LOCUS       sequence-199285-        7407 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9-HF1 mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7407)
  AUTHORS   Kim S, Bae T, Hwang J, Kim JS
  TITLE     Rescue of high-specificity Cas9 variants using sgRNAs with matched 
            5' nucleotides.
  JOURNAL   Genome Biol. 2017 Nov 15;18(1):218. doi: 10.1186/s13059-017-1355-3.
  PUBMED    29141659
REFERENCE   2  (bases 1 to 7407)
  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: "Genome 
            Biol."; date: "2017-11-15"; volume: "18(1):218. doi"; pages: " 
            10.1186/s13059-017-1355-3"
COMMENT     Sequence Label: p3s-Cas9-HF1
FEATURES             Location/Qualifiers
     source          1..7407
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        145..524
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        525..728
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     679..699
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     773..792
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        773..791
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      805..814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             817..843
                     /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             817..843
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             853..873
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(901..2385,2386..2388,2389..2877,2878..2880,2881..2979,
                     2980..2982,2983..3672,3673..3675,3676..5001)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus pyogenes
                     Type II CRISPR/Cas system, mutated to improve targeting 
                     specificity (Kleinstiver et al., 2016)"
                     /label=SpCas9-HF1
                     /note="carries the mutations N497A, R661A, Q695A, and 
                     Q926A"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     promoter        complement(5035..5053)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5036..5053)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5073..5090)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5079..5303
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5391..5408)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5562..6150)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5642..5661)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6321..7112,7113..7181))
                     /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     6944..6963
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7182..7286)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7361..7380)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
       61 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
      121 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
      181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
      241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
      301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
      361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
      421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
      481 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
      541 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
      601 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
      661 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
      721 gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga
      781 ctcactatag ggagacccaa gcttgccacc atggtgtacc cctacgacgt gcccgactac
      841 gccgaattgc ctccaaaaaa gaagagaaag gtagggatcc gaattcccgg ggaaaaaccg
      901 gacaagaagt acagcatcgg cctggacatc ggtaccaaca gcgtgggctg ggccgtgatc
      961 accgacgagt acaaggtgcc cagcaagaag ttcaaggtgc tgggcaacac cgaccgccac
     1021 agcatcaaga agaacctgat cggcgccctg ctgttcgaca gcggcgagac cgccgaggcc
     1081 acccgcctga agcgcaccgc ccgccgccgc tacacccgcc gcaagaaccg catctgctac
     1141 ctgcaggaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccaccgcctg
     1201 gaggagagct tcctggtgga ggaggacaag aagcacgagc gccaccccat cttcggcaac
     1261 atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gcgcaagaag
     1321 ctggtggaca gcaccgacaa ggccgacctg cgcctgatct acctggccct ggcccacatg
     1381 atcaagttcc gcggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg
     1441 gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga gaaccccatc
     1501 aacgccagcg gcgtggacgc caaggccatc ctgagcgccc gcctgagcaa gagccgccgc
     1561 ctggagaacc tgatcgccca gctgcccggc gagaagaaga acggcctgtt cggcaacctg
     1621 atcgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggac
     1681 gccaagctgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag
     1741 atcggcgacc agtacgccga cctgttcctg gccgccaaga acctgagcga cgccatcctg
     1801 ctgagcgaca tcctgcgcgt gaacaccgag atcaccaagg cccccctgag cgccagcatg
     1861 atcaagcgct acgacgagca ccaccaggac ctgaccctgc tgaaggccct ggtgcgccag
     1921 cagctgcccg agaagtacaa ggagatcttc ttcgaccaga gcaagaacgg ctacgccggc
     1981 tacatcgacg gcggcgccag ccaggaggag ttctacaagt tcatcaagcc catcctggag
     2041 aagatggacg gcaccgagga gctgctggtg aagctgaacc gcgaggacct gctgcgcaag
     2101 cagcgcacct tcgacaacgg cagcatcccc caccagatcc acctgggcga gctgcacgcc
     2161 atcctgcgcc gccaggagga cttctacccc ttcctgaagg acaaccgcga gaagatcgag
     2221 aagatcctga ccttccgcat cccctactac gtgggccccc tggcccgcgg caacagccgc
     2281 ttcgcctgga tgacccgcaa gagcgaggag accatcaccc cctggaactt cgaggaggtg
     2341 gtggacaagg gcgccagcgc ccagagcttc atcgagcgca tgaccgcctt cgacaagaac
     2401 ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtac
     2461 aacgagctga ccaaggtgaa gtacgtgacc gagggcatgc gcaagcccgc cttcctgagc
     2521 ggcgagcaga agaaggccat cgtggacctg ctgttcaaga ccaaccgcaa ggtgaccgtg
     2581 aagcagctga aggaggacta cttcaagaag atcgagtgct tcgacagcgt ggagatcagc
     2641 ggcgtggagg accgcttcaa cgccagcctg ggcacctacc acgacctgct gaagatcatc
     2701 aaggacaagg acttcctgga caacgaggag aacgaggaca tcctggagga catcgtgctg
     2761 accctgaccc tgttcgagga ccgcgagatg atcgaggagc gcctgaagac ctacgcccac
     2821 ctgttcgacg acaaggtgat gaagcagctg aagcgccgcc gctacaccgg ctggggcgcc
     2881 ctgagccgca agcttatcaa cggcatccgc gacaagcaga gcggcaagac catcctggac
     2941 ttcctgaaga gcgacggctt cgccaaccgc aacttcatgg ccctgatcca cgacgacagc
     3001 ctgaccttca aggaggacat ccagaaggcc caggtgagcg gccagggcga cagcctgcac
     3061 gagcacatcg ccaacctggc cggcagcccc gccatcaaga agggcatcct gcagaccgtg
     3121 aaggtggtgg acgagctggt gaaggtgatg ggccgccaca agcccgagaa catcgtgatc
     3181 gagatggccc gcgagaacca gaccacccag aagggccaga agaacagccg cgagcgcatg
     3241 aagcgcatcg aggagggcat caaggagctg ggcagccaga tcctgaagga gcaccccgtg
     3301 gagaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa cggccgcgac
     3361 atgtacgtgg accaggagct ggacatcaac cgcctgagcg actacgacgt ggaccacatc
     3421 gtgccccaga gcttcctgaa ggacgacagc atcgacaaca aggtgctgac ccgcagcgac
     3481 aagaaccgcg gcaagagcga caacgtgccc agcgaggagg tggtgaagaa gatgaagaac
     3541 tactggcgcc agctgctgaa cgccaagctg atcacccagc gcaagttcga caacctgacc
     3601 aaggccgagc gcggcggcct gagcgagctg gacaaggccg gcttcatcaa gcgccagctg
     3661 gtggagaccc gcgccatcac caagcacgtg gcccagatcc tggacagccg catgaacacc
     3721 aagtacgacg agaacgacaa gctgatccgc gaggtgaagg tgatcaccct gaagagcaag
     3781 ctggtgagcg acttccgcaa ggacttccag ttctacaagg tgcgcgagat caacaactac
     3841 caccacgccc acgacgccta cctgaacgcc gtggtgggca ccgccctgat caagaagtac
     3901 cccaagctgg agagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcgcaagatg
     3961 atcgccaaga gcgagcagga gatcggcaag gccaccgcca agtacttctt ctacagcaac
     4021 atcatgaact tcttcaagac cgagatcacc ctggccaacg gcgagatccg caagcgcccc
     4081 ctgatcgaga ccaacggcga gaccggcgag atcgtgtggg acaagggccg cgacttcgcc
     4141 accgtgcgca aggtgctgag catgccccag gtgaacatcg tgaagaagac cgaggtgcag
     4201 accggcggct tcagcaagga gagcatcctg cccaagcgca acagcgacaa gctgatcgcc
     4261 cgcaagaagg actgggaccc caagaagtac ggcggcttcg acagccccac cgtggcctac
     4321 agcgtgctgg tggtggccaa ggtggagaag ggcaagagca agaagctgaa gagcgtgaag
     4381 gagctgctgg gcatcaccat catggagcgc agcagcttcg agaagaaccc catcgacttc
     4441 ctggaggcca agggctacaa ggaggtgaag aaggacctga tcatcaagct gcccaagtac
     4501 agcctgttcg agctggagaa cggccgcaag cgcatgctgg ccagcgccgg cgagctgcag
     4561 aagggcaacg agctggccct gcccagcaag tacgtgaact tcctgtacct ggccagccac
     4621 tacgagaagc tgaagggcag ccccgaggac aacgagcaga agcagctgtt cgtggagcag
     4681 cacaagcact acctggacga gatcatcgag cagatcagcg agttcagcaa gcgcgtgatc
     4741 ctggccgacg ccaacctgga caaggtgctg agcgcctaca acaagcaccg cgacaagccc
     4801 atccgcgagc aggccgagaa catcatccac ctgttcaccc tgaccaacct gggcgccccc
     4861 gccgccttca agtacttcga caccaccatc gaccgcaagc gctacaccag caccaaggag
     4921 gtgctggacg ccaccctgat ccaccagagc atcaccggtc tgtacgagac ccgcatcgac
     4981 ctgagccagc tgggcggcga ctaataactc gagcatgcat ctagagggcc ctattctata
     5041 gtgtcaccta aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc
     5101 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     5161 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     5221 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     5281 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctgca ttaatgaatc
     5341 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5401 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5461 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5521 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5581 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5641 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5701 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5761 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5821 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5881 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5941 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     6001 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     6061 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     6121 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6181 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6241 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6301 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6361 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6421 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6481 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6541 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6601 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6661 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6721 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6781 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6841 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6901 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6961 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     7021 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     7081 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     7141 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7201 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7261 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     7321 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     7381 catagttaag ccagtatctg ctccctg
//
