LOCUS       sequence-486583-        8112 bp    DNA     circular SYN 17-SEP-2025
DEFINITION  Mammalian expression of catalytically inactive SpRY Cas9 (dSpRY) .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8112)
  AUTHORS   Shi H, Al-Sayyad N, Wasko KM, Trinidad MI, Doherty EE, Vohra K, 
            Boger RS, Colognori D, Cofsky JC, Skopintsev P, Bryant Z, Doudna JA
  TITLE     Rapid two-step target capture ensures efficient CRISPR-Cas9-guided 
            genome editing.
  JOURNAL   Mol Cell. 2025 May 1;85(9):1730-1742.e9. doi: 
            10.1016/j.molcel.2025.03.024. Epub 2025 Apr 23.
  PUBMED    40273916
REFERENCE   2  (bases 1 to 8112)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2025.03.024"; journalName: "Mol Cell"; date: 
            "2025-05-1- 1"; volume: "85"; issue: "9"; pages: "1730-1742.e9"
COMMENT     Sequence Label: pKMW281_dSpRY
FEATURES             Location/Qualifiers
     source          1..8112
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        51..336
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        338..613
                     /label=chicken beta-actin promoter
     intron          614..841
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      853..862
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             862..927
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             934..954
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             979..5079
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5080..5127
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(5158..5175)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5164..5371
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   5380..5520
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5380..5509
                     /label=AAV2 ITR
     rep_origin      5595..6050
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5682..5701)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5892..5913
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6067..6086)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6186..6208
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6246..6264)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6332..6436
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6437..6505,6506..7297)
                     /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(6655..6674)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7468..8056
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7957..7976
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtttagc gcgtgcgcca attctgcaga caaatggctc tagaggtacc cgttacataa
       61 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
      121 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
      181 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
      241 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
      301 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
      361 ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt tattttttaa
      421 ttattttgtg cagcgatggg ggcggggggg gggggggcgc gcgccaggcg gggcggggcg
      481 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
      541 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      601 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
      661 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
      721 cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta agggatggtt
      781 ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca
      841 ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa ggatcatgat
      901 attgattaca aagacgatga cgataagatg gccccaaaga agaagcggaa ggtcggtatc
      961 cacggagtcc cagcagccga caagaagtac agcatcggcc tggccatcgg caccaactct
     1021 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaaatt caaggtgctg
     1081 ggcaacaccg accggcacag catcaagaag aacctgatcg gagccctgct gttcgacagc
     1141 ggcgaaacag ccgagagaac ccggctgaag agaaccgcca gaagaagata caccagacgg
     1201 aagaaccgga tctgctatct gcaagagatc ttcagcaacg agatggccaa ggtggacgac
     1261 agcttcttcc acagactgga agagtccttc ctggtggaag aggataagaa gcacgagcgg
     1321 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
     1381 taccacctga gaaagaaact ggtggacagc accgacaagg ccgacctgcg gctgatctat
     1441 ctggccctgg cccacatgat caagttccgg ggccacttcc tgatcgaggg cgacctgaac
     1501 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
     1561 ttcgaggaaa accccatcaa cgccagcggc gtggacgcca aggccatcct gtctgccaga
     1621 ctgagcaaga gcagacggct ggaaaatctg atcgcccagc tgcccggcga gaagaagaat
     1681 ggcctgttcg gaaacctgat tgccctgagc ctgggcctga cccccaactt caagagcaac
     1741 ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg acacctacga cgacgacctg
     1801 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgtttctggc cgccaagaac
     1861 ctgtccgacg ccatcctgct gagcgacatc ctgagagtga acaccgagat caccaaggcc
     1921 cccctgagcg cctctatgat caagagatac gacgagcacc accaggacct gaccctgctg
     1981 aaagctctcg tgcggcagca gctgcctgag aagtacaaag agattttctt cgaccagagc
     2041 aagaacggct acgccggcta cattgacggc ggagccagcc aggaagagtt ctacaagttc
     2101 atcaagccca tcctggaaaa gatggacggc accgaggaac tgctcgtgaa gctgaacaga
     2161 gaggacctgc tgcggaagca gcggaccttc gacaacggca gcatccccca ccagatccac
     2221 ctgggagagc tgcacgccat tctgcggcgg caggaagatt tttacccatt cctgaaggac
     2281 aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccctctg
     2341 gccaggggaa acagcagatt cgcctggatg accagaaaga gcgaggaaac catcaccccc
     2401 tggaacttcg aggaagtggt ggacaagggc gcttccgccc agagcttcat cgagcggatg
     2461 accaacttcg ataagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
     2521 gagtacttca ccgtgtataa cgagctgacc aaagtgaaat acgtgaccga gggaatgaga
     2581 aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg tggacctgct gttcaagacc
     2641 aaccggaaag tgaccgtgaa gcagctgaaa gaggactact tcaagaaaat cgagtgcttc
     2701 gactccgtgg aaatctccgg cgtggaagat cggttcaacg cctccctggg cacataccac
     2761 gatctgctga aaattatcaa ggacaaggac ttcctggaca atgaggaaaa cgaggacatt
     2821 ctggaagata tcgtgctgac cctgacactg tttgaggaca gagagatgat cgaggaacgg
     2881 ctgaaaacct atgcccacct gttcgacgac aaagtgatga agcagctgaa gcggcggaga
     2941 tacaccggct ggggcaggct gagccggaag ctgatcaacg gcatccggga caagcagtcc
     3001 ggcaagacaa tcctggattt cctgaagtcc gacggcttcg ccaacagaaa cttcatgcag
     3061 ctgatccacg acgacagcct gacctttaaa gaggacatcc agaaagccca ggtgtccggc
     3121 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     3181 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     3241 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     3301 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     3361 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     3421 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     3481 tacgatgtgg acgccatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     3541 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     3601 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     3661 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     3721 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     3781 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     3841 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     3901 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     3961 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     4021 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     4081 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     4141 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     4201 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     4261 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcagacc caagaggaac
     4321 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcctg
     4381 tggcccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     4441 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     4501 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     4561 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     4621 tctgccaagc agctgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     4681 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     4741 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     4801 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     4861 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     4921 accagactgg gagcccctag agccttcaag tactttgaca ccaccatcga ccccaagcag
     4981 tacagaagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     5041 tacgagacac ggatcgacct gtctcagctg ggaggcgaca aaaggccggc ggccacgaaa
     5101 aaggccggcc aggcaaaaaa gaaaaagtaa gaattctaac tagagctcgc tgatcagcct
     5161 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     5221 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     5281 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5341 attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta gtgatggagt
     5401 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     5461 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     5521 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     5581 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5641 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     5701 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5761 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     5821 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5881 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     5941 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     6001 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     6061 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     6121 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     6181 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     6241 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     6301 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     6361 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6421 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6481 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6541 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6601 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6661 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6721 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6781 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6841 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6901 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6961 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     7021 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     7081 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     7141 cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     7201 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     7261 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     7321 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     7381 ttttgataat ctcatgacca aaatccatta acgtgagttt tcgttccact gagcgtcaga
     7441 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     7501 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     7561 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     7621 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     7681 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     7741 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     7801 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7861 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7921 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7981 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     8041 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     8101 gccttttgct ca
//
