LOCUS       sequence-231042-        6807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pUC19_BsaI-dCas9-BsaI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6807)
  AUTHORS   Oakes BL, Nadler DC, Flamholz A, Fellmann C, Staahl BT, Doudna JA, 
            Savage DF
  TITLE     Profiling of engineering hotspots identifies an allosteric 
            CRISPR-Cas9 switch.
  JOURNAL   Nat Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528.
  PUBMED    27136077
REFERENCE   2  (bases 1 to 6807)
  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 
            Biotechnol. 2016 May 2. doi: 10.1038/nbt.3528."
COMMENT     Sequence Label: pBLO5.01
FEATURES             Location/Qualifiers
     source          1..6807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(429..455,456..458,459..2945,2946..2948,2949..4532)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(4586..4602)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4586..4602)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4599..4621)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4610..4626
                     /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(4634..4640,4641..4658,4659..4664))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4679..4700
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4817..4834)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4988..5576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5068..5087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5747..6538,6539..6607))
                     /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     6370..6389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6608..6712)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6780..6798
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gtgacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccgggggt
      421 ctcctggtat ggataagaaa tactcaatag gcttagctat cggcacaaat agcgtcggat
      481 gggcggtgat cactgatgaa tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata
      541 cagaccgcca cagtatcaaa aaaaatctta taggggctct tttatttgac agtggagaga
      601 cagcggaagc gactcgtctc aaacggacag ctcgtagaag gtatacacgt cggaagaatc
      661 gtatttgtta tctacaggag attttttcaa atgagatggc gaaagtagat gatagtttct
      721 ttcatcgact tgaagagtct tttttggtgg aagaagacaa gaagcatgaa cgtcatccta
      781 tttttggaaa tatagtagat gaagttgctt atcatgagaa atatccaact atctatcatc
      841 tgcgaaaaaa attggtagat tctactgata aagcggattt gcgcttaatc tatttggcct
      901 tagcgcatat gattaagttt cgtggtcatt ttttgattga gggagattta aatcctgata
      961 atagtgatgt ggacaaacta tttatccagt tggtacaaac ctacaatcaa ttatttgaag
     1021 aaaaccctat taacgcaagt ggagtagatg ctaaagcgat tctttctgca cgattgagta
     1081 aatcaagacg attagaaaat ctcattgctc agctccccgg tgagaagaaa aatggcttat
     1141 ttgggaatct cattgctttg tcattgggtt tgacccctaa ttttaaatca aattttgatt
     1201 tggcagaaga tgctaaatta cagctttcaa aagatactta cgatgatgat ttagataatt
     1261 tattggcgca aattggagat caatatgctg atttgttttt ggcagctaag aatttatcag
     1321 atgctatttt actttcagat atcctaagag taaatactga aataactaag gctcccctat
     1381 cagcttcaat gattaaacgc tacgatgaac atcatcaaga cttgactctt ttaaaagctt
     1441 tagttcgaca acaacttcca gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg
     1501 gatatgcagg ttatattgat gggggagcta gccaagaaga attttataaa tttatcaaac
     1561 caattttaga aaaaatggat ggtactgagg aattattggt gaaactaaat cgtgaagatt
     1621 tgctgcgcaa gcaacggacc tttgacaacg gctctattcc ccatcaaatt cacttgggtg
     1681 agctgcatgc tattttgaga agacaagaag acttttatcc atttttaaaa gacaatcgtg
     1741 agaagattga aaaaatcttg acttttcgaa ttccttatta tgttggtcca ttggcgcgtg
     1801 gcaatagtcg ttttgcatgg atgactcgga agtctgaaga aacaattacc ccatggaatt
     1861 ttgaagaagt tgtcgataaa ggtgcttcag ctcaatcatt tattgaacgc atgacaaact
     1921 ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca tagtttgctt tatgagtatt
     1981 ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg cgaaaaccag
     2041 catttctttc aggtgaacag aagaaagcca ttgttgattt actcttcaaa acaaatcgaa
     2101 aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatgt tttgatagtg
     2161 ttgaaatttc aggagttgaa gatagattta atgcttcatt aggtacctac catgatttgc
     2221 taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat atcttagagg
     2281 atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa agacttaaaa
     2341 catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc cgttatactg
     2401 gttggggacg tttgtctcga aaattgatta atggtattag ggataagcaa tctggcaaaa
     2461 caatattaga ttttttgaaa tcagatggtt ttgccaatcg caattttatg cagctgatcc
     2521 atgatgatag tttgacattt aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg
     2581 atagtttaca tgaacatatt gcaaatttag ctggtagccc tgctattaaa aaaggtattt
     2641 tacagactgt aaaagttgtt gatgaattgg tcaaagtaat ggggcggcat aagccagaaa
     2701 atatcgttat tgaaatggca cgtgaaaatc agacaactca aaagggccag aaaaattcgc
     2761 gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt aggaagtcag attcttaaag
     2821 agcatcctgt tgaaaatact caattgcaaa atgaaaagct ctatctctat tatctccaaa
     2881 atggaagaga catgtatgtg gaccaagaat tagatattaa tcgtttaagt gattatgatg
     2941 tcgatgccat tgttccacaa agtttcctta aagacgattc aatagacaat aaggtcttaa
     3001 cgcgttctga taaaaatcgt ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa
     3061 agatgaaaaa ctattggaga caacttctaa acgccaagtt aatcactcaa cgtaagtttg
     3121 ataatttaac gaaagctgaa cgtggaggtt tgagtgaact tgataaagct ggttttatca
     3181 aacgccaatt ggttgaaact cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc
     3241 gcatgaatac taaatacgat gaaaatgata aacttattcg agaggttaaa gtgattacct
     3301 taaaatctaa attagtttct gacttccgaa aagatttcca attctataaa gtacgtgaga
     3361 ttaacaatta ccatcatgcc catgatgcgt atctaaatgc cgtcgttgga actgctttga
     3421 ttaagaaata tccaaaactt gaatcggagt ttgtctatgg tgattataaa gtttatgatg
     3481 ttcgtaaaat gattgctaag tctgagcaag aaataggcaa agcaaccgca aaatatttct
     3541 tttactctaa tatcatgaac ttcttcaaaa cagaaattac acttgcaaat ggagagattc
     3601 gcaaacgccc tctaatcgaa actaatgggg aaactggaga aattgtctgg gataaagggc
     3661 gagattttgc cacagtgcgc aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa
     3721 cagaagtaca gacaggcgga ttctccaagg agtcaatttt accaaaaaga aattcggaca
     3781 agcttattgc tcgtaaaaaa gactgggatc caaaaaaata tggtggtttt gatagtccaa
     3841 cggtagctta ttcagtccta gtggttgcta aggtggaaaa agggaaatcg aagaagttaa
     3901 aatccgttaa agagttacta gggatcacaa ttatggaaag aagttccttt gaaaaaaatc
     3961 cgattgactt tttagaagct aaaggatata aggaagttaa aaaagactta atcattaaac
     4021 tacctaaata tagtcttttt gagttagaaa acggtcgtaa acggatgctg gctagtgccg
     4081 gagaattaca aaaaggaaat gagctggctc tgccaagcaa atatgtgaat tttttatatt
     4141 tagctagtca ttatgaaaag ttgaagggta gtccagaaga taacgaacaa aaacaattgt
     4201 ttgtggagca gcataagcat tatttagatg agattattga gcaaatcagt gaattttcta
     4261 agcgtgttat tttagcagat gccaatttag ataaagttct tagtgcatat aacaaacata
     4321 gagacaaacc aatacgtgaa caagcagaaa atattattca tttatttacg ttgacgaatc
     4381 ttggagctcc cgctgctttt aaatattttg atacaacaat tgatcgtaaa cgatatacgt
     4441 ctacaaaaga agttttagat gccactctta tccatcaatc catcactggt ctttatgaaa
     4501 cacgcattga tttgagtcag ctaggaggtg acggcgagag acctctagag tcgacctgca
     4561 ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4621 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4681 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4741 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4801 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4861 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4921 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4981 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5041 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5101 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5161 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5221 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5281 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5341 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5401 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5461 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5521 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5581 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5641 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5701 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5761 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5821 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5881 taccgcgaga gccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5941 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6001 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6061 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6121 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6181 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6241 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6301 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6361 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6421 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6481 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6541 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6601 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6661 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6721 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     6781 ataggcgtat cacgaggccc tttcatc
//
