LOCUS       sequence-412372-        8036 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression plasmid for human codon-optimized increased fidelity 
            Hypa2SpCas9 (without U6-sgRNA coding sequence, with silent 
            mutations).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8036)
  AUTHORS   Kulcsar PI, Talas A, Ligeti Z, Toth E, Rakvacs Z, Bartos Z, Krausz 
            SL, Welker A, Vegi VL, Huszar K, Welker E
  TITLE     A cleavage rule for selection of increased-fidelity SpCas9 variants 
            with high efficiency and no detectable off-targets.
  JOURNAL   Nat Commun. 2023 Sep 16;14(1):5746. doi: 10.1038/s41467-023-41393-5.
  PUBMED    37717069
REFERENCE   2  (bases 1 to 8036)
  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: "2023-09-16"; volume: "14(1):5746. doi"; pages: " 
            10.1038/s41467-023-41393-5"
COMMENT     Sequence Label: HypaR-SpCas9
FEATURES             Location/Qualifiers
     source          1..8036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          127..354
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      366..375
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             375..440
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             447..467
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             492..4592
                     /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
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGALSRKLINGIRDKQSGKTILDFLKSDGFANRAFAA
                     LIADDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4593..4640
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(4668..4685)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4674..4881
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4890..5030
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4890..5019
                     /label=AAV2 ITR
     rep_origin      5105..5560
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5192..5211)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5402..5423
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5577..5596)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5696..5718
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5756..5774)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5842..5946
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5947..6015,6016..6807)
                     /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(6165..6184)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6978..7566
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7467..7486
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        7638..7923
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
ORIGIN
        1 gggggggggg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
       61 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      121 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      181 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      241 tctcctccgg gctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt
      301 ggggtattaa tgtttaatta cctggagcac ctgcctgaaa tcactttttt tcaggttgga
      361 ccggtgccac catggactat aaggaccacg acggagacta caaggatcat gatattgatt
      421 acaaagacga tgacgataag atggccccaa agaagaagcg gaaggtcggt atccacggag
      481 tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct
      541 gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca
      601 ccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa
      661 cagccgaggc cacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc
      721 ggatctgcta tctgcaagag atcttcagca acgagatggc caaggtggac gacagcttct
      781 tccacagact ggaagagtcc ttcctggtgg aagaggataa gaagcacgag cggcacccca
      841 tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc
      901 tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc
      961 tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca
     1021 acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg
     1081 aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca
     1141 agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt
     1201 tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc
     1261 tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc
     1321 tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg
     1381 acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga
     1441 gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc
     1501 tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg
     1561 gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc
     1621 ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc
     1681 tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag
     1741 agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg
     1801 aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg
     1861 gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact
     1921 tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact
     1981 tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact
     2041 tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg
     2101 ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga
     2161 aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg
     2221 tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc
     2281 tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag
     2341 atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa
     2401 cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg
     2461 gctggggcgc gctgagccgg aagctgatca acggcatccg cgacaagcag tccggcaaga
     2521 caatcctgga tttcctgaag tccgacggct tcgccaacag agcctttgca gccctgatcg
     2581 ctgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg
     2641 atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcc
     2701 tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga
     2761 acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc
     2821 gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag
     2881 aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga
     2941 atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg
     3001 tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga
     3061 ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga
     3121 agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg
     3181 acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca
     3241 agagacagct ggtggaaacc cggcagatca caaagcacgt ggcacagatc ctggactccc
     3301 ggatgaacac taagtacgac gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc
     3361 tgaagtccaa gctggtgtcc gatttccgga aggatttcca gttttacaaa gtgcgcgaga
     3421 tcaacaacta ccaccacgcc cacgacgcgt acctgaacgc cgtcgtggga accgccctga
     3481 tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg
     3541 tacggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct
     3601 tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc
     3661 ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc
     3721 gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga
     3781 ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata
     3841 agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca
     3901 ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga
     3961 agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc
     4021 ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc
     4081 tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg
     4141 gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc
     4201 tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt
     4261 ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca
     4321 agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc
     4381 gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc
     4441 tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca
     4501 gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga
     4561 cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg
     4621 gccaggcaaa aaagaaaaag taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     4681 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     4741 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     4801 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     4861 gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc
     4921 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4981 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     5041 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     5101 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     5161 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     5221 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     5281 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     5341 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5401 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     5461 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     5521 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     5581 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5641 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5701 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5761 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5821 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5881 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5941 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     6001 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6061 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6121 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6181 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6241 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6301 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6361 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6421 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6481 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6541 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6601 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6661 ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6721 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6781 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6841 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6901 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6961 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     7021 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     7081 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     7141 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7201 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7261 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7321 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7381 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7441 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7501 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7561 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7621 cacatgctag aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     7681 aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa
     7741 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     7801 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac
     7861 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     7921 atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc
     7981 ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc gggggg
//
