggcc cccctgcaga
     1861 gatcacggtg gagttgacta gggatcttaa acttagtcca cggcgcctgg cagagctgga
     1921 gagagaacag gcagaaaatc agaggaaaaa tgataagcga acctcacttc tccgaaaact
     1981 cgggctgcca gcttccacac ataacctcct gaaactgagg ttgtgggacg agcagggtga
     2041 tgtggccagc gagtgcccgt ataccgggga ggctatcggg ctcgaacggt tggtgtctga
     2101 tgatgtagat attgatcatt tgatcccttt ctccatttca tgggacgata gtgccgcgaa
     2161 caaggtggtg tgcatgagat atgccaatag agagaaggga aatcggaccc cttttgaggc
     2221 ctttggtcat aggcaggggc gtccctacga ctgggccgat attgccgaac gggctgccag
     2281 attgcccagg ggaaagcgat ggaggtttgg gcctggggcc cgagcgcagt tcgaggaact
     2341 gggagacttt caggccagac tgctcaacga gacttcatgg ctggccaggg tagccaagca
     2401 gtacttggcc gcggtgacgc atccgcatcg cattcacgtg ctccccggac gcctcacagc
     2461 actgcttcgg gcaacatggg agctgaatga tcttctgcca gggagtgatg atcgggctgc
     2521 caagtcacgc aaggatcatc gacatcacgc aattgacgca ttggttgctg ccctcactga
     2581 tcaggctctg ctgagacgga tggccaacgc ccacgacgat actagacgca agatcgaggt
     2641 gcttctgcct tggccaactt tccggataga tctggaaacc cgacttaagg ccatgctggt
     2701 ttcccacaaa ccagatcacg gcctgcaggc aagactccac gaggataccg cttatggcac
     2761 tgtggagcac ccggagacag aagacggagc aaacctcgtg tacaggaaga cttttgtgga
     2821 catttcagag aaagagatcg ataggatccg ggatcggcga cttagggacc tggttcgcgc
     2881 tcatgtagct ggagagaggc agcagggaaa aactttgaag gcagcggtat tgagcttcgc
     2941 acagagacga gatatcgctg gccatcccaa tggcatcagg cacgtgcggc tgacgaagtc
     3001 catcaaacca gattacctgg tacctatcag agataaggct ggcagaatct acaagtctta
     3061 taatgccgga gagaacgctt tcgtggacat cttgcaggct gaatctggca ggtggatcgc
     3121 tcgagccaca accgtcttcc aggcgaacca ggctaacgaa tctcacgacg caccagctgc
     3181 gcagccaatt atgagggtct tcaagggaga catgctgagg atcgaccatg caggtgccga
     3241 gaagttcgtc aagatcgtgc gcttgtctcc atcaaacaac ctgctgtatc tcgtggagca
     3301 ccaccaggcc ggggtgttcc agactcgaca cgatgaccct gaagacagtt tccgctggct
     3361 gttcgcgtct ttcgataaac tgagagaatg gaacgctgaa cttgttcgca tcgatacgct
     3421 cggacagccg tggcgaagaa aaaggggttt ggaaactggt tcagaggacg ccacgcgcat
     3481 cggatggacc cgcccaaaaa aatggccaag cagggctgac cccaagaaga agaggaaggt
     3541 gaggtccatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3601 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3661 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3721 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3781 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg tcttccgctt cctcgctcac
     3841 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3901 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3961 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4021 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4081 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4141 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4201 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4261 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4321 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4381 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4441 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4501 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4561 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4621 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4681 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     4741 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4801 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4861 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4921 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4981 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5041 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5101 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5161 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5221 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5281 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5341 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5401 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5461 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5521 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5581 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5641 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     5701 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     5761 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     5821 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5881 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5941 tgt
//
