LOCUS       sequence-103506-        5043 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  t1 gRNA (ATGAGAATCAAGGCGGTCGA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 5043)
  AUTHORS   Moore R, Spinhirne A, Lai MJ, Preisser S, Li Y, Kang T, Bleris L
  TITLE     CRISPR-based self-cleaving mechanism for controllable gene delivery 
            in human cells.
  JOURNAL   Nucleic Acids Res. 2015 Jan 30;43(2):1297-303. doi: 
            10.1093/nar/gku1326. Epub 2014 Dec 18.
  PUBMED    25527740
REFERENCE   2  (bases 1 to 5043)
  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"; doi: 
            "10.1093/nar/gku1326"; journalName: "Nucleic Acids Res"; date: 
            "2015-01-30- 30"; volume: "43"; issue: "2"; pages: "1297-303"
COMMENT     Sequence Label: pCas9–mKate2ps–T1gRNA
FEATURES             Location/Qualifiers
     source          1..5043
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             1..4104
                     /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             1..4104
                     /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             1..4104
                     /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             4117..4137
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4141..4194
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             4207..4902
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             4921..5040
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
ORIGIN
        1 atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc
       61 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc
      121 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa
      181 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc
      241 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg
      301 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc
      361 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag
      421 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat
      481 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat
      541 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg
      601 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg
      661 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat
      721 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa
      781 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc
      841 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt
      901 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt
      961 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga
     1021 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc
     1081 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg
     1141 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc
     1201 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac
     1261 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt
     1321 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc
     1381 agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa
     1441 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa
     1501 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt
     1561 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg
     1621 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc
     1681 gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc
     1741 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc
     1801 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc
     1861 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct
     1921 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg
     1981 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg
     2041 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac
     2101 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt
     2161 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
     2221 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
     2281 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
     2341 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
     2401 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
     2461 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat
     2521 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc
     2581 gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
     2641 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
     2701 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
     2761 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
     2821 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
     2881 aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
     2941 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
     3001 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
     3061 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
     3121 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
     3181 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
     3241 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
     3301 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
     3361 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct
     3421 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
     3481 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac
     3541 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag
     3601 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg
     3661 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
     3721 cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa
     3781 caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg
     3841 atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag
     3901 cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg
     3961 cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag
     4021 gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc
     4081 gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtggct
     4141 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg cccttccgga
     4201 gggcccatgg tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc
     4261 accgtgaaca accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc
     4321 acccagacca tgagaatcaa ggcggtcgag ggcggccctc tccccttcgc cttcgacatc
     4381 ctggctacca gcttcatgta cggcagcaaa accttcatca accacaccca gggcatcccc
     4441 gacttcttta agcagtcctt ccccgagggc ttcacatggg agagagtcac cacatacgaa
     4501 gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac
     4561 aacgtcaaga tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca
     4621 ctcggctggg aggcctccac cgagaccctg taccccgctg acggcggcct ggaaggcaga
     4681 gccgacatgg ccctgaagct cgtgggcggg ggccacctga tctgcaactt gaagaccaca
     4741 tacagatcca agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggacaga
     4801 agactggaaa gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct
     4861 gtggccagat actgcgacct ccctagcaaa ctggggcaca gaggtggagg aggttccgga
     4921 tctcacggct tccctcccga ggtggaggag caggccgccg gcaccctgcc catgagctgc
     4981 gcccaggaga gcggcatgga tagacaccct gctgcttgcg ccagcgccag gatcaacgtc
     5041 taa
//
