LOCUS       sequence-221991-        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             18..29
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(36..62,63..65,66..2552,2553..2555,2556..4139)
                     /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             4146..4418
                     /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      4442..4513
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4529..4556
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4584..4601)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4718..5263)
                     /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(4798..4817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5377..5471)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5492..6151)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     6066..6085
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(6152..6254)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(6284..6910)
                     /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     6815..6834
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        6926..6981
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    6962..6980
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 aagaattcaa aagatctaaa gaggagaaag gatctatgga taagaaatac tcaataggct
       61 tagctatcgg cacaaatagc gtcggatggg cggtgatcac tgatgaatat aaggttccgt
      121 ctaaaaagtt caaggttctg ggaaatacag accgccacag tatcaaaaaa aatcttatag
      181 gggctctttt atttgacagt ggagagacag cggaagcgac tcgtctcaaa cggacagctc
      241 gtagaaggta tacacgtcgg aagaatcgta tttgttatct acaggagatt ttttcaaatg
      301 agatggcgaa agtagatgat agtttctttc atcgacttga agagtctttt ttggtggaag
      361 aagacaagaa gcatgaacgt catcctattt ttggaaatat agtagatgaa gttgcttatc
      421 atgagaaata tccaactatc tatcatctgc gaaaaaaatt ggtagattct actgataaag
      481 cggatttgcg cttaatctat ttggccttag cgcatatgat taagtttcgt ggtcattttt
      541 tgattgaggg agatttaaat cctgataata gtgatgtgga caaactattt atccagttgg
      601 tacaaaccta caatcaatta tttgaagaaa accctattaa cgcaagtgga gtagatgcta
      661 aagcgattct ttctgcacga ttgagtaaat caagacgatt agaaaatctc attgctcagc
      721 tccccggtga gaagaaaaat ggcttatttg ggaatctcat tgctttgtca ttgggtttga
      781 cccctaattt taaatcaaat tttgatttgg cagaagatgc taaattacag ctttcaaaag
      841 atacttacga tgatgattta gataatttat tggcgcaaat tggagatcaa tatgctgatt
      901 tgtttttggc agctaagaat ttatcagatg ctattttact ttcagatatc ctaagagtaa
      961 atactgaaat aactaaggct cccctatcag cttcaatgat taaacgctac gatgaacatc
     1021 atcaagactt gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag
     1081 aaatcttttt tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc
     1141 aagaagaatt ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat
     1201 tattggtgaa actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct
     1261 ctattcccca tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact
     1321 tttatccatt tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc
     1381 cttattatgt tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt
     1441 ctgaagaaac aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc
     1501 aatcatttat tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac
     1561 caaaacatag tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat
     1621 atgttactga aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg
     1681 ttgatttact cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt
     1741 tcaaaaaaat agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg
     1801 cttcattagg tacctaccat gatttgctaa aaattattaa agataaagat tttttggata
     1861 atgaagaaaa tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata
     1921 gggagatgat tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga
     1981 aacagcttaa acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg
     2041 gtattaggga taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg
     2101 ccaatcgcaa ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc
     2161 aaaaagcaca agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg
     2221 gtagccctgc tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca
     2281 aagtaatggg gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga
     2341 caactcaaaa gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca
     2401 aagaattagg aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg
     2461 aaaagctcta tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag
     2521 atattaatcg tttaagtgat tatgatgtcg atgccattgt tccacaaagt ttccttaaag
     2581 acgattcaat agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata
     2641 acgttccaag tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg
     2701 ccaagttaat cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga
     2761 gtgaacttga taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta
     2821 agcatgtggc acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac
     2881 ttattcgaga ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag
     2941 atttccaatt ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc
     3001 taaatgccgt cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg
     3061 tctatggtga ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa
     3121 taggcaaagc aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag
     3181 aaattacact tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa
     3241 ctggagaaat tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca
     3301 tgccccaagt caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt
     3361 caattttacc aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa
     3421 aaaaatatgg tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg
     3481 tggaaaaagg gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta
     3541 tggaaagaag ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg
     3601 aagttaaaaa agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg
     3661 gtcgtaaacg gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc
     3721 caagcaaata tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc
     3781 cagaagataa cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga
     3841 ttattgagca aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata
     3901 aagttcttag tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata
     3961 ttattcattt atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata
     4021 caacaattga tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc
     4081 atcaatccat cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgacg
     4141 cagctgcacg cgtaactgtt caggacgctg tagagaaaat tggtaaccgt tttgacctgg
     4201 tactggtcgc cgcgcgtcgc gctcgtcaga tgcaggtagg cggaaaggat ccgctggtac
     4261 cggaagaaaa cgataaaacc actgtaatcg cgctgcgcga aatcgaagaa ggtctgatca
     4321 acaaccagat cctcgacgtt cgcgaacgcc aggaacagca agagcaggaa gccgctgaat
     4381 tacaagccgt taccgctatt gctgaaggtc gtcgttaact cgagtaagga tctccaggca
     4441 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     4501 ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta
     4561 tacctaggga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg
     4621 cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata
     4681 cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg
     4741 acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa
     4801 gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt
     4861 ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt
     4921 tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac
     4981 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca
     5041 ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg
     5101 ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg
     5161 gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc
     5221 gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa
     5281 tcagataaaa tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt
     5341 cagccccata cgatataagt tgttactagt gcttggattc tcaccaataa aaaacgcccg
     5401 gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat
     5461 caacaggagt ccaagcgagc tcgatatcaa attacgcccc gccctgccac tcatcgcagt
     5521 actgttgtaa ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa
     5581 cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga
     5641 aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca
     5701 cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca
     5761 ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt
     5821 cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac
     5881 aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cgaaattccg
     5941 gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat
     6001 ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac
     6061 attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa
     6121 cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg
     6181 ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc
     6241 ttacgtgccg atcaacgtct cattttcgcc agatatcgac gtcttaagac ccactttcac
     6301 atttaagttg tttttctaat ccgcatatga tcaattcaag gccgaataag aaggctggct
     6361 ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa taatggcggc atactatcag
     6421 tagtaggtgt ttccctttct tctttagcga cttgatgctc ttgatcttcc aatacgcaac
     6481 ctaaagtaaa atgccccaca gcgctgagtg catataatgc attctctagt gaaaaacctt
     6541 gttggcataa aaaggctaat tgattttcga gagtttcata ctgtttttct gtaggccgtg
     6601 tacctaaatg tacttttgct ccatcgcgat gacttagtaa agcacatcta aaacttttag
     6661 cgttattacg taaaaaatct tgccagcttt ccccttctaa agggcaaaag tgagtatggt
     6721 gcctatctaa catctcaatg gctaaggcgt cgagcaaagc ccgcttattt tttacatgcc
     6781 aatacaatgt aggctgctct acacctagct tctgggcgag tttacgggtt gttaaacctt
     6841 cgattccgac ctcattaagc agctctaatg cgctgttaat cactttactt ttatctaatc
     6901 tagacatcat taattcctaa tttttgttga cactctatcg ttgatagagt tattttacca
     6961 ctccctatca gtgatagaga a
//
