LOCUS       sequence-231640-        8360 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses S. pyogenes dCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8360)
  AUTHORS   Collias D, Marshall R, Collins SP, Beisel CL, Noireaux V
  TITLE     An educational module to explore CRISPR technologies with a 
            cell-free transcription-translation system.
  JOURNAL   Synth Biol (Oxf). 2019 Jan 21;4(1):ysz005. doi: 
            10.1093/synbio/ysz005. eCollection 2019.
  PUBMED    32995532
REFERENCE   2  (bases 1 to 8360)
  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: "Synth Biol 
            (Oxf)."; date: "2019-01-21"; volume: "4(1):ysz005. doi"; pages: " 
            10.1093/synbio/ysz005. eCollection 2019"
COMMENT     Sequence Label: p15A_J23108_SpydCas9_CmR
FEATURES             Location/Qualifiers
     source          1..8360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..135
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        226..260
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     RBS             276..287
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(294..320,321..323,324..2810,2811..2813,2814..4400)
                     /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"
     terminator      4424..4495
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4511..4538
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(4613..4630)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4783..4869
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4961..4988
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5007..5098
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5556..6011
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5643..5662)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5853..5874
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6567..7226)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7141..7160
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(7227..7329)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(7721..7738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(7855..8360,1..40))
                     /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(7935..7954)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcagagcaa gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca
       61 gataaaatat ttctaggctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
      121 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
      181 gtccggcgta gaggatctgc tcatgtttga cagcttatca tcgatctgac agctagctca
      241 gtcctaggta taatgctagc cccgggaaaa gatctaaaga ggagaaagga tctatggata
      301 agaaatactc aataggctta gctatcggca caaatagcgt cggatgggcg gtgatcactg
      361 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta
      421 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc
      481 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac
      541 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag
      601 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag
      661 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg
      721 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta
      781 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca
      841 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg
      901 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag
      961 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg
     1021 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta
     1081 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg
     1141 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt
     1201 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta
     1261 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac
     1321 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata
     1381 ttgatggggg agctagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa
     1441 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac
     1501 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg catgctattt
     1561 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa
     1621 tcttgacttt tcgaattcct tattatgttg gtccattggc gcgtggcaat agtcgttttg
     1681 catggatgac tcggaagtct gaagaaacaa ttaccccatg gaattttgaa gaagttgtcg
     1741 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc
     1801 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg
     1861 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg
     1921 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc
     1981 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag
     2041 ttgaagatag atttaatgct tcattaggta cctaccatga tttgctaaaa attattaaag
     2101 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat
     2161 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct
     2221 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt
     2281 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt
     2341 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga
     2401 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac
     2461 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag
     2521 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa
     2581 tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac
     2641 gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa
     2701 atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt
     2761 atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat gccattgttc
     2821 cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa
     2881 atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt
     2941 ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag
     3001 ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg
     3061 aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat
     3121 acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag
     3181 tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc
     3241 atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa
     3301 aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg
     3361 ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca
     3421 tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaaa cgccctctaa
     3481 tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag
     3541 tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagacag
     3601 gcggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta
     3661 aaaaagactg ggatccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag
     3721 tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt
     3781 tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag
     3841 aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc
     3901 tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag
     3961 gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg
     4021 aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata
     4081 agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag
     4141 cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac
     4201 gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga gctcccgctg
     4261 cttttaaata ttttgataca acaattgatc gtaaacgata tacgtctaca aaagaagttt
     4321 tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga
     4381 gtcagctagg aggtgactaa ctcgagtaag gatctccagg catcaaataa aacgaaaggc
     4441 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     4501 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatacctagg gagacgtcgg
     4561 cgctgcaggc atgcaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
     4621 agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg
     4681 cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
     4741 gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     4801 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     4861 aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
     4921 gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat
     4981 ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta
     5041 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5101 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5161 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcagc
     5221 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5281 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5341 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5401 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     5461 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     5521 gaccaagttt actcatatat actttagatt gatttacgcg ccctgtagcg gcgcattaag
     5581 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5641 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5701 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5761 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5821 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5881 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     5941 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     6001 gtttacaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     6061 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     6121 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     6181 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     6241 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     6301 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtcaggc
     6361 atttgagaag cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata
     6421 gacataagcg gctatttaac gaccctgccc tgaaccgacg accgggtcga atttgctttc
     6481 gaatttctgc cattcatccg cttattatca cttattcagg cgtagcaacc aggcgtttaa
     6541 gggcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc gcagtactgt
     6601 tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga
     6661 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
     6721 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     6781 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     6841 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     6901 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     6961 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa ttccggatga
     7021 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     7081 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     7141 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     7201 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     7261 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     7321 tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg gcttcccggt atcaacaggg
     7381 acaccaggat ttatttattc tgcgaagtga tcttccgtca caggtattta ttcggcgcaa
     7441 agtgcgtcgg gtgatgctgc caacttactg atttagtgta tgatggtgtt tttgaggtgc
     7501 tccagtggct tctgtttcta tcagctgtcc ctcctgttca gctactgacg gggtggtgcg
     7561 taacggcaaa agcaccgccg gacatcagcg ctagcggagt gtatactggc ttactatgtt
     7621 ggcactgatg agggtgtcag tgaagtgctt catgtggcag gagaaaaaag gctgcaccgg
     7681 tgcgtcagca gaatatgtga tacaggatat attccgcttc ctcgctcact gactcgctac
     7741 gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga acggggcgga gatttcctgg
     7801 aagatgccag gaagatactt aacagggaag tgagagggcc gcggcaaagc cgtttttcca
     7861 taggctccgc ccccctgaca agcatcacga aatctgacgc tcaaatcagt ggtggcgaaa
     7921 cccgacagga ctataaagat accaggcgtt tccccctggc ggctccctcg tgcgctctcc
     7981 tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt tgtctcattc
     8041 cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt atgcacgaac
     8101 cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     8161 aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga ggagttagtc
     8221 ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga ctgcgctcct
     8281 ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga aaaaccgccc
     8341 tgcaaggcgg ttttttcgtt
//
