LOCUS       sequence-310948-        6245 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 6245)
  TITLE     Orzaez lab plasmids
REFERENCE   2  (bases 1 to 6245)
  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..6245
                     /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      459..516
                     /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(540..557)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(711..1299)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(791..810)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1482..1576)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1597..2256)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2171..2190
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     2418..2436
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2440..2483)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2486..2507
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             join(2524..6245,1..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="MDKKYSIGLAIGTNSVGWAVITAEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETDEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     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 aggttggttc
      421 gtgagaccac tcatcgcgac tagtagcggc cgctgcagtc cggcaaaaaa gggcaaggtg
      481 tcaccaccct gccctttttc tttaaaaccg aaaagattac ttcgcgttat gcaggcttcc
      541 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
      601 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
      661 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccacagg
      721 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
      781 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
      841 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      901 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      961 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1021 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1081 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1141 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     1201 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     1261 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     1321 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     1381 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1441 agtatatatg agtaaacttg gtctgacagc tcgaggcttg gattctcacc aataaaaaac
     1501 gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga
     1561 tctatcaaca ggagtccaag cgagctcgat atcaaattac gccccgccct gccactcgtc
     1621 gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg
     1681 atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat
     1741 ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa
     1801 actcacccag ggattggctg atacgaaaaa catattctca ataaaccctt tagggaaata
     1861 ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa
     1921 atcgtcgtgg tattcactcc agagtgatga aaacgtttca gtttgctcat ggaaaacggt
     1981 gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa
     2041 ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg
     2101 cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata
     2161 ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat
     2221 atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa
     2281 tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga
     2341 acctcttacg tgcccgatca actcgagtgc cacctgacgt ctaagaaacc attattatca
     2401 tgacattaac ctataaaaat aggcgtatca cgaggcagaa tttcagataa aaaaaatcct
     2461 tagctttcgc taaggatgat ttctggaatt cgcggccgct tctagagcga tgagtggtct
     2521 cgaatggaca agaagtactc cattgggctc gctatcggca caaacagcgt cggctgggcc
     2581 gtcattacgg ccgagtacaa ggtgccgagc aaaaaattca aagttctggg caataccgat
     2641 cgccacagca taaagaagaa cctcattggc gccctcctgt tcgactccgg ggaaacggac
     2701 gaagccacgc ggctcaaaag aacagcacgg cgcagatata cccgcagaaa gaatcggatc
     2761 tgctacctgc aggagatctt tagtaatgag atggctaagg tggatgactc tttcttccat
     2821 aggctggagg agtccttttt ggtggaggag gataaaaagc acgagcgcca cccaatcttt
     2881 ggcaatatcg tggacgaggt ggcgtaccat gaaaagtacc caaccatata tcatctgagg
     2941 aagaagcttg tagacagtac tgataaggct gacttgcggt tgatctatct cgcgctggcg
     3001 catatgatca aatttcgggg acacttcctc atcgaggggg acctgaaccc agacaacagc
     3061 gatgtcgaca aactctttat ccaactggtt cagacttaca atcagctttt cgaagagaac
     3121 ccgatcaacg catccggagt tgacgccaaa gcaatcctga gcgctaggct gtccaaatcc
     3181 cggcggctcg aaaacctcat cgcacagctc cctggggaga agaagaacgg cctgtttggt
     3241 aatcttatcg ccctgtcact cgggctgacc cccaacttta aatctaactt cgacctggcc
     3301 gaagatgcca agcttcaact gagcaaagac acctacgatg atgatctcga caatctgctg
     3361 gcccagatcg gcgaccagta cgcagacctt tttttggcgg caaagaacct gtcagacgcc
     3421 attctgctga gtgatattct gcgagtgaac acggagatca ccaaagctcc gctgagcgct
     3481 agtatgatca agcgctatga tgagcaccac caagacttga ctttgctgaa ggcccttgtc
     3541 agacagcaac tgcctgagaa gtacaaggaa attttcttcg atcagtctaa aaatggctac
     3601 gccggataca ttgacggcgg agcaagccag gaggaatttt acaaatttat taagcccatc
     3661 ttggaaaaaa tggacggcac cgaggagctg ctggtaaagc ttaacagaga agatctgttg
     3721 cgcaaacagc gcactttcga caatggaagc atcccccacc agattcacct gggcgaactg
     3781 cacgctatcc tcaggcggca agaggatttc tacccctttt tgaaagataa cagggaaaag
     3841 attgagaaaa tcctcacatt tcggataccc tactatgtag gccccctcgc ccggggaaat
     3901 tccagattcg cgtggatgac tcgcaaatca gaagagacta tcactccctg gaacttcgag
     3961 gaagtcgtgg ataagggggc ctctgcccag tccttcatcg aaaggatgac taactttgat
     4021 aaaaatctgc ctaacgaaaa ggtgcttcct aaacactctc tgctgtacga gtacttcaca
     4081 gtttataacg agctcaccaa ggtcaaatac gtcacagaag ggatgagaaa gccagcattc
     4141 ctgtctggag agcagaagaa agctatcgtg gacctcctct tcaagacgaa ccggaaagtt
     4201 accgtgaaac agctcaaaga agattatttc aaaaagattg aatgtttcga ctctgttgaa
     4261 atcagcggag tggaggatcg cttcaacgca tccctgggaa cgtatcacga tctcctgaaa
     4321 atcattaaag acaaggactt cctggacaat gaggagaacg aggacattct tgaggacatt
     4381 gtcctcaccc ttacgttgtt tgaagatagg gagatgattg aagaacgctt gaaaacttac
     4441 gctcatctct tcgacgacaa agtcatgaaa cagctcaaga ggcgccgata tacaggatgg
     4501 gggcggctgt caagaaaact gatcaatggg atccgagaca agcagagtgg aaagacaatc
     4561 ctggattttc ttaagtccga tggatttgcc aaccggaact tcatgcagtt gatccatgat
     4621 gactctctca cctttaagga ggacatccag aaagcacaag tttctggcca gggggacagt
     4681 ctccacgagc acatcgctaa tcttgcaggt agcccagcta tcaaaaaggg aatactgcag
     4741 accgttaagg tcgtggatga actcgtcaaa gtaatgggaa ggcataagcc cgagaatatc
     4801 gttatcgaga tggcccgaga gaaccaaact acccagaagg gacagaagaa cagtagggaa
     4861 aggatgaaga ggattgaaga gggtataaaa gaactggggt cccaaatcct taaggaacac
     4921 ccagttgaaa acacccagct tcagaatgag aagctctacc tgtactacct gcagaacggc
     4981 agggacatgt acgtggatca ggaactggac atcaatcggc tctccgacta cgacgtggat
     5041 catatcgtgc cccagtcttt tctcaaagat gattctattg ataataaagt gttgacaaga
     5101 tccgataaaa atagagggaa gagtgataac gtcccctcag aagaagttgt caagaaaatg
     5161 aaaaattatt ggcggcagct gctgaacgcc aaactgatca cacaacggaa gttcgataat
     5221 ctgactaagg ctgaacgagg tggcctgtct gagttggata aagccggctt catcaaaagg
     5281 cagcttgttg agacacgcca gatcaccaag cacgtggccc aaattctcga ttcacgcatg
     5341 aacaccaagt acgatgaaaa tgacaaactg attcgagagg tgaaagttat tactctgaag
     5401 tctaagctgg tttcagattt cagaaaggac tttcagtttt ataaggtgag agagatcaac
     5461 aattaccacc atgcgcatga tgcctacctg aatgcagtgg taggcactgc acttatcaaa
     5521 aaatatccca agcttgaatc tgaatttgtt tacggagact ataaagtgta cgatgttagg
     5581 aaaatgatcg caaagtctga gcaggaaata ggcaaggcca ccgctaagta cttcttttac
     5641 agcaatatta tgaatttttt caagaccgag attacactgg ccaatggaga gattcggaag
     5701 cgaccactta tcgaaacaaa cggagaaaca ggagaaatcg tgtgggacaa gggtagggat
     5761 ttcgcgacag tccggaaggt cctgtccatg ccgcaggtga acatcgttaa aaagaccgaa
     5821 gtacagaccg gaggcttctc caaggaaagt atcctcccga aaaggaacag cgacaagctg
     5881 atcgcacgca aaaaagattg ggaccccaag aaatacggcg gattcgattc tcctacagtc
     5941 gcttacagtg tactggttgt ggccaaagtg gagaaaggga agtctaaaaa actcaaaagc
     6001 gtcaaggaac tgctgggcat cacaatcatg gagcgatcaa gcttcgaaaa aaaccccatc
     6061 gactttctcg aggcgaaagg atataaagag gtcaaaaaag acctcatcat taagcttccc
     6121 aagtactctc tctttgagct tgaaaacggc cggaaacgaa tgctcgctag tgcgggcgag
     6181 ctgcagaaag gtaacgagct ggcactgccc tctaaatacg ttaatttctt gtatctggcc
     6241 agcca
//
