LOCUS Exported 7926 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Module A, Promoter: 35S, Gene: Csy4-P2A-AtCas9_dead (D10A + H840A), Terminator: HSP. ACCESSION . VERSION . KEYWORDS pMOD_A0801 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7926) AUTHORS Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF TITLE A multi-purpose toolkit to enable advanced genome engineering in plants. JOURNAL Plant Cell. 2017 May 18. pii: tpc.00922.2016. doi: 10.1105/tpc.16.00922. PUBMED 28522548 REFERENCE 2 (bases 1 to 7926) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7926 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 80..668 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 569..588 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 822..839 /label=L4440 /note="L4440 vector, forward primer" promoter 1366..1711 /label=CaMV 35S promoter /note="strong constitutive promoter from cauliflower mosaic virus" primer_bind 1654..1674 /label=35S promoter /note="CaMV 35S promoter, forward primer" CDS 2303..2359 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 2360..6463 /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 6476..6496 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" promoter 6847..6938 /gene="bla" /label=AmpR promoter ORIGIN 1 gccttgacgg caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 61 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 121 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ctaactcttt 181 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 241 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 301 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 361 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgctcacagc 421 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 481 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 541 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 601 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 661 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 721 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 781 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 841 aagcggaaga gcgcccacct gctcatgatc ggcgcgccag atttgccttt tcaatttcag 901 aaagaatgct aacccacaga tggttagaga ggcttacgca gcaggtatca tcaagacgat 961 ctacccgagc aataatctcc aggaaatcaa ataccttccc aagaaggtta aagatgcagt 1021 caaaagattc aggactaact gcatcaagaa cacagagaaa gatatatttc tcaagatcag 1081 aagtactatt ccagtatgga cgattcaagg cttgcttcac aaaccaaggc aagtaataga 1141 gattggagtc tctaaaaagg tagttcccac tgaatcaaag gccatggagt caaagattca 1201 aatagaggac ctaacagaac tcgccgtaaa gactggcgaa cagttcatac agagtctctt 1261 acgactcaat gacaagaaga aaatcttcgt caacatggtg gagcacgaca cacttgtcta 1321 ctccaaaaat atcaaagata cagtctcaga agaccaaagg gcaattgaga cttttcaaca 1381 aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc actttattgt 1441 gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata aaggaaaggc 1501 catcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac ccacgaggag 1561 catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt gatgtgatat 1621 ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc cttcctctat 1681 ataaggaagt tcatttcatt tggagagaac acgggggact cctgcaggaa aaatggatca 1741 ttatcttgat attagactta gacctgatcc agaatttcca ccagctcaac ttatgtctgt 1801 tctttttgga aaacttcatc aagctcttgt tgctcaagga ggagatagaa ttggagtttc 1861 ttttcctgat cttgatgaat caagatcaag acttggagaa agacttagaa ttcatgcttc 1921 tgctgatgat cttagagctt tgcttgctag accttggctt gaaggactta gagatcatct 1981 tcaatttgga gaaccagctg ttgttccaca tccaactcct tatagacaag tttcaagagt 2041 tcaagctaaa tctaatccag aaagacttag aagaagactt atgagaagac atgatctttc 2101 tgaagaagaa gctagaaaaa gaattcctga tactgttgct agagctttgg atttgccttt 2161 tgttacactt agatcacaat ctactggaca acattttaga ctttttatta gacatggacc 2221 acttcaagtt actgctgaag aaggaggatt tacttgttat ggactttcta agggaggttt 2281 tgttccttgg tttggatctg gagctactaa tttttctctt cttaagcaag ctggagatgt 2341 tgaagaaaat cctggaccca tggataagaa gtactctatc ggactcgcta tcggaactaa 2401 ctctgtggga tgggctgtga tcaccgatga gtacaaggtg ccatctaaga agttcaaggt 2461 tctcggaaac accgataggc actctatcaa gaaaaacctt atcggtgctc tcctcttcga 2521 ttctggtgaa actgctgagg ctaccagact caagagaacc gctagaagaa ggtacaccag 2581 aagaaagaac aggatctgct acctccaaga gatcttctct aacgagatgg ctaaagtgga 2641 tgattcattc ttccacaggc tcgaagagtc attcctcgtg gaagaagata agaagcacga 2701 gaggcaccct atcttcggaa acatcgttga tgaggtggca taccacgaga agtaccctac 2761 tatctaccac ctcagaaaga agctcgttga ttctactgat aaggctgatc tcaggctcat 2821 ctacctcgct ctcgctcaca tgatcaagtt cagaggacac ttcctcatcg agggtgatct 2881 caaccctgat aactctgatg tggataagtt gttcatccag ctcgtgcaga cctacaacca 2941 gcttttcgaa gagaacccta tcaacgcttc aggtgtggat gctaaggcta tcctctctgc 3001 taggctctct aagtcaagaa ggcttgagaa cctcattgct cagctccctg gtgagaagaa 3061 gaacggactt ttcggaaact tgatcgctct ctctctcgga ctcaccccta acttcaagtc 3121 taacttcgat ctcgctgagg atgcaaagct ccagctctca aaggatacct acgatgatga 3181 tctcgataac ctcctcgctc agatcggaga tcagtacgct gatttgttcc tcgctgctaa 3241 gaacctctct gatgctatcc tcctcagtga tatcctcaga gtgaacaccg agatcaccaa 3301 ggctccactc tcagcttcta tgatcaagag atacgatgag caccaccagg atctcacact 3361 tctcaaggct cttgttagac agcagctccc agagaagtac aaagagattt tcttcgatca 3421 gtctaagaac ggatacgctg gttacatcga tggtggtgca tctcaagaag agttctacaa 3481 gttcatcaag cctatcctcg agaagatgga tggaaccgag gaactcctcg tgaagctcaa 3541 tagagaggat cttctcagaa agcagaggac cttcgataac ggatctatcc ctcatcagat 3601 ccacctcgga gagttgcacg ctatccttag aaggcaagag gatttctacc cattcctcaa 3661 ggataacagg gaaaagattg agaagattct caccttcaga atcccttact acgtgggacc 3721 tctcgctaga ggaaactcaa gattcgcttg gatgaccaga aagtctgagg aaaccatcac 3781 cccttggaac ttcgaagagg tggtggataa gggtgctagt gctcagtctt tcatcgagag 3841 gatgaccaac ttcgataaga accttccaaa cgagaaggtg ctccctaagc actctttgct 3901 ctacgagtac ttcaccgtgt acaacgagtt gaccaaggtt aagtacgtga ccgagggaat 3961 gaggaagcct gcttttttgt caggtgagca aaagaaggct atcgttgatc tcttgttcaa 4021 gaccaacaga aaggtgaccg tgaagcagct caaagaggat tacttcaaga aaatcgagtg 4081 cttcgattca gttgagattt ctggtgttga ggataggttc aacgcatctc tcggaaccta 4141 ccacgatctc ctcaagatca ttaaggataa ggatttcttg gataacgagg aaaacgagga 4201 tatcttggag gatatcgttc ttaccctcac cctctttgaa gatagagaga tgattgaaga 4261 aaggctcaag acctacgctc atctcttcga tgataaggtg atgaagcagt tgaagagaag 4321 aagatacact ggttggggaa ggctctcaag aaagctcatt aacggaatca gggataagca 4381 gtctggaaag acaatccttg atttcctcaa gtctgatgga ttcgctaaca gaaacttcat 4441 gcagctcatc cacgatgatt ctctcacctt taaagaggat atccagaagg ctcaggtttc 4501 aggacagggt gatagtctcc atgagcatat cgctaacctc gctggatctc ctgcaatcaa 4561 gaagggaatc ctccagactg tgaaggttgt ggatgagttg gtgaaggtga tgggaaggca 4621 taagcctgag aacatcgtga tcgaaatggc tagagagaac cagaccactc agaagggaca 4681 gaagaactct agggaaagga tgaagaggat cgaggaaggt atcaaagagc ttggatctca 4741 gatcctcaaa gagcaccctg ttgagaacac tcagctccag aatgagaagc tctacctcta 4801 ctacctccag aacggaaggg atatgtatgt ggatcaagag ttggatatca acaggctctc 4861 tgattacgat gttgatgcta tcgtgccaca gtcattcttg aaggatgatt ctatcgataa 4921 caaggtgctc accaggtctg ataagaacag gggtaagagt gataacgtgc caagtgaaga 4981 ggttgtgaag aaaatgaaga actattggag gcagctcctc aacgctaagc tcatcactca 5041 gagaaagttc gataacttga ctaaggctga gaggggagga ctctctgaat tggataaggc 5101 aggattcatc aagaggcagc ttgtggaaac caggcagatc actaagcacg ttgcacagat 5161 cctcgattct aggatgaaca ccaagtacga tgagaacgat aagttgatca gggaagtgaa 5221 ggttatcacc ctcaagtcaa agctcgtgtc tgatttcaga aaggatttcc aattctacaa 5281 ggtgagggaa atcaacaact accaccacgc tcacgatgct taccttaacg ctgttgttgg 5341 aaccgctctc atcaagaagt atcctaagct cgagtcagag ttcgtgtacg gtgattacaa 5401 ggtgtacgat gtgaggaaga tgatcgctaa gtctgagcaa gagatcggaa aggctaccgc 5461 taagtatttc ttctactcta acatcatgaa tttcttcaag accgagatta ccctcgctaa 5521 cggtgagatc agaaagaggc cactcatcga gacaaacggt gaaacaggtg agatcgtgtg 5581 ggataaggga agggatttcg ctaccgttag aaaggtgctc tctatgccac aggtgaacat 5641 cgttaagaaa accgaggtgc agaccggtgg attctctaaa gagtctatcc tccctaagag 5701 gaactctgat aagctcattg ctaggaagaa ggattgggac cctaagaaat acggtggttt 5761 cgattctcct accgtggctt actctgttct cgttgtggct aaggttgaga agggaaagag 5821 taagaagctc aagtctgtta aggaacttct cggaatcact atcatggaaa ggtcatcttt 5881 cgagaagaac ccaatcgatt tcctcgaggc taagggatac aaagaggtta agaaggatct 5941 catcatcaag ctcccaaagt actcactctt cgaactcgag aacggtagaa agaggatgct 6001 cgcttctgct ggtgagcttc aaaagggaaa cgagcttgct ctcccatcta agtacgttaa 6061 ctttctttac ctcgcttctc actacgagaa gttgaaggga tctccagaag ataacgagca 6121 gaagcaactt ttcgttgagc agcacaagca ctacttggat gagatcatcg agcagatctc 6181 tgagttctct aaaagggtga tcctcgctga tgcaaacctc gataaggtgt tgtctgctta 6241 caacaagcac agagataagc ctatcaggga acaggcagag aacatcatcc atctcttcac 6301 ccttaccaac ctcggtgctc ctgctgcttt caagtacttc gatacaacca tcgataggaa 6361 gagatacacc tctaccaaag aagtgctcga tgctaccctc atccatcagt ctatcactgg 6421 actctacgag actaggatcg atctctcaca gctcggtggt gattcaaggg ctgatcctaa 6481 gaagaagagg aaggtttgac gtcgacgata tgaagatgaa gatgaaatat ttggtgtgtc 6541 aaataaaaag cttgtgtgct taagtttgtg tttttttctt ggcttgttgt gttatgaatt 6601 tgtggctttt tctaatatta aatgaatgta agatcacatt ataatgaata aacaaatgtt 6661 tctataatcc attgtgaatg ttttgttgga tctcttctgc agcatataac tactgtatgt 6721 gctatggtat ggactatgga atatgattaa agataagcca gagctctggt gacggacgtg 6781 ggcaggtgtg atgaaaagcc ttagggtcag gtggcacttt tcggggaaat gtgcgatgaa 6841 cccctatttg tttatttttc taaatacatt caaatatgta tccgctaatg agacaataac 6901 cctgataaat gcttcaatta tattgaaaaa ggaagagtat gagtatccaa catttccgtg 6961 tcgcccttat tccctttttt gcggccttct gccttcctgt ttttgctcac ccagaaacgc 7021 tggtgaaagt aaaagatgct gaagatcagt tgggtgcccg agtgggttac attgaactgg 7081 atctcaacag cggcaaaatc ttagagagtt ttcgccccga agaacgcttc ccaatgatga 7141 gcactttcaa agttctgcta tgtggcgcgg tgttatcccg tattgatgct gggcaagagc 7201 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag 7261 aaaagcatct tacggatggc atgacagtaa gagaattatg tagtgctgcc ataaccatga 7321 gtgataacac tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg 7381 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 7441 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaactc 7501 tccgcaaact attaactggc gaactactta ctcttgcttc acgccagcaa ctcattgact 7561 ggatggaggc ggacaaagtt gcaggaccac ttctgcgctc ggcacttccg gctggctggt 7621 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 7681 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 7741 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 7801 tgtcagacga agggaaataa atagtagccc gcctgatgtg cgggcttttt tttggtggtg 7861 gttaccagtg tcgcttagct accgtaggtc ctcttaggcg gtccgattag cgacatagtc 7921 atcaat //