LOCUS       sequence-262486-        8099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Campylobacter jejuni-derived Cas9 nuclease in mammalian 
            cells. Used to modify genome of mouse embroys.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8099)
  AUTHORS   Fujii W, Ikeda A, Sugiura K, Naito K
  TITLE     Efficient Generation of Genome-Modified Mice Using Campylobacter 
            jejuni-Derived CRISPR/Cas.
  JOURNAL   Int J Mol Sci. 2017 Oct 31;18(11). pii: ijms18112286. doi: 
            10.3390/ijms18112286.
  PUBMED    29088065
REFERENCE   2  (bases 1 to 8099)
  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: "Int J Mol 
            Sci."; date: "2017-10-31"; volume: "18(11). pii: ijms18112286. doi";
            pages: " 10.3390/ijms18112286"
COMMENT     Sequence Label: pCAG-T3-CjCAS9-pA
FEATURES             Location/Qualifiers
     source          1..8099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             76..99
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             109..129
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3100..3120
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(3406..3425)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3471..3526
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3525..3544)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3887..3903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3887..3903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3900..3922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3911..3927
                     /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(3935..3941,3942..3959,3960..3965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3980..4001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4060..4255
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4106..4241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4261..4395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4298..4317)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4352..4371
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4463..4480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4634..5222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4714..4733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5393..6184,6185..6253))
                     /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     6016..6035
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6254..6358)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6389..6768
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6770..7026
                     /label=chicken beta-actin promoter
                     /note="contains a 21-bp deletion relative to the standard 
                     chicken beta-actin promoter"
     intron          7027..8043
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     7967..7989
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     8051..8070
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg gccgcgggaa
       61 ttcgattcca ccatggacta taaggacgac gacgataaga taatggcccc taagaagaaa
      121 agaaaggtgg gatccatggc ccgcattctt gctttcgata ttggcatcag ctcaatagga
      181 tgggcatttt ccgagaatga cgaattgaag gactgcggag ttaggatctt cactaaagta
      241 gagaacccta agactggtga atccctggca ctgcccagaa gactggctag gagcgcacgg
      301 aagcgactcg ctcgcagaaa ggcaagactt aatcacctta aacaccttat tgcaaacgag
      361 ttcaagctca attacgaaga ttatcagtct tttgacgaaa gccttgccaa ggcctacaaa
      421 ggtagtctca tcagccccta cgaactcagg ttcagggcac tgaatgaact cctgtccaag
      481 caggatttcg caagagtcat acttcacata gctaaacgga ggggctatga tgacataaag
      541 aactccgacg acaaggaaaa aggggctata cttaaggcaa tcaaacagaa cgaagagaaa
      601 cttgctaact accaatcagt aggagaatac ctgtataagg aatattttca gaagtttaag
      661 gaaaactcca aggaattcac taacgttcga aacaagaagg agtcttatga acgctgcatc
      721 gcacagagtt tcttgaagga tgagctgaaa cttatattca aaaagcaaag ggagttcgga
      781 ttctccttta gcaaaaaatt tgaagaggag gtgttgagcg tcgcttttta caaacgggca
      841 ctcaaagact tttcacatct ggttggcaat tgctctttct ttaccgatga gaaacgcgta
      901 cctaagaata gtccactcgc ttttatgttt gtcgctttga cacgaattat aaacctgctg
      961 aataacctta aaaatacaga gggcatcctg tacacaaagg atgatctcaa tgctctgttg
     1021 aatgaagttc tcaagaacgg tactctgact tataaacaaa caaagaagct gctcggtctg
     1081 tcagacgatt atgagtttaa gggcgaaaag gggacctact ttatcgagtt taagaagtat
     1141 aaagaattca tcaaagccct gggagagcat aacttgtcac aagatgatct taatgagatt
     1201 gcaaaagata taaccctcat aaaggatgag attaaactga aaaaggccct cgccaagtac
     1261 gatcttaatc agaaccaaat tgactcattg agcaagctgg aattcaaaga tcacctgaac
     1321 atttcattta aggccctcaa gcttattaca cccttgatgc tggaaggcaa aaaatatgac
     1381 gaagcttgca acgagctcaa tctgaaagtc gccataaacg aggataaaaa agatttcctt
     1441 cccgcattta acgagaccta ttacaaggat gaagtgacta atcccgtggt tcttcgagca
     1501 ataaaggaat acaggaaagt tcttaatgca ctcctcaaaa aatatggcaa agtacataaa
     1561 attaacatag agctcgcccg agaggtagga aagaatcatt cccaaagggc aaaaatcgaa
     1621 aaagagcaaa atgaaaatta caaggcaaag aaggatgccg agctcgagtg cgaaaaactg
     1681 gggctgaaga tcaattccaa gaacatattg aagctgcgac tcttcaagga acagaaggaa
     1741 ttttgtgctt acagcggaga aaaaataaaa ataagcgatc tgcaagatga aaagatgctc
     1801 gagatcgacc acatctaccc atattcaaga tctttcgacg actcctacat gaataaagtc
     1861 ctcgttttta ccaaacagaa tcaagagaag ctcaaccaga ccccctttga ggccttcggc
     1921 aacgacagtg caaagtggca aaaaatagaa gtcctcgcta aaaatctccc cacaaaaaaa
     1981 caaaagagga tattggacaa gaactataaa gataaggagc aaaaaaactt taaagacagg
     2041 aatcttaacg acactagata catcgctaga ctggtattga attataccaa agactacctg
     2101 gactttcttc cactgagcga cgacgaaaac acaaaactca acgacacaca gaaaggtagt
     2161 aaggtacatg ttgaggccaa aagtggaatg ttgacttcag cccttcgaca tacctggggt
     2221 ttttctgcta aagatcgcaa taaccacctt caccatgcca ttgatgctgt tattattgca
     2281 tacgctaaca actcaatagt taaggctttc tctgatttta agaaagaaca ggaaagcaat
     2341 tctgctgagc tctatgcaaa aaagatcagc gagttggatt acaagaacaa gaggaaattt
     2401 tttgagccct tttccggatt cagacaaaag gtcttggaca aaattgatga gatttttgtg
     2461 tcaaagcctg agagaaagaa gcctagtgga gccctgcacg aggaaacctt taggaaggag
     2521 gaggaatttt atcaaagtta cggtggtaaa gagggtgttc ttaaagcact cgagcttgga
     2581 aagatccgga aggtgaacgg gaagatagtc aaaaatgggg acatgttccg ggtcgatatt
     2641 tttaagcaca agaagacaaa caagttctac ggtgtaccca tatacaccat ggattttgct
     2701 cttaaagtat tgccaaataa ggctgtggct agaagcaaga aaggagaaat caaggactgg
     2761 atactcatgg atgaaaacta cgaattctgc ttctcacttt acaaggactc cctcattctc
     2821 atccaaacca aagatatgca ggaacctgag ttcgtttact ataatgcttt taccagctct
     2881 accgtgtccc ttatcgtctc caagcacgac aataaattcg aaacactgtc taagaatcaa
     2941 aaaattttgt ttaagaatgc taacgagaag gaggtgattg caaagagcat tggcattcaa
     3001 aacctgaaag tgtttgaaaa atacatcgtt tcagcactgg gagaagtaac aaaggctgaa
     3061 tttaggcaaa gagaggactt caagaaagga tccatggctc ctaagaaaaa acggaaggtt
     3121 tgaatcgatg gtacctatgg gcaccaaaga acctgtaaac gttatctttt taaattgaat
     3181 gtgcacaaat aaaagtttgg aaaaraaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3241 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaact
     3301 cgaggtagca tgcaagtgta cggaattcac tcctcaggtg caggctgcct atcagaaggt
     3361 ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt ttccctctgc
     3421 caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa
     3481 atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata
     3541 tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata
     3601 tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa
     3661 acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt
     3721 ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt
     3781 tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct
     3841 cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca tagctgtttc
     3901 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3961 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4021 ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt agtcagcaac
     4081 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4141 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4201 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     4261 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4321 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4381 tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4441 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4501 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4561 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4621 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4681 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4741 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4801 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4861 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4921 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4981 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     5041 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     5101 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     5161 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5221 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5281 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5341 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5401 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5461 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5521 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5581 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5641 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5701 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5761 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5821 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5881 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5941 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6001 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6061 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6121 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6181 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6241 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6301 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6361 catttccccg aaaagtgcca cctgggtcga cattgattat tgactagtta ttaatagtaa
     6421 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     6481 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     6541 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     6601 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     6661 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     6721 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc
     6781 cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta
     6841 tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg gsggsgcgmg
     6901 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
     6961 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
     7021 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
     7081 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
     7141 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
     7201 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
     7261 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
     7321 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     7381 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     7441 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     7501 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     7561 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     7621 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     7681 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     7741 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     7801 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     7861 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     7921 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     7981 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     8041 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcga
//
