LOCUS       sequence-201919-        6996 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 6996)
  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 6996)
  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-K1LM04
FEATURES             Location/Qualifiers
     source          1..6996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(9..31)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     131..150
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     344..366
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     358..375
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     359..375
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4675..4695
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4705..4721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4705..4721)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4718..4740)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4729..4745
                     /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(4753..4759,4760..4777,4778..4783))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4798..4819
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5157..5745)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5237..5256)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5916..6707,6708..6776))
                     /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     6539..6558
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6777..6881)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6949..6967
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
       61 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
      121 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata
      181 ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc
      241 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
      301 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
      361 aaaacgacgg ccagtgtgct gtctcatcat tttggcaaag aattcgcggc cgccaccatg
      421 aaacatatcc tggggctcga cctggggact aacagtattg gatgggctct cattgaacgc
      481 aacatcgagg agaagtatgg taagatcatc gggatgggct ctagaattgt gcctatgggc
      541 gccgaactct ccaagttcga gcagggacaa gcacagacga aaaacgccga tagacggact
      601 aaccggggag cccggcggct gaacaagagg tacaagcaac gccgcaataa gcttatatac
      661 atcttgcaga aactggacat gctgccctcc cagatcaaat tgaaggagga tttctccgac
      721 cctaataaga tagacaagat taccattctg ccaatctcaa aaaagcagga acagctcacc
      781 gctttcgacc ttgttagtct cagggtgaaa gccctgactg agaaggtcgg actcgaggat
      841 ctgggaaaga taatttacaa atataaccaa cttcgcggtt acgccggagg ctccttggag
      901 cccgaaaaag aagatatatt cgatgaggag cagtctaaag ataaaaaaaa caaatccttc
      961 atagcctttt ccaaaatagt gttcctggga gaacctcagg aagaaatttt taagaataaa
     1021 aagctcaacc gccgcgccat cattgtcgaa acggaggagg gtaattttga gggcagtacg
     1081 tttctggaga acattaaggt tggcgactca ctcgaattgc tgattaatat ctctgcctct
     1141 aagtccggtg acacaattac cataaaactg cccaacaaaa ccaattggag aaagaaaatg
     1201 gagaacatcg agaaccagct caaagaaaag tccaaagaaa tgggcagaga gttctacatt
     1261 tcagagttcc tgttggaact tctgaaggag aacagatggg ccaaaatccg caacaacact
     1321 attcttaggg cccgatacga gtccgagttt gaggcaatat ggaacgagca ggtgaagcac
     1381 tatcccttcc tggagaatct cgacaagaaa actctgatcg agatcgtgtc ctttatattc
     1441 ccaggcgaaa aggagagtca aaaaaaatat agggaactgg gattggagaa aggcttgaag
     1501 tatatcatca aaaaccaagt ggtattctac cagcgggagc ttaaggacca aagccacctc
     1561 attagtgatt gccggtacga gccgaatgag aaagcgattg ctaagtctca tcccgtgttt
     1621 caggaatata aagtgtggga acaaataaat aagctgatcg tgaataccaa aattgaggcc
     1681 ggcacaaacc ggaaaggcga gaaaaagtac aaatacattg atagacctat cccaaccgcc
     1741 cttaaagagt ggatatttga ggagctccag aataagaaag agatcacctt ttcagccatt
     1801 ttcaagaaac tcaaggcaga gttcgatctg cgagaaggta ttgattttct caatggcatg
     1861 agccctaagg acaagcttaa aggtaacgag actaaactgc agttgcagaa gtccctcggg
     1921 gaactgtggg atgtcttggg tctggacagc attaatagac agattgaatt gtggaatatt
     1981 ctctacaacg agaagggcaa cgagtatgat ctgacttcag atcggactag caaggttctg
     2041 gagtttataa acaagtatgg taataatatt gtcgacgata acgctgaaga aaccgcaatt
     2101 cgaatctcaa agattaagtt cgcacgggcc tatagttccc tcagcctcaa agctgtcgaa
     2161 cgaattctgc cattggtgcg ggcgggaaag tattttaata acgatttttc ccagcagctg
     2221 caatctaaaa ttctgaagct ccttaatgag aacgtggagg atccttttgc caaggccgct
     2281 cagacgtacc ttgataacaa tcagtcagtg ctgagcgagg gaggtgttgg caactccatc
     2341 gccacaatcc tggtatatga caagcacact gctaaggaat acagtcatga tgaactgtat
     2401 aagtcttaca aggagattaa cctcctcaag cagggcgatc tcaggaaccc cctcgtggag
     2461 cagatcatta acgaagccct cgtgttgatc agagatatct ggaaaaacta tgggataaag
     2521 ccgaacgaga ttagggtcga gctggccaga gatctcaaga attcagctaa ggagagagcc
     2581 actattcaca aacggaacaa ggataatcag actatcaata acaaaatcaa ggagactctc
     2641 gtaaaaaata agaaagagct gtcactggca aatatcgaga aggtcaaact gtgggaagcc
     2701 cagcggcatc tgtcccctta tacagggcag cccatccccc tttctgattt gttcgacaaa
     2761 gagaaatatg acgtagacca catcatacct atctctaggt atttcgatga ttcatttacc
     2821 aataaagtga tatccgaaaa gtccgtaaac caggagaaag cgaatcgcac cgctatggag
     2881 tattttgagg tgggaagcct taaatacagc atttttacga aagagcaatt catcgctcac
     2941 gtcaatgagt atttctccgg ggtgaaaagg aaaaatctcc tcgcaacttc aattccagag
     3001 gatcccgtgc agcgacagat aaaggacacc caatatattg cgattcgcgt taaagaagaa
     3061 ctgaataaga ttgtgggcaa cgagaatgtc aagacaacca ctggttcaat caccgactat
     3121 cttcggaatc attggggtct gactgataag tttaagctcc tgctcaagga acgatacgag
     3181 gccttgctgg agagtgagaa gtttctcgag gctgagtacg acaactacaa gaaggacttc
     3241 gactcccgca aaaaggaata tgaagaaaag gaggtgctct ttgaagaaca ggagctgaca
     3301 agagaagaat tcattaagga atataaagag aactatatcc ggtataaaaa gaacaaactg
     3361 attattaagg gctggagcaa acgaattgat catcggcacc acgccattga tgcgctcatc
     3421 gttgcatgca ccgaacccgc gcacatcaag aggctcaatg acctgaataa ggtcctgcag
     3481 gattggctcg ttgagcacaa atccgagttc atgcccaatt tcgagggatc caacagtgag
     3541 ctccttgaag aaattctgag tttgcctgag aacgaaagaa ccgagatttt cacccagata
     3601 gagaagttcc gggccataga aatgccgtgg aaggggtttc cagagcaggt ggaacagaag
     3661 ctcaaggaaa ttatcatttc tcataaaccc aaagataagc tgttgcttca gtacaataag
     3721 gctggggacc gacaaattaa actgagaggg cagctgcacg aagggaccct ctatgggatt
     3781 tcccagggta aagaagcata tagaatacct ctgaccaagt tcggcggcag caagtttgca
     3841 acagaaaaaa acattcagaa gatcgtgagt ccttttctgt caggattcat tgcgaatcac
     3901 ctgaaagagt ataacaacaa aaaggaggaa gcgttctctg ccgagggaat catggatctt
     3961 aacaacaagc tcgctcagta ccggaatgaa aagggcgaac tgaagcctca tacccctatt
     4021 tccaccgtga agatttatta taaagaccca tcaaaaaata aaaagaaaaa ggatgaggag
     4081 gatctgagtc tgcagaagct ggacagagag aaagccttca atgaaaaact ttacgtgaag
     4141 accggcgaca actatctttt cgccgtgctc gagggcgaaa tcaaaactaa aaagacttca
     4201 cagataaaga gactttatga tatcatctct ttcttcgacg ctacaaattt tctgaaggag
     4261 gagtttagga acgctccaga caagaagaca tttgataaag atctgctgtt caggcagtac
     4321 ttcgaggaac ggaataaggc caaactgctg ttcactctga agcagggaga cttcgtttat
     4381 cttccaaatg aaaatgagga ggtgattttg gacaaggagt cacctctcta caaccagtac
     4441 tggggagatc tcaaagagag gggaaagaac atctacgtgg ttcagaagtt ttccaagaag
     4501 cagatttatt ttattaagca cacgattgca gacatcatta aaaaggacgt tgagttcggg
     4561 tcccagaatt gctatgaaac cgttgagggc cgatccatca aggagaactg ctttaagctc
     4621 gagatcgacc gcttgggcaa cattgtgaag gtgataaaga gaagcagggc tgaccccaag
     4681 aagaagagga aggtgaggtc catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4741 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4801 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4921 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4981 ggtatctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5041 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5101 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5161 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5221 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5281 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5341 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5401 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5461 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5521 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5581 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5641 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5701 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5761 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5821 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5881 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5941 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     6001 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     6061 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     6121 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     6181 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     6241 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     6301 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     6361 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6421 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6481 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6541 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6601 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6661 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6721 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6781 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6841 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6901 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     6961 acgaggccct ttcgtctcgc gcgtttcggt gatgac
//
