LOCUS       sequence-157087-        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"
     rep_origin      27..615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     516..535
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     769..786
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     817..836
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        817..835
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             843..908
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             915..935
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             960..5060
                     /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             960..5060
                     /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             5061..5108
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     promoter        5288..5392
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5393..5461,5462..6253)
                     /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(5611..5630)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
       61 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      121 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
      181 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
      241 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      301 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      361 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      421 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      481 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      541 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      601 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      661 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      721 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      781 agcgaggaag cggaagacct gcaggaccca tctagataat acgactcact atagggagaa
      841 tggactataa ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg
      901 acgataagat ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg
      961 acaagaagta cagcatcggc ctggacatcg gcaccaactc tgtgggctgg gccgtgatca
     1021 ccgacgagta caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca
     1081 gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca
     1141 cccggctgaa gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc
     1201 tgcaagagat cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg
     1261 aagagtcctt cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca
     1321 tcgtggacga ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac
     1381 tggtggacag caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga
     1441 tcaagttccg gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg
     1501 acaagctgtt catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca
     1561 acgccagcgg cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc
     1621 tggaaaatct gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggaaacctga
     1681 ttgccctgag cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg
     1741 ccaaactgca gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga
     1801 tcggcgacca gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc
     1861 tgagcgacat cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga
     1921 tcaagagata cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc
     1981 agctgcctga gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct
     2041 acattgacgg cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa
     2101 agatggacgg caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc
     2161 agcggacctt cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca
     2221 ttctgcggcg gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga
     2281 agatcctgac cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat
     2341 tcgcctggat gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg
     2401 tggacaaggg cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc
     2461 tgcccaacga gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata
     2521 acgagctgac caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg
     2581 gcgagcagaa aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga
     2641 agcagctgaa agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg
     2701 gcgtggaaga tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca
     2761 aggacaagga cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga
     2821 ccctgacact gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc
     2881 tgttcgacga caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc
     2941 tgagccggaa gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt
     3001 tcctgaagtc cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc
     3061 tgacctttaa agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg
     3121 agcacattgc caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga
     3181 aggtggtgga cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg
     3241 aaatggccag agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga
     3301 agcggatcga agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg
     3361 aaaacaccca gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata
     3421 tgtacgtgga ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gaccatatcg
     3481 tgcctcagag ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca
     3541 agaaccgggg caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact
     3601 actggcggca gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca
     3661 aggccgagag aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg
     3721 tggaaacccg gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta
     3781 agtacgacga gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc
     3841 tggtgtccga tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc
     3901 accacgccca cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc
     3961 ctaagctgga aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga
     4021 tcgccaagag cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca
     4081 tcatgaactt tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc
     4141 tgatcgagac aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca
     4201 ccgtgcggaa agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga
     4261 caggcggctt cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca
     4321 gaaagaagga ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt
     4381 ctgtgctggt ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag
     4441 agctgctggg gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc
     4501 tggaagccaa gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact
     4561 ccctgttcga gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga
     4621 agggaaacga actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact
     4681 atgagaagct gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc
     4741 acaagcacta cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc
     4801 tggccgacgc taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca
     4861 tcagagagca ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg
     4921 ccgccttcaa gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg
     4981 tgctggacgc caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc
     5041 tgtctcagct gggaggcgac aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     5101 agaaaaagta agaattccta gagctcgcta tccaaaaaaa caaaaaaaaa aaaaaaaaaa
     5161 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     5221 aaaaaaaaaa aagaagagcg cggccgctta attaatttaa ataggtggca cttttcgggg
     5281 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5341 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5401 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5461 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5521 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5581 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5641 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5701 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5761 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     5821 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5881 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5941 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6001 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6061 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6121 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6181 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6241 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6301 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6361 cccttaacgt gagttttcgt tccactgagc gtcagac
//
