LOCUS       sequence-178965-        6231 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CjCas9 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6231)
  AUTHORS   Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, Song DW, Lee KJ, Jung 
            MH, Kim S, Kim JH, Kim JH, Kim JS
  TITLE     In vivo genome editing with a small Cas9 orthologue derived from 
            Campylobacter jejuni.
  JOURNAL   Nat Commun. 2017 Feb 21;8:14500. doi: 10.1038/ncomms14500.
  PUBMED    28220790
REFERENCE   2  (bases 1 to 6231)
  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: "Nat 
            Commun."; date: "2017-02-21"; volume: "8:14500. doi"; pages: " 
            10.1038/ncomms14500"
COMMENT     Sequence Label: pRGEN-CMV-CjCas9
FEATURES             Location/Qualifiers
     source          1..6231
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             7..33
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             7..33
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(67..85)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(68..85)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(105..122)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    111..335
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(423..440)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(594..1182)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(674..693)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1353..2144,2145..2213))
                     /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     1976..1995
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2214..2318)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(2393..2412)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2584..2963
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2964..3167
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3118..3138
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3212..3231
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3212..3230
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3256..6207
                     /codon_start=1
                     /product="Cas9 endonuclease from the Campylobacter jejuni 
                     Type II CRISPR/Cas system (Kim et al., 2016)"
                     /label=CjCas9
                     /translation="MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLA
                     LPRRLARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYEL
                     RFRALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVG
                     EYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKF
                     EEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNT
                     EGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIK
                     ALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFK
                     ALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKE
                     YRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGL
                     KINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVL
                     VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDR
                     NLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTW
                     GFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKR
                     KFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKAL
                     ELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGE
                     IKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFET
                     LSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK"
     CDS             join(6217..6231,1..6)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 aaagtatacc cctacgacgt gcccgactac gcctaataac tcgagcatgc atctagaggg
       61 ccctattcta tagtgtcacc taaatgctag agctcgctga tcagcctcga ctgtgccttc
      121 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
      181 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
      241 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
      301 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
      361 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
      421 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      481 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      541 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      601 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      661 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      721 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      781 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      841 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      901 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      961 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1021 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1081 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1141 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1201 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1261 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1321 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1381 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1441 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1501 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1561 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1621 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1681 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1741 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     1801 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     1861 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     1921 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     1981 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2041 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2101 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2161 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2221 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     2281 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     2341 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     2401 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     2461 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
     2521 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
     2581 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     2641 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     2701 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     2761 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     2821 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     2881 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     2941 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     3001 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     3061 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     3121 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
     3181 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
     3241 cttgggatcc gaaccatggc cagaatcctg gccttcgaca tcggcatcag cagcatcggc
     3301 tgggccttca gcgagaacga cgagctgaag gactgcggcg tgcggatctt caccaaggtg
     3361 gaaaacccca agaccggcga gagcctggcc ctgcccagaa ggctggccag aagcgcccgg
     3421 aagagactgg ccagacggaa ggcccggctg aaccacctga agcacctgat cgccaacgag
     3481 ttcaagctga actacgagga ctaccagagc ttcgacgagt ccctggccaa ggcctacaag
     3541 ggcagcctga tcagccccta cgagctgcgg ttccgggccc tgaacgagct gctgagcaag
     3601 caggacttcg ccagagtgat cctgcacatt gccaagcgga gaggctacga cgacatcaag
     3661 aacagcgacg acaaagagaa gggcgccatc ctgaaggcca tcaagcagaa cgaggaaaag
     3721 ctggccaact accagtccgt gggcgagtac ctgtacaaag agtacttcca gaagttcaaa
     3781 gagaacagca aagaattcac caacgtgcgg aacaagaaag aaagctacga gcggtgtatc
     3841 gcccagagct tcctgaagga tgagctgaag ctgatcttca agaagcagag agagttcggc
     3901 ttcagcttca gcaagaaatt cgaggaagag gtgctgagcg tcgccttcta caagagagcc
     3961 ctgaaggact tcagccacct cgtgggcaac tgcagcttct tcaccgacga gaagagagcc
     4021 cccaagaaca gccccctggc cttcatgttc gtggccctga cccggatcat caacctgctg
     4081 aacaatctga agaacaccga gggcatcctg tacaccaagg acgacctgaa cgccctgctg
     4141 aatgaggtgc tgaagaacgg caccctgacc tacaagcaga ccaagaagct gctgggcctg
     4201 agcgacgact acgagtttaa gggcgagaag ggcacctact tcatcgagtt caagaagtac
     4261 aaagagttca tcaaggccct gggcgagcac aacctgagcc aggacgatct gaatgagatc
     4321 gccaaggaca tcaccctgat caaggacgag attaagctga agaaggccct ggccaaatac
     4381 gacctgaatc agaaccagat cgacagcctg agcaagctgg aattcaagga tcacctgaac
     4441 atcagcttca aggctctgaa gctggtcacc cccctgatgc tggaaggcaa gaagtacgac
     4501 gaggcctgca acgagctgaa cctgaaggtg gccatcaacg aggacaagaa ggacttcctg
     4561 cccgccttca acgaaaccta ctacaaggac gaagtgacca accccgtggt gctgcgggcc
     4621 atcaaagaat accggaaggt gctgaatgcc ctgctcaaga aatacggcaa ggtgcacaag
     4681 atcaacatcg agctggcccg ggaagtgggc aagaaccaca gccagcgggc caagatcgag
     4741 aaagagcaga acgaaaacta caaggccaag aaggacgctg agctggaatg cgagaagctg
     4801 ggactgaaga tcaacagcaa gaacatcctg aagctgcggc tgttcaaaga acagaaagag
     4861 ttctgcgcct acagcggcga gaagatcaag atcagcgatc tgcaggacga gaagatgctg
     4921 gaaatcgacc acatctaccc ctacagccgg tccttcgacg acagctacat gaacaaggtg
     4981 ctggtgttca ccaaacagaa ccaggaaaaa ctgaaccaga cccccttcga ggccttcggc
     5041 aacgacagcg ccaagtggca gaaaatcgag gtgctggcca agaacctgcc caccaagaaa
     5101 cagaagagaa tcctggacaa gaattacaag gacaaagagc agaagaactt caaggaccgg
     5161 aacctgaacg acacccggta tatcgcccgg ctggtgctga actacacaaa ggactacctg
     5221 gatttcctgc ccctgtccga cgacgagaac accaagctga acgataccca gaaaggctcc
     5281 aaggtgcacg tggaagccaa gagcggcatg ctgaccagcg ccctgagaca cacctggggc
     5341 ttcagcgcca aggatcggaa caaccatctg caccacgcca tcgacgccgt gatcattgcc
     5401 tacgccaaca acagcatcgt gaaggccttc tccgacttca agaaagaaca ggaaagcaac
     5461 agcgccgagc tgtacgccaa gaagatctct gagctggact acaagaacaa gcggaagttc
     5521 ttcgagccct tcagcggctt ccggcagaag gtgctggata agatcgacga gatcttcgtg
     5581 tccaagcccg agcggaagaa gccctctggc gccctgcacg aggaaacctt cagaaaagag
     5641 gaagagttct accagtccta cggcggcaaa gaaggcgtgc tgaaggccct cgagctgggc
     5701 aagatcagaa aagtgaacgg caagatcgtg aagaacgggg acatgttccg ggtggacatc
     5761 ttcaagcaca aaaagaccaa caagttctac gccgtgccca tctacaccat ggacttcgcc
     5821 ctgaaggtgc tgcccaacaa ggccgtggcc cggtccaaga agggcgagat caaggactgg
     5881 attctgatgg acgagaacta cgagttctgc tttagcctgt acaaggactc cctgatcctg
     5941 atccagacca aggacatgca ggaacccgag ttcgtctact acaacgcctt caccagcagc
     6001 accgtgtccc tgatcgtgtc taagcacgac aacaagttcg agacactgag caagaaccag
     6061 aagatcctgt tcaagaacgc caacgagaaa gaagtgatcg ccaagagcat cggcatccag
     6121 aatctgaagg tgttcgagaa gtacatcgtg tccgccctgg gagaagtgac aaaggccgag
     6181 ttccggcaga gagaggactt caaaaagtcc ggacctccaa agaaaaagag a
//
