LOCUS       sequence-201718-        6189 bp    DNA     circular SYN 15-APR-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 6189)
  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 6189)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-J9E534
FEATURES             Location/Qualifiers
     source          1..6189
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..120
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     314..336
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     328..345
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     329..345
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      382..391
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3838..3858
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3868..3884)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3868..3884)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3881..3903)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3892..3908
                     /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(3916..3922,3923..3940,3941..3946))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3961..3982
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4320..4908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4400..4419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5079..5870,5871..5939))
                     /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     5702..5721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5940..6044)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6112..6130
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(6168..6189,1))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
       61 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
      121 ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
      181 atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      241 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      301 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgtgct gtctcatcat
      361 tttggcaaag aattcgcggc cgccaccatg gcatacaggc tgggactcga tatcggcatc
      421 acaagtgtgg gatgggcagt ggtcgcgttg gagaaggacg agagcggcct caaaccagtt
      481 aggattcagg acctcggcgt acgcattttc gacaaagccg aagatagcaa aaccggagcg
      541 agtctggctc ttcctcgacg agaggcaaga agcgccagaa ggcggaccag acgccgcagg
      601 catcgactgt ggagggttaa acggctgctg gagcagcacg gcattttgtc aatggagcag
      661 attgaagccc tctatgccca gcgcacatcc tcacccgacg tgtacgccct cagagtggcg
      721 gggctcgaca ggtgtctgat tgccgaggag atcgctcgcg tcctgatcca tattgcgcac
      781 cgacggggat tccagagcaa caggaagagc gaaatcaagg atagtgacgc gggcaagctc
      841 ttgaaggctg ttcaggagaa cgagaatctg atgcagagta aggggtacag gaccgtcgct
      901 gaaatgttgg tgagtgaggc caccaagacc gacgccgaag gaaaactcgt tcacgggaag
      961 aagcacggtt acgtgtccaa tgtgagaaac aaggccgggg agtatagaca cacggtgtcc
     1021 aggcaggcca ttgtggacga agtgcgcaaa atctttgccg cacaacgggc ccttgggaat
     1081 gatgtcatgt ccgaagaact cgaggacagc tacctgaaaa tcctgtgttc acagcggaac
     1141 ttcgatgatg gaccgggagg tgacagtccc tatggccacg gaagtgtgtc acctgatggc
     1201 gtccgacagt ctatttacga gcggatggtt ggatcctgca ccttcgagac tggcgagaaa
     1261 agagccccac ggtcctccta cagtttcgag aggttccagc ttctgactaa agtagtgaac
     1321 cttagaatct atcgccaaca agaggatggg ggcaggtatc cctgtgaatt gactcaaaca
     1381 gaaagggccc gcgttatcga ctgcgcctat gaacaaacta agattacgta tgggaaactg
     1441 cgaaagcttc tggacatgaa ggacactgag agtttcgccg gcctcactta tgggctgaac
     1501 cggtcaagga acaaaacgga ggacactgtc tttgtagaga tgaaatttta ccacgaagtg
     1561 cgcaaagccc tgcagcgggc aggcgttttt attcaggact tgagtattga gacactggac
     1621 caaatcgggt ggatactcag cgtttggaaa tcagacgaca accgccggaa aaaactctca
     1681 acactggggc tgtctgacaa tgttattgaa gagcttctgc cattgaatgg atccaagttc
     1741 ggtcatctgt ccctcaaagc catcagaaag atcctgccct ttctcgagga tggatactct
     1801 tacgatgttg cctgtgagct ggcgggttac caatttcagg ggaagaccga atatgttaag
     1861 cagcggttgc tgcctccttt gggggaggga gaagtgacca acccagtagt aagaagggca
     1921 ctgtctcaag ccatcaaagt tgtgaatgct gtcatccgca aacacggtag tcctgaaagc
     1981 atccacattg aattggctag agagctttcc aagaatctcg acgagcgccg gaagattgag
     2041 aaagcccaga aggaaaacca aaaaaacaat gagcagatta aggacgagat tagggagatc
     2101 ctcggatctg cacacgtgac aggacgggat attgttaaat acaagctgtt caagcagcag
     2161 caagaattct gtatgtatag tggcgagaag ctcgatgtca cacgcttgtt cgaacccggt
     2221 tatgcagagg tggatcacat catcccctat ggtatttcct tcgatgatag ttatgataat
     2281 aaggtcctgg tgaagaccga gcagaacagg cagaaaggca atcggacacc cctcgagtat
     2341 ctgcgagata agccggagca gaaagccaag ttcattgcat tggtagaatc aatccctttg
     2401 tcccagaaga aaaagaacca tctgctgatg gataagcgcg ccatcgattt ggagcaagaa
     2461 gggtttagag agagaaacct ttcagacacg cgatatatta ctcgagcgtt gatgaaccac
     2521 attcaggcat ggcttctttt cgatgaaact gcgtctacca gatccaagcg ggtggtttgc
     2581 gttaacggag ccgtgacggc ctacatgcgg gcgcgctggg gattgactaa agaccgagat
     2641 gccggcgata agcatcacgc agccgacgcc gtcgttgtcg cttgtatagg agactccctg
     2701 attcagagag ttaccaagta cgataaattc aagaggaatg ccctggcaga taggaatcgg
     2761 tatgtgcagc aggtttctaa atccgagggc attacacagt acgttgataa agagacaggt
     2821 gaggtgttca catgggaaag cttcgatgag cggaagttcc tgccgaacga gcccctggaa
     2881 ccttggccat tcttccgcga cgaactgctt gctcgactga gcgatgaccc gtctaaaaat
     2941 attcgggcga tcggcttgct cacatactcc gaaacagaac agatagatcc tatctttgtg
     3001 tcccgaatgc ctactagaaa ggttactggc gcagctcata aggagactat ccgaagtccg
     3061 cgcattgtta aggtggacga taataagggc accgagattc aggtggtggt aagcaaggtc
     3121 gctctcacag agctgaagct gacaaaagat ggggagatca aggactactt tagacccgaa
     3181 gacgacccgc gcctgtacaa tacattgaga gagcgcctgg tacagttcgg gggagacgcc
     3241 aaggctgctt tcaaggaacc agtgtacaag atcagcaagg acgggtccgt gcgaactcca
     3301 gtgaggaaag tgaaaattca agaaaaactt acactgggcg tgcctgtgca tggtggtcgc
     3361 ggtatagccg agaatggggg catggtccgc atcgatgttt ttgcaaaggg cggcaaatat
     3421 tactttgtgc ccatttatgt ggccgatgtt ctgaagaggg agttgcctaa tcggttggca
     3481 actgcacata agccatacag tgaatggcgg gtcgtggatg actcatacca attcaagttc
     3541 tccctgtatc ccaacgatgc cgtcatgatc aagcccagca gagaggtaga catcacttac
     3601 aaggaccgaa aggagcctgt gggctgccgg atcatgtact tcgtgtccgc caacatcgcc
     3661 tcagcatcca tctccttgag gacacacgat aattccgggg aattggaggg tttgggaatt
     3721 caaggactgg aggtgttcga aaagtacgtg gtcggaccgc tgggcgatac ccatcccgtc
     3781 tacaaagagc gaaggatgcc gttccgagtt gagcgaaaga tgaacagcag ggctgacccc
     3841 aagaagaaga ggaaggtgag gtccatggtc atagctgttt cctgtgtgaa attgttatcc
     3901 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3961 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4021 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4081 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcgtct
     4141 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4201 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4261 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4321 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4381 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4441 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4501 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4561 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4621 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4681 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4741 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4801 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4861 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4921 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4981 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5041 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5101 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5161 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5221 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5281 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5341 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5401 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5461 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5521 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5581 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5641 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5701 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5761 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5821 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5881 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5941 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6001 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6061 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     6121 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     6181 cagctcccg
//
