LOCUS       sequence-71727-e        6806 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial nuclease-null SP Cas9 expression.
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-SPcasN-
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             join(377..403,404..406,407..2890,2891..2896,2897..2962,
                     2963..2965,2966..4480)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     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 acaagaagta ctccattggg ctcgctatcg gcacaaacag
      421 cgtcggctgg gccgtcatta cggacgagta caaggtgccg agcaaaaaat tcaaagttct
      481 gggcaatacc gatcgccaca gcataaagaa gaacctcatt ggcgccctcc tgttcgactc
      541 cggggagacg gccgaagcca cgcggctcaa aagaacagca cggcgcagat atacccgcag
      601 aaagaatcgg atctgctacc tgcaggagat ctttagtaat gagatggcta aggtggatga
      661 ctctttcttc cataggctgg aggagtcctt tttggtggag gaggataaaa agcacgagcg
      721 ccacccaatc tttggcaata tcgtggacga ggtggcgtac catgaaaagt acccaaccat
      781 atatcatctg aggaagaagc ttgtagacag tactgataag gctgacttgc ggttgatcta
      841 tctcgcgctg gcgcatatga tcaaatttcg gggacacttc ctcatcgagg gggacctgaa
      901 cccagacaac agcgatgtcg acaaactctt tatccaactg gttcagactt acaatcagct
      961 tttcgaagag aacccgatca acgcatccgg agttgacgcc aaagcaatcc tgagcgctag
     1021 gctgtccaaa tcccggcggc tcgaaaacct catcgcacag ctccctgggg agaagaagaa
     1081 cggcctgttt ggtaatctta tcgccctgtc actcgggctg acccccaact ttaaatctaa
     1141 cttcgacctg gccgaagatg ccaagcttca actgagcaaa gacacctacg atgatgatct
     1201 cgacaatctg ctggcccaga tcggcgacca gtacgcagac ctttttttgg cggcaaagaa
     1261 cctgtcagac gccattctgc tgagtgatat tctgcgagtg aacacggaga tcaccaaagc
     1321 tccgctgagc gctagtatga tcaagcgcta tgatgagcac caccaagact tgactttgct
     1381 gaaggccctt gtcagacagc aactgcctga gaagtacaag gaaattttct tcgatcagtc
     1441 taaaaatggc tacgccggat acattgacgg cggagcaagc caggaggaat tttacaaatt
     1501 tattaagccc atcttggaaa aaatggacgg caccgaggag ctgctggtaa agcttaacag
     1561 agaagatctg ttgcgcaaac agcgcacttt cgacaatgga agcatccccc accagattca
     1621 cctgggcgaa ctgcacgcta tcctcaggcg gcaagaggat ttctacccct ttttgaaaga
     1681 taacagggaa aagattgaga aaatcctcac atttcggata ccctactatg taggccccct
     1741 cgcccgggga aattccagat tcgcgtggat gactcgcaaa tcagaagaga ccatcactcc
     1801 ctggaacttc gaggaagtcg tggataaggg ggcctctgcc cagtccttca tcgaaaggat
     1861 gactaacttt gataaaaatc tgcctaacga aaaggtgctt cctaaacact ctctgctgta
     1921 cgagtacttc acagtttata acgagctcac caaggtcaaa tacgtcacag aagggatgag
     1981 aaagccagca ttcctgtctg gagagcagaa gaaagctatc gtggacctcc tcttcaagac
     2041 gaaccggaaa gttaccgtga aacagctcaa agaagactat ttcaaaaaga ttgaatgttt
     2101 cgactctgtt gaaatcagcg gagtggagga tcgcttcaac gcatccctgg gaacgtatca
     2161 cgatctcctg aaaatcatta aagacaagga cttcctggac aatgaggaga acgaggacat
     2221 tcttgaggac attgtcctca cccttacgtt gtttgaagat agggagatga ttgaagaacg
     2281 cttgaaaact tacgctcatc tcttcgacga caaagtcatg aaacagctca agaggcgccg
     2341 atatacagga tgggggcggc tgtcaagaaa actgatcaat gggatccgag acaagcagag
     2401 tggaaagaca atcctggatt ttcttaagtc cgatggattt gccaaccgga acttcatgca
     2461 gttgatccat gatgactctc tcacctttaa ggaggacatc cagaaagcac aagtttctgg
     2521 ccagggggac agtcttcacg agcacatcgc taatcttgca ggtagcccag ctatcaaaaa
     2581 gggaatactg cagaccgtta aggtcgtgga tgaactcgtc aaagtaatgg gaaggcataa
     2641 gcccgagaat atcgttatcg agatggcccg agagaaccaa actacccaga agggacagaa
     2701 gaacagtagg gaaaggatga agaggattga agagggtata aaagaactgg ggtcccaaat
     2761 ccttaaggaa cacccagttg aaaacaccca gcttcagaat gagaagctct acctgtacta
     2821 cctgcagaac ggcagggaca tgtacgtgga tcaggaactg gacatcaatc ggctctccga
     2881 ctacgacgtg gctgctatcg tgccccagtc ttttctcaaa gatgattcta ttgataataa
     2941 agtgttgaca agatccgata aagctagagg gaagagtgat aacgtcccct cagaagaagt
     3001 tgtcaagaaa atgaaaaatt attggcggca gctgctgaac gccaaactga tcacacaacg
     3061 gaagttcgat aatctgacta aggctgaacg aggtggcctg tctgagttgg ataaagccgg
     3121 cttcatcaaa aggcagcttg ttgagacacg ccagatcacc aagcacgtgg cccaaattct
     3181 cgattcacgc atgaacacca agtacgatga aaatgacaaa ctgattcgag aggtgaaagt
     3241 tattactctg aagtctaagc tggtctcaga tttcagaaag gactttcagt tttataaggt
     3301 gagagagatc aacaattacc accatgcgca tgatgcctac ctgaatgcag tggtaggcac
     3361 tgcacttatc aaaaaatatc ccaagcttga atctgaattt gtttacggag actataaagt
     3421 gtacgatgtt aggaaaatga tcgcaaagtc tgagcaggaa ataggcaagg ccaccgctaa
     3481 gtacttcttt tacagcaata ttatgaattt tttcaagacc gagattacac tggccaatgg
     3541 agagattcgg aagcgaccac ttatcgaaac aaacggagaa acaggagaaa tcgtgtggga
     3601 caagggtagg gatttcgcga cagtccggaa ggtcctgtcc atgccgcagg tgaacatcgt
     3661 taaaaagacc gaagtacaga ccggaggctt ctccaaggaa agtatcctcc cgaaaaggaa
     3721 cagcgacaag ctgatcgcac gcaaaaaaga ttgggacccc aagaaatacg gcggattcga
     3781 ttctcctaca gtcgcttaca gtgtactggt tgtggccaaa gtggagaaag ggaagtctaa
     3841 aaaactcaaa agcgtcaagg aactgctggg catcacaatc atggagcgat caagcttcga
     3901 aaaaaacccc atcgactttc tcgaggcgaa aggatataaa gaggtcaaaa aagacctcat
     3961 cattaagctt cccaagtact ctctctttga gcttgaaaac ggccggaaac gaatgctcgc
     4021 tagtgcgggc gagctgcaga aaggtaacga gctggcactg ccctctaaat acgttaattt
     4081 cttgtatctg gccagccact atgaaaagct caaagggtct cccgaagata atgagcagaa
     4141 gcagctgttc gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga
     4201 attctccaaa agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa
     4261 taagcacagg gataagccca tcagggagca ggcagaaaac attatccact tgtttactct
     4321 gaccaacttg ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg
     4381 gtacacctct acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct
     4441 ctatgaaaca agaatcgacc tctctcagct cggtggagac taaccggctg 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
//
