LOCUS       sequence-71746-e        7270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial SP Cas9 targeting filamentous phage gene III at five 
            protospacers.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 4722 to 4756)
  AUTHORS   Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM
  TITLE     Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
  JOURNAL   Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681.
  PUBMED    24076762
REFERENCE   2  (bases 1 to 7270)
  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: "Nat 
            Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681."
COMMENT     Sequence Label: EE-SP!gIII
FEATURES             Location/Qualifiers
     source          1..7270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      81..108
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             305..4411
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             305..4408
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     misc_RNA        4479..4557
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        4722..4756
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     repeat_region   4762..4797
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   4828..4863
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   4894..4929
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   4960..4995
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   5026..5061
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   5092..5127
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     terminator      5284..5370
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(5535..6123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5615..5634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6211..6305)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        complement(7078..7182)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7250..7268
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctgccgtgcc tgcaggaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
       61 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
      121 aactctgcta gcttctagag cacagctaac accacgtcgt ccctatctgc tgccctaggt
      181 ctatgagtgg ttgctggata actttacggg catgcataag gctcgtatga tatattcagg
      241 gagaccacaa cggtttccct ctacaaataa ttttgtttaa cttttactag aggaggaggc
      301 aaaaatggat aagaaatact caataggctt agatatcggc acaaatagcg tcggatgggc
      361 ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg gaaatacaga
      421 ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg gagagacagc
      481 ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga agaatcgtat
      541 ttgttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata gtttctttca
      601 tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc atcctatttt
      661 tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct atcatctgcg
      721 aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt tggccttagc
      781 gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc ctgataatag
      841 tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat ttgaagaaaa
      901 ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat tgagtaaatc
      961 aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg gcttatttgg
     1021 gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt ttgatttggc
     1081 agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag ataatttatt
     1141 ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt tatcagatgc
     1201 tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc ccctatcagc
     1261 ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa aagctttagt
     1321 tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatcaa aaaacggata
     1381 tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta tcaaaccaat
     1441 tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg aagatttgct
     1501 gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact tgggtgagct
     1561 gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca atcgtgagaa
     1621 gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg cgcgtggcaa
     1681 tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat ggaattttga
     1741 agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga caaactttga
     1801 taaaaatctt ccaaatgaaa aagtactacc aaaacatagt ttgctttatg agtattttac
     1861 ggtttataac gaattgacaa aggtcaaata tgttactgaa ggaatgcgaa aaccagcatt
     1921 tctttcaggt gaacagaaga aagccattgt tgatttactc ttcaaaacaa atcgaaaagt
     1981 aaccgttaag caattaaaag aagattattt caaaaaaata gaatgttttg atagtgttga
     2041 aatttcagga gttgaagata gatttaatgc ttcattaggt acctaccatg atttgctaaa
     2101 aattattaaa gataaagatt ttttggataa tgaagaaaat gaagatatct tagaggatat
     2161 tgttttaaca ttgaccttat ttgaagatag ggagatgatt gaggaaagac ttaaaacata
     2221 tgctcacctc tttgatgata aggtgatgaa acagcttaaa cgtcgccgtt atactggttg
     2281 gggacgtttg tctcgaaaat tgattaatgg tattagggat aagcaatctg gcaaaacaat
     2341 attagatttt ttgaaatcag atggttttgc caatcgcaat tttatgcagc tgatccatga
     2401 tgatagtttg acatttaaag aagacattca aaaagcacaa gtgtctggac aaggcgatag
     2461 tttacatgaa catattgcaa atttagctgg tagccctgct attaaaaaag gtattttaca
     2521 gactgtaaaa gttgttgatg aattggtcaa agtaatgggg cggcataagc cagaaaatat
     2581 cgttattgaa atggcacgtg aaaatcagac aactcaaaag ggccagaaaa attcgcgaga
     2641 gcgtatgaaa cgaatcgaag aaggtatcaa agaattagga agtcagattc ttaaagagca
     2701 tcctgttgaa aatactcaat tgcaaaatga aaagctctat ctctattatc tccaaaatgg
     2761 aagagacatg tatgtggacc aagaattaga tattaatcgt ttaagtgatt atgatgtcga
     2821 tcacattgtt ccacaaagtt tccttaaaga cgattcaata gacaataagg tcttaacgcg
     2881 ttctgataaa aatcgtggta aatcggataa cgttccaagt gaagaagtag tcaaaaagat
     2941 gaaaaactat tggagacaac ttctaaacgc caagttaatc actcaacgta agtttgataa
     3001 tttaacgaaa gctgaacgtg gaggtttgag tgaacttgat aaagctggtt ttatcaaacg
     3061 ccaattggtt gaaactcgcc aaatcactaa gcatgtggca caaattttgg atagtcgcat
     3121 gaatactaaa tacgatgaaa atgataaact tattcgagag gttaaagtga ttaccttaaa
     3181 atctaaatta gtttctgact tccgaaaaga tttccaattc tataaagtac gtgagattaa
     3241 caattaccat catgcccatg atgcgtatct aaatgccgtc gttggaactg ctttgattaa
     3301 gaaatatcca aaacttgaat cggagtttgt ctatggtgat tataaagttt atgatgttcg
     3361 taaaatgatt gctaagtctg agcaagaaat aggcaaagca accgcaaaat atttctttta
     3421 ctctaatatc atgaacttct tcaaaacaga aattacactt gcaaatggag agattcgcaa
     3481 acgccctcta atcgaaacta atggggaaac tggagaaatt gtctgggata aagggcgaga
     3541 ttttgccaca gtgcgcaaag tattgtccat gccccaagtc aatattgtca agaaaacaga
     3601 agtacagaca ggcggattct ccaaggagtc aattttacca aaaagaaatt cggacaagct
     3661 tattgctcgt aaaaaagact gggatccaaa aaaatatggt ggttttgata gtccaacggt
     3721 agcttattca gtcctagtgg ttgctaaggt ggaaaaaggg aaatcgaaga agttaaaatc
     3781 cgttaaagag ttactaggga tcacaattat ggaaagaagt tcctttgaaa aaaatccgat
     3841 tgacttttta gaagctaaag gatataagga agttaaaaaa gacttaatca ttaaactacc
     3901 taaatatagt ctttttgagt tagaaaacgg tcgtaaacgg atgctggcta gtgccggaga
     3961 attacaaaaa ggaaatgagc tggctctgcc aagcaaatat gtgaattttt tatatttagc
     4021 tagtcattat gaaaagttga agggtagtcc agaagataac gaacaaaaac aattgtttgt
     4081 ggagcagcat aagcattatt tagatgagat tattgagcaa atcagtgaat tttctaagcg
     4141 tgttatttta gcagatgcca atttagataa agttcttagt gcatataaca aacatagaga
     4201 caaaccaata cgtgaacaag cagaaaatat tattcattta tttacgttga cgaatcttgg
     4261 agctcccgct gcttttaaat attttgatac aacaattgat cgtaaacgat atacgtctac
     4321 aaaagaagtt ttagatgcca ctcttatcca tcaatccatc actggtcttt atgaaacacg
     4381 cattgatttg agtcagctag gaggtgactg accggctgat aaatttcttt gaatttctcc
     4441 ttgattattt gttataaatg ttataaaata atcttgttgg aaccattcaa aacagcatag
     4501 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     4561 ttttgatact tctattctac tctgactgca aaccaaaaaa acaagcgctt tcaaaacgct
     4621 tgttttatca tttttaggga aattaatctc ttaatccttt tatcattcta catttaggcg
     4681 ctgccatcta gcgacaatga aaacgttagt catggcgcgc cttgacggct agctcagtcc
     4741 taggtacagt gctagcttaa tgttttagag ctatgctgtt ttgaatggtc ccaaaacaag
     4801 cttacgttag taaatgaatt ttctgtagtt ttagagctat gctgttttga atggtcccaa
     4861 aacaccctca ttttcaggga tagcaagccc aatgttttag agctatgctg ttttgaatgg
     4921 tcccaaaacg gatccccatc ttttcataat caaaatcacg ttttagagct atgctgtttt
     4981 gaatggtccc aaaaccagac tgtagcgcgt tttcatcggc attttgtttt agagctatgc
     5041 tgttttgaat ggtcccaaaa cctcgagtag caccattacc attagcaagg cgttttagag
     5101 ctatgctgtt ttgaatggtc ccaaaaccca cccaccataa aaaccggcat cctgccggtt
     5161 tttattgcgg ccgcacccac gtagagcatc ctgcagacaa gcccgcatgg acgagctgta
     5221 caagtaaaag cttgatatcg aattcctgca gcccggcgga tttgtcccgc gtgctagagg
     5281 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     5341 tcggtgaacg ctctcctgag taggacaaat ccgccgccct agacctaggg cgttcggctg
     5401 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5461 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5521 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5581 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5641 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5701 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     5761 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5821 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5881 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5941 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     6001 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6061 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6121 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6181 taagggattt tggtcatgac tagtgcttgg attctcacca ataaaaaacg cccggcggca
     6241 accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag
     6301 gagtccaagc gagctcgtaa acttggtctg acagccctta acttacttat taaataattt
     6361 atagctattg aaaagagata agaattgttc aaagctaata ttgtttaaat cgtcaattcc
     6421 tgcatgtttt aaggaattgt taaattgatt ttttgtaaat attttcttgt attctttgtt
     6481 aacccatttc ataacgaaat aattatactt ttgtttatct ttgtgtgata ttcttgattt
     6541 ttttctactt aatctgataa gtgagctatt cactttaggt ttaggatgaa aatattctct
     6601 tggaaccata cttaatatag aaatatcaac ttctgccatt aaaagtaatg ccaatgagcg
     6661 ttttgtattt aataatcttt tagcaaaccc gtattccacg attaaataaa tctcattagc
     6721 tatactatca aaaacaattt tgcgtattat atccgtactt atgttataag gtatattacc
     6781 atatatttta taggattggt ttttaggaaa tttaaactgc aatatatcct tgtttaaaac
     6841 ttggaaatta tcgtgatcaa caagtttatt ttctgtagtt ttgcataatt tatggtctat
     6901 ttcaatggca gttacgaaat tacacctctt tactaattca agggtaaaat ggccttttcc
     6961 tgagccgatt tcaaagatat tatcatgttc atttaatctt atatttgtca ttattttatc
     7021 tatattatgt tttgaagtaa taaagttttg actgtgtttt atatttttct cgttcatact
     7081 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7141 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7201 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     7261 cacgaggccc
//
