LOCUS Exported 7729 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Gateway entry vector with pco-dCas9-3X(SRDX). ACCESSION . VERSION . KEYWORDS pYPQ153 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7729) AUTHORS Lowder LG, Paul JW, Baltes NJ, Voytas DF, Zhang Y, Zhang D, Tang X, Zheng X, Hsieh TF, Qi Y TITLE A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. JOURNAL Plant Physiol. 2015 Aug 21. pii: pp.00636.2015. PUBMED 26297141 REFERENCE 2 (bases 1 to 7729) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7729 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 3..255 /label=NOS terminator /note="nopaline synthase terminator and poly(A) signal" protein_bind 300..423 /gene="mutant version of attR" /label=attR5 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(440..459) /label=T7 /note="T7 promoter, forward primer" promoter complement(441..459) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(464..480) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(464..480) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 911..1702 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK" rep_origin 1795..2383 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2284..2303 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2537..2554 /label=L4440 /note="L4440 vector, forward primer" terminator 2713..2740 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 2832..2918 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 2967..2989 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 2981..2998 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 2982..2998 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 3014..3113 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS 3135..3182 /codon_start=1 /product="two tandem FLAG(R) epitope tags" /label=2xFLAG /translation="DYKDDDDKDYKDDDDK" CDS 3189..3209 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS join(3234..4670,4860..7523) /codon_start=1 /product="Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=pcoCas9 /note="plant codon-optimized Cas9 gene containing the potato IV2 intron" /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" intron 4671..4859 /label=IV2 intron /note="modified second intron of the potato ST-LS1 gene (Vancanneyt et al., 1990)" CDS 7524..7571 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" ORIGIN 1 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 61 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 121 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 181 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 241 ctatgttact agatcgggaa ttgatccccc ctcgacagct tccggaaagg gcgaattcgc 301 aactttgtat acaaaagttg aacgagaaac gtaaaatgat ataaatatca atatattaaa 361 ttagattttg cataaaaaac agactacata atactgtaaa acacaacata tccagtcact 421 atgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg 481 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc 541 atcatgcctc ctctggacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt 601 tgccgggtga cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg 661 ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt 721 gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt 781 ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg 841 atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa 901 gttaaacatc atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt 961 tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc 1021 agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag 1081 gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc 1141 tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat 1201 tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat 1261 tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa 1321 agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt 1381 tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc 1441 cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc 1501 agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc 1561 ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga 1621 tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat 1681 caccaaggta gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag 1741 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 1801 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 1861 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 1921 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 1981 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 2041 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 2101 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 2161 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 2221 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 2281 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 2341 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 2401 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 2461 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 2521 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 2581 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 2641 ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgcgagcc aggaagagtt 2701 tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca 2761 gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc 2821 gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga 2881 aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct 2941 cgcgttaacg cttgcatgga tgttttccca gtcacgacgt tgtaaaacga cggccagtct 3001 taagctcggg ccccaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca 3061 aattgatgag caatgctttt ttataatgcc aactttgtac aaaaaagcag gctccgaatt 3121 cgcccttcac catggattac aaggatgatg atgataagga ttacaaggat gatgatgata 3181 agatggctcc aaagaagaag agaaaggttg gaatccacgg agttccagct gctgataaga 3241 agtactctat cggacttgcc atcggaacca actctgttgg atgggctgtt atcaccgatg 3301 agtacaaggt tccatctaag aagttcaagg ttcttggaaa caccgataga cactctatca 3361 agaagaacct tatcggtgct cttcttttcg attctggaga gaccgctgag gctaccagat 3421 tgaagagaac cgctagaaga agatacacca gaagaaagaa cagaatctgc taccttcagg 3481 aaatcttctc taacgagatg gctaaggttg atgattcttt cttccacaga cttgaggagt 3541 ctttccttgt tgaggaggat aagaagcacg agagacaccc aatcttcgga aacatcgttg 3601 atgaggttgc ttaccacgag aagtacccaa ccatctacca ccttagaaag aagttggttg 3661 attctaccga taaggctgat cttagactta tctaccttgc tcttgctcac atgatcaagt 3721 tcagaggaca cttccttatc gagggagacc ttaacccaga taactctgat gttgataagt 3781 tgttcatcca gcttgttcag acctacaacc agcttttcga ggagaaccca atcaacgctt 3841 ctggagttga tgctaaggct atcctttctg ctagactttc taagtctcgt agacttgaga 3901 accttatcgc tcagcttcca ggagagaaga agaacggact tttcggaaac cttatcgctc 3961 tttctcttgg acttacccca aacttcaagt ctaacttcga tcttgctgag gatgctaagt 4021 tgcagctttc taaggatacc tacgatgatg atcttgataa ccttcttgct cagatcggag 4081 atcagtacgc tgatcttttc cttgctgcta agaacctttc tgatgctatc cttctttctg 4141 acatccttag agttaacacc gagatcacca aggctccact ttctgcttct atgatcaaga 4201 gatacgatga gcaccaccag gatcttaccc ttttgaaggc tcttgttaga cagcagcttc 4261 cagagaagta caaggaaatc ttcttcgatc agtctaagaa cggatacgct ggatacatcg 4321 atggaggagc ttctcaggag gagttctaca agttcatcaa gccaatcctt gagaagatgg 4381 atggaaccga ggagcttctt gttaagttga acagagagga tcttcttaga aagcagagaa 4441 ccttcgataa cggatctatc ccacaccaga tccaccttgg agagcttcac gctatccttc 4501 gtagacagga ggatttctac ccattcttga aggataacag agagaagatc gagaagatcc 4561 ttaccttcag aatcccatac tacgttggac cacttgctag aggaaactct cgtttcgctt 4621 ggatgaccag aaagtctgag gagaccatca ccccttggaa cttcgaggag gtaagtttct 4681 gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca 4741 aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa 4801 gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcagg 4861 ttgttgataa gggagcttct gctcagtctt tcatcgagag aatgaccaac ttcgataaga 4921 accttccaaa cgagaaggtt cttccaaagc actctcttct ttacgagtac ttcaccgttt 4981 acaacgagct taccaaggtt aagtacgtta ccgagggaat gagaaagcca gctttccttt 5041 ctggagagca gaagaaggct atcgttgatc ttcttttcaa gaccaacaga aaggttaccg 5101 ttaagcagtt gaaggaggat tacttcaaga agatcgagtg cttcgattct gttgaaatct 5161 ctggagttga ggatagattc aacgcttctc ttggaaccta ccacgatctt ttgaagatca 5221 tcaaggataa ggatttcctt gataacgagg agaacgagga catccttgag gacatcgttc 5281 ttacccttac ccttttcgag gatagagaga tgatcgagga gagactcaag acctacgctc 5341 accttttcga tgataaggtt atgaagcagt tgaagagaag aagatacacc ggatggggta 5401 gactttctcg taagttgatc aacggaatca gagataagca gtctggaaag accatccttg 5461 atttcttgaa gtctgatgga ttcgctaaca gaaacttcat gcagcttatc cacgatgatt 5521 ctcttacctt caaggaggac atccagaagg ctcaggtttc tggacaggga gattctcttc 5581 acgagcacat cgctaacctt gctggatctc cagctatcaa gaagggaatc cttcagaccg 5641 ttaaggttgt tgatgagctt gttaaggtta tgggtagaca caagccagag aacatcgtta 5701 tcgagatggc tagagagaac cagaccaccc agaagggaca gaagaactct cgtgagagaa 5761 tgaagagaat cgaggaggga atcaaggagc ttggatctca aatcttgaag gagcacccag 5821 ttgagaacac ccagcttcag aacgagaagt tgtaccttta ctaccttcag aacggaagag 5881 atatgtacgt tgatcaggag cttgacatca acagactttc tgattacgat gttgatgcca 5941 tcgttccaca gtctttcttg aaggatgatt ctatcgataa caaggttctt acccgttctg 6001 ataagaacag aggaaagtct gataacgttc catctgagga ggttgttaag aagatgaaga 6061 actactggag acagcttctt aacgctaagt tgatcaccca gagaaagttc gataacctta 6121 ccaaggctga gagaggagga ctttctgagc ttgataaggc tggattcatc aagagacagc 6181 ttgttgagac cagacagatc accaagcacg ttgctcagat ccttgattct cgtatgaaca 6241 ccaagtacga tgagaacgat aagttgatca gagaggttaa ggttatcacc ttgaagtcta 6301 agttggtttc tgatttcaga aaggatttcc agttctacaa ggttagagag atcaacaact 6361 accaccacgc tcacgatgct taccttaacg ctgttgttgg aaccgctctt atcaagaagt 6421 acccaaagtt ggagtctgag ttcgtttacg gagattacaa ggtttacgat gttagaaaga 6481 tgatcgctaa gtctgagcag gagatcggaa aggctaccgc taagtacttc ttctactcta 6541 acatcatgaa cttcttcaag accgagatca cccttgctaa cggagagatc agaaagagac 6601 cacttatcga gaccaacgga gagaccggag agatcgtttg ggataaggga agagatttcg 6661 ctaccgttag aaaggttctt tctatgccac aggttaacat cgttaagaaa accgaggttc 6721 agaccggagg attctctaag gagtctatcc ttccaaagag aaactctgat aagttgatcg 6781 ctagaaagaa ggattgggac ccaaagaagt acggaggatt cgattctcca accgttgctt 6841 actctgttct tgttgttgct aaggttgaga agggaaagtc taagaagttg aagtctgtta 6901 aggagcttct tggaatcacc atcatggagc gttcttcttt cgagaagaac ccaatcgatt 6961 tccttgaggc taagggatac aaggaggtta agaaggatct tatcatcaag ttgccaaagt 7021 actctctttt cgagcttgag aacggaagaa agagaatgct tgcttctgct ggagagcttc 7081 agaagggaaa cgagcttgct cttccatcta agtacgttaa cttcctttac cttgcttctc 7141 actacgagaa gttgaaggga tctccagagg ataacgagca gaagcagctt ttcgttgagc 7201 agcacaagca ctaccttgat gagatcatcg agcaaatctc tgagttctct aagagagtta 7261 tccttgctga tgctaacctt gataaggttc tttctgctta caacaagcac agagataagc 7321 caatcagaga gcaggctgag aacatcatcc accttttcac ccttaccaac cttggtgctc 7381 cagctgcttt caagtacttc gataccacca tcgatagaaa aagatacacc tctaccaagg 7441 aggttcttga tgctaccctt atccaccagt ctatcaccgg actttacgag accagaatcg 7501 atctttctca gcttggagga gataagagac cagctgctac caagaaggct ggacaggcta 7561 agaagaagaa gggagacggc tctggatcgg ggtcgggttc tggctcagtc gaccttgatc 7621 ttgacctcga actcagactt ggatttgctc tcgatctcga ccttgaactt agactcggat 7681 ttgctcttga cctcgatctt gagcttagac tcggattcgc ttaggacgt //