LOCUS       sequence-486306-        6246 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  dCas9 with Stop codon as Level-0 part (Golden Gate cloning).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6246)
  AUTHORS   Khan MA, Herring G, Zhu JY, Oliva M, Fourie E, Johnston B, Zhang Z, 
            Potter J, Pineda L, Pflueger J, Swain T, Pflueger C, Lloyd JPB, 
            Secco D, Small I, Kidd BN, Lister R
  TITLE     CRISPRi-based circuits to control gene expression in plants.
  JOURNAL   Nat Biotechnol. 2025 Mar;43(3):416-430. doi: 
            10.1038/s41587-024-02236-w. Epub 2024 May 20.
  PUBMED    38769424
REFERENCE   2  (bases 1 to 6246)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41587-024-02236-w"; journalName: "Nat Biotechnol"; date: 
            "2025-03"; volume: "43"; issue: "3"; pages: "416-430"
COMMENT     Sequence Label: LLP1010
FEATURES             Location/Qualifiers
     source          1..6246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             285..1076
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1417..2005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1906..1925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             2061..2081
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2094..2117,2118..2120,2121..4607,4608..4610,
                     4611..6194)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             6207..6227
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gtgccgaggc acgaacccag tggacataag cctgttcggt tcgtaagctg taatgcaagt
       61 agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg
      121 cagtggcggt tttcatggct tgttatgact gtttttttgg ggtacagtct atgcctcggg
      181 catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg
      241 atgttacgca gcagggcagt cgccctaaaa caaagttaaa catcatgggg gaagcggtga
      301 tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac
      361 cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca
      421 gcgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt
      481 tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg
      541 tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg
      601 aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca
      661 cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg
      721 taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc
      781 taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg
      841 tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg
      901 atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg
      961 aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt
     1021 tggaagaatt tgtccattac gtgaaaggcg agatcaccaa ggtagtcggc aaataatgtc
     1081 tagctagaaa ttcgttcaag ccgacgccgc ttcgcggcgc ggcttaactc aagcgttaga
     1141 tgcactaagc acataattgc tcacagccaa actatcaggt caagtctgct tttattattt
     1201 ttaagcgtgc ataataagcc ctacacaaat tgggagatat atcatgctgt cagaccaagt
     1261 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     1321 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     1381 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     1441 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     1501 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     1561 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     1621 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     1681 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     1741 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     1801 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     1861 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     1921 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     1981 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctgca
     2041 ctctgtggtc tcaaatgatg ccaaagaaga agagaaaggt cgggggtggt tctgataaga
     2101 agtactctat cggactcgct atcggaacta actctgtggg atgggctgtg atcaccgatg
     2161 agtacaaggt gccatctaag aagttcaagg ttctcggaaa caccgatagg cactctatca
     2221 agaaaaacct tatcggtgct ctcctcttcg attctggtga aactgctgag gctaccagac
     2281 tcaagagaac cgctagaaga aggtacacca gaagaaagaa caggatctgc tacctccaag
     2341 agatcttctc taacgagatg gctaaagtgg atgattcatt cttccacagg ctcgaagagt
     2401 cattcctcgt ggaagaagat aagaagcacg agaggcaccc tatcttcgga aacatcgttg
     2461 atgaggtggc ataccacgag aagtacccta ctatctacca cctcagaaag aagctcgttg
     2521 attctactga taaggctgat ctcaggctca tctacctcgc tctcgctcac atgatcaagt
     2581 tcagaggaca cttcctcatc gagggtgatc tcaaccctga taactctgat gtggataagt
     2641 tgttcatcca gctcgtgcag acctacaacc agcttttcga agagaaccct atcaacgctt
     2701 caggtgtgga tgctaaggct atcctctctg ctaggctctc taagtcaaga aggcttgaga
     2761 acctcattgc tcagctccct ggtgagaaga agaacggact tttcggaaac ttgatcgctc
     2821 tctctctcgg actcacccct aacttcaagt ctaacttcga tctcgctgag gatgcaaagc
     2881 tccagctctc aaaggatacc tacgatgatg atctcgataa cctcctcgct cagatcggag
     2941 atcagtacgc tgatttgttc ctcgctgcta agaacctctc tgatgctatc ctcctcagtg
     3001 atatcctcag agtgaacacc gagatcacca aggctccact ctcagcttct atgatcaaga
     3061 gatacgatga gcaccaccag gatctcacac ttctcaaggc tcttgttaga cagcagctcc
     3121 cagagaagta caaagagatt ttcttcgatc agtctaagaa cggatacgct ggttacatcg
     3181 atggtggtgc atctcaagaa gagttctaca agttcatcaa gcctatcctc gagaagatgg
     3241 atggaaccga ggaactcctc gtgaagctca atagagagga tcttctcaga aagcagagga
     3301 ccttcgataa cggatctatc cctcatcaga tccacctcgg agagttgcac gctatcctta
     3361 gaaggcaaga ggatttctac ccattcctca aggataacag ggaaaagatt gagaagattc
     3421 tcaccttcag aatcccttac tacgtgggac ctctcgctag aggaaactca agattcgctt
     3481 ggatgaccag aaagtctgag gaaaccatca ccccttggaa cttcgaagag gtggtggata
     3541 agggtgctag tgctcagtct ttcatcgaga ggatgaccaa cttcgataag aaccttccaa
     3601 acgagaaggt gctccctaag cactctttgc tctacgagta cttcaccgtg tacaacgagt
     3661 tgaccaaggt taagtacgtg accgagggaa tgaggaagcc tgcttttttg tcaggtgagc
     3721 aaaagaaggc tatcgttgat ctcttgttca agaccaacag aaaggtgacc gtgaagcagc
     3781 tcaaagagga ttacttcaag aaaatcgagt gcttcgattc agttgagatt tctggtgttg
     3841 aggataggtt caacgcatct ctcggaacct accacgatct cctcaagatc attaaggata
     3901 aggatttctt ggataacgag gaaaacgagg atatcttgga ggatatcgtt cttaccctca
     3961 ccctctttga agatagagag atgattgaag aaaggctcaa gacctacgct catctcttcg
     4021 atgataaggt gatgaagcag ttgaagagaa gaagatacac tggttgggga aggctctcaa
     4081 gaaagctcat taacggaatc agggataagc agtctggaaa gacaatcctt gatttcctca
     4141 agtctgatgg attcgctaac agaaacttca tgcagctcat ccacgatgat tctctcacct
     4201 ttaaagagga tatccagaag gctcaggttt caggacaggg tgatagtctc catgagcata
     4261 tcgctaacct cgctggatct cctgcaatca agaagggaat cctccagact gtgaaggttg
     4321 tggatgagtt ggtgaaggtg atgggaaggc ataagcctga gaacatcgtg atcgaaatgg
     4381 ctagagagaa ccagaccact cagaagggac agaagaactc tagggaaagg atgaagagga
     4441 tcgaggaagg tatcaaagag cttggatctc agatcctcaa agagcaccct gttgagaaca
     4501 ctcagctcca gaatgagaag ctctacctct actacctcca gaacggaagg gatatgtatg
     4561 tggatcaaga gttggatatc aacaggctct ctgattacga tgttgatgct atcgtgccac
     4621 agtcattctt gaaggatgat tctatcgata acaaggtgct caccaggtct gataagaaca
     4681 ggggtaagag tgataacgtg ccaagtgaag aggttgtgaa gaaaatgaag aactattgga
     4741 ggcagctcct caacgctaag ctcatcactc agagaaagtt cgataacttg actaaggctg
     4801 agaggggagg actctctgaa ttggataagg caggattcat caagaggcag cttgtggaaa
     4861 ccaggcagat cactaagcac gttgcacaga tcctcgattc taggatgaac accaagtacg
     4921 atgagaacga taagttgatc agggaagtga aggttatcac cctcaagtca aagctcgtgt
     4981 ctgatttcag aaaggatttc caattctaca aggtgaggga aatcaacaac taccaccacg
     5041 ctcacgatgc ttaccttaac gctgttgttg gaaccgctct catcaagaag tatcctaagc
     5101 tcgagtcaga gttcgtgtac ggtgattaca aggtgtacga tgtgaggaag atgatcgcta
     5161 agtctgagca agagatcgga aaggctaccg ctaagtattt cttctactct aacatcatga
     5221 atttcttcaa gaccgagatt accctcgcta acggtgagat cagaaagagg ccactcatcg
     5281 agacaaacgg tgaaacaggt gagatcgtgt gggataaggg aagggatttc gctaccgtta
     5341 gaaaggtgct ctctatgcca caggtgaaca tcgttaagaa aaccgaggtg cagaccggtg
     5401 gattctctaa agagtctatc ctccctaaga ggaactctga taagctcatt gctaggaaga
     5461 aggattggga ccctaagaaa tacggtggtt tcgattctcc taccgtggct tactctgttc
     5521 tcgttgtggc taaggttgag aagggaaaga gtaagaagct caagtctgtt aaggaacttc
     5581 tcggaatcac tatcatggaa aggtcatctt tcgagaagaa cccaatcgat ttcctcgagg
     5641 ctaagggata caaagaggtt aagaaggatc tcatcatcaa gctcccaaag tactcactct
     5701 tcgaactcga gaacggtaga aagaggatgc tcgcttctgc tggtgagctt caaaagggaa
     5761 acgagcttgc tctcccatct aagtacgtta actttcttta cctcgcttct cactacgaga
     5821 agttgaaggg atctccagaa gataacgagc agaagcaact tttcgttgag cagcacaagc
     5881 actacttgga tgagatcatc gagcagatct ctgagttctc taaaagggtg atcctcgctg
     5941 atgcaaacct cgataaggtg ttgtctgctt acaacaagca cagagataag cctatcaggg
     6001 aacaggcaga gaacatcatc catctcttca cccttaccaa cctcggtgct cctgctgctt
     6061 tcaagtactt cgatacaacc atcgatagga agagatacac ctctaccaaa gaagtgctcg
     6121 atgctaccct catccatcag tctatcactg gactctacga gactaggatc gatctctcac
     6181 agctcggtgg tgatggtggg ggcagtccta aaaagaagag aaaggtgtga gcttcgagac
     6241 cacgaa
//
