LOCUS       sequence-329428-        7722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  miHiFiCas9 expressing plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7722)
  AUTHORS   Ma L, Ruan J, Song J, Wen L, Yang D, Zhao J, Xia X, Chen YE, Zhang 
            J, Xu J
  TITLE     MiCas9 increases large size gene knock-in rates and reduces 
            undesirable on-target and off-target indel edits.
  JOURNAL   Nat Commun. 2020 Nov 27;11(1):6082. doi: 10.1038/s41467-020-19842-2.
  PUBMED    33247137
REFERENCE   2  (bases 1 to 7722)
  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: "2020-11-27"; volume: "11(1):6082. doi"; pages: " 
            10.1038/s41467-020-19842-2"
COMMENT     Sequence Label: pmiHiFiCas9
FEATURES             Location/Qualifiers
     source          1..7722
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        162..541
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        542..745
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     696..716
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     787..806
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        787..805
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      813..822
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(819..4220,4221..4223,4224..4820,4821,4822..4824,
                     4825..4826,4827..4829,4830..4922)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANANFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4929..4949
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4956..5021
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5147..5164
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5187..5204)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5193..5417
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5488..5504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5488..5504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5501..5523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5512..5528
                     /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(5536..5542,5543..5560,5561..5566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5581..5602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5719..5736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5890..6478)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5970..5989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6649..7440,7441..7509))
                     /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     7272..7291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7510..7614)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7693..7712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
       61 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
      121 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
      181 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      241 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      301 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      361 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      421 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      481 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      541 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      601 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      661 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      721 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agagatccgc
      781 ggccgctaat acgactcact atagggagag ccgccaccat ggataaaaag tattctattg
      841 gtttagatat cggcactaat tccgttggat gggctgtcat aaccgatgaa tacaaagtac
      901 cttcaaagaa atttaaggtg ttggggaaca cagaccgtca ttcgattaaa aagaatctta
      961 tcggtgccct cctattcgat agtggcgaaa cggcagaggc gactcgcctg aaacgaaccg
     1021 ctcggagaag gtatacacgt cgcaagaacc gaatatgtta cttacaagaa atttttagca
     1081 atgagatggc caaagttgac gattctttct ttcaccgttt ggaagagtcc ttccttgtcg
     1141 aagaggacaa gaaacatgaa cggcacccca tctttggaaa catagtagat gaggtggcat
     1201 atcatgaaaa gtacccaacg atttatcacc tcagaaaaaa gctagttgac tcaactgata
     1261 aagcggacct gaggttaatc tacttggctc ttgcccatat gataaagttc cgtgggcact
     1321 ttctcattga gggtgatcta aatccggaca actcggatgt cgacaaactg ttcatccagt
     1381 tagtacaaac ctataatcag ttgtttgaag agaaccctat aaatgcaagt ggcgtggatg
     1441 cgaaggctat tcttagcgcc cgcctctcta aatcccgacg gctagaaaac ctgatcgcac
     1501 aattacccgg agagaagaaa aatgggttgt tcggtaacct tatagcgctc tcactaggcc
     1561 tgacaccaaa ttttaagtcg aacttcgact tagctgaaga tgccaaattg cagcttagta
     1621 aggacacgta cgatgacgat ctcgacaatc tactggcaca aattggagat cagtatgcgg
     1681 acttattttt ggctgccaaa aaccttagcg atgcaatcct cctatctgac atactgagag
     1741 ttaatactga gattaccaag gcgccgttat ccgcttcaat gatcaaaagg tacgatgaac
     1801 atcaccaaga cttgacactt ctcaaggccc tagtccgtca gcaactgcct gagaaatata
     1861 aggaaatatt ctttgatcag tcgaaaaacg ggtacgcagg ttatattgac ggcggagcga
     1921 gtcaagagga attctacaag tttatcaaac ccatattaga gaagatggat gggacggaag
     1981 agttgcttgt aaaactcaat cgcgaagatc tactgcgaaa gcagcggact ttcgacaacg
     2041 gtagcattcc acatcaaatc cacttaggcg aattgcatgc tatacttaga aggcaggagg
     2101 atttttatcc gttcctcaaa gacaatcgtg aaaagattga gaaaatccta acctttcgca
     2161 taccttacta tgtgggaccc ctggcccgag ggaactctcg gttcgcatgg atgacaagaa
     2221 agtccgaaga aacgattact ccatggaatt ttgaggaagt tgtcgataaa ggtgcgtcag
     2281 ctcaatcgtt catcgagagg atgaccaact ttgacaagaa tttaccgaac gaaaaagtat
     2341 tgcctaagca cagtttactt tacgagtatt tcacagtgta caatgaactc acgaaagtta
     2401 agtatgtcac tgagggcatg cgtaaacccg cctttctaag cggagaacag aagaaagcaa
     2461 tagtagatct gttattcaag accaaccgca aagtgacagt taagcaattg aaagaggact
     2521 actttaagaa aattgaatgc ttcgattctg tcgagatctc cggggtagaa gatcgattta
     2581 atgcgtcact tggtacgtat catgacctcc taaagataat taaagataag gacttcctgg
     2641 ataacgaaga gaatgaagat atcttagaag atatagtgtt gactcttacc ctctttgaag
     2701 atcgggaaat gattgaggaa agactaaaaa catacgctca cctgttcgac gataaggtta
     2761 tgaaacagtt aaagaggcgt cgctatacgg gctggggacg attgtcgcgg aaacttatca
     2821 acgggataag agacaagcaa agtggtaaaa ctattctcga ttttctaaag agcgacggct
     2881 tcgccaatgc gaactttatg cagctgatcc atgatgactc tttaaccttc aaagaggata
     2941 tacaaaaggc acaggtttcc ggacaagggg actcattgca cgaacatatt gcgaatcttg
     3001 ctggttcgcc agccatcaaa aagggcatac tccagacagt caaagtagtg gatgagctag
     3061 ttaaggtcat gggacgtcac aaaccggaaa acattgtaat cgagatggca cgcgaaaatc
     3121 aaacgactca gaaggggcaa aaaaacagtc gagagcggat gaagagaata gaagagggta
     3181 ttaaagaact gggcagccag atcttaaagg agcatcctgt ggaaaatacc caattgcaga
     3241 acgagaaact ttacctctat tacctacaaa atggaaggga catgtatgtt gatcaggaac
     3301 tggacataaa ccgtttatct gattacgacg tcgatcatat tgtaccccaa tcctttttga
     3361 aggacgattc aatcgacaat aaagtgctta cacgctcgga taagaaccga gggaaaagtg
     3421 acaatgttcc aagcgaggaa gtcgtaaaga aaatgaagaa ctattggcgg cagctcctaa
     3481 atgcgaaact gataacgcaa agaaagttcg ataacttaac taaagctgag aggggtggct
     3541 tgtctgaact tgacaaggcc ggatttatta aacgtcagct cgtggaaacc cgccaaatca
     3601 caaagcatgt tgcacagata ctagattccc gaatgaatac gaaatacgac gagaacgata
     3661 agctgattcg ggaagtcaaa gtaatcactt taaagtcaaa attggtgtcg gacttcagaa
     3721 aggattttca attctataaa gttagggaga taaataacta ccaccatgcg cacgacgctt
     3781 atcttaatgc cgtcgtaggg accgcactca ttaagaaata cccgaagcta gaaagtgagt
     3841 ttgtgtatgg tgattacaaa gtttatgacg tccgtaagat gatcgcgaaa agcgaacagg
     3901 agataggcaa ggctacagcc aaatacttct tttattctaa cattatgaat ttctttaaga
     3961 cggaaatcac tctggcaaac ggagagatac gcaaacgacc tttaattgaa accaatgggg
     4021 agacaggtga aatcgtatgg gataagggcc gggacttcgc gacggtgaga aaagttttgt
     4081 ccatgcccca agtcaacata gtaaagaaaa ctgaggtgca gaccggaggg ttttcaaagg
     4141 aatcgattct tccaaaaagg aatagtgata agctcatcgc tcgtaaaaag gactgggacc
     4201 cgaaaaagta cggtggcttc gatagcccta cagttgccta ttctgtccta gtagtggcaa
     4261 aagttgagaa gggaaaatcc aagaaactga agtcagtcaa agaattattg gggataacga
     4321 ttatggagcg ctcgtctttt gaaaagaacc ccatcgactt ccttgaggcg aaaggttaca
     4381 aggaagtaaa aaaggatctc ataattaaac taccaaagta tagtctgttt gagttagaaa
     4441 atggccgaaa acggatgttg gctagcgccg gagagcttca aaaggggaac gaactcgcac
     4501 taccgtctaa atacgtgaat ttcctgtatt tagcgtccca ttacgagaag ttgaaaggtt
     4561 cacctgaaga taacgaacag aagcaacttt ttgttgagca gcacaaacat tatctcgacg
     4621 aaatcataga gcaaatttcg gaattcagta agagagtcat cctagctgat gccaatctgg
     4681 acaaagtatt aagcgcatac aacaagcaca gggataaacc catacgtgag caggcggaaa
     4741 atattatcca tttgtttact cttaccaacc tcggcgctcc agccgcattc aagtattttg
     4801 acacaacgat agatcgcaaa cgatacactt ctaccaagga ggtgctagac gcgacactga
     4861 ttcaccaatc catcacggga ttatatgaaa ctcggataga tttgtcacag cttgggggtg
     4921 acggatcccc caagaagaag aggaaagtct cgagcgacta caaagaccat gacggtgatt
     4981 ataaagatca tgacatcgat tacaaggatg acgatgacaa ggctgcagga gcattggatt
     5041 tccttagtag actccccttg ccacctccgg tgtcccccat ctgcacgttt gtgagcccag
     5101 cggcacagaa ggcatttcaa ccgccacgga gttgcgggta accggtcatc atcaccatca
     5161 ccattgagtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     5221 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     5281 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     5341 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     5401 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga taccgtcgac
     5461 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     5521 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct agggtgccta
     5581 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5641 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5701 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5761 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5821 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5881 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5941 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     6001 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6061 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6121 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6181 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6241 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6301 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6361 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6421 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6481 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6541 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6601 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6661 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6721 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6781 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6841 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6901 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6961 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7021 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7081 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7141 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7201 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7261 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7321 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7381 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7441 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7501 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7561 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7621 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat cgatctcccg atcccctagg
     7681 gtcgactctc agtacaatct gctctgatgc cgcatagtta ag
//
