LOCUS       sequence-322079-        6242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nickase D10A Cas9 for Nt and Ct fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6242)
  TITLE     Orzaez lab plasmids
REFERENCE   2  (bases 1 to 6242)
  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 (GB1690)
FEATURES             Location/Qualifiers
     source          1..6242
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             395..415
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      456..513
                     /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(537..554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(708..1296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(788..807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1479..1573)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1594..2253)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2168..2187
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2415..2433
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2437..2480)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2483..2504
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             join(2521..2547,2548..2550,2551..6242,1..382)
                     /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(2521..6242,1..382)
                     /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 ctatgaaaag ctcaaaggat ctcccgaaga taatgagcag aagcagctgt tcgtggaaca
       61 acacaaacac taccttgatg agatcatcga gcaaataagc gaattctcca aaagagtgat
      121 cctcgccgac gctaacctcg ataaggtgct ttctgcttac aataagcaca gggataagcc
      181 catcagggag caggcagaaa acattatcca cttgtttact ctgaccaact tgggcgcgcc
      241 tgcagccttc aagtacttcg acaccaccat agacagaaag cggtacacct ctacaaagga
      301 ggtcctggac gccacactga ttcatcagtc aattacgggg ctctatgaaa caagaatcga
      361 cctctctcag ctcggtggag acagcagggc tgaccccaag aagaagagga aggtttcgtg
      421 agaccactca tcgcgactag tagcggccgc tgcagtccgg caaaaaaggg caaggtgtca
      481 ccaccctgcc ctttttcttt aaaaccgaaa agattacttc gcgttatgca ggcttcctcg
      541 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      601 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      661 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccacaggctc
      721 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      781 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      841 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      901 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      961 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1021 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1081 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1141 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1201 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1261 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1321 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1381 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1441 atatatgagt aaacttggtc tgacagctcg aggcttggat tctcaccaat aaaaaacgcc
     1501 cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat tactggatct
     1561 atcaacagga gtccaagcga gctcgatatc aaattacgcc ccgccctgcc actcgtcgca
     1621 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg
     1681 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt
     1741 gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact
     1801 cacccaggga ttggctgata cgaaaaacat attctcaata aaccctttag ggaaataggc
     1861 caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc
     1921 gtcgtggtat tcactccaga gtgatgaaaa cgtttcagtt tgctcatgga aaacggtgta
     1981 acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacgaaattc
     2041 cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt
     2101 atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggttataggt
     2161 acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt gggatatatc
     2221 aacggtggta tatccagtga tttttttctc cattttagct tccttagctc ctgaaaatct
     2281 cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc
     2341 tcttacgtgc ccgatcaact cgagtgccac ctgacgtcta agaaaccatt attatcatga
     2401 cattaaccta taaaaatagg cgtatcacga ggcagaattt cagataaaaa aaatccttag
     2461 ctttcgctaa ggatgatttc tggaattcgc ggccgcttct agagcgatga gtggtctcga
     2521 atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc
     2581 attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc
     2641 cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga aacggccgaa
     2701 gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc
     2761 tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg
     2821 ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc
     2881 aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag
     2941 aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat
     3001 atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat
     3061 gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg
     3121 atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg
     3181 cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat
     3241 cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa
     3301 gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc
     3361 cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt
     3421 ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt
     3481 atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga
     3541 cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc
     3601 ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg
     3661 gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc
     3721 aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac
     3781 gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt
     3841 gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc
     3901 agattcgcgt ggatgactcg caaatcagaa gagactatca ctccctggaa cttcgaggaa
     3961 gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa
     4021 aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt
     4081 tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg
     4141 tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc
     4201 gtgaaacagc tcaaagaaga ttatttcaaa aagattgaat gtttcgactc tgttgaaatc
     4261 agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc
     4321 attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc
     4381 ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct
     4441 catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg
     4501 cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg
     4561 gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac
     4621 tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctc
     4681 cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc
     4741 gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt
     4801 atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg
     4861 atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca
     4921 gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg
     4981 gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat
     5041 atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc
     5101 gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa
     5161 aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg
     5221 actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag
     5281 cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac
     5341 accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct
     5401 aagctggttt cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat
     5461 taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa
     5521 tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa
     5581 atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc
     5641 aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga
     5701 ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc
     5761 gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta
     5821 cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc
     5881 gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct
     5941 tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc
     6001 aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac
     6061 tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag
     6121 tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg
     6181 cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc
     6241 ca
//
