LOCUS       sequence-322078-        6246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nickase D10A Cas9 for Nt fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6246)
  TITLE     Orzaez lab plasmids
REFERENCE   2  (bases 1 to 6246)
  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"
COMMENT     Sequence Label: pUPD2 nickase D10A Cas9 (GB1691)
FEATURES             Location/Qualifiers
     source          1..6246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             403..423
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      468..525
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(549..566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(720..1308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(800..819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1491..1585)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1606..2265)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2180..2199
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2427..2445
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2449..2492)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2495..2516
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             join(2533..2559,2560..2562,2563..6246,1..390)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(2533..6246,1..390)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gccagccact atgaaaagct caaaggatct cccgaagata atgagcagaa gcagctgttc
       61 gtggaacaac acaaacacta ccttgatgag atcatcgagc aaataagcga attctccaaa
      121 agagtgatcc tcgccgacgc taacctcgat aaggtgcttt ctgcttacaa taagcacagg
      181 gataagccca tcagggagca ggcagaaaac attatccact tgtttactct gaccaacttg
      241 ggcgcgcctg cagccttcaa gtacttcgac accaccatag acagaaagcg gtacacctct
      301 acaaaggagg tcctggacgc cacactgatt catcagtcaa ttacggggct ctatgaaaca
      361 agaatcgacc tctctcagct cggtggagac agcagggctg accccaagaa gaagaggaag
      421 gtgtgagctt tgagaccact catcgcgact agtagcggcc gctgcagtcc ggcaaaaaag
      481 ggcaaggtgt caccaccctg ccctttttct ttaaaaccga aaagattact tcgcgttatg
      541 caggcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
      601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
      661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
      721 ttccacaggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
      781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
      841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
      901 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
      961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     1021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     1081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     1141 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     1201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     1261 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     1321 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     1381 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     1441 tcaatctaaa gtatatatga gtaaacttgg tctgacagct cgaggcttgg attctcacca
     1501 ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc
     1561 attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg
     1621 ccactcgtcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca
     1681 aacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata
     1741 tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa
     1801 actggtgaaa ctcacccagg gattggctga tacgaaaaac atattctcaa taaacccttt
     1861 agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa
     1921 ctgccggaaa tcgtcgtggt attcactcca gagtgatgaa aacgtttcag tttgctcatg
     1981 gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc
     2041 catacgaaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata
     2101 aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt
     2161 ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca
     2221 ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc
     2281 tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg
     2341 aaagttggaa cctcttacgt gcccgatcaa ctcgagtgcc acctgacgtc taagaaacca
     2401 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcagaat ttcagataaa
     2461 aaaaatcctt agctttcgct aaggatgatt tctggaattc gcggccgctt ctagagcgat
     2521 gagtggtctc gaatggacaa gaagtactcc attgggctcg ctatcggcac aaacagcgtc
     2581 ggctgggccg tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc
     2641 aataccgatc gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg
     2701 gaaacggccg aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag
     2761 aatcggatct gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct
     2821 ttcttccata ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac
     2881 ccaatctttg gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat
     2941 catctgagga agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc
     3001 gcgctggcgc atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca
     3061 gacaacagcg atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc
     3121 gaagagaacc cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg
     3181 tccaaatccc ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc
     3241 ctgtttggta atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc
     3301 gacctggccg aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac
     3361 aatctgctgg cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg
     3421 tcagacgcca ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg
     3481 ctgagcgcta gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag
     3541 gcccttgtca gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa
     3601 aatggctacg ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt
     3661 aagcccatct tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa
     3721 gatctgttgc gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg
     3781 ggcgaactgc acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac
     3841 agggaaaaga ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc
     3901 cggggaaatt ccagattcgc gtggatgact cgcaaatcag aagagactat cactccctgg
     3961 aacttcgagg aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact
     4021 aactttgata aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag
     4081 tacttcacag tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag
     4141 ccagcattcc tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac
     4201 cggaaagtta ccgtgaaaca gctcaaagaa gattatttca aaaagattga atgtttcgac
     4261 tctgttgaaa tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat
     4321 ctcctgaaaa tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt
     4381 gaggacattg tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg
     4441 aaaacttacg ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat
     4501 acaggatggg ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga
     4561 aagacaatcc tggattttct taagtccgat ggatttgcca accggaactt catgcagttg
     4621 atccatgatg actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag
     4681 ggggacagtc tccacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga
     4741 atactgcaga ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc
     4801 gagaatatcg ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac
     4861 agtagggaaa ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt
     4921 aaggaacacc cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg
     4981 cagaacggca gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac
     5041 gacgtggatc atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg
     5101 ttgacaagat ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc
     5161 aagaaaatga aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag
     5221 ttcgataatc tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc
     5281 atcaaaaggc agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat
     5341 tcacgcatga acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt
     5401 actctgaagt ctaagctggt ttcagatttc agaaaggact ttcagtttta taaggtgaga
     5461 gagatcaaca attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca
     5521 cttatcaaaa aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac
     5581 gatgttagga aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac
     5641 ttcttttaca gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag
     5701 attcggaagc gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag
     5761 ggtagggatt tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa
     5821 aagaccgaag tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc
     5881 gacaagctga tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct
     5941 cctacagtcg cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa
     6001 ctcaaaagcg tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa
     6061 aaccccatcg actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt
     6121 aagcttccca agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt
     6181 gcgggcgagc tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg
     6241 tatctg
//
