LOCUS       sequence-201807-        6846 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 6846)
  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 6846)
  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-D6GRK4
FEATURES             Location/Qualifiers
     source          1..6846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     252..274
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     266..283
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     267..283
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             4433..4453
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4463..4479)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4463..4479)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4476..4498)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4487..4503
                     /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(4511..4517,4518..4535,4536..4541))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4556..4577
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4915..5503)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4995..5014)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5674..6465,6466..6534))
                     /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     6297..6316
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6535..6639)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6707..6725
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6763..6785)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
       61 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
      121 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
      181 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
      241 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgtgctgt ctcatcattt
      301 tggcaaagaa ttcgcggccg ccaccatgac aaaagaatac tatttgggtt tggacgtggg
      361 aacaaacagc gtagggtggg ccgtgactga tagccagtat aacctttgca aatttaagaa
      421 gaaagacatg tggggcattc ggttgttcga atcagccaac acagcaaaag acagaagact
      481 gcaaaggggc aaccgaaggc gcctggagag gaagaaacaa agaattgact tgctccagga
      541 aatcttctcc cccgaaattt gcaagatcga ccctactttt ttcattcgcc tcaatgagtc
      601 ccgccttcat ctggaggaca aaagcaacga ttttaagtac cctctcttca ttgagaaaga
      661 ctactccgac atagagtact ataaggaatt cccaacaata ttccatctgc ggaagcacct
      721 catagaatcc gaagaaaagc aggacatccg cctgatttat ctggctctcc ataatatcat
      781 taaaacacgc ggtcacttcc tgatcgacgg cgacttgcag tccgcgaaac agctccgacc
      841 tattcttgat actttcctgc tgtccctgca ggaagagcaa aacctcagcg tgagcctcag
      901 cgagaaccag aaggacgaat acgaggagat cctgaaaaac agaagcatcg ccaaatcaga
      961 aaaggtgaag aagctgaaaa atttgttcga aatttccgac gagttggaaa aggaggaaaa
     1021 gaaggcgcag agtgccgtca tcgagaattt ttgcaagttt atagtgggga acaaaggaga
     1081 tgtctgcaag ttcctgaggg tcagtaagga ggagcttgaa atcgactctt tttcattttc
     1141 agagggaaag tacgaggatg acattgtgaa gaaccttgaa gaaaaagtgc cagagaaggt
     1201 gtacctcttc gagcagatga aagcgatgta cgactggaac atcctggtgg atattctgga
     1261 aaccgaggaa tatatctcct ttgccaaagt gaagcagtat gagaagcata agaccaacct
     1321 ccgcctcctc agggatatca ttcttaagta ttgtacgaag gatgaatata accggatgtt
     1381 caacgacgag aaggaggctg gctcttacac ggcatacgtg gggaaactga agaaaaacaa
     1441 taagaaatac tggattgaaa agaagcgcaa ccctgaagag ttctataaat ctctggggaa
     1501 gctgttggac aagatcgagc ccctgaagga ggatctggaa gtgctgacaa tgatgataga
     1561 agagtgcaag aatcacaccc tgctgcccat ccagaagaat aaggacaacg gggtcatccc
     1621 gcatcaggtt cacgaagtcg agcttaagaa aattctggag aacgcaaaaa agtactactc
     1681 ttttcttact gaaacagata aagacggata ctcagttgtc cagaaaatcg agtccatctt
     1741 tcggttccgg atcccttact atgttggccc tctgagcacc aggcaccaag agaagggtag
     1801 taatgtttgg atggtgcgca agcctgggag ggaggaccgc atctacccgt ggaacatgga
     1861 agagatcatt gacttcgaga agtccaacga gaacttcatt accagaatga caaacaagtg
     1921 cacatacctt attggcgagg acgtcttgcc aaagcatagc cttctgtact ccaaatacat
     1981 ggtgctgaac gagctgaata atgtgaaggt gcggggaaaa aaattgccca ctagtctgaa
     2041 acagaaagtc ttcgaagacc tgtttgagaa caaatcaaaa gtgacgggga aaaacctctt
     2101 ggaatatctg cagattcagg ataaggacat ccagatcgac gacttgagtg gctttgataa
     2161 ggacttcaag accagcctga agtcatatct tgatttcaag aaacagatct ttggagagga
     2221 aattgaaaaa gaatcaatac agaatatgat cgaggatata atcaaatgga tcactatcta
     2281 cggtaatgat aaagaaatgt tgaagcgcgt aattcgcgca aattacagca accaattgac
     2341 tgaggaacaa atgaaaaaga tcactggttt ccaatatagt gggtggggta acttctctaa
     2401 gatgtttctt aaggggatct ctggtagcga tgtgtcaact ggtgaaacct ttgacatcat
     2461 cactgccatg tgggaaaccg ataacaacct catgcaaatc cttagcaaga aatttacgtt
     2521 catggataac gtggaggact tcaactccgg aaaagtgggc aaaattgaca agattactta
     2581 tgactccacc gtgaaagaaa tgtttctttc ccctgagaat aaaagggcag tgtggcaaac
     2641 catccaagtg gccgaggaaa tcaaaaaggt gatgggctgc gaaccaaaaa agatctttat
     2701 tgagatggca aggggtggag aaaaggtgaa gaagcggacc aaatctagga aggcccaact
     2761 gctcgaactg tatgctgcgt gcgaggaaga ctgtagagag ctgatcaaag agattgagga
     2821 ccgggatgag cgcgatttca acagcatgaa attgtttctc tactatacac aatttggaaa
     2881 gtgcatgtat agcggggacg atattgatat caatgagctc atccggggaa atagtaagtg
     2941 ggatcgcgac cacatttacc cccaatctaa aattaaagac gactctattg acaacctggt
     3001 gctcgttaat aaaacctata atgcaaagaa gtccaacgag cttctgtctg aagatattca
     3061 gaagaagatg cactctttct ggcttagtct gctgaataag aaactgatta ccaagtctaa
     3121 gtacgacagg ctgactagga aaggggactt cacagacgag gagcttagtg ggtttattgc
     3181 tcggcagctc gtagagacga ggcagtctac caaagccatt gcagatattt tcaaacagat
     3241 ttacagtagc gaggtggtgt acgtgaagag ttccttggtc agtgatttta ggaagaaacc
     3301 tctgaactac cttaagagta ggagagtcaa cgactatcac catgccaagg atgcatacct
     3361 taatattgtg gtcggtaacg tttataacaa gaagtttact agcaacccta ttcaatggat
     3421 gaaaaagaat cgcgacacca actatagcct caataaggtt tttgagcacg atgtggtgat
     3481 caacggcgaa gtgatatggg aaaaatgtac atatcacgag gacaccaata catatgatgg
     3541 aggaacgctg gatcggatta ggaaaatcgt ggaacgggac aatatcctgt atactgagta
     3601 tgcctattgc gagaaaggcg aattgttcaa cgcaaccata cagaataaga atggaaactc
     3661 aacagttagc ctgaagaagg gtttggatgt gaagaagtat ggcggctatt ttagtgctaa
     3721 tacgtcttat ttcagtctca ttgaattcga agataaaaag ggagatcgcg cacgacacat
     3781 tatcggcgtt cctatctata tcgcaaacat gctggagcac tctccctcag ccttcctgga
     3841 gtactgtgag caaaaaggtt atcaaaatgt gcggatcctc gtggagaaaa tcaagaagaa
     3901 ttctcttctg attatcaacg gatatcccct gcgcataaga ggagagaacg aagtggatac
     3961 gtcatttaaa cgggcgattc agctgaaact cgatcagaag aactacgagc tcgtgagaaa
     4021 tattgaaaag ttcctcgaga agtacgttga aaagaaaggt aattatccta tcgatgagaa
     4081 ccgcgatcac attacgcacg agaaaatgaa tcaactgtat gaagtactgc tctccaaaat
     4141 gaagaagttc aataaaaagg gcatggccga tccctcagac cgaattgaga aatccaagcc
     4201 aaagtttatt aagttggagg atctgatcga caaaatcaac gtgataaaca aaatgctgaa
     4261 tttgctccgc tgtgacaatg atactaaagc cgacttgtca cttattgagc ttcccaaaaa
     4321 cgccggctca tttgttgtca aaaagaatac gatcggtaag tccaagatta tcctggttaa
     4381 ccagagcgtg acagggttgt acgagaaccg gagggagctg agcagggctg accccaagaa
     4441 gaagaggaag gtgaggtcca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4501 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4561 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4621 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4681 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cgtcttccgc
     4741 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     4801 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     4861 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     4921 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     4981 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5041 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     5101 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     5161 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     5221 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     5281 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     5341 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     5401 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     5461 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     5521 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     5581 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     5641 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     5701 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     5761 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     5821 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     5881 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     5941 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6001 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6061 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     6121 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     6181 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     6241 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     6301 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     6361 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     6421 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     6481 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     6541 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     6601 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     6661 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     6721 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     6781 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     6841 cgcgtc
//
