LOCUS       sequence-359664-        7351 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cas9 cassette (TU1).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7351)
  AUTHORS   Novarina D, Koutsoumpa A, Milias-Argeitis A
  TITLE     A user-friendly and streamlined protocol for CRISPR/Cas9 genome 
            editing in budding yeast.
  JOURNAL   STAR Protoc. 2022 Jun 10;3(2):101358. doi: 
            10.1016/j.xpro.2022.101358. eCollection 2022 Jun 17.
  PUBMED    35712010
REFERENCE   2  (bases 1 to 7351)
  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: "STAR 
            Protoc."; date: "2022-06-10"; volume: "3(2):101358. doi"; pages: " 
            10.1016/j.xpro.2022.101358. eCollection 2022 Jun 17"
COMMENT     Sequence Label: pYTK-DN1
FEATURES             Location/Qualifiers
     source          1..7351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             887..4990
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             887..4990
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5003..5023
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5027..5047
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(join(5570..6361,6362..6430))
                     /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     6193..6212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6431..6535)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6638..7226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7127..7146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atggtagagc cacaaacagc cggtacaagc aacgatctcc aggaccatct gaatcatgcg
       61 cggatgacac gaactcacga cggcgatcac agacattaac ccacagtaca gacactgcga
      121 caacgtggca attcgtcgca ataccgtctc actgaactgg ccgataattg cagacgaacg
      181 gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc
      241 gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt
      301 ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga
      361 aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc
      421 tcgacttcct gtcatcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag
      481 cgacggctca caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt
      541 agtaccacat gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg
      601 ttactctctc tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca
      661 cacactcttt tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac
      721 atttacatat atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt
      781 tctaattcgt agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc
      841 aaggaagtaa ttatctactt tttacaacaa atataaaaca agatctatgg acaagaagta
      901 ttctatcgga ctggatatcg ggactaatag cgtcgggtgg gccgtgatca ctgacgagta
      961 caaggtgccc tctaagaagt tcaaggtgct cgggaacacc gaccggcatt ccatcaagaa
     1021 aaatctgatc ggagctctcc tctttgattc aggggaaacc gctgaagcaa cccgcctcaa
     1081 gcggactgct agacggcggt acaccaggag gaagaaccgg atttgttacc ttcaagagat
     1141 attctccaac gaaatggcaa aggtcgacga cagcttcttc cataggctgg aagaatcatt
     1201 cctcgtggaa gaggataaga agcatgaacg gcatcccatc ttcggtaata tcgtcgacga
     1261 ggtggcctat cacgagaaat acccaaccat ctaccatctt cgcaaaaagc tggtggactc
     1321 aaccgacaag gcagacctcc ggcttatcta cctggccctg gcccacatga tcaagttcag
     1381 aggccacttc ctgatcgagg gcgacctcaa tcctgacaat agcgatgtgg ataaactgtt
     1441 catccagctg gtgcagactt acaaccagct ctttgaagag aaccccatca atgcaagcgg
     1501 agtcgatgcc aaggccattc tgtcagcccg gctgtcaaag agccgcagac ttgagaatct
     1561 tatcgctcag ctgccgggtg aaaagaaaaa tggactgttc gggaacctga ttgctctttc
     1621 acttgggctg actcccaatt tcaagtctaa tttcgacctg gcagaggatg ccaagctgca
     1681 actgtccaag gacacctatg atgacgatct cgacaacctc ctggcccaga tcggtgacca
     1741 atacgccgac cttttccttg ctgctaagaa tctttctgac gccatcctgc tgtctgacat
     1801 tctccgcgtg aacactgaaa tcaccaaggc ccctctttca gcttcaatga ttaagcggta
     1861 tgatgagcac caccaggacc tgaccctgct taaggcactc gtccggcagc agcttccgga
     1921 gaagtacaag gaaatcttct ttgaccagtc aaagaatgga tacgccggct acatcgacgg
     1981 aggtgcctcc caagaggaat tttataagtt tatcaaacct atccttgaga agatggacgg
     2041 caccgaagag ctcctcgtga aactgaatcg ggaggatctg ctgcggaagc agcgcacttt
     2101 cgacaatggg agcattcccc accagatcca tcttggggag cttcacgcca tccttcggcg
     2161 ccaagaggac ttctacccct ttcttaagga caacagggag aagattgaga aaattctcac
     2221 tttccgcatc ccctactacg tgggacccct cgccagagga aatagccggt ttgcttggat
     2281 gaccagaaag tcagaagaaa ctatcactcc ctggaacttc gaagaggtgg tggacaaggg
     2341 agccagcgct cagtcattca tcgaacggat gactaacttc gataagaacc tccccaatga
     2401 gaaggtcctg ccgaaacatt ccctgctcta cgagtacttt accgtgtaca acgagctgac
     2461 caaggtgaaa tatgtcaccg aagggatgag gaagcccgca ttcctgtcag gcgaacaaaa
     2521 gaaggcaatt gtggaccttc tgttcaagac caatagaaag gtgaccgtga agcagctgaa
     2581 ggaggactat ttcaagaaaa ttgaatgctt cgactctgtg gagattagcg gggtcgaaga
     2641 tcggttcaac gcaagcctgg gtacttacca tgatctgctt aagatcatca aggacaagga
     2701 ttttctggac aatgaggaga acgaggacat ccttgaggac attgtcctga ctctcactct
     2761 gttcgaggac cgggaaatga tcgaggagag gcttaagacc tacgcccatc tgttcgacga
     2821 taaagtgatg aagcaactta aacggagaag atataccgga tggggacgcc ttagccgcaa
     2881 actcatcaac ggaatccggg acaaacagag cggaaagacc attcttgatt tccttaagag
     2941 cgacggattc gctaatcgca acttcatgca acttatccat gatgattccc tgacctttaa
     3001 ggaggacatc cagaaggccc aagtgtctgg acaaggtgac tcactgcacg agcatatcgc
     3061 aaatctggct ggttcacccg ctattaagaa gggtattctc cagaccgtga aagtcgtgga
     3121 cgagctggtc aaggtgatgg gtcgccataa accagagaac attgtcatcg agatggccag
     3181 ggaaaaccag actacccaga agggacagaa gaacagcagg gagcggatga aaagaattga
     3241 ggaagggatt aaggagctcg ggtcacagat ccttaaagag cacccggtgg aaaacaccca
     3301 gcttcagaat gagaagctct atctgtacta ccttcaaaat ggacgcgata tgtatgtgga
     3361 ccaagagctt gatatcaaca ggctctcaga ctacgacgtg gaccatatcg tccctcagag
     3421 cttcctcaaa gacgactcaa ttgacaataa ggtgctgact cgctcagaca agaaccgggg
     3481 aaagtcagat aacgtgccct cagaggaagt cgtgaaaaag atgaagaact attggcgcca
     3541 gcttctgaac gcaaagctga tcactcagcg gaagttcgac aatctcacta aggctgagag
     3601 gggcggactg agcgaactgg acaaagcagg attcattaaa cggcaacttg tggagactcg
     3661 gcagattact aaacatgtcg cccaaatcct tgactcacgc atgaatacca agtacgacga
     3721 aaacgacaaa cttatccgcg aggtgaaggt gattaccctg aagtccaagc tggtcagcga
     3781 tttcagaaag gactttcaat tctacaaagt gcgggagatc aataactatc atcatgctca
     3841 tgacgcatat ctgaatgccg tggtgggaac cgccctgatc aagaagtacc caaagctgga
     3901 aagcgagttc gtgtacggag actacaaggt ctacgacgtg cgcaagatga ttgccaaatc
     3961 tgagcaggag atcggaaagg ccaccgcaaa gtacttcttc tacagcaaca tcatgaattt
     4021 cttcaagacc gaaatcaccc ttgcaaacgg tgagatccgg aagaggccgc tcatcgagac
     4081 taatggggag actggcgaaa tcgtgtggga caagggcaga gatttcgcta ccgtgcgcaa
     4141 agtgctttct atgcctcaag tgaacatcgt gaagaaaacc gaggtgcaaa ccggaggctt
     4201 ttctaaggaa tcaatcctcc ccaagcgcaa ctccgacaag ctcattgcaa ggaagaagga
     4261 ttgggaccct aagaagtacg gcggattcga ttcaccaact gtggcttatt ctgtcctggt
     4321 cgtggctaag gtggaaaaag gaaagtctaa gaagctcaag agcgtgaagg aactgctggg
     4381 tatcaccatt atggagcgca gctccttcga gaagaaccca attgactttc tcgaagccaa
     4441 aggttacaag gaagtcaaga aggaccttat catcaagctc ccaaagtata gcctgttcga
     4501 actggagaat gggcggaagc ggatgctcgc ctccgctggc gaacttcaga agggtaatga
     4561 gctggctctc ccctccaagt acgtgaattt cctctacctt gcaagccatt acgagaagct
     4621 gaaggggagc cccgaggaca acgagcaaaa gcaactgttt gtggagcagc ataagcatta
     4681 tctggacgag atcattgagc agatttccga gttttctaaa cgcgtcattc tcgctgatgc
     4741 caacctcgat aaagtcctta gcgcatacaa taagcacaga gacaaaccaa ttcgggagca
     4801 ggctgagaat atcatccacc tgttcaccct caccaatctt ggtgcccctg ccgcattcaa
     4861 gtacttcgac accaccatcg accggaaacg ctatacctcc accaaagaag tgctggacgc
     4921 caccctcatc caccagagca tcaccggact ttacgaaact cggattgacc tctcacagct
     4981 cggaggggat gagggagctg atccaaaaaa gaagagaaag gtagatccaa aaaagaagag
     5041 aaaggtataa ggatcctaac tcgagattga attgaattga aatcgataga tcaatttttt
     5101 tcttttctct ttccccatcc tttacgctaa aataatagtt tattttattt tttgaatatt
     5161 ttttatttat atacgtatat atagactatt atttatcttt taatgattat taagattttt
     5221 attaaaaaaa aattcgctcc tcttttaatg cctttatgca gttttttttt cccattcgat
     5281 atttctatgt gctgccaatg agacgacggg gtcatcacgg ctcatcatgc gccaaacaaa
     5341 tgtgtgcaat acacgctcgg atgactgcat gatgaccgca ctgactgggg acagcagatc
     5401 cacctaagcc tgtgagagaa gcagacaccc gacagatcaa ggcagttaac tagtgcactg
     5461 cagtacagcg gccgcgatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5521 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5581 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5641 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5701 tgataccgcg ggacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5761 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5821 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5881 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5941 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6001 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6061 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6121 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6181 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6241 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6301 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6361 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6421 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6481 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6541 ttccccgaaa agtgccacct gtcatgacca aaatccctta acgtgagttt tcgttccact
     6601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6781 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7261 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     7321 accgtgcggc cgcccctgaa ttcgcatcta g
//
