LOCUS       sequence-217181-        7045 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 7045)
  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 7045)
  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: Native promoter dCas9
FEATURES             Location/Qualifiers
     source          1..7045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(509..535,536..538,539..3025,3026..3028,3029..4615)
                     /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    4636..4767
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   4768..4803
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     terminator      4819..4867
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(join(5278..6069,6070..6138))
                     /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     5901..5920
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6139..6243)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      6346..6934
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6835..6854
                     /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 aacggctgtt ttgaatggtt ccaacaagat tattttataa cttttataac
      241 aaataatcaa ggagaaattc aaagaaattt atcagccata aaacaatact taatactata
      301 gaatgataac aaaataaact actttttaaa agaattttgt gttataatct atttattatt
      361 aagtattggg taatattttt tgaagagata ttttgaaaaa gaaaaattaa agcatattaa
      421 actaatttcg gaggtcatta aaactattat tgaaatcatc aaactcatta tggatttaat
      481 ttaaactttt tattttagga ggcaaaatat ggataagaaa tactcaatag gcttagctat
      541 cggcacaaat agcgtcggat gggcggtgat cactgatgaa tataaggttc cgtctaaaaa
      601 gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa aaaaatctta taggggctct
      661 tttatttgac agtggagaga cagcggaagc gactcgtcat aaacggacag ctcgtagaag
      721 gtatacacgt cggaagaatc gtatttgtta tctacaggag attttttcaa atgagatggc
      781 gaaagtagat gatagtttct ttcatcgact tgaagagtct tttttggtgg aagaagacaa
      841 gaagcatgaa cgtcatccta tttttggaaa tatagtagat gaagttgctt atcatgagaa
      901 atatccaact atctatcatc tgcgaaaaaa attggtagat tctactgata aagcggattt
      961 gcgcttaatc tatttggcct tagcgcatat gattaagttt cgtggtcatt ttttgattga
     1021 gggagattta aatcctgata atagtgatgt ggacaaacta tttatccagt tggtacaaac
     1081 ctacaatcaa ttatttgaag aaaaccctat taacgcaagt ggagtagatg ctaaagcgat
     1141 tctttctgca cgattgagta aatcaagacg attagaaaat ctcattgctc agctccccgg
     1201 tgagaagaaa aatggcttat ttgggaatct cattgctttg tcattgggtt tgacccctaa
     1261 ttttaaatca aattttgatt tggcagaaga tgctaaatta cagctttcaa aagatactta
     1321 cgatgatgat ttagataatt tattggcgca aattggagat caatatgctg atttgttttt
     1381 ggcagctaag aatttatcag atgctatttt actttcagat atcctaagag taaatactga
     1441 aataactaag gctcccctat cagcttcaat gattaaacgc tacgatgaac atcatcaaga
     1501 cttgactctt ttaaaagctt tagttcgaca acaacttcca gaaaagtata aagaaatctt
     1561 ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat gggggagcta gccaagaaga
     1621 attttataaa tttatcaaac caattttaga aaaaatggat ggtactgagg aattattggt
     1681 gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc tttgacaacg gctctattcc
     1741 ccatcaaatt cacttgggtg agctgcatgc tattttgaga agacaagaag acttttatcc
     1801 atttttaaaa gacaatcgtg agaagattga aaaaatcttg acttttcgaa ttccttatta
     1861 tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg atgactcgga agtctgaaga
     1921 aacaattacc ccatggaatt ttgaagaagt tgtcgataaa ggtgcttcag ctcaatcatt
     1981 tattgaacgc atgacaaact ttgataaaaa tcttccaaat gaaaaagtac taccaaaaca
     2041 tagtttgctt tatgagtatt ttacggttta taacgaattg acaaaggtca aatatgttac
     2101 tgaaggaatg cgaaaaccag catttctttc aggtgaacag aagaaagcca ttgttgattt
     2161 actcttcaaa acaaatcgaa aagtaaccgt taagcaatta aaagaagatt atttcaaaaa
     2221 aatagaatgt tttgatagtg ttgaaatttc aggagttgaa gatagattta atgcttcatt
     2281 aggtacctac catgatttgc taaaaattat taaagataaa gattttttgg ataatgaaga
     2341 aaatgaagat atcttagagg atattgtttt aacattgacc ttatttgaag atagggagat
     2401 gattgaggaa agacttaaaa catatgctca cctctttgat gataaggtga tgaaacagct
     2461 taaacgtcgc cgttatactg gttggggacg tttgtctcga aaattgatta atggtattag
     2521 ggataagcaa tctggcaaaa caatattaga ttttttgaaa tcagatggtt ttgccaatcg
     2581 caattttatg cagctgatcc atgatgatag tttgacattt aaagaagaca ttcaaaaagc
     2641 acaagtgtct ggacaaggcg atagtttaca tgaacatatt gcaaatttag ctggtagccc
     2701 tgctattaaa aaaggtattt tacagactgt aaaagttgtt gatgaattgg tcaaagtaat
     2761 ggggcggcat aagccagaaa atatcgttat tgaaatggca cgtgaaaatc agacaactca
     2821 aaagggccag aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta tcaaagaatt
     2881 aggaagtcag attcttaaag agcatcctgt tgaaaatact caattgcaaa atgaaaagct
     2941 ctatctctat tatctccaaa atggaagaga catgtatgtg gaccaagaat tagatattaa
     3001 tcgtttaagt gattatgatg tcgatgccat tgttccacaa agtttcctta aagacgattc
     3061 aatagacaat aaggtcttaa cgcgttctga taaaaatcgt ggtaaatcgg ataacgttcc
     3121 aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga caacttctaa acgccaagtt
     3181 aatcactcaa cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt tgagtgaact
     3241 tgataaagct ggttttatca aacgccaatt ggttgaaact cgccaaatca ctaagcatgt
     3301 ggcacaaatt ttggatagtc gcatgaatac taaatacgat gaaaatgata aacttattcg
     3361 agaggttaaa gtgattacct taaaatctaa attagtttct gacttccgaa aagatttcca
     3421 attctataaa gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt atctaaatgc
     3481 cgtcgttgga actgctttga ttaagaaata tccaaaactt gaatcggagt ttgtctatgg
     3541 tgattataaa gtttatgatg ttcgtaaaat gattgctaag tctgagcaag aaataggcaa
     3601 agcaaccgca aaatatttct tttactctaa tatcatgaac ttcttcaaaa cagaaattac
     3661 acttgcaaat ggagagattc gcaaacgccc tctaatcgaa actaatgggg aaactggaga
     3721 aattgtctgg gataaagggc gagattttgc cacagtgcgc aaagtattgt ccatgcccca
     3781 agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg agtcaatttt
     3841 accaaaaaga aattcggaca agcttattgc tcgtaaaaaa gactgggatc caaaaaaata
     3901 tggtggtttt gatagtccaa cggtagctta ttcagtccta gtggttgcta aggtggaaaa
     3961 agggaaatcg aagaagttaa aatccgttaa agagttacta gggatcacaa ttatggaaag
     4021 aagttccttt gaaaaaaatc cgattgactt tttagaagct aaaggatata aggaagttaa
     4081 aaaagactta atcattaaac tacctaaata tagtcttttt gagttagaaa acggtcgtaa
     4141 acggatgctg gctagtgccg gagaattaca aaaaggaaat gagctggctc tgccaagcaa
     4201 atatgtgaat tttttatatt tagctagtca ttatgaaaag ttgaagggta gtccagaaga
     4261 taacgaacaa aaacaattgt ttgtggagca gcataagcat tatttagatg agattattga
     4321 gcaaatcagt gaattttcta agcgtgttat tttagcagat gccaatttag ataaagttct
     4381 tagtgcatat aacaaacata gagacaaacc aatacgtgaa caagcagaaa atattattca
     4441 tttatttacg ttgacgaatc ttggagctcc cgctgctttt aaatattttg atacaacaat
     4501 tgatcgtaaa cgatatacgt ctacaaaaga agttttagat gccactctta tccatcaatc
     4561 catcactggt ctttatgaaa cacgcattga tttgagtcag ctaggaggtg actgaagtat
     4621 attttagatg aagattattt cttaataact aaaaatatgg tataatactc ttaataaatg
     4681 cagtaataca ggggcttttc aagactgaag tctagctgag acaaatagtg cgattacgaa
     4741 attttttaga caaaaatagt ctacgaggtt ttagagctat gctgttttga atggtcccaa
     4801 aacaaagcaa gctgataaac cgatacaatt aaaggctcct tttggagcct ttttttttgg
     4861 agattttcaa catgaagaaa ttattatttg atgatcagat agcggcgggg aactgccaga
     4921 catcaaataa aacaaaaggc tcagtcggaa gactgggcct tttgttttat ctgttgtttg
     4981 tcggtgaaca ctctcccggc tgagcatgag acggaaatct gctcgtcagt ggtgctcaca
     5041 ctgacgaatc atgtacagat cataccgatg actgcctggc gactcacaac taagcaagac
     5101 agccggaacc agcgccggcg aacaccactg catatatggc atatcacaac agtccaacta
     5161 gtgcactgca gtacagcggc cgcgattatc aaaaaggatc ttcacctaga tccttttaaa
     5221 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5281 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5341 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5401 tgctgcaatg ataccgcggg acccacgctc accggctcca gatttatcag caataaacca
     5461 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5521 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5581 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5641 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5701 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5761 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5821 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5881 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5941 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6001 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6061 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6121 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6181 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6241 gcgcacattt ccccgaaaag tgccacctgt catgaccaaa atcccttaac gtgagttttc
     6301 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6361 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6421 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6481 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6541 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6601 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6661 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6721 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6781 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6841 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6901 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6961 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     7021 tgtggataac cgtgcggccg cccct
//
