LOCUS       sequence-405523-        8214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of vCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8214)
  AUTHORS   Chauhan VP, Sharp PA, Langer R
  TITLE     Altered DNA repair pathway engagement by engineered CRISPR-Cas9 
            nucleases.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Mar 14;120(11):e2300605120. doi: 
            10.1073/pnas.2300605120. Epub 2023 Mar 7.
  PUBMED    36881621
REFERENCE   2  (bases 1 to 8214)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-03-14"; volume: "120(11):e2300605120. 
            doi"; pages: " 10.1073/pnas.2300605120. Epub 2023 Mar 7"
COMMENT     Sequence Label: pV132-vCas9
FEATURES             Location/Qualifiers
     source          1..8214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        149..434
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        436..713
                     /label=chicken beta-actin promoter
     intron          714..941
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      953..962
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             962..1027
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1034..1054
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1079..5179
                     /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
                     LIGALLFDRGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVAEINNYHHAHDAYLN
                     AVVGTALIKKYPALESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFRLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5180..5227
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5255..5272)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5261..5468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5477..5617
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5477..5606
                     /label=AAV2 ITR
     rep_origin      5692..6147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5779..5798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5989..6010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6164..6183)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6283..6305
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6343..6361)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6429..6533
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6534..6602,6603..7394)
                     /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(6752..6771)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7565..8153
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8054..8073
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gaggtacccg ttacataact tacggtaaat ggcccgcctg
      181 gctgaccgcc caacgacccc cgcccattga cgtcaatagt aacgccaata gggactttcc
      241 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      301 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      361 gtgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      421 tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc atctcccccc
      481 cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca gcgatggggg
      541 cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg cggggcgggg
      601 cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta
      661 tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg
      721 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc
      781 tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct
      841 gtaattagct gagcaagagg taagggttta agggatggtt ggttggtggg gtattaatgt
      901 ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggaccg gtgccaccat
      961 ggactataag gaccacgacg gagactacaa ggatcatgat attgattaca aagacgatga
     1021 cgataagatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga
     1081 caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac
     1141 cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag
     1201 catcaagaag aacctgatcg gagccctgct gttcgaccgg ggcgaaacag ccgaggccac
     1261 ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct
     1321 gcaagagatc ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga
     1381 agagtccttc ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat
     1441 cgtggacgag gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact
     1501 ggtggacagc accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat
     1561 caagttccgg ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga
     1621 caagctgttc atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa
     1681 cgccagcggc gtggacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct
     1741 ggaaaatctg atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat
     1801 tgccctgagc ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc
     1861 caaactgcag ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat
     1921 cggcgaccag tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct
     1981 gagcgacatc ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat
     2041 caagagatac gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca
     2101 gctgcctgag aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta
     2161 cattgacggc ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa
     2221 gatggacggc accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca
     2281 gcggaccttc gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat
     2341 tctgcggcgg caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa
     2401 gatcctgacc ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt
     2461 cgcctggatg accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt
     2521 ggacaagggc gcttccgccc agagcttcat cgagcggatg accaacttcg ataagaacct
     2581 gcccaacgag aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa
     2641 cgagctgacc aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg
     2701 cgagcagaaa aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa
     2761 gcagctgaaa gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg
     2821 cgtggaagat cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa
     2881 ggacaaggac ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac
     2941 cctgacactg tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct
     3001 gttcgacgac aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct
     3061 gagccggaag ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt
     3121 cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct
     3181 gacctttaaa gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga
     3241 gcacattgcc aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa
     3301 ggtggtggac gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga
     3361 aatggccaga gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa
     3421 gcggatcgaa gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga
     3481 aaacacccag ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat
     3541 gtacgtggac caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt
     3601 gcctcagagc tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa
     3661 gaaccggggc aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta
     3721 ctggcggcag ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa
     3781 ggccgagaga ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt
     3841 ggaaacccgg cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa
     3901 gtacgacgag aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct
     3961 ggtgtccgat ttccggaagg atttccagtt ttacaaagtg gccgagatca acaactacca
     4021 ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc
     4081 tgccctggaa agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat
     4141 cgccaagagc gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat
     4201 catgaacttt ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct
     4261 gatcgagaca aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac
     4321 cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac
     4381 aggcggcttc agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag
     4441 aaagaaggac tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc
     4501 tgtgctggtg gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga
     4561 gctgctgggg atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct
     4621 ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc
     4681 cctgttcgag ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa
     4741 gggaaacgaa ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta
     4801 tgagaagctg aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca
     4861 caagcactac ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct
     4921 ggccgacgct aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat
     4981 cagagagcag gccgagaata tcatccacct gtttcggctg accaatctgg gagcccctgc
     5041 cgccttcaag tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt
     5101 gctggacgcc accctgatcc accagagcat caccggcctg tacgagacac ggatcgacct
     5161 gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     5221 gaaaaagtaa gaattcctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     5281 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     5341 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     5401 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     5461 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     5521 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     5581 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     5641 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     5701 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5761 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5821 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5881 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     5941 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     6001 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     6061 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     6121 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     6181 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     6241 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     6301 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     6361 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     6421 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     6481 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     6541 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     6601 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     6661 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     6721 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     6781 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     6841 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     6901 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6961 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     7021 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     7081 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     7141 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     7201 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta
     7261 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     7321 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     7381 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     7441 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     7501 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7561 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7621 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7681 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     7741 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7801 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     7861 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     7921 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     7981 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     8041 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     8101 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     8161 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgt
//
