LOCUS       sequence-162048-        8191 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dCas9 without a promoter in acsA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8191)
  AUTHORS   Gordon GC, Korosh TC, Cameron JC, Markley AL, Begemann MB, Pfleger 
            BF
  TITLE     CRISPR interference as a titratable, trans-acting regulatory tool 
            for metabolic engineering in the cyanobacterium Synechococcus sp. 
            strain PCC 7002.
  JOURNAL   Metab Eng. 2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 
            10.1016/j.ymben.2016.07.007.
  PUBMED    27481676
REFERENCE   2  (bases 1 to 8191)
  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: "Metab Eng. 
            2016 Jul 29. pii: S1096-7176(16)30062-3. doi: 
            10.1016/j.ymben.2016.07.007."
COMMENT     Sequence Label: pCas8
FEATURES             Location/Qualifiers
     source          1..8191
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..83)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     274..295
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        510..614
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(615..683,684..1475)
                     /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     complement(833..852)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1646..2234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2135..2154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2388..2405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2522..2543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2558..2563,2564..2581,2582..2588)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2596..2612
                     /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)."
     primer_bind     2601..2623
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2620..2636
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2620..2636
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2654..2672
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2654..2671
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             join(3275..3301,3302..3304,3305..5791,5792..5794,
                     5795..7381)
                     /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(7417..7435)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7431..7478
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(7985..8004)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7986..8004)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8011..8028)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8011..8027)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8020..8042)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(8168..8191,1..432)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
       61 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
      121 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
      181 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      241 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      301 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
      361 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      421 cgcttacaat ttcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      481 catcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      541 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      601 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
      661 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
      721 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
      781 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
      841 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
      901 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
      961 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1021 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1081 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1141 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1201 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1261 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1321 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1381 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1441 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1501 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     1801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     1861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     1921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     1981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2341 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2401 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2461 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     2521 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     2581 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     2641 attacgccaa gctatttagg tgacactata gaatactcaa gctatgaggt gactgccgca
     2701 ctcaatgttc aaccagatct agtgattatt tcggggttag cagccgatgc gggcaacctg
     2761 gtgaagcaac tgcgagaatt aggttacaac ggcattattg ttggggggaa cggcttaaat
     2821 acttctaata ttttccccgt ctgccaagca aaatgtgatg gggtgttggt ggcccaagcc
     2881 tacagtgccg agttagataa tgagattaac cgcgcgtttc gggacgccta ttttcaacaa
     2941 aaccaaaaag agccgcccca atttagtgcc caggctttta cggcgatcca agtttttgtt
     3001 gaagccctca gcagcctcga tgaaaaaacg cccttagaaa ctcttgctct accggacttg
     3061 cgacgacaac tgcgggacga aatttttgca ggtacctacg tcacgccttt gggtgaaatt
     3121 tccttcacag aggaagggga aattgtccag aaggaatttt ttgtggccca aattgaaatg
     3181 gatgaatcgg gtcaacaggg gcgtttcgcc ttcattgaaa cgaacaaagg aattcgagct
     3241 cccaactcat aaagtcaagt aggagattaa ttccatggat aagaaatact caataggctt
     3301 agctatcggc acaaatagcg tcggatgggc ggtgatcact gatgaatata aggttccgtc
     3361 taaaaagttc aaggttctgg gaaatacaga ccgccacagt atcaaaaaaa atcttatagg
     3421 ggctctttta tttgacagtg gagagacagc ggaagcgact cgtctcaaac ggacagctcg
     3481 tagaaggtat acacgtcgga agaatcgtat ttgttatcta caggagattt tttcaaatga
     3541 gatggcgaaa gtagatgata gtttctttca tcgacttgaa gagtcttttt tggtggaaga
     3601 agacaagaag catgaacgtc atcctatttt tggaaatata gtagatgaag ttgcttatca
     3661 tgagaaatat ccaactatct atcatctgcg aaaaaaattg gtagattcta ctgataaagc
     3721 ggatttgcgc ttaatctatt tggccttagc gcatatgatt aagtttcgtg gtcatttttt
     3781 gattgaggga gatttaaatc ctgataatag tgatgtggac aaactattta tccagttggt
     3841 acaaacctac aatcaattat ttgaagaaaa ccctattaac gcaagtggag tagatgctaa
     3901 agcgattctt tctgcacgat tgagtaaatc aagacgatta gaaaatctca ttgctcagct
     3961 ccccggtgag aagaaaaatg gcttatttgg gaatctcatt gctttgtcat tgggtttgac
     4021 ccctaatttt aaatcaaatt ttgatttggc agaagatgct aaattacagc tttcaaaaga
     4081 tacttacgat gatgatttag ataatttatt ggcgcaaatt ggagatcaat atgctgattt
     4141 gtttttggca gctaagaatt tatcagatgc tattttactt tcagatatcc taagagtaaa
     4201 tactgaaata actaaggctc ccctatcagc ttcaatgatt aaacgctacg atgaacatca
     4261 tcaagacttg actcttttaa aagctttagt tcgacaacaa cttccagaaa agtataaaga
     4321 aatctttttt gatcaatcaa aaaacggata tgcaggttat attgatgggg gagctagcca
     4381 agaagaattt tataaattta tcaaaccaat tttagaaaaa atggatggta ctgaggaatt
     4441 attggtgaaa ctaaatcgtg aagatttgct gcgcaagcaa cggacctttg acaacggctc
     4501 tattccccat caaattcact tgggtgagct gcatgctatt ttgagaagac aagaagactt
     4561 ttatccattt ttaaaagaca atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc
     4621 ttattatgtt ggtccattgg cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc
     4681 tgaagaaaca attaccccat ggaattttga agaagttgtc gataaaggtg cttcagctca
     4741 atcatttatt gaacgcatga caaactttga taaaaatctt ccaaatgaaa aagtactacc
     4801 aaaacatagt ttgctttatg agtattttac ggtttataac gaattgacaa aggtcaaata
     4861 tgttactgaa ggaatgcgaa aaccagcatt tctttcaggt gaacagaaga aagccattgt
     4921 tgatttactc ttcaaaacaa atcgaaaagt aaccgttaag caattaaaag aagattattt
     4981 caaaaaaata gaatgttttg atagtgttga aatttcagga gttgaagata gatttaatgc
     5041 ttcattaggt acctaccatg atttgctaaa aattattaaa gataaagatt ttttggataa
     5101 tgaagaaaat gaagatatct tagaggatat tgttttaaca ttgaccttat ttgaagatag
     5161 ggagatgatt gaggaaagac ttaaaacata tgctcacctc tttgatgata aggtgatgaa
     5221 acagcttaaa cgtcgccgtt atactggttg gggacgtttg tctcgaaaat tgattaatgg
     5281 tattagggat aagcaatctg gcaaaacaat attagatttt ttgaaatcag atggttttgc
     5341 caatcgcaat tttatgcagc tgatccatga tgatagtttg acatttaaag aagacattca
     5401 aaaagcacaa gtgtctggac aaggcgatag tttacatgaa catattgcaa atttagctgg
     5461 tagccctgct attaaaaaag gtattttaca gactgtaaaa gttgttgatg aattggtcaa
     5521 agtaatgggg cggcataagc cagaaaatat cgttattgaa atggcacgtg aaaatcagac
     5581 aactcaaaag ggccagaaaa attcgcgaga gcgtatgaaa cgaatcgaag aaggtatcaa
     5641 agaattagga agtcagattc ttaaagagca tcctgttgaa aatactcaat tgcaaaatga
     5701 aaagctctat ctctattatc tccaaaatgg aagagacatg tatgtggacc aagaattaga
     5761 tattaatcgt ttaagtgatt atgatgtcga tgccattgtt ccacaaagtt tccttaaaga
     5821 cgattcaata gacaataagg tcttaacgcg ttctgataaa aatcgtggta aatcggataa
     5881 cgttccaagt gaagaagtag tcaaaaagat gaaaaactat tggagacaac ttctaaacgc
     5941 caagttaatc actcaacgta agtttgataa tttaacgaaa gctgaacgtg gaggtttgag
     6001 tgaacttgat aaagctggtt ttatcaaacg ccaattggtt gaaactcgcc aaatcactaa
     6061 gcatgtggca caaattttgg atagtcgcat gaatactaaa tacgatgaaa atgataaact
     6121 tattcgagag gttaaagtga ttaccttaaa atctaaatta gtttctgact tccgaaaaga
     6181 tttccaattc tataaagtac gtgagattaa caattaccat catgcccatg atgcgtatct
     6241 aaatgccgtc gttggaactg ctttgattaa gaaatatcca aaacttgaat cggagtttgt
     6301 ctatggtgat tataaagttt atgatgttcg taaaatgatt gctaagtctg agcaagaaat
     6361 aggcaaagca accgcaaaat atttctttta ctctaatatc atgaacttct tcaaaacaga
     6421 aattacactt gcaaatggag agattcgcaa acgccctcta atcgaaacta atggggaaac
     6481 tggagaaatt gtctgggata aagggcgaga ttttgccaca gtgcgcaaag tattgtccat
     6541 gccccaagtc aatattgtca agaaaacaga agtacagaca ggcggattct ccaaggagtc
     6601 aattttacca aaaagaaatt cggacaagct tattgctcgt aaaaaagact gggatccaaa
     6661 aaaatatggt ggttttgata gtccaacggt agcttattca gtcctagtgg ttgctaaggt
     6721 ggaaaaaggg aaatcgaaga agttaaaatc cgttaaagag ttactaggga tcacaattat
     6781 ggaaagaagt tcctttgaaa aaaatccgat tgacttttta gaagctaaag gatataagga
     6841 agttaaaaaa gacttaatca ttaaactacc taaatatagt ctttttgagt tagaaaacgg
     6901 tcgtaaacgg atgctggcta gtgccggaga attacaaaaa ggaaatgagc tggctctgcc
     6961 aagcaaatat gtgaattttt tatatttagc tagtcattat gaaaagttga agggtagtcc
     7021 agaagataac gaacaaaaac aattgtttgt ggagcagcat aagcattatt tagatgagat
     7081 tattgagcaa atcagtgaat tttctaagcg tgttatttta gcagatgcca atttagataa
     7141 agttcttagt gcatataaca aacatagaga caaaccaata cgtgaacaag cagaaaatat
     7201 tattcattta tttacgttga cgaatcttgg agctcccgct gcttttaaat attttgatac
     7261 aacaattgat cgtaaacgat atacgtctac aaaagaagtt ttagatgcca ctcttatcca
     7321 tcaatccatc actggtcttt atgaaacacg cattgatttg agtcagctag gaggtgacta
     7381 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     7441 cccttggggc ctctaaacgg gtcttgacgg gttttttgtc tagagcgctc aagttgatcg
     7501 aatgtatgcc ccggatatga cctagggaga ttcttctaaa gcgagcaggg tttgccaagt
     7561 ggcgatcgcc tcagggaacc ataaccaact ttgttcgagg cggcgggggt tgagctggga
     7621 acgatcgaga tctagggcga tcgcccggag tttttcggct ttccctacaa gaatttcctg
     7681 gtcaacgtcg gtgggttgcc tttgggccaa atcgtacata ctaatcgcca ggcccgcata
     7741 aatctccgcc tggagggctg gggggaaatc ctgggtcgtt gccgccgcta aggcttcata
     7801 ccaacgatca ttggcggctt cgtaatctgc attgaggtaa tggacaaaac ccagggccat
     7861 caaaatttcg gggtcgttgg gctgttgggc tagggccatg tcccaattgt cttgcaccgt
     7921 ggcgatcgcc gttgcttggg ttggcgatgg tgggacggca gccaaagttg tttgccagca
     7981 tgcgccctat agtgagtcgt attacaattc actggccgtc gttttacaac gtcgtgactg
     8041 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     8101 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     8161 cgaatggacg cgccctgtag cggcgcatta a
//
