LOCUS Exported 5858 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Level0 Golden Gate part: CDS, SaCas9. ACCESSION . VERSION . KEYWORDS pEPOR0SP0020 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5858) 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 5858) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5858 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 278..300 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 292..309 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 293..309 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 320..339 /label=T7 /note="T7 promoter, forward primer" promoter 320..338 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 384..404 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 429..3584 /codon_start=1 /product="Cas9 endonuclease from the Staphylococcus aureus Type II CRISPR/Cas system" /label=SaCas9 /note="generates RNA-guided double strand breaks in DNA" /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI IKKG" CDS 3585..3632 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 3639..3719 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA" primer_bind complement(3848..3865) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3958..4546) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4038..4057) /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter complement(5731..5835) /gene="bla" /label=AmpR promoter ORIGIN 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt 181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgagtg aaggccgtca 361 aggccgcatg gtctcaaatg gccccaaaga agaagcggaa ggtcggtatc cacggagtcc 421 cagcagccaa gcggaactac atcctgggcc tggacatcgg catcaccagc gtgggctacg 481 gcatcatcga ctacgagaca cgggacgtga tcgatgccgg cgtgcggctg ttcaaagagg 541 ccaacgtgga aaacaacgag ggcaggcgga gcaagagagg cgccagaagg ctgaagcggc 601 ggaggcggca tagaatccag agagtgaaga agctgctgtt cgactacaac ctgctgaccg 661 accacagcga gctgagcggc atcaacccct acgaggccag agtgaagggc ctgagccaga 721 agctgagcga ggaagagttc tctgccgccc tgctgcacct ggccaagaga agaggcgtgc 781 acaacgtgaa cgaggtggaa gaggacaccg gcaacgagct gtccaccaaa gagcagatca 841 gccggaacag caaggccctg gaagagaaat acgtggccga actgcagctg gaacggctga 901 agaaagacgg cgaagtgcgg ggcagcatca acagattcaa gaccagcgac tacgtgaaag 961 aagccaaaca gctgctgaag gtgcagaagg cctaccacca gctggaccag agcttcatcg 1021 acacctacat cgacctgctg gaaacccggc ggacctacta tgagggacct ggcgagggca 1081 gccccttcgg ctggaaggac atcaaagaat ggtacgagat gctgatgggc cactgcacct 1141 acttccccga ggaactgcgg agcgtgaagt acgcctacaa cgccgacctg tacaacgccc 1201 tgaacgacct gaacaatctc gtgatcacca gggacgagaa cgagaagctg gaatattacg 1261 agaagttcca gatcatcgag aacgtgttca agcagaagaa gaagcccacc ctgaagcaga 1321 tcgccaaaga aatcctcgtg aacgaagagg atattaaggg ctacagagtg accagcaccg 1381 gcaagcccga gttcaccaac ctgaaggtgt accacgacat caaggacatt accgcccgga 1441 aagagattat tgagaacgcc gagctgctgg atcagattgc caagatcctg accatctacc 1501 agagcagcga ggacatccag gaagaactga ccaatctgaa ctccgagctg acccaggaag 1561 agatcgagca gatctctaat ctgaagggct ataccggcac ccacaacctg agcctgaagg 1621 ccatcaacct gatcctggac gagctgtggc acaccaacga caaccagatc gctatcttca 1681 accggctgaa gctggtgccc aagaaggtgg acctgtccca gcagaaagag atccccacca 1741 ccctggtgga cgacttcatc ctgagccccg tcgtgaagag aagcttcatc cagagcatca 1801 aagtgatcaa cgccatcatc aagaagtacg gcctgcccaa cgacatcatt atcgagctgg 1861 cccgcgagaa gaactccaag gacgcccaga aaatgatcaa cgagatgcag aagcggaacc 1921 ggcagaccaa cgagcggatc gaggaaatca tccggaccac cggcaaagag aacgccaagt 1981 acctgatcga gaagatcaag ctgcacgaca tgcaggaagg caagtgcctg tacagcctgg 2041 aagccatccc tctggaagat ctgctgaaca accccttcaa ctatgaggtg gaccacatca 2101 tccccagaag cgtgtccttc gacaacagct tcaacaacaa ggtgctcgtg aagcaggaag 2161 aaaacagcaa gaagggcaac cggaccccat tccagtacct gagcagcagc gacagcaaga 2221 tcagctacga aaccttcaag aagcacatcc tgaatctggc caagggcaag ggcagaatca 2281 gcaagaccaa gaaagagtat ctgctggaag aacgggacat caacaggttc tccgtgcaga 2341 aagacttcat caaccggaac ctggtggata ccagatacgc caccagaggc ctgatgaacc 2401 tgctgcggag ctacttcaga gtgaacaacc tggacgtgaa agtgaagtcc atcaatggcg 2461 gcttcaccag ctttctgcgg cggaagtgga agtttaagaa agagcggaac aaggggtaca 2521 agcaccacgc cgaggacgcc ctgatcattg ccaacgccga tttcatcttc aaagagtgga 2581 agaaactgga caaggccaaa aaagtgatgg aaaaccagat gttcgaggaa aagcaggccg 2641 agagcatgcc cgagatcgaa accgagcagg agtacaaaga gatcttcatc accccccacc 2701 agatcaagca cattaaggac ttcaaggact acaagtacag ccaccgggtg gacaagaagc 2761 ctaatagaga gctgattaac gacaccctgt actccacccg gaaggacgac aagggcaaca 2821 ccctgatcgt gaacaatctg aacggcctgt acgacaagga caatgacaag ctgaaaaagc 2881 tgatcaacaa gagccccgaa aagctgctga tgtaccacca cgacccccag acctaccaga 2941 aactgaagct gattatggaa cagtacggcg acgagaagaa tcccctgtac aagtactacg 3001 aggaaaccgg gaactacctg accaagtact ccaaaaagga caacggcccc gtgatcaaga 3061 agattaagta ttacggcaac aaactgaacg cccatctgga catcaccgac gactacccca 3121 acagcagaaa caaggtcgtg aagctgtccc tgaagcccta cagattcgac gtgtacctgg 3181 acaatggcgt gtacaagttc gtgaccgtga agaatctgga tgtgatcaaa aaagaaaact 3241 actacgaagt gaatagcaag tgctatgagg aagctaagaa gctgaagaag atcagcaacc 3301 aggccgagtt tatcgcctcc ttctacaaca acgatctgat caagatcaac ggcgagctgt 3361 atagagtgat cggcgtgaac aacgacctgc tgaaccggat cgaagtgaac atgatcgaca 3421 tcacctaccg cgagtacctg gaaaacatga acgacaagag gccccccagg atcattaaga 3481 caatcgcctc caagacccag agcattaaga agtacagcac agacattctg ggcaacctgt 3541 atgaagtgaa atctaagaag caccctcaga tcatcaaaaa gggcaaaagg ccggcggcca 3601 cgaaaaaggc cggccaggca aaaaagaaaa agggatccta cccatacgat gttccagatt 3661 acgcttaccc atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt 3721 aagctttgag accctgggcc tcatgggcct tcacttcact gcccgctttc cagtcgggaa 3781 acctgtcgtg ccagctgcat taacatggtc atagctgttt ccttgcgtat tgggcgctct 3841 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg ggtaaagcct ggggtgccta 3901 atgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 3961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4381 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 4561 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4621 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 4681 aatctaaagt atatatgagt aaacttggtc tgacagttat tatttaccca ccactttggt 4741 aatttcgcct ttcacgtaat gcacaaattc ttccagctga tccgcacggc tggccagacg 4801 atcttcttcc tggcccagat acgcctgacg cgcttccaga atcaccggct gatactgcgc 4861 cggcagacgt tccatggccc aatccgccgc cacatctttc ggcgcaattt tgccggtcac 4921 cgcgctatac caaatacggc tcagggtcag caccacgttg cgttcatcac ccgcccaatc 4981 cggcgggctg ttccacaggg tcagggtttc gttcagcgct tcaaacaggt cctgttccgg 5041 caccggatca aacagttctt ccgctgccgg accaaccagc gccacgctat gttcacgcgc 5101 tttggtcagc agaatggcca gatcgatatc aatggtcgcc ggttcaaaaa tgccggccag 5161 aatatcgtta cgctgccatt cgccaaactg cagctcacgt ttcgccggat aacgccacgg 5221 aataatgtca tcatgcacca cgatggtcac ttccaccgca cgcagaattt cgctttcacc 5281 cgggctcgcg ctggtttcca gcagatcgtt aatcagcgca cgacgggtgg tttcatccag 5341 acgcacggtc acggtcacca gcagatcaat atcgctatgc ggtttcaggc cgccatccac 5401 cgcgctgcca tacagatgca cggccagcag ggtcggttcc agatgacgtt caatcacgcc 5461 caccacttca gacagctggg tgctcacttc cgcaatcacc gcttcacgca taatgttcag 5521 tttggttttc ggacggctgc cctgctgggt cacatcgttg ctgctccaca gcatcaggca 5581 acggcccacc gcataacgcg cctgctgttt gctcgcacgc ggcatgctct gcacgccaaa 5641 aaagcaatca tagcacgcca taaacaccgc caccgcacca ttaccgccgc tacgttcggt 5701 cagggtacgg ctccaattac ggctacgcat actcttcctt tttcaatatt attgaagcat 5761 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca 5821 aataggggtt ccgcgcacat ttccccgaaa agtgccac //