LOCUS       sequence-268569-        7895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CAG promoter expression plasmid for human codon optimized CjeCas9 
            nuclease with C-terminal NLS (SV40).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7895)
  AUTHORS   Mahendra C, Christie KA, Osuna BA, Pinilla-Redondo R, Kleinstiver 
            BP, Bondy-Denomy J
  TITLE     Broad-spectrum anti-CRISPR proteins facilitate horizontal gene 
            transfer.
  JOURNAL   Nat Microbiol. 2020 Apr;5(4):620-629. doi: 
            10.1038/s41564-020-0692-2. Epub 2020 Mar 26.
  PUBMED    32218510
REFERENCE   2  (bases 1 to 7895)
  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"; doi: 
            "10.1038/s41564-020-0692-2"; journalName: "Nat Microbiol"; date: 
            "2020-04"; volume: "5"; issue: "4"; pages: "620-629"
COMMENT     Sequence Label: pCAG-hCjeCas9-NLS(SV40)-3xFLAG (KAC579)
FEATURES             Location/Qualifiers
     source          1..7895
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1771..1780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1777..4728
                     /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             4735..4755
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4762..4827
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(4917..4936)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4982..5037
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5036..5055)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5398..5414)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5398..5414)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5411..5433)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5422..5438
                     /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(5446..5452,5453..5470,5471..5476))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5491..5512
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5571..5766
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5617..5752
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5772..5906
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5809..5828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5863..5882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5974..5991)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6145..6733)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6225..6244)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6904..7695,7696..7764))
                     /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     7527..7546
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7765..7869)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tctgcagtcg
     1741 acggtaccgc gggcccggga tccaccggtc gccaccatgg ccagaatcct ggccttcgac
     1801 atcggcatca gcagcatcgg ctgggccttc agcgagaacg acgagctgaa ggactgcggc
     1861 gtgcggatct tcaccaaggt ggaaaacccc aagaccggcg agagcctggc cctgcccaga
     1921 aggctggcca gaagcgcccg gaagagactg gccagacgga aggcccggct gaaccacctg
     1981 aagcacctga tcgccaacga gttcaagctg aactacgagg actaccagag cttcgacgag
     2041 tccctggcca aggcctacaa gggcagcctg atcagcccct acgagctgcg gttccgggcc
     2101 ctgaacgagc tgctgagcaa gcaggacttc gccagagtga tcctgcacat tgccaagcgg
     2161 agaggctacg acgacatcaa gaacagcgac gacaaagaga agggcgccat cctgaaggcc
     2221 atcaagcaga acgaggaaaa gctggccaac taccagtccg tgggcgagta cctgtacaaa
     2281 gagtacttcc agaagttcaa agagaacagc aaagaattca ccaacgtgcg gaacaagaaa
     2341 gaaagctacg agcggtgtat cgcccagagc ttcctgaagg atgagctgaa gctgatcttc
     2401 aagaagcaga gagagttcgg cttcagcttc agcaagaaat tcgaggaaga ggtgctgagc
     2461 gtcgccttct acaagagagc cctgaaggac ttcagccacc tcgtgggcaa ctgcagcttc
     2521 ttcaccgacg agaagagagc ccccaagaac agccccctgg ccttcatgtt cgtggccctg
     2581 acccggatca tcaacctgct gaacaatctg aagaacaccg agggcatcct gtacaccaag
     2641 gacgacctga acgccctgct gaatgaggtg ctgaagaacg gcaccctgac ctacaagcag
     2701 accaagaagc tgctgggcct gagcgacgac tacgagttta agggcgagaa gggcacctac
     2761 ttcatcgagt tcaagaagta caaagagttc atcaaggccc tgggcgagca caacctgagc
     2821 caggacgatc tgaatgagat cgccaaggac atcaccctga tcaaggacga gattaagctg
     2881 aagaaggccc tggccaaata cgacctgaat cagaaccaga tcgacagcct gagcaagctg
     2941 gaattcaagg atcacctgaa catcagcttc aaggctctga agctggtcac ccccctgatg
     3001 ctggaaggca agaagtacga cgaggcctgc aacgagctga acctgaaggt ggccatcaac
     3061 gaggacaaga aggacttcct gcccgccttc aacgaaacct actacaagga cgaagtgacc
     3121 aaccccgtgg tgctgcgggc catcaaagaa taccggaagg tgctgaatgc cctgctcaag
     3181 aaatacggca aggtgcacaa gatcaacatc gagctggccc gggaagtggg caagaaccac
     3241 agccagcggg ccaagatcga gaaagagcag aacgaaaact acaaggccaa gaaggacgct
     3301 gagctggaat gcgagaagct gggactgaag atcaacagca agaacatcct gaagctgcgg
     3361 ctgttcaaag aacagaaaga gttctgcgcc tacagcggcg agaagatcaa gatcagcgat
     3421 ctgcaggacg agaagatgct ggaaatcgac cacatctacc cctacagccg gtccttcgac
     3481 gacagctaca tgaacaaggt gctggtgttc accaaacaga accaggaaaa actgaaccag
     3541 acccccttcg aggccttcgg caacgacagc gccaagtggc agaaaatcga ggtgctggcc
     3601 aagaacctgc ccaccaagaa acagaagaga atcctggaca agaattacaa ggacaaagag
     3661 cagaagaact tcaaggaccg gaacctgaac gacacccggt atatcgcccg gctggtgctg
     3721 aactacacaa aggactacct ggatttcctg cccctgtccg acgacgagaa caccaagctg
     3781 aacgataccc agaaaggctc caaggtgcac gtggaagcca agagcggcat gctgaccagc
     3841 gccctgagac acacctgggg cttcagcgcc aaggatcgga acaaccatct gcaccacgcc
     3901 atcgacgccg tgatcattgc ctacgccaac aacagcatcg tgaaggcctt ctccgacttc
     3961 aagaaagaac aggaaagcaa cagcgccgag ctgtacgcca agaagatctc tgagctggac
     4021 tacaagaaca agcggaagtt cttcgagccc ttcagcggct tccggcagaa ggtgctggat
     4081 aagatcgacg agatcttcgt gtccaagccc gagcggaaga agccctctgg cgccctgcac
     4141 gaggaaacct tcagaaaaga ggaagagttc taccagtcct acggcggcaa agaaggcgtg
     4201 ctgaaggccc tcgagctggg caagatcaga aaagtgaacg gcaagatcgt gaagaacggg
     4261 gacatgttcc gggtggacat cttcaagcac aaaaagacca acaagttcta cgccgtgccc
     4321 atctacacca tggacttcgc cctgaaggtg ctgcccaaca aggccgtggc ccggtccaag
     4381 aagggcgaga tcaaggactg gattctgatg gacgagaact acgagttctg ctttagcctg
     4441 tacaaggact ccctgatcct gatccagacc aaggacatgc aggaacccga gttcgtctac
     4501 tacaacgcct tcaccagcag caccgtgtcc ctgatcgtgt ctaagcacga caacaagttc
     4561 gagacactga gcaagaacca gaagatcctg ttcaagaacg ccaacgagaa agaagtgatc
     4621 gccaagagca tcggcatcca gaatctgaag gtgttcgaga agtacatcgt gtccgccctg
     4681 ggagaagtga caaaggccga gttccggcag agagaggact tcaaaaaggg atcccccaag
     4741 aagaagagga aagtctcgag cgactacaaa gaccatgacg gtgattataa agatcatgac
     4801 atcgattaca aggatgacga tgacaagtaa agcggccgca ctcctcaggt gcaggctgcc
     4861 tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatctt
     4921 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     4981 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     5041 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     5101 ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat
     5161 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga
     5221 ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt
     5281 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct
     5341 gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc
     5401 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5461 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5521 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat
     5581 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5641 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5701 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     5761 tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5821 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5881 tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag
     5941 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6001 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6061 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6121 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6181 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6241 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6301 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6361 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6421 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     6481 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     6541 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     6601 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     6661 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     6721 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     6781 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     6841 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6901 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6961 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7021 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7081 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7141 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7201 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7261 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7321 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7381 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7441 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7501 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7561 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7621 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7681 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7741 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7801 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7861 ggttccgcgc acatttcccc gaaaagtgcc acctg
//
