LOCUS       sequence-114423-        6735 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial constitutive S. pyogenes Cas9 expression plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6735)
  TITLE     Bacterial constitutive gRNA expression plasmid
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6735)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCMK
FEATURES             Location/Qualifiers
     source          1..6735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(160..177)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(294..839)
                     /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(374..393)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(951..956,957..973,974..979)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     misc_RNA        complement(1297..1375)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             1671..5774
                     /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             1671..5774
                     /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             5811..6626
                     /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="SHIQRETSCSSRPRLNSNMDADLYGYKWARDNVGQSGATIY*LYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHNLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5884..5903)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg tatactggct tactatgttg
       61 gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg ctgcaccggt
      121 gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg actcgctacg
      181 ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga
      241 agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat
      301 aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac
      361 ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct
      421 gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc
      481 acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc
      541 ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga
      601 aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct
      661 tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc
      721 caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct
      781 gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag
      841 aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa
      901 atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt ttgacagctt
      961 atcatcgata agctttaatg cggtagttta tcacagttaa attgctaacg cagtcaggca
     1021 ccgtgtatga aatctaacaa tgcgctcatc gtcatcctcg gcaccgtcac cctggatgct
     1081 gtaggcatag gcttggttat gccggtactg ccgggcctct tgcgggatta cgaaatcatc
     1141 ctgtggagct tagtaggttt agcaagatgg cagcgcctaa atgtagaatg ataaaaggat
     1201 taagagatta atttccctaa aaatgataaa acaagcgttt tgaaagcgct tgtttttttg
     1261 gtttgcagtc agagtagaat agaagtatca aaaaaagcac cgactcggtg ccactttttc
     1321 aagttgataa cggactagcc ttattttaac ttgctatgct gttttgaatg gttccaacaa
     1381 gattatttta taacttttat aacaaataat caaggagaaa ttcaaagaaa tttatcagcc
     1441 ataaaacaat acttaatact atagaatgat aacaaaataa actacttttt aaaagaattt
     1501 tgtgttataa tctatttatt attaagtatt gggtaatatt ttttgaagag atattttgaa
     1561 aaagaaaaat taaagcatat taaactaatt tcggaggtca ttaaaactat tattgaaatc
     1621 atcaaactca ttatggattt aatttaaact ttttatttta ggaggcaaaa atggataaga
     1681 aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg atcactgatg
     1741 aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc cacagtatca
     1801 aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa gcgactcgtc
     1861 tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt tatctacagg
     1921 agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga cttgaagagt
     1981 cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga aatatagtag
     2041 atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa aaattggtag
     2101 attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat atgattaagt
     2161 ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat gtggacaaac
     2221 tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct attaacgcaa
     2281 gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga cgattagaaa
     2341 atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat ctcattgctt
     2401 tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa gatgctaaat
     2461 tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg caaattggag
     2521 atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt ttactttcag
     2581 atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca atgattaaac
     2641 gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga caacaacttc
     2701 cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca ggttatattg
     2761 atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta gaaaaaatgg
     2821 atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc aagcaacgga
     2881 cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat gctattttga
     2941 gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt gaaaaaatct
     3001 tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt cgttttgcat
     3061 ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa gttgtcgata
     3121 aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa aatcttccaa
     3181 atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt tataacgaat
     3241 tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt tcaggtgaac
     3301 agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc gttaagcaat
     3361 taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt tcaggagttg
     3421 aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt attaaagata
     3481 aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt ttaacattga
     3541 ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct cacctctttg
     3601 atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga cgtttgtctc
     3661 gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta gattttttga
     3721 aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat agtttgacat
     3781 ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta catgaacata
     3841 ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact gtaaaagttg
     3901 ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt attgaaatgg
     3961 cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt atgaaacgaa
     4021 tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct gttgaaaata
     4081 ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga gacatgtatg
     4141 tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac attgttccac
     4201 aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct gataaaaatc
     4261 gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa aactattgga
     4321 gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta acgaaagctg
     4381 aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa ttggttgaaa
     4441 ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat actaaatacg
     4501 atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct aaattagttt
     4561 ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat taccatcatg
     4621 cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa tatccaaaac
     4681 ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa atgattgcta
     4741 agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct aatatcatga
     4801 acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc cctctaatcg
     4861 aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt gccacagtgc
     4921 gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta cagacaggcg
     4981 gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa
     5041 aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct tattcagtcc
     5101 tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt aaagagttac
     5161 tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac tttttagaag
     5221 ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa tatagtcttt
     5281 ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta caaaaaggaa
     5341 atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt cattatgaaa
     5401 agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag cagcataagc
     5461 attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt attttagcag
     5521 atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa ccaatacgtg
     5581 aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct cccgctgctt
     5641 ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa gaagttttag
     5701 atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt gatttgagtc
     5761 agctaggagg tgacgaagaa cagaaactga tctctgaagg aagacctgtg agccatattc
     5821 aacgggaaac gtcttgctcg tcgaggccgc gattaaattc caacatggat gctgatttat
     5881 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tattgattgt
     5941 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     6001 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     6061 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccctggga
     6121 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     6181 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     6241 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     6301 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     6361 accttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     6421 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     6481 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     6541 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     6601 atttgatgct cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga
     6661 gcattacgct gacccgcttc ggcgggtttt tttatcagct gtccctcctg ttcagctact
     6721 gacggggtgg tgcgt
//
