LOCUS       sequence-209053-        6027 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 6027)
  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 6027)
  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-E3HCA8
FEATURES             Location/Qualifiers
     source          1..6027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2031..2053)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2153..2172
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2366..2388
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2380..2397
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2381..2397
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             5728..5748
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5758..5774)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5758..5774)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5771..5793)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5782..5798
                     /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(5806..5812,5813..5830,5831..5836))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5851..5872
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2041 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2101 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     2161 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     2221 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     2281 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     2341 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgtg
     2401 ctgtctcatc attttggcaa agaattcgcg gccgccacca tgaagtattc tatcggactg
     2461 gacattggga tcgcgtcagt gggttggagc gtcatcaaca aggacaagga gagaatagag
     2521 gacatggggg tgagaatctt ccagaaagca gagaacccga aggatggatc cagccttgca
     2581 tcctcacgcc gcgaaaagag gggatctcgg cgccgaaacc gacggaaaaa gcatcggctg
     2641 gataggatca agaatattct ctgtgagtcc ggccttgtga agaaaaatga aatcgagaag
     2701 atttataaaa atgcgtacct gaaaagcccg tgggagttgc gcgccaagtc tctcgaggcc
     2761 aagatatcca acaaagagat cgctcagatt ctgctccaca ttgccaaaag gcggggcttt
     2821 aaatcctttc ggaaaaccga ccggaacgct gacgatactg gtaagctctt gtctggaata
     2881 caggagaaca agaaaattat ggaagagaaa ggatacctga ccatcggtga tatggtggcg
     2941 aaggatccaa agtttaatac ccacgttcga aataaggccg gctcatacct gttttctttt
     3001 agccgcaaac tgctggaaga cgaggtaagg aagatccagg ccaagcagaa agagctggga
     3061 aacacgcact tcacagacga tgtgctcgaa aaatatattg aagtctttaa tagccagagg
     3121 aactttgacg aaggacctag caaaccctcc ccttactact cagaaatcgg ccagatagcc
     3181 aaaatgatag gcaactgcac ctttgaatct tctgaaaaaa gaaccgcaaa aaatacttgg
     3241 agcggcgaac gattcgtgtt tctgcagaaa ttgaacaact tcagaatcgt gggactgagc
     3301 ggtaaaaggc ccctgaccga agaagaacgg gacatagtgg agaaagaggt gtatctgaag
     3361 aaggaggtgc ggtatgagaa actgaggaaa atcctctacc tcaaggagga ggagcgattc
     3421 ggggacctca actacagcaa agatgaaaag caggacaaaa aaactgaaaa gaccaaattt
     3481 atctcattga tcggcaatta taccatcaaa aaactgaatc tctctgagaa gcttaagagc
     3541 gaaatcgagg aagacaaatc taagttggat aagatcatcg agatactcac ttttaacaag
     3601 tccgacaaaa ccattgaatc caatctgaag aagctcgagc tcagcagaga agatattgaa
     3661 attctcctga gcgaggagtt ttcaggcacc ctgaatttgt ccctgaaggc aattaagaaa
     3721 attctcccct atctcgaaaa gggcctgtca tacaatgagg cgtgcgagaa ggccgattat
     3781 gattataaaa acaacgggat aaagttcaag cgcggtgagc tcctgcccgt cgtcgacaag
     3841 gatctgatcg caaaccctgt cgtgcttaga gctatcagtc aaacaaggaa ggttgttaac
     3901 gccatcatcc ggaagtacgg aacaccgcac accattcatg tcgaggtagc acgggacttg
     3961 gcaaaatcat atgacgaccg gcagactatc attaaggaaa acaagaagcg ggagctggag
     4021 aacgaaaaaa caaagaagtt tattagcgaa gaatttggca ttaagaatgt gaagggcaag
     4081 ctcctgttga aatacagact gtaccaggaa caagagggca ggtgtgccta cagtcggaaa
     4141 gagctgagct tgtctgaggt gattctggat gagagtatga ctgatattga tcacattata
     4201 ccctatagca gatccatgga tgactcctat tctaacaaag tcctggtgct ttctggtgaa
     4261 aaccggaaga aaagcaatct cctgcccaag gaatatttcg acagacaggg gcgcgactgg
     4321 gataccttcg tccttaacgt taaggccatg aagattcacc caaggaaaaa atccaatctc
     4381 ctcaaagaga aattcacacg ggaagataac aaggactgga aatcacgcgc cctcaacgac
     4441 actcgataca ttagtcgctt cgtggctaac tacctggaga acgcactcga gtacagggat
     4501 gatagtccta aaaagagagt gtttatgatc cccggtcagc tgacagccca actccgcgcc
     4561 agatggcgcc tgaataaggt acgcgagaac ggcgatctgc atcatgcact cgatgcggct
     4621 gtggtcgccg taaccgacca aaaagctatc aataacattt caaatatttc ccgctacaag
     4681 gagctcaaga attgcaagga cgtcatccct tccatagagt accatgccga tgaggagact
     4741 ggggaggtgt acttcgaaga ggtgaaagat acacgcttcc caatgccttg gagcgggttt
     4801 gaccttgagc tgcagaagag actggagagc gagaatcctc gcgaggagtt ttataatctt
     4861 ctttccgaca agaggtatct cgggtggttt aactacgaag agggattcat cgaaaaactg
     4921 cgccccgtgt ttgtaagtag gatgcccaac agaggggtga aggggcaggc acatcaggag
     4981 acaattcgct ctagtaaaaa gatctccaac cagatagctg tgagtaagaa accgctcaat
     5041 agcatcaaac tgaaagatct ggagaaaatg cagggccggg acaccgacag gaaactctat
     5101 gaagccctga aaaaccgcct ggaggaatac gacgacaaac ccgagaaagc cttcgctgaa
     5161 ccattctata agcccaccaa ttctggcaag cgcgggcccc tggtccgcgg tataaaggtg
     5221 gaggaaaagc agaatgttgg cgtgtacgtt aatggaggac aggctagcaa cggaagcatg
     5281 gtccgaattg acgtatttcg caagaacggc aagttctaca ccgtccctat atacgtgcac
     5341 cagaccctgc tcaaggaact ccctaaccgg gctatcaatg gcaagccata taaagactgg
     5401 gacctgatcg atggctcctt cgagttcttg tatagcttct atcccaatga tctcattgag
     5461 attgagtttg gaaagtccaa aagcattaaa aacgataata aattgactaa aacagagata
     5521 cccgaagtaa acctgtctga agtgttgggt tattatcggg gcatggacac aagcaccggc
     5581 gccgcaacca tagacactca agatggcaag atccagatgc gaataggaat taagacagtt
     5641 aagaatatta agaaatacca agttgatgta ctgggaaacg tgtataaggt gaagcgggaa
     5701 aaaaggcaga ccttcagcag ggctgacccc aagaagaaga ggaaggtgag gtccatggtc
     5761 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5821 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5881 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5941 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     6001 ctcgctgcgc tcggtcgttc ggctgcg
//
