LOCUS       sequence-163056-        8280 bp    DNA     circular SYN 15-APR-2025
DEFINITION  dCas9 expressed from low constituve promoter, c225, in acsA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8280)
  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 8280)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pCas7
FEATURES             Location/Qualifiers
     source          1..8280
                     /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(3364..3390,3391..3393,3394..5880,5881..5883,
                     5884..7470)
                     /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(7506..7524)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7520..7567
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(8074..8093)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8075..8093)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8100..8117)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8100..8116)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8109..8131)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      join(8257..8280,1..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 aattcgtaac
     3241 aaaaaagcag gaataaaatt aacaagacgt aatagacata agtcctatca ccgttgttaa
     3301 aagttaactg tgggattaca aaaggagctc ccaactcata aagtcaagta ggagattaat
     3361 tccatggata agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg
     3421 gtgatcactg atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac
     3481 cgccacagta tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg
     3541 gaagcgactc gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt
     3601 tgttatctac aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat
     3661 cgacttgaag agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt
     3721 ggaaatatag tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga
     3781 aaaaaattgg tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg
     3841 catatgatta agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt
     3901 gatgtggaca aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac
     3961 cctattaacg caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca
     4021 agacgattag aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg
     4081 aatctcattg ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca
     4141 gaagatgcta aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg
     4201 gcgcaaattg gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct
     4261 attttacttt cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct
     4321 tcaatgatta aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt
     4381 cgacaacaac ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat
     4441 gcaggttata ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt
     4501 ttagaaaaaa tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg
     4561 cgcaagcaac ggacctttga caacggctct attccccatc aaattcactt gggtgagctg
     4621 catgctattt tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag
     4681 attgaaaaaa tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat
     4741 agtcgttttg catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa
     4801 gaagttgtcg ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat
     4861 aaaaatcttc caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg
     4921 gtttataacg aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt
     4981 ctttcaggtg aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta
     5041 accgttaagc aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa
     5101 atttcaggag ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa
     5161 attattaaag ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt
     5221 gttttaacat tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat
     5281 gctcacctct ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg
     5341 ggacgtttgt ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata
     5401 ttagattttt tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat
     5461 gatagtttga catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt
     5521 ttacatgaac atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag
     5581 actgtaaaag ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc
     5641 gttattgaaa tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag
     5701 cgtatgaaac gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat
     5761 cctgttgaaa atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga
     5821 agagacatgt atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat
     5881 gccattgttc cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt
     5941 tctgataaaa atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg
     6001 aaaaactatt ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat
     6061 ttaacgaaag ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc
     6121 caattggttg aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg
     6181 aatactaaat acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa
     6241 tctaaattag tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac
     6301 aattaccatc atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag
     6361 aaatatccaa aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt
     6421 aaaatgattg ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac
     6481 tctaatatca tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa
     6541 cgccctctaa tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat
     6601 tttgccacag tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa
     6661 gtacagacag gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt
     6721 attgctcgta aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta
     6781 gcttattcag tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc
     6841 gttaaagagt tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt
     6901 gactttttag aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct
     6961 aaatatagtc tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa
     7021 ttacaaaaag gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct
     7081 agtcattatg aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg
     7141 gagcagcata agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt
     7201 gttattttag cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac
     7261 aaaccaatac gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga
     7321 gctcccgctg cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca
     7381 aaagaagttt tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc
     7441 attgatttga gtcagctagg aggtgactaa caaagcccga aaggaagctg agttggctgc
     7501 tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgacggg
     7561 ttttttgtct agagcgctca agttgatcga atgtatgccc cggatatgac ctagggagat
     7621 tcttctaaag cgagcagggt ttgccaagtg gcgatcgcct cagggaacca taaccaactt
     7681 tgttcgaggc ggcgggggtt gagctgggaa cgatcgagat ctagggcgat cgcccggagt
     7741 ttttcggctt tccctacaag aatttcctgg tcaacgtcgg tgggttgcct ttgggccaaa
     7801 tcgtacatac taatcgccag gcccgcataa atctccgcct ggagggctgg ggggaaatcc
     7861 tgggtcgttg ccgccgctaa ggcttcatac caacgatcat tggcggcttc gtaatctgca
     7921 ttgaggtaat ggacaaaacc cagggccatc aaaatttcgg ggtcgttggg ctgttgggct
     7981 agggccatgt cccaattgtc ttgcaccgtg gcgatcgccg ttgcttgggt tggcgatggt
     8041 gggacggcag ccaaagttgt ttgccagcat gcgccctata gtgagtcgta ttacaattca
     8101 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     8161 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     8221 ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa
//
