LOCUS       sequence-216332-        6981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Addgene plasmid 44249 where dCas9 has been replaced with dCas9 fused
            to the omega subunit of RNAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6981)
  AUTHORS   Otoupal PB, Cordell WT, Bachu V, Sitton MJ, Chatterjee A
  TITLE     Multiplexed deactivated CRISPR-Cas9 gene expression perturbations 
            deter bacterial adaptation by inducing negative epistasis.
  JOURNAL   Commun Biol. 2018 Sep 3;1:129. doi: 10.1038/s42003-018-0135-2. 
            eCollection 2018.
  PUBMED    30272008
REFERENCE   2  (bases 1 to 6981)
  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: "Commun 
            Biol."; date: "2018-09-3"; volume: "1:129. doi"; pages: " 
            10.1038/s42003-018-0135-2. eCollection 2018"
COMMENT     Sequence Label: pdCas9-omega
FEATURES             Location/Qualifiers
     source          1..6981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             7..18
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(25..51,52..54,55..2541,2542..2544,2545..4128)
                     /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"
     CDS             4135..4407
                     /codon_start=1
                     /gene="E. coli rpoZ"
                     /product="DNA-directed RNA polymerase subunit omega"
                     /label=RNAP omega
                     /note="promotes RNA polymerase assembly"
                     /translation="ARVTVQDAVEKIGNRFDLVLVAARRARQMQVGGKDPLVPEENDKT
                     TVIALREIEEGLINNQILDVRERQEQQEQEAAELQAVTAIAEGRR"
     terminator      4431..4502
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4518..4545
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4573..4590)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4707..5252)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(4787..4806)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5366..5460)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5481..6140)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6055..6074
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6141..6243)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6273..6899)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     6804..6823
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        6915..6970
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6951..6969
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agatctaaag aggagaaagg atctatggat aagaaatact caataggctt agctatcggc
       61 acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc taaaaagttc
      121 aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg ggctctttta
      181 tttgacagtg gagagacagc ggaagcgact cgtctcaaac ggacagctcg tagaaggtat
      241 acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga gatggcgaaa
      301 gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga agacaagaag
      361 catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca tgagaaatat
      421 ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc ggatttgcgc
      481 ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt gattgaggga
      541 gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt acaaacctac
      601 aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa agcgattctt
      661 tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct ccccggtgag
      721 aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac ccctaatttt
      781 aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga tacttacgat
      841 gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt gtttttggca
      901 gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa tactgaaata
      961 actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca tcaagacttg
     1021 actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga aatctttttt
     1081 gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca agaagaattt
     1141 tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt attggtgaaa
     1201 ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc tattccccat
     1261 caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt ttatccattt
     1321 ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc ttattatgtt
     1381 ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca
     1441 attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca atcatttatt
     1501 gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc aaaacatagt
     1561 ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata tgttactgaa
     1621 ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt tgatttactc
     1681 ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt caaaaaaata
     1741 gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc ttcattaggt
     1801 acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa tgaagaaaat
     1861 gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag ggagatgatt
     1921 gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa acagcttaaa
     1981 cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg tattagggat
     2041 aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc caatcgcaat
     2101 tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca aaaagcacaa
     2161 gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg tagccctgct
     2221 attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa agtaatgggg
     2281 cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac aactcaaaag
     2341 ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga
     2401 agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga aaagctctat
     2461 ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga tattaatcgt
     2521 ttaagtgatt atgatgtcga tgccattgtt ccacaaagtt tccttaaaga cgattcaata
     2581 gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa cgttccaagt
     2641 gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc caagttaatc
     2701 actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat
     2761 aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa gcatgtggca
     2821 caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact tattcgagag
     2881 gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga tttccaattc
     2941 tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct aaatgccgtc
     3001 gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt ctatggtgat
     3061 tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat aggcaaagca
     3121 accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga aattacactt
     3181 gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac tggagaaatt
     3241 gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat gccccaagtc
     3301 aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc aattttacca
     3361 aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa aaaatatggt
     3421 ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg
     3481 aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat ggaaagaagt
     3541 tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga agttaaaaaa
     3601 gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg
     3661 atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc aagcaaatat
     3721 gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc agaagataac
     3781 gaacaaaaac aattgtttgt ggagcagcat aagcattatt tagatgagat tattgagcaa
     3841 atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa agttcttagt
     3901 gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat tattcattta
     3961 tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac aacaattgat
     4021 cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca tcaatccatc
     4081 actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacgc agctgcacgc
     4141 gtaactgttc aggacgctgt agagaaaatt ggtaaccgtt ttgacctggt actggtcgcc
     4201 gcgcgtcgcg ctcgtcagat gcaggtaggc ggaaaggatc cgctggtacc ggaagaaaac
     4261 gataaaacca ctgtaatcgc gctgcgcgaa atcgaagaag gtctgatcaa caaccagatc
     4321 ctcgacgttc gcgaacgcca ggaacagcaa gagcaggaag ccgctgaatt acaagccgtt
     4381 accgctattg ctgaaggtcg tcgttaactc gagtaaggat ctccaggcat caaataaaac
     4441 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     4501 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acctagggat
     4561 atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga
     4621 aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac ttaacaggga
     4681 agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac
     4741 gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg
     4801 tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc
     4861 attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc
     4921 agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt
     4981 atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc
     5041 agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa
     5101 ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt
     5161 tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc
     5221 aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat cagataaaat
     5281 atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     5341 gatataagtt gttactagtg cttggattct caccaataaa aaacgcccgg cggcaaccga
     5401 gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc aacaggagtc
     5461 caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat
     5521 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc
     5581 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg
     5641 aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg
     5701 gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg
     5761 taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca
     5821 ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca
     5881 ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg atgagcattc
     5941 atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg
     6001 gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact
     6061 gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat
     6121 ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa
     6181 aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga
     6241 tcaacgtctc attttcgcca gatatcgacg tcttaagacc cactttcaca tttaagttgt
     6301 ttttctaatc cgcatatgat caattcaagg ccgaataaga aggctggctc tgcaccttgg
     6361 tgatcaaata attcgatagc ttgtcgtaat aatggcggca tactatcagt agtaggtgtt
     6421 tccctttctt ctttagcgac ttgatgctct tgatcttcca atacgcaacc taaagtaaaa
     6481 tgccccacag cgctgagtgc atataatgca ttctctagtg aaaaaccttg ttggcataaa
     6541 aaggctaatt gattttcgag agtttcatac tgtttttctg taggccgtgt acctaaatgt
     6601 acttttgctc catcgcgatg acttagtaaa gcacatctaa aacttttagc gttattacgt
     6661 aaaaaatctt gccagctttc cccttctaaa gggcaaaagt gagtatggtg cctatctaac
     6721 atctcaatgg ctaaggcgtc gagcaaagcc cgcttatttt ttacatgcca atacaatgta
     6781 ggctgctcta cacctagctt ctgggcgagt ttacgggttg ttaaaccttc gattccgacc
     6841 tcattaagca gctctaatgc gctgttaatc actttacttt tatctaatct agacatcatt
     6901 aattcctaat ttttgttgac actctatcgt tgatagagtt attttaccac tccctatcag
     6961 tgatagagaa aagaattcaa a
//
