LOCUS       sequence-410966-        8219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-opt. increased fidelity 
            SpCas9-HF1-plus (w/o U6-sgRNA). Cleaves both 20nt and 5'-extended 
            21nt spacer containing sgRNAs with close to the same fidelity as 
            SpCas9-HF1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8219)
  AUTHORS   Kulcsar PI, Talas A, Toth E, Nyeste A, Ligeti Z, Welker Z, Welker E
  TITLE     Blackjack mutations improve the on-target activities of increased 
            fidelity variants of SpCas9 with 5'G-extended sgRNAs.
  JOURNAL   Nat Commun. 2020 Mar 6;11(1):1223. doi: 10.1038/s41467-020-15021-5.
  PUBMED    32144253
REFERENCE   2  (bases 1 to 8219)
  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: "2020-03-6"; volume: "11(1):1223. doi"; pages: " 
            10.1038/s41467-020-15021-5"
COMMENT     Sequence Label: SpCas9-HF1-plus
FEATURES             Location/Qualifiers
     source          1..8219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          181..408
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      420..429
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             429..494
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             501..521
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             546..4625
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMA
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRAITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLGGKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
                     IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRN
                     SDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF
                     EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYV
                     NFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLS
                     AYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS
                     ITGLYETRIDLSQLGGD"
     CDS             4626..4673
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4701..4718)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4707..4914
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4923..5063
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4923..5052
                     /label=AAV2 ITR
     rep_origin      5138..5593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5225..5244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5435..5456
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5610..5629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5729..5751
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5789..5807)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5875..5979
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5980..6048,6049..6840)
                     /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     complement(6198..6217)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7011..7599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7500..7519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7671..7956
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggggggggg gggggggggg ggggcgsgcg ssrggsggsg gggggcgggg cgrggggcgg
       61 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      121 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      181 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      241 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      301 ccgggctgta attagctgag caagaggtaa gggtttaagg gatggttggt tggtggggta
      361 ttaatgttta attacctgga gcacctgcct gaaatcactt tttttcaggt tggaccggtg
      421 ccaccatgga ctataaggac cacgacggag actacaagga tcatgatatt gattacaaag
      481 acgatgacga taagatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag
      541 cagccgacaa gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg
      601 tgatcaccga cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc
      661 ggcacagcat caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg
      721 aggccacccg gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct
      781 gctatctgca agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca
      841 gactggaaga gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg
      901 gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa
      961 agaaactggt ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc
     1021 acatgatcaa gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg
     1081 acgtggacaa gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc
     1141 ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca
     1201 gacggctgga aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa
     1261 acctgattgc cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg
     1321 aggatgccaa actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg
     1381 cccagatcgg cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca
     1441 tcctgctgag cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct
     1501 ctatgatcaa gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc
     1561 ggcagcagct gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg
     1621 ccggctacat tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc
     1681 tggaaaagat ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc
     1741 ggaagcagcg gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc
     1801 acgccattct gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga
     1861 tcgagaagat cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca
     1921 gcagattcgc ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg
     1981 aagtggtgga caagggcgct tccgcccaga gcttcatcga gcggatgacc gccttcgata
     2041 agaacctgcc caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg
     2101 tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc
     2161 tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga
     2221 ccgtgaagca gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa
     2281 tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa
     2341 ttatcaagga caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg
     2401 tgctgaccct gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg
     2461 cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg
     2521 gcaggctgag ccggaagctg atcaacggca tccgcgacaa gcagtccggc aagacaatcc
     2581 tggatttcct gaagtccgac ggcttcgcca acagaaactt catggcgctg atccacgacg
     2641 acagcctgac ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc
     2701 tgcacgagca cattgccaat ctcgccggca gccccgccat taagaagggc atcctccaga
     2761 cagtgaaggt ggtggacgag ctggtgaaag tgatgggccg gcacaagccc gagaacatcg
     2821 tgatcgaaat ggccagagag aaccagacca cccagaaggg acagaagaac agtcgcgaga
     2881 gaatgaagcg gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc
     2941 ccgtggaaaa cacccagctc cagaacgaga agctgtacct gtactacctc cagaatgggc
     3001 gggatatgta cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc
     3061 atatcgtgcc tcagagcttt cttaaggacg actccatcga caacaaggtg ctgaccagaa
     3121 gcgacaagaa ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga
     3181 agaactactg gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc
     3241 tgaccaaggc cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac
     3301 agctggtgga aacccgggcg atcacaaagc acgtggcaca gatcctggac tcccggatga
     3361 acactaagta cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt
     3421 ccaagctggt gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca
     3481 actaccacca cgcccacgac gcgtacctga acgccgtcgt gggaaccgcc ctgatcaaaa
     3541 agtaccctaa gctgggtggc aaggtgtacg atgtacggaa gatgatcgcc aagagcgagc
     3601 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     3661 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     3721 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     3781 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     3841 aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     3901 atcctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg
     3961 ccaaagtgga aaagggcaag tccaagaagc ttaagagtgt gaaagagctg ctggggatca
     4021 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     4081 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactcgctc ttcgagctgg
     4141 aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga aacgaactgg
     4201 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     4261 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     4321 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     4381 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     4441 agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact
     4501 ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc
     4561 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     4621 gcgacaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat
     4681 tcctagagct cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     4741 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4801 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4861 ggggcaggac agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc
     4921 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4981 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5041 gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg
     5101 cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta
     5161 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg
     5221 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     5281 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     5341 aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     5401 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     5461 acactcaact ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc
     5521 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     5581 acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5641 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5701 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5761 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5821 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     5881 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5941 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     6001 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     6061 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6121 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6181 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6241 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6301 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6361 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6421 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6481 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6541 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6601 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6661 tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg
     6721 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6781 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6841 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6901 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6961 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     7021 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     7081 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7141 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7201 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     7261 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     7321 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     7381 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     7441 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7501 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7561 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7621 ttacggttcc tggccttttg ctggcctttt gctcacatgc tagaggtacc cgttacataa
     7681 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     7741 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     7801 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     7861 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
     7921 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     7981 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
     8041 ttattttgtg cagcgatggg ggcggggggg gggggggggg ggcsmgncgg gggggggggg
     8101 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggngggggg
     8161 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg ggggggggg
//
