LOCUS       sequence-227538-        5870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro expression of S. pyogenes Cas9 from the T7 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5870)
  AUTHORS   Stark JC, Huang A, Hsu KJ, Dubner RS, Forbrook J, Marshalla S, 
            Rodriguez F, Washington M, Rybnicky GA, Nguyen PQ, Hasselbacher B, 
            Jabri R, Kamran R, Koralewski V, Wightkin W, Martinez T, Jewett MC
  TITLE     BioBits Health: Classroom Activities Exploring Engineering, Biology,
            and Human Health with Fluorescent Readouts.
  JOURNAL   ACS Synth Biol. 2019 May 7. doi: 10.1021/acssynbio.8b00381.
  PUBMED    30925042
REFERENCE   2  (bases 1 to 5870)
  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: "ACS Synth 
            Biol. 2019 May 7. doi: 10.1021/acssynbio.8b00381."
COMMENT     Sequence Label: pJL1-SpCas9
FEATURES             Location/Qualifiers
     source          1..5870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      65..653
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     554..573
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     681..700
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        681..699
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             731..753
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             761..4867
                     /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             761..4864
                     /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"
     primer_bind     complement(4919..4937)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4933..4980
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4998..5813)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSKPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5721..5740
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5805..5824
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtgag atcaaaggat
       61 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
      121 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
      181 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
      241 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
      301 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
      361 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
      421 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
      481 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
      541 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
      601 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
      661 gcaacgcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc
      721 tagaaataat tttgtttaac tttaagaagg agatatacat atggataaga aatactcaat
      781 aggcttagat atcggcacaa atagcgtcgg atgggcggtg atcactgatg aatataaggt
      841 tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc cacagtatca aaaaaaatct
      901 tataggggct cttttatttg acagtggaga gacagcggaa gcgactcgtc tcaaacggac
      961 agctcgtaga aggtatacac gtcggaagaa tcgtatttgt tatctacagg agattttttc
     1021 aaatgagatg gcgaaagtag atgatagttt ctttcatcga cttgaagagt cttttttggt
     1081 ggaagaagac aagaagcatg aacgtcatcc tatttttgga aatatagtag atgaagttgc
     1141 ttatcatgag aaatatccaa ctatctatca tctgcgaaaa aaattggtag attctactga
     1201 taaagcggat ttgcgcttaa tctatttggc cttagcgcat atgattaagt ttcgtggtca
     1261 ttttttgatt gagggagatt taaatcctga taatagtgat gtggacaaac tatttatcca
     1321 gttggtacaa acctacaatc aattatttga agaaaaccct attaacgcaa gtggagtaga
     1381 tgctaaagcg attctttctg cacgattgag taaatcaaga cgattagaaa atctcattgc
     1441 tcagctcccc ggtgagaaga aaaatggctt atttgggaat ctcattgctt tgtcattggg
     1501 tttgacccct aattttaaat caaattttga tttggcagaa gatgctaaat tacagctttc
     1561 aaaagatact tacgatgatg atttagataa tttattggcg caaattggag atcaatatgc
     1621 tgatttgttt ttggcagcta agaatttatc agatgctatt ttactttcag atatcctaag
     1681 agtaaatact gaaataacta aggctcccct atcagcttca atgattaaac gctacgatga
     1741 acatcatcaa gacttgactc ttttaaaagc tttagttcga caacaacttc cagaaaagta
     1801 taaagaaatc ttttttgatc aatcaaaaaa cggatatgca ggttatattg atgggggagc
     1861 tagccaagaa gaattttata aatttatcaa accaatttta gaaaaaatgg atggtactga
     1921 ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc aagcaacgga cctttgacaa
     1981 cggctctatt ccccatcaaa ttcacttggg tgagctgcat gctattttga gaagacaaga
     2041 agacttttat ccatttttaa aagacaatcg tgagaagatt gaaaaaatct tgacttttcg
     2101 aattccttat tatgttggtc cattggcgcg tggcaatagt cgttttgcat ggatgactcg
     2161 gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa gttgtcgata aaggtgcttc
     2221 agctcaatca tttattgaac gcatgacaaa ctttgataaa aatcttccaa atgaaaaagt
     2281 actaccaaaa catagtttgc tttatgagta ttttacggtt tataacgaat tgacaaaggt
     2341 caaatatgtt actgaaggaa tgcgaaaacc agcatttctt tcaggtgaac agaagaaagc
     2401 cattgttgat ttactcttca aaacaaatcg aaaagtaacc gttaagcaat taaaagaaga
     2461 ttatttcaaa aaaatagaat gttttgatag tgttgaaatt tcaggagttg aagatagatt
     2521 taatgcttca ttaggtacct accatgattt gctaaaaatt attaaagata aagatttttt
     2581 ggataatgaa gaaaatgaag atatcttaga ggatattgtt ttaacattga ccttatttga
     2641 agatagggag atgattgagg aaagacttaa aacatatgct cacctctttg atgataaggt
     2701 gatgaaacag cttaaacgtc gccgttatac tggttgggga cgtttgtctc gaaaattgat
     2761 taatggtatt agggataagc aatctggcaa aacaatatta gattttttga aatcagatgg
     2821 ttttgccaat cgcaatttta tgcagctgat ccatgatgat agtttgacat ttaaagaaga
     2881 cattcaaaaa gcacaagtgt ctggacaagg cgatagttta catgaacata ttgcaaattt
     2941 agctggtagc cctgctatta aaaaaggtat tttacagact gtaaaagttg ttgatgaatt
     3001 ggtcaaagta atggggcggc ataagccaga aaatatcgtt attgaaatgg cacgtgaaaa
     3061 tcagacaact caaaagggcc agaaaaattc gcgagagcgt atgaaacgaa tcgaagaagg
     3121 tatcaaagaa ttaggaagtc agattcttaa agagcatcct gttgaaaata ctcaattgca
     3181 aaatgaaaag ctctatctct attatctcca aaatggaaga gacatgtatg tggaccaaga
     3241 attagatatt aatcgtttaa gtgattatga tgtcgatcac attgttccac aaagtttcct
     3301 taaagacgat tcaatagaca ataaggtctt aacgcgttct gataaaaatc gtggtaaatc
     3361 ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa aactattgga gacaacttct
     3421 aaacgccaag ttaatcactc aacgtaagtt tgataattta acgaaagctg aacgtggagg
     3481 tttgagtgaa cttgataaag ctggttttat caaacgccaa ttggttgaaa ctcgccaaat
     3541 cactaagcat gtggcacaaa ttttggatag tcgcatgaat actaaatacg atgaaaatga
     3601 taaacttatt cgagaggtta aagtgattac cttaaaatct aaattagttt ctgacttccg
     3661 aaaagatttc caattctata aagtacgtga gattaacaat taccatcatg cccatgatgc
     3721 gtatctaaat gccgtcgttg gaactgcttt gattaagaaa tatccaaaac ttgaatcgga
     3781 gtttgtctat ggtgattata aagtttatga tgttcgtaaa atgattgcta agtctgagca
     3841 agaaataggc aaagcaaccg caaaatattt cttttactct aatatcatga acttcttcaa
     3901 aacagaaatt acacttgcaa atggagagat tcgcaaacgc cctctaatcg aaactaatgg
     3961 ggaaactgga gaaattgtct gggataaagg gcgagatttt gccacagtgc gcaaagtatt
     4021 gtccatgccc caagtcaata ttgtcaagaa aacagaagta cagacaggcg gattctccaa
     4081 ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa aagactggga
     4141 tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct tattcagtcc tagtggttgc
     4201 taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt aaagagttac tagggatcac
     4261 aattatggaa agaagttcct ttgaaaaaaa tccgattgac tttttagaag ctaaaggata
     4321 taaggaagtt aaaaaagact taatcattaa actacctaaa tatagtcttt ttgagttaga
     4381 aaacggtcgt aaacggatgc tggctagtgc cggagaatta caaaaaggaa atgagctggc
     4441 tctgccaagc aaatatgtga attttttata tttagctagt cattatgaaa agttgaaggg
     4501 tagtccagaa gataacgaac aaaaacaatt gtttgtggag cagcataagc attatttaga
     4561 tgagattatt gagcaaatca gtgaattttc taagcgtgtt attttagcag atgccaattt
     4621 agataaagtt cttagtgcat ataacaaaca tagagacaaa ccaatacgtg aacaagcaga
     4681 aaatattatt catttattta cgttgacgaa tcttggagct cccgctgctt ttaaatattt
     4741 tgatacaaca attgatcgta aacgatatac gtctacaaaa gaagttttag atgccactct
     4801 tatccatcaa tccatcactg gtctttatga aacacgcatt gatttgagtc agctaggagg
     4861 tgactgagtc gaccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     4921 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     4981 ctgaaagcca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc
     5041 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     5101 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     5161 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     5221 tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag
     5281 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     5341 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     5401 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     5461 tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg
     5521 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     5581 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     5641 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc
     5701 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     5761 ttggaattta atcgcggctt cgagcaagac gtttcccgtt gaatatggct cataacaccc
     5821 cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat
//
