LOCUS Exported 5594 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level0 Golden Gate part: CDS, eSaCas9 (no stop codon). ACCESSION . VERSION . KEYWORDS pEPOR0SP0012 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5594) AUTHORS Raitskin O, Schudoma C, West A, Patron NJ TITLE Comparison of efficiency and specificity of CRISPR-associated (Cas) nucleases in plants: An expanded toolkit for precision genome engineering. JOURNAL PLoS One. 2019 Feb 27;14(2):e0211598. doi: 10.1371/journal.pone.0211598. eCollection 2019. PUBMED 30811422 REFERENCE 2 (bases 1 to 5594) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5594 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(24..42) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 110..214 /gene="bla" /label=AmpR promoter CDS 436..1227 /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 1398..1986 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1887..1906 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2140..2157 /label=L4440 /note="L4440 vector, forward primer" CDS 2241..2261 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5442..5489 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5496..5576 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" ORIGIN 1 gtggctcttc agtggacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 61 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 121 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 181 ataaatgctt caataatatt gaaaaaggaa gagtatgcgc tcacgcaact ggtccagaac 241 cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg 301 tttttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg 361 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa 421 acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag 481 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc 541 tccgcagtgg atggcggcct gaagccacac agcgatattg atttgctggt tacggtgacc 601 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct 661 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac 721 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat 781 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg 841 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat 901 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg 961 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac 1021 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc 1081 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa 1141 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccatta cgtaaaaggc 1201 gagatcacca aggtagtcgg caaataactg tcagaccaag tttactcata tatactttag 1261 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 1321 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 1381 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 1441 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 1501 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 1561 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 1621 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 1681 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 1741 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 1801 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 1861 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 1921 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 1981 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 2041 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 2101 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 2161 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatca 2221 ctctgtggtc tcaaatggcc ccaaagaaga agcggaaggt cggtatccac ggagtcccag 2281 cagccaagcg gaactacatc ctgggcctgg acatcggcat caccagcgtg ggctacggca 2341 tcatcgacta cgagacacgg gacgtgatcg atgccggcgt gcggctgttc aaagaggcca 2401 acgtggaaaa caacgagggc aggcggagca agagaggcgc cagaaggctg aagcggcgga 2461 ggcggcatag aatccagaga gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc 2521 acagcgagct gagcggcatc aacccctacg aggccagagt gaagggcctg agccagaagc 2581 tgagcgagga agagttctct gccgccctgc tgcacctggc caagagaaga ggcgtgcaca 2641 acgtgaacga ggtggaagag gacaccggca acgagctgtc caccaaagag cagatcagcc 2701 ggaacagcaa ggccctggaa gagaaatacg tggccgaact gcagctggaa cggctgaaga 2761 aagacggcga agtgcggggc agcatcaaca gattcaagac cagcgactac gtgaaagaag 2821 ccaaacagct gctgaaggtg cagaaggcct accaccagct ggaccagagc ttcatcgaca 2881 cctacatcga cctgctggaa acccggcgga cctactatga gggacctggc gagggcagcc 2941 ccttcggctg gaaggacatc aaagaatggt acgagatgct gatgggccac tgcacctact 3001 tccccgagga actgcggagc gtgaagtacg cctacaacgc cgacctgtac aacgccctga 3061 acgacctgaa caatctcgtg atcaccaggg acgagaacga gaagctggaa tattacgaga 3121 agttccagat catcgagaac gtgttcaagc agaagaagaa gcccaccctg aagcagatcg 3181 ccaaagaaat cctcgtgaac gaagaggata ttaagggcta cagagtgacc agcaccggca 3241 agcccgagtt caccaacctg aaggtgtacc acgacatcaa ggacattacc gcccggaaag 3301 agattattga gaacgccgag ctgctggatc agattgccaa gatcctgacc atctaccaga 3361 gcagcgagga catccaggaa gaactgacca atctgaactc cgagctgacc caggaagaga 3421 tcgagcagat ctctaatctg aagggctata ccggcaccca caacctgagc ctgaaggcca 3481 tcaacctgat cctggacgag ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc 3541 ggctgaagct ggtgcccaag aaggtggacc tgtcccagca gaaagagatc cccaccaccc 3601 tggtggacga cttcatcctg agccccgtcg tgaagagaag cttcatccag agcatcaaag 3661 tgatcaacgc catcatcaag aagtacggcc tgcccaacga catcattatc gagctggccc 3721 gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga gatgcagaag cggaacgcga 3781 aaaccaacga gcggatcgag gaaatcatcc ggaccaccgg caaagagaac gccaagtacc 3841 tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa gtgcctgtac agcctggaag 3901 ccatccctct ggaagatctg ctgaacaacc ccttcaacta tgaggtggac cacatcatcc 3961 ccagaagcgt gtccttcgac aacagcttca acaacaaggt gctcgtgaag caggaagaaa 4021 acagcaagaa gggcaaccgg accccattcc agtacctgag cagcagcgac agcaagatca 4081 gctacgaaac cttcaagaag cacatcctga atctggccaa gggcaagggc agaatcagca 4141 agaccaagaa agagtatctg ctggaagaac gggacatcaa caggttctcc gtgcagaaag 4201 acttcatcaa ccggaacctg gtggatacca gatacgccac cgcacgtctg atgaacctgc 4261 tgcggagcta cttcagagtg aacaacctgg acgtgaaagt gaagtccatc aatggcggct 4321 tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga gcggaacaag gggtacaagc 4381 accacgccga ggacgccctg atcattgcca acgccgattt catcttcaaa gagtggaaga 4441 aactggacaa ggccaaaaaa gtgatggaaa accagatgtt cgaggaaaag caggccgaga 4501 gcatgcccga gatcgaaacc gagcaggagt acaaagagat cttcatcacc ccccaccaga 4561 tcaagcacat taaggacttc aaggactaca agtacagcca ccgggtggac aagaagccta 4621 atagagagct gattaacgac accctgtact ccacccggaa ggacgacaag ggcaacaccc 4681 tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa tgacaagctg aaaaagctga 4741 tcaacaagag ccccgaaaag ctgctgatgt accaccacga cccccagacc taccagaaac 4801 tgaagctgat tatggaacag tacggcgacg agaagaatcc cctgtacaag tactacgagg 4861 aaaccgggaa ctacctgacc aagtactcca aaaaggacaa cggccccgtg atcaagaaga 4921 ttaagtatta cggcaacaaa ctgaacgccc atctggacat caccgacgac taccccaaca 4981 gcagaaacaa ggtcgtgaag ctgtccctga agccctacag attcgacgtg tacctggaca 5041 atggcgtgta caagttcgtg accgtgaaga atctggatgt gatcaaaaaa gaaaactact 5101 acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct gaagaagatc agcaaccagg 5161 ccgagtttat cgcctccttc tacaacaacg atctgatcaa gatcaacggc gagctgtata 5221 gagtgatcgg cgtgaacaac gacctgctga accggatcga agtgaacatg atcgacatca 5281 cctaccgcga gtacctggaa aacatgaacg acaagaggcc ccccaggatc attaagacaa 5341 tcgcctccaa gacccagagc attaagaagt acagcacaga cattctgggc aacctgtatg 5401 aagtgaaatc taagaagcac cctcagatca tcaaaaaggg caaaaggccg gcggccacga 5461 aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc atacgatgtt ccagattacg 5521 cttacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgctggtt 5581 cgtgagacca cgaa //