LOCUS       sequence-233276-        6794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 expression unit plasmid. The dCas9 expression unit plasmids 
            contain connector ConL1, ConRE, and one dCas9 transcriptional unit. 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6794)
  AUTHORS   Geng P, Leonard SP, Mishler DM, Barrick JE
  TITLE     Synthetic Genome Defenses against Selfish DNA Elements Stabilize 
            Engineered Bacteria against Evolutionary Failure.
  JOURNAL   ACS Synth Biol. 2019 Mar 15;8(3):521-531. doi: 
            10.1021/acssynbio.8b00426. Epub 2019 Feb 15.
  PUBMED    30703321
REFERENCE   2  (bases 1 to 6794)
  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"; doi: 
            "10.1021/acssynbio.8b00426"; journalName: "ACS Synth Biol"; date: 
            "2019-03-15- 15"; volume: "8"; issue: "3"; pages: "521-531"
COMMENT     Sequence Label: TDK promoter dCas9
FEATURES             Location/Qualifiers
     source          1..6794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             238..249
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(258..284,285..287,288..2774,2775..2777,2778..4364)
                     /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
                     NLIGALLFDSGETAEATRHKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     misc_feature    4385..4516
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   4517..4552
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     terminator      4568..4616
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(join(5027..5818,5819..5887))
                     /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     5650..5669
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5888..5992)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6095..6683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6584..6603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcgcat ctagaacggg gtcatcacgg ctcatcatgc gccaaacaaa tgtgtgcaat
       61 acacgctcgg atgactgcat gatgaccgca ctgactgggg acagcagatc cacctaagcc
      121 tgtgagagaa gcagacaccc gacagatcaa ggcagttacg tctcaccaaa ccagatgtca
      181 acacagctac aacgcccagc ctccaattca aatcataaaa aatttatttg ctttattaaa
      241 gaggagaaat taattatatg gataagaaat actcaatagg cttagctatc ggcacaaata
      301 gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag ttcaaggttc
      361 tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt ttatttgaca
      421 gtggagagac agcggaagcg actcgtcata aacggacagc tcgtagaagg tatacacgtc
      481 ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg aaagtagatg
      541 atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag aagcatgaac
      601 gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa tatccaacta
      661 tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg cgcttaatct
      721 atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag ggagatttaa
      781 atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc tacaatcaat
      841 tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt ctttctgcac
      901 gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt gagaagaaaa
      961 atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat tttaaatcaa
     1021 attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac gatgatgatt
     1081 tagataattt attggcgcaa attggagatc aatatgctga tttgtttttg gcagctaaga
     1141 atttatcaga tgctatttta ctttcagata tcctaagagt aaatactgaa ataactaagg
     1201 ctcccctatc agcttcaatg attaaacgct acgatgaaca tcatcaagac ttgactcttt
     1261 taaaagcttt agttcgacaa caacttccag aaaagtataa agaaatcttt tttgatcaat
     1321 caaaaaacgg atatgcaggt tatattgatg ggggagctag ccaagaagaa ttttataaat
     1381 ttatcaaacc aattttagaa aaaatggatg gtactgagga attattggtg aaactaaatc
     1441 gtgaagattt gctgcgcaag caacggacct ttgacaacgg ctctattccc catcaaattc
     1501 acttgggtga gctgcatgct attttgagaa gacaagaaga cttttatcca tttttaaaag
     1561 acaatcgtga gaagattgaa aaaatcttga cttttcgaat tccttattat gttggtccat
     1621 tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa gtctgaagaa acaattaccc
     1681 catggaattt tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt attgaacgca
     1741 tgacaaactt tgataaaaat cttccaaatg aaaaagtact accaaaacat agtttgcttt
     1801 atgagtattt tacggtttat aacgaattga caaaggtcaa atatgttact gaaggaatgc
     1861 gaaaaccagc atttctttca ggtgaacaga agaaagccat tgttgattta ctcttcaaaa
     1921 caaatcgaaa agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa atagaatgtt
     1981 ttgatagtgt tgaaatttca ggagttgaag atagatttaa tgcttcatta ggtacctacc
     2041 atgatttgct aaaaattatt aaagataaag attttttgga taatgaagaa aatgaagata
     2101 tcttagagga tattgtttta acattgacct tatttgaaga tagggagatg attgaggaaa
     2161 gacttaaaac atatgctcac ctctttgatg ataaggtgat gaaacagctt aaacgtcgcc
     2221 gttatactgg ttggggacgt ttgtctcgaa aattgattaa tggtattagg gataagcaat
     2281 ctggcaaaac aatattagat tttttgaaat cagatggttt tgccaatcgc aattttatgc
     2341 agctgatcca tgatgatagt ttgacattta aagaagacat tcaaaaagca caagtgtctg
     2401 gacaaggcga tagtttacat gaacatattg caaatttagc tggtagccct gctattaaaa
     2461 aaggtatttt acagactgta aaagttgttg atgaattggt caaagtaatg gggcggcata
     2521 agccagaaaa tatcgttatt gaaatggcac gtgaaaatca gacaactcaa aagggccaga
     2581 aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat caaagaatta ggaagtcaga
     2641 ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc tatctctatt
     2701 atctccaaaa tggaagagac atgtatgtgg accaagaatt agatattaat cgtttaagtg
     2761 attatgatgt cgatgccatt gttccacaaa gtttccttaa agacgattca atagacaata
     2821 aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca agtgaagaag
     2881 tagtcaaaaa gatgaaaaac tattggagac aacttctaaa cgccaagtta atcactcaac
     2941 gtaagtttga taatttaacg aaagctgaac gtggaggttt gagtgaactt gataaagctg
     3001 gttttatcaa acgccaattg gttgaaactc gccaaatcac taagcatgtg gcacaaattt
     3061 tggatagtcg catgaatact aaatacgatg aaaatgataa acttattcga gaggttaaag
     3121 tgattacctt aaaatctaaa ttagtttctg acttccgaaa agatttccaa ttctataaag
     3181 tacgtgagat taacaattac catcatgccc atgatgcgta tctaaatgcc gtcgttggaa
     3241 ctgctttgat taagaaatat ccaaaacttg aatcggagtt tgtctatggt gattataaag
     3301 tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa gcaaccgcaa
     3361 aatatttctt ttactctaat atcatgaact tcttcaaaac agaaattaca cttgcaaatg
     3421 gagagattcg caaacgccct ctaatcgaaa ctaatgggga aactggagaa attgtctggg
     3481 ataaagggcg agattttgcc acagtgcgca aagtattgtc catgccccaa gtcaatattg
     3541 tcaagaaaac agaagtacag acaggcggat tctccaagga gtcaatttta ccaaaaagaa
     3601 attcggacaa gcttattgct cgtaaaaaag actgggatcc aaaaaaatat ggtggttttg
     3661 atagtccaac ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa gggaaatcga
     3721 agaagttaaa atccgttaaa gagttactag ggatcacaat tatggaaaga agttcctttg
     3781 aaaaaaatcc gattgacttt ttagaagcta aaggatataa ggaagttaaa aaagacttaa
     3841 tcattaaact acctaaatat agtctttttg agttagaaaa cggtcgtaaa cggatgctgg
     3901 ctagtgccgg agaattacaa aaaggaaatg agctggctct gccaagcaaa tatgtgaatt
     3961 ttttatattt agctagtcat tatgaaaagt tgaagggtag tccagaagat aacgaacaaa
     4021 aacaattgtt tgtggagcag cataagcatt atttagatga gattattgag caaatcagtg
     4081 aattttctaa gcgtgttatt ttagcagatg ccaatttaga taaagttctt agtgcatata
     4141 acaaacatag agacaaacca atacgtgaac aagcagaaaa tattattcat ttatttacgt
     4201 tgacgaatct tggagctccc gctgctttta aatattttga tacaacaatt gatcgtaaac
     4261 gatatacgtc tacaaaagaa gttttagatg ccactcttat ccatcaatcc atcactggtc
     4321 tttatgaaac acgcattgat ttgagtcagc taggaggtga ctgaagtata ttttagatga
     4381 agattatttc ttaataacta aaaatatggt ataatactct taataaatgc agtaatacag
     4441 gggcttttca agactgaagt ctagctgaga caaatagtgc gattacgaaa ttttttagac
     4501 aaaaatagtc tacgaggttt tagagctatg ctgttttgaa tggtcccaaa acaaagcaag
     4561 ctgataaacc gatacaatta aaggctcctt ttggagcctt tttttttgga gattttcaac
     4621 atgaagaaat tattatttga tgatcagata gcggcgggga actgccagac atcaaataaa
     4681 acaaaaggct cagtcggaag actgggcctt ttgttttatc tgttgtttgt cggtgaacac
     4741 tctcccggct gagcatgaga cggaaatctg ctcgtcagtg gtgctcacac tgacgaatca
     4801 tgtacagatc ataccgatga ctgcctggcg actcacaact aagcaagaca gccggaacca
     4861 gcgccggcga acaccactgc atatatggca tatcacaaca gtccaactag tgcactgcag
     4921 tacagcggcc gcgattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4981 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5041 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5101 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5161 taccgcggga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5221 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5281 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5341 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5401 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5461 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5521 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5581 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5641 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5701 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5761 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5821 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5881 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5941 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6001 cccgaaaagt gccacctgtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6061 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6121 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6181 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6241 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6301 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6361 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6421 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6481 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6541 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6601 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6661 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6721 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6781 gtgcggccgc ccct
//
