LOCUS       sequence-217972-        7383 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sniper-Cas9 with CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7383)
  AUTHORS   Lee JK, Jeong E, Lee J, Jung M, Shin E, Kim YH, Lee K, Jung I, Kim 
            D, Kim S, Kim JS
  TITLE     Directed evolution of CRISPR-Cas9 to increase its specificity.
  JOURNAL   Nat Commun. 2018 Aug 6;9(1):3048. doi: 10.1038/s41467-018-05477-x.
  PUBMED    30082838
REFERENCE   2  (bases 1 to 7383)
  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: "2018-08-6"; volume: "9(1):3048. doi"; pages: " 
            10.1038/s41467-018-05477-x"
COMMENT     Sequence Label: p3s-Sniper-Cas9
FEATURES             Location/Qualifiers
     source          1..7383
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      895..904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5020..5040
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5041..5067
                     /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             5041..5067
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        complement(5101..5119)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5102..5119)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5139..5156)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5145..5369
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5457..5474)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5628..6216)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5708..5727)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6387..7178,7179..7247))
                     /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     7010..7029
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7248..7352)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgccacc
      901 atggacaaga agtacagcat cggcctggac atcggtacca acagcgtggg ctgggccgtg
      961 atcaccgacg agtacaaggt gcccagcaag aagttcaagg tgctgggcaa caccgaccgc
     1021 cacagcatca agaagaacct gatcggcgcc ctgctgttcg acagcggcga gaccgccgag
     1081 gccacccgcc tgaagcgcac cgcccgccgc cgctacaccc gccgcaagaa ccgcatctgc
     1141 tacctgcagg agatcttcag caacgagatg gccaaggtgg acgacagctt cttccaccgc
     1201 ctggaggaga gcttcctggt ggaggaggac aagaagcacg agcgccaccc catcttcggc
     1261 aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgcgcaag
     1321 aagctggtgg acagcaccga caaggccgac ctgcgcctga tctacctggc cctggcccac
     1381 atgatcaagt tccgcggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac
     1441 gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggagaacccc
     1501 atcaacgcca gcggcgtgga cgccaaggcc atcctgagcg cccgcctgag caagagccgc
     1561 cgcctggaga acctgatcgc ccagctgccc ggcgagaaga agaacggcct gttcggcaac
     1621 ctgatcgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag
     1681 gacgccaagc tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc
     1741 cagatcggcg accagtacgc cgacctgttc ctggccgcca agaacctgag cgacgccatc
     1801 ctgctgagcg acatcctgcg cgtgaacacc gagatcacca aggcccccct gagcgccagc
     1861 atgatcaagc gctacgacga gcaccaccag gacctgaccc tgctgaaggc cctggtgcgc
     1921 cagcagctgc ccgagaagta caaggagatc ttcttcgacc agagcaagaa cggctacgcc
     1981 ggctacatcg acggcggcgc cagccaggag gagttctaca agttcatcaa gcccatcctg
     2041 gagaagatgg acggcaccga ggagctgctg gtgaagctga accgcgagga cctgctgcgc
     2101 aagcagcgca ccttcgacaa cggcagcatc ccccaccaga tccacctggg cgagctgcac
     2161 gccatcctgc gccgccagga ggacttctac cccttcctga aggacaaccg cgagaagatc
     2221 gagaagatcc tgaccttccg catcccctac tacgtgggcc ccctggcccg cggcaacagc
     2281 cgcttcgcct ggatgacccg caagagcgag gagaccatca ccccctggaa cttcgaggag
     2341 gtggtggaca agggcgccag cgcccagagc ttcatcgagc gcatgaccaa cttcgacaag
     2401 aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
     2461 tacaacgagc tgaccaaggt gaagtacgtg accgagggca tgcgcaagcc cgcctccctg
     2521 agcggcgagc agaagaaggc catcgtggac ctgctgttca agaccaaccg caaggtgacc
     2581 gtgaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgacag cgtggagatc
     2641 agcggcgtgg aggaccgctt caacgccagc ctgggcacct accacgacct gctgaagatc
     2701 atcaaggaca aggacttcct ggacaacgag gagaacgagg acatcctgga ggacatcgtg
     2761 ctgaccctga ccctgttcga ggaccgcgag atgatcgagg agcgcctgaa gacctacgcc
     2821 cacctgttcg acgacaaggt gatgaagcag ctgaagcgcc gccgctacac cggctggggc
     2881 cgcctgagcc gcaagcttat caacggcatc cgcgacaagc agagcggcaa gaccatcctg
     2941 gacttcctga agagcgacgg cttcgccaac cgcaacttca tgcagctgat ccacgacgac
     3001 agcctgacct tcaaggagga catccagaag gcccaggtga gcggccaggg cgacagcctg
     3061 cacgagcaca tcgccaacct ggccggcagc cccgccatca agaagggcat cctgcagacc
     3121 gtgaaggtgg tggacgagct ggtgaaggtg atgggccgcc acaagcccga gaacatcgtg
     3181 atcgagatag cccgcgagaa ccagaccacc cagaagggcc agaagaacag ccgcgagcgc
     3241 atgaagcgca tcgaggaggg catcaaggag ctgggcagcc agatcctgaa ggagcacccc
     3301 gtggagaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaacggccgc
     3361 gacatgtacg tggaccagga gctggacatc aaccgcctga gcgactacga cgtggaccac
     3421 atcgtgcccc agagcttcct gaaggacgac agcatcgaca acaaggtgct gacccgcagc
     3481 gacaagaacc gcggcaagag cgacaacgtg cccagcgagg aggtggtgaa gaagatgaag
     3541 aactactggc gccagctgct gaacgccaat ctgatcaccc agcgcaagtt cgacaacctg
     3601 accaaggccg agcgcggcgg cctgagcgag ctggacaagg ccggcttcat caagcgccag
     3661 ctggtggaga cccgccagat caccaagcac gtggcccaga tcctggacag ccgcatgaac
     3721 accaagtacg acgagaacga caagctgatc cgcgaggtga aggtgatcac cctgaagagc
     3781 aagctggtga gcgacttccg caaggacttc cagttctaca aggtgcgcga gatcaacaac
     3841 taccaccacg cccacgacgc ctacctgaac gccgtggtgg gcaccgccct gatcaagaag
     3901 taccccaagc tggagagcga gttcgtgtac ggcgactaca aggtatacga cgtgcgcaag
     3961 atgatcgcta agagcgagca ggagatcggc aaggccaccg ccaagtactt cttctacagc
     4021 aacatcatga acttcttcaa gaccgagatc accctggcca acggcgagat ccgcaagcgc
     4081 cccctgatcg agaccaacgg cgagaccggc gagatcgtgt gggacaaggg ccgcgacttc
     4141 gccaccgtgc gcaaggtgct gagcatgccc caggtgaaca tcgtgaagaa gaccgaggtg
     4201 cagaccggcg gcttcagcaa ggagagcatc ctgcccaagc gcaacagcga caagctgatc
     4261 gcccgcaaga aggactggga ccccaagaag tacggcggct tcgacagccc caccgtggcc
     4321 tacagcgtgc tggtggtggc caaggtggag aagggcaaga gcaagaagct gaagagcgtg
     4381 aaggagctgc tgggcatcac catcatggag cgcagcagct tcgagaagaa ccccatcgac
     4441 ttcctggagg ccaagggcta caaggaggtg aagaaggacc tgatcatcaa gctgcccaag
     4501 tacagcctgt tcgagctgga gaacggccgc aagcgcatgc tggccagcgc cggcgagctg
     4561 cagaagggca acgagctggc cctgcccagc aagtacgtga acttcctgta cctggccagc
     4621 cactacgaga agctgaaggg cagccccgag gacaacgagc agaagcagct gttcgtggag
     4681 cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttcag caagcgcgtg
     4741 atcctggccg acgccaacct ggacaaggtg ctgagcgcct acaacaagca ccgcgacaag
     4801 cccatccgcg agcaggccga gaacatcatc cacctgttca ccctgaccaa cctgggcgcc
     4861 cccgccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac cagcaccaag
     4921 gaggtgctgg acgccaccct gatccaccag agcatcaccg gtctgtacga gacccgcatc
     4981 gacctgagcc agctgggcgg cgacggcggc tccggacctc caaagaaaaa gagaaaagta
     5041 tacccctacg acgtgcccga ctacgcctaa taactcgagc atgcatctag agggccctat
     5101 tctatagtgt cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg
     5161 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     5221 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     5281 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     5341 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctgcattaa
     5401 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     5461 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5521 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5581 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5641 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5701 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5761 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5821 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5881 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5941 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     6001 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     6061 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     6121 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6181 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6241 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6301 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6361 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6421 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6481 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6541 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6601 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6661 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6721 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6781 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6841 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6901 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6961 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     7021 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     7081 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     7141 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7201 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7261 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7321 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7381 gtc
//
