LOCUS       sequence-70445-e        6806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial S. pyogenes Cas9 (SP) + tracrRNA expression, 
            cloDF13/spectinomycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 4794 to 4828)
  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 6806)
  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: DS-SPcas
FEATURES             Location/Qualifiers
     source          1..6806
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      153..180
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             377..4483
                     /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             377..4480
                     /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        4551..4629
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        4794..4828
                     /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   4845..4880
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     rep_origin      5062..5800
                     /direction=RIGHT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     promoter        5848..5939
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5940..6731
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 accaggcacg cctaaccgtc agtgagattg gatgagtgaa cgatattgat cgagaagagc
       61 cctgcgcagc cgctgccgtg cctgcaggaa gcaacggccc ggagggtggc gggcaggacg
      121 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
      181 tgcgtttcta caaactctgc tagcttctag agcacagcta acaccacgtc gtccctatct
      241 gctgccctag gtctatgagt ggttgctgga taactttacg ggcatgcata aggctcgtat
      301 gatatattca gggagaccac aacggtttcc ctctacaaat aattttgttt aacttttact
      361 agaggaggag gcaaaaatgg ataagaaata ctcaataggc ttagatatcg gcacaaatag
      421 cgtcggatgg gcggtgatca ctgatgaata taaggttccg tctaaaaagt tcaaggttct
      481 gggaaataca gaccgccaca gtatcaaaaa aaatcttata ggggctcttt tatttgacag
      541 tggagagaca gcggaagcga ctcgtctcaa acggacagct cgtagaaggt atacacgtcg
      601 gaagaatcgt atttgttatc tacaggagat tttttcaaat gagatggcga aagtagatga
      661 tagtttcttt catcgacttg aagagtcttt tttggtggaa gaagacaaga agcatgaacg
      721 tcatcctatt tttggaaata tagtagatga agttgcttat catgagaaat atccaactat
      781 ctatcatctg cgaaaaaaat tggtagattc tactgataaa gcggatttgc gcttaatcta
      841 tttggcctta gcgcatatga ttaagtttcg tggtcatttt ttgattgagg gagatttaaa
      901 tcctgataat agtgatgtgg acaaactatt tatccagttg gtacaaacct acaatcaatt
      961 atttgaagaa aaccctatta acgcaagtgg agtagatgct aaagcgattc tttctgcacg
     1021 attgagtaaa tcaagacgat tagaaaatct cattgctcag ctccccggtg agaagaaaaa
     1081 tggcttattt gggaatctca ttgctttgtc attgggtttg acccctaatt ttaaatcaaa
     1141 ttttgatttg gcagaagatg ctaaattaca gctttcaaaa gatacttacg atgatgattt
     1201 agataattta ttggcgcaaa ttggagatca atatgctgat ttgtttttgg cagctaagaa
     1261 tttatcagat gctattttac tttcagatat cctaagagta aatactgaaa taactaaggc
     1321 tcccctatca gcttcaatga ttaaacgcta cgatgaacat catcaagact tgactctttt
     1381 aaaagcttta gttcgacaac aacttccaga aaagtataaa gaaatctttt ttgatcaatc
     1441 aaaaaacgga tatgcaggtt atattgatgg gggagctagc caagaagaat tttataaatt
     1501 tatcaaacca attttagaaa aaatggatgg tactgaggaa ttattggtga aactaaatcg
     1561 tgaagatttg ctgcgcaagc aacggacctt tgacaacggc tctattcccc atcaaattca
     1621 cttgggtgag ctgcatgcta ttttgagaag acaagaagac ttttatccat ttttaaaaga
     1681 caatcgtgag aagattgaaa aaatcttgac ttttcgaatt ccttattatg ttggtccatt
     1741 ggcgcgtggc aatagtcgtt ttgcatggat gactcggaag tctgaagaaa caattacccc
     1801 atggaatttt gaagaagttg tcgataaagg tgcttcagct caatcattta ttgaacgcat
     1861 gacaaacttt gataaaaatc ttccaaatga aaaagtacta ccaaaacata gtttgcttta
     1921 tgagtatttt acggtttata acgaattgac aaaggtcaaa tatgttactg aaggaatgcg
     1981 aaaaccagca tttctttcag gtgaacagaa gaaagccatt gttgatttac tcttcaaaac
     2041 aaatcgaaaa gtaaccgtta agcaattaaa agaagattat ttcaaaaaaa tagaatgttt
     2101 tgatagtgtt gaaatttcag gagttgaaga tagatttaat gcttcattag gtacctacca
     2161 tgatttgcta aaaattatta aagataaaga ttttttggat aatgaagaaa atgaagatat
     2221 cttagaggat attgttttaa cattgacctt atttgaagat agggagatga ttgaggaaag
     2281 acttaaaaca tatgctcacc tctttgatga taaggtgatg aaacagctta aacgtcgccg
     2341 ttatactggt tggggacgtt tgtctcgaaa attgattaat ggtattaggg ataagcaatc
     2401 tggcaaaaca atattagatt ttttgaaatc agatggtttt gccaatcgca attttatgca
     2461 gctgatccat gatgatagtt tgacatttaa agaagacatt caaaaagcac aagtgtctgg
     2521 acaaggcgat agtttacatg aacatattgc aaatttagct ggtagccctg ctattaaaaa
     2581 aggtatttta cagactgtaa aagttgttga tgaattggtc aaagtaatgg ggcggcataa
     2641 gccagaaaat atcgttattg aaatggcacg tgaaaatcag acaactcaaa agggccagaa
     2701 aaattcgcga gagcgtatga aacgaatcga agaaggtatc aaagaattag gaagtcagat
     2761 tcttaaagag catcctgttg aaaatactca attgcaaaat gaaaagctct atctctatta
     2821 tctccaaaat ggaagagaca tgtatgtgga ccaagaatta gatattaatc gtttaagtga
     2881 ttatgatgtc gatcacattg ttccacaaag tttccttaaa gacgattcaa tagacaataa
     2941 ggtcttaacg cgttctgata aaaatcgtgg taaatcggat aacgttccaa gtgaagaagt
     3001 agtcaaaaag atgaaaaact attggagaca acttctaaac gccaagttaa tcactcaacg
     3061 taagtttgat aatttaacga aagctgaacg tggaggtttg agtgaacttg ataaagctgg
     3121 ttttatcaaa cgccaattgg ttgaaactcg ccaaatcact aagcatgtgg cacaaatttt
     3181 ggatagtcgc atgaatacta aatacgatga aaatgataaa cttattcgag aggttaaagt
     3241 gattacctta aaatctaaat tagtttctga cttccgaaaa gatttccaat tctataaagt
     3301 acgtgagatt aacaattacc atcatgccca tgatgcgtat ctaaatgccg tcgttggaac
     3361 tgctttgatt aagaaatatc caaaacttga atcggagttt gtctatggtg attataaagt
     3421 ttatgatgtt cgtaaaatga ttgctaagtc tgagcaagaa ataggcaaag caaccgcaaa
     3481 atatttcttt tactctaata tcatgaactt cttcaaaaca gaaattacac ttgcaaatgg
     3541 agagattcgc aaacgccctc taatcgaaac taatggggaa actggagaaa ttgtctggga
     3601 taaagggcga gattttgcca cagtgcgcaa agtattgtcc atgccccaag tcaatattgt
     3661 caagaaaaca gaagtacaga caggcggatt ctccaaggag tcaattttac caaaaagaaa
     3721 ttcggacaag cttattgctc gtaaaaaaga ctgggatcca aaaaaatatg gtggttttga
     3781 tagtccaacg gtagcttatt cagtcctagt ggttgctaag gtggaaaaag ggaaatcgaa
     3841 gaagttaaaa tccgttaaag agttactagg gatcacaatt atggaaagaa gttcctttga
     3901 aaaaaatccg attgactttt tagaagctaa aggatataag gaagttaaaa aagacttaat
     3961 cattaaacta cctaaatata gtctttttga gttagaaaac ggtcgtaaac ggatgctggc
     4021 tagtgccgga gaattacaaa aaggaaatga gctggctctg ccaagcaaat atgtgaattt
     4081 tttatattta gctagtcatt atgaaaagtt gaagggtagt ccagaagata acgaacaaaa
     4141 acaattgttt gtggagcagc ataagcatta tttagatgag attattgagc aaatcagtga
     4201 attttctaag cgtgttattt tagcagatgc caatttagat aaagttctta gtgcatataa
     4261 caaacataga gacaaaccaa tacgtgaaca agcagaaaat attattcatt tatttacgtt
     4321 gacgaatctt ggagctcccg ctgcttttaa atattttgat acaacaattg atcgtaaacg
     4381 atatacgtct acaaaagaag ttttagatgc cactcttatc catcaatcca tcactggtct
     4441 ttatgaaaca cgcattgatt tgagtcagct aggaggtgac tgaccggctg ataaatttct
     4501 ttgaatttct ccttgattat ttgttataaa tgttataaaa taatcttgtt ggaaccattc
     4561 aaaacagcat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     4621 agtcggtgct ttttttgata cttctattct actctgactg caaaccaaaa aaacaagcgc
     4681 tttcaaaacg cttgttttat catttttagg gaaattaatc tcttaatcct tttatcattc
     4741 tacatttagg cgctgccatc ttgggacaat gaaaacgtta gtcatggcgc gccttgacgg
     4801 ctagctcagt cctaggtaca gtgctagctt aattagtcta cgaggtttta gagctatgct
     4861 gttttgaatg gtcccaaaac ctgcagacaa gcccggccgg cctaaggcga tgccccctcg
     4921 acctcgatca gggaggcgtt caggacgact cacaaagaaa gccgggcaat gcccggcttt
     4981 ttccacgcct cctgggctga cttcaggtgc tacatttgaa gagataaatt gcactgaaat
     5041 ctagagcggt tcagtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     5101 aatcttttgc cctgtaaacg aaaaaaccac ctggggaggt ggtttgatcg aaggttaagt
     5161 cagttgggga actgcttaac cgtggtaact ggctttcgca gagcacagca accaaatctg
     5221 tccttccagt gtagccggac tttggcgcac acttcaagag caaccgcgtg tttagctaaa
     5281 caaatcctct gcgaactccc agttaccaat ggctgctgcc agtggcgttt taccgtgctt
     5341 ttccgggttg gactcaagtg aacagttacc ggataaggcg cagcagtcgg gctgaacggg
     5401 gagttcttgc ttacagccca gcttggagcg aacgacctac accgagccga gataccagtg
     5461 tgtgagctat gagaaagcgc cacacttccc gtaagggaga aaggcggaac aggtatccgg
     5521 taaacggcag ggtcggaaca ggagagcgca agagggagcg acccgccgga aacggtgggg
     5581 atctttaagt cctgtcgggt ttcgcccgta ctgtcagatt catggttgag cctcacggct
     5641 cccacagatg caccggaaaa gcgtctgttt atgtgaactc tggcaggagg gcggagccta
     5701 tggaaaaacg ccaccggcgc ggccctgctg ttttgcctca catgttagtc ccctgcttat
     5761 ccacggaatc tgtgggtaac tttgtatgtg tccgcagcgc ccgccgcagt ctcacgcccg
     5821 gagcgtagcg accgagtgag ctagctattt gtttattttt ctaaatacat tcaaatatgt
     5881 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     5941 tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg
     6001 agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg
     6061 gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa
     6121 caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg
     6181 agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt
     6241 atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta
     6301 tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac
     6361 atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg
     6421 atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg
     6481 gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca
     6541 aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc
     6601 agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct
     6661 cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc accaaggtag
     6721 tcggcaaata atgtctaaca attcgttcaa gccgaggggc cgcaagatcc ggccacgatg
     6781 acccggtcgt cggttcaggg cagggt
//
