LOCUS       sequence-228465-        6397 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The plasmid encodes the T7 promoter, Cas9 (from pX330 #42230) and a 
            stretch of poly-A. After linearization with restriction enzyme SapI 
            It is used to produce in vitro transcribed mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6397)
  AUTHORS   Pritchard CEJ, Kroese LJ, Huijbers IJ
  TITLE     Direct Generation of Conditional Alleles Using CRISPR/Cas9 in Mouse 
            Zygotes.
  JOURNAL   Methods Mol Biol. 2017;1642:21-35. doi: 10.1007/978-1-4939-7169-5_2.
  PUBMED    28815491
REFERENCE   2  (bases 1 to 6397)
  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: "Methods Mol
            Biol."; date: "2017"; volume: "1642:21-35. doi"; pages: " 
            10.1007/978-1-4939-7169-5_2"
COMMENT     Sequence Label: bbCas9pluspAAA
FEATURES             Location/Qualifiers
     source          1..6397
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             27..92
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             99..119
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             144..4244
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             144..4244
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4245..4292
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        4472..4576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4577..4645,4646..5437)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4795..4814)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5608..6196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6097..6116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6350..6367
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg agaatggact ataaggacca cgacggagac tacaaggatc
       61 atgatattga ttacaaagac gatgacgata agatggcccc aaagaagaag cggaaggtcg
      121 gtatccacgg agtcccagca gccgacaaga agtacagcat cggcctggac atcggcacca
      181 actctgtggg ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg
      241 tgctgggcaa caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg
      301 acagcggcga aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca
      361 gacggaagaa ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg
      421 acgacagctt cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg
      481 agcggcaccc catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca
      541 ccatctacca cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga
      601 tctatctggc cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc
      661 tgaaccccga caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc
      721 agctgttcga ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg
      781 ccagactgag caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga
      841 agaatggcct gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga
      901 gcaacttcga cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg
      961 acctggacaa cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca
     1021 agaacctgtc cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca
     1081 aggcccccct gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc
     1141 tgctgaaagc tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc
     1201 agagcaagaa cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca
     1261 agttcatcaa gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga
     1321 acagagagga cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga
     1381 tccacctggg agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga
     1441 aggacaaccg ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc
     1501 ctctggccag gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca
     1561 ccccctggaa cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc
     1621 ggatgaccaa cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc
     1681 tgtacgagta cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa
     1741 tgagaaagcc cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca
     1801 agaccaaccg gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt
     1861 gcttcgactc cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat
     1921 accacgatct gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg
     1981 acattctgga agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg
     2041 aacggctgaa aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
     2101 ggagatacac cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc
     2161 agtccggcaa gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca
     2221 tgcagctgat ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt
     2281 ccggccaggg cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta
     2341 agaagggcat cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
     2401 acaagcccga gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac
     2461 agaagaacag ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc
     2521 agatcctgaa agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt
     2581 actacctgca gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt
     2641 ccgactacga tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca
     2701 acaaggtgct gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag
     2761 aggtcgtgaa gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc
     2821 agagaaagtt cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg
     2881 ccggcttcat caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga
     2941 tcctggactc ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga
     3001 aagtgatcac cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca
     3061 aagtgcgcga gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg
     3121 gaaccgccct gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca
     3181 aggtgtacga cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg
     3241 ccaagtactt cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca
     3301 acggcgagat ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt
     3361 gggataaggg ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata
     3421 tcgtgaaaaa gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga
     3481 ggaacagcga taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct
     3541 tcgacagccc caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt
     3601 ccaagaaact gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct
     3661 tcgagaagaa tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc
     3721 tgatcatcaa gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc
     3781 tggcctctgc cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga
     3841 acttcctgta cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc
     3901 agaaacagct gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca
     3961 gcgagttctc caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct
     4021 acaacaagca ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta
     4081 ccctgaccaa tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga
     4141 agaggtacac cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg
     4201 gcctgtacga gacacggatc gacctgtctc agctgggagg cgacaaaagg ccggcggcca
     4261 cgaaaaaggc cggccaggca aaaaagaaaa agtaagaatt cctagagctc gctaaaaaaa
     4321 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     4381 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagaag agcgcggccg cttaattaat
     4441 ttaaataggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4501 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4561 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4621 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4681 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4741 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4801 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4861 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4921 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4981 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5041 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5101 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5161 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5221 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5281 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5341 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5401 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5461 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5521 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5581 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5641 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5701 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5761 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5821 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5881 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5941 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     6001 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     6061 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     6121 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     6181 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     6241 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     6301 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     6361 gagcgaggaa gcggaagacc tgcaggaccc atctaga
//
