LOCUS Exported 6389 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Level0 Golden Gate part: CDS, SpCas9-p (no stop codon). ACCESSION . VERSION . KEYWORDS pEPOR0SP0009 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6389) 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 6389) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6389 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(36..54) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 122..226 /gene="bla" /label=AmpR promoter CDS 448..1239 /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 1410..1998 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1899..1918 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2152..2169 /label=L4440 /note="L4440 vector, forward primer" CDS 2247..6350 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 6363..6383 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" ORIGIN 1 tgagaccacg aagtggctct tcagtggacg aaagggcctc gtgatacgcc tatttttata 61 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt 121 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 181 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatgc gctcacgcaa 241 ctggtccaga accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc 301 ttgttatgac tgtttttttt tggggtacag tctatgcctc gggcatccaa gcagcaagcg 361 cgttacgccg tgggtcgatg tttgatgtta tggagcagca acgatgttac gcagcagggc 421 agtcgcccta aaacaaagtt aaacatcatg agggaagcgg tgatcgccga agtatcgact 481 caactatcag aggtagttgg cgtcatcgag cgccatctcg aaccgacgtt gctggccgta 541 catttgtacg gctccgcagt ggatggcggc ctgaagccac acagcgatat tgatttgctg 601 gttacggtga ccgtaaggct tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg 661 gaaacttcgg cttcccctgg agagagcgag attctccgcg ctgtagaagt caccattgtt 721 gtgcacgacg acatcattcc gtggcgttat ccagctaagc gcgaactgca atttggagaa 781 tggcagcgca atgacattct tgcaggtatc ttcgagccag ccacgatcga cattgatctg 841 gctatcttgc tgacaaaagc aagagaacat agcgttgcct tggtaggtcc agcggcggag 901 gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaaatga aaccttaacg 961 ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc 1021 cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg 1081 gcaatggagc gcctgccggc ccagtatcag cccgtcatac ttgaagctag acaggcttat 1141 cttggacaag aagaagatcg cttggcctcg cgcgcagatc agttggaaga atttgtccat 1201 tacgtaaaag gcgagatcac caaggtagtc ggcaaataac tgtcagacca agtttactca 1261 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 1321 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 1381 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 1441 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 1501 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 1561 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 1621 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 1681 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 1741 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 1801 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 1861 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 1921 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 1981 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 2041 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 2101 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 2161 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 2221 attcattaat cactctgtgg tctcaaatgg ataagaagta ctctatcgga ctcgatatcg 2281 gaactaactc tgtgggatgg gctgtgatca ccgatgagta caaggtgcca tctaagaagt 2341 tcaaggttct cggaaacacc gataggcact ctatcaagaa aaaccttatc ggtgctctcc 2401 tcttcgattc tggtgaaact gctgaggcta ccagactcaa gagaaccgct agaagaaggt 2461 acaccagaag aaagaacagg atctgctacc tccaagagat cttctctaac gagatggcta 2521 aagtggatga ttcattcttc cacaggctcg aagagtcatt cctcgtggaa gaagataaga 2581 agcacgagag gcaccctatc ttcggaaaca tcgttgatga ggtggcatac cacgagaagt 2641 accctactat ctaccacctc agaaagaagc tcgttgattc tactgataag gctgatctca 2701 ggctcatcta cctcgctctc gctcacatga tcaagttcag aggacacttc ctcatcgagg 2761 gtgatctcaa ccctgataac tctgatgtgg ataagttgtt catccagctc gtgcagacct 2821 acaaccagct tttcgaagag aaccctatca acgcttcagg tgtggatgct aaggctatcc 2881 tctctgctag gctctctaag tcaagaaggc ttgagaacct cattgctcag ctccctggtg 2941 agaagaagaa cggacttttc ggaaacttga tcgctctctc tctcggactc acccctaact 3001 tcaagtctaa cttcgatctc gctgaggatg caaagctcca gctctcaaag gatacctacg 3061 atgatgatct cgataacctc ctcgctcaga tcggagatca gtacgctgat ttgttcctcg 3121 ctgctaagaa cctctctgat gctatcctcc tcagtgatat cctcagagtg aacaccgaga 3181 tcaccaaggc tccactctca gcttctatga tcaagagata cgatgagcac caccaggatc 3241 tcacacttct caaggctctt gttagacagc agctcccaga gaagtacaaa gagattttct 3301 tcgatcagtc taagaacgga tacgctggtt acatcgatgg tggtgcatct caagaagagt 3361 tctacaagtt catcaagcct atcctcgaga agatggatgg aaccgaggaa ctcctcgtga 3421 agctcaatag agaggatctt ctcagaaagc agaggacctt cgataacgga tctatccctc 3481 atcagatcca cctcggagag ttgcacgcta tccttagaag gcaagaggat ttctacccat 3541 tcctcaagga taacagggaa aagattgaga agattctcac cttcagaatc ccttactacg 3601 tgggacctct cgctagagga aactcaagat tcgcttggat gaccagaaag tctgaggaaa 3661 ccatcacccc ttggaacttc gaagaggtgg tggataaggg tgctagtgct cagtctttca 3721 tcgagaggat gaccaacttc gataagaacc ttccaaacga gaaggtgctc cctaagcact 3781 ctttgctcta cgagtacttc accgtgtaca acgagttgac caaggttaag tacgtgaccg 3841 agggaatgag gaagcctgct tttttgtcag gtgagcaaaa gaaggctatc gttgatctct 3901 tgttcaagac caacagaaag gtgaccgtga agcagctcaa agaggattac ttcaagaaaa 3961 tcgagtgctt cgattcagtt gagatttctg gtgttgagga taggttcaac gcatctctcg 4021 gaacctacca cgatctcctc aagatcatta aggataagga tttcttggat aacgaggaaa 4081 acgaggatat cttggaggat atcgttctta ccctcaccct ctttgaagat agagagatga 4141 ttgaagaaag gctcaagacc tacgctcatc tcttcgatga taaggtgatg aagcagttga 4201 agagaagaag atacactggt tggggaaggc tctcaagaaa gctcattaac ggaatcaggg 4261 ataagcagtc tggaaagaca atccttgatt tcctcaagtc tgatggattc gctaacagaa 4321 acttcatgca gctcatccac gatgattctc tcacctttaa agaggatatc cagaaggctc 4381 aggtttcagg acagggtgat agtctccatg agcatatcgc taacctcgct ggatctcctg 4441 caatcaagaa gggaatcctc cagactgtga aggttgtgga tgagttggtg aaggtgatgg 4501 gaaggcataa gcctgagaac atcgtgatcg aaatggctag agagaaccag accactcaga 4561 agggacagaa gaactctagg gaaaggatga agaggatcga ggaaggtatc aaagagcttg 4621 gatctcagat cctcaaagag caccctgttg agaacactca gctccagaat gagaagctct 4681 acctctacta cctccagaac ggaagggata tgtatgtgga tcaagagttg gatatcaaca 4741 ggctctctga ttacgatgtt gatcatatcg tgccacagtc attcttgaag gatgattcta 4801 tcgataacaa ggtgctcacc aggtctgata agaacagggg taagagtgat aacgtgccaa 4861 gtgaagaggt tgtgaagaaa atgaagaact attggaggca gctcctcaac gctaagctca 4921 tcactcagag aaagttcgat aacttgacta aggctgagag gggaggactc tctgaattgg 4981 ataaggcagg attcatcaag aggcagcttg tggaaaccag gcagatcact aagcacgttg 5041 cacagatcct cgattctagg atgaacacca agtacgatga gaacgataag ttgatcaggg 5101 aagtgaaggt tatcaccctc aagtcaaagc tcgtgtctga tttcagaaag gatttccaat 5161 tctacaaggt gagggaaatc aacaactacc accacgctca cgatgcttac cttaacgctg 5221 ttgttggaac cgctctcatc aagaagtatc ctaagctcga gtcagagttc gtgtacggtg 5281 attacaaggt gtacgatgtg aggaagatga tcgctaagtc tgagcaagag atcggaaagg 5341 ctaccgctaa gtatttcttc tactctaaca tcatgaattt cttcaagacc gagattaccc 5401 tcgctaacgg tgagatcaga aagaggccac tcatcgagac aaacggtgaa acaggtgaga 5461 tcgtgtggga taagggaagg gatttcgcta ccgttagaaa ggtgctctct atgccacagg 5521 tgaacatcgt taagaaaacc gaggtgcaga ccggtggatt ctctaaagag tctatcctcc 5581 ctaagaggaa ctctgataag ctcattgcta ggaagaagga ttgggaccct aagaaatacg 5641 gtggtttcga ttctcctacc gtggcttact ctgttctcgt tgtggctaag gttgagaagg 5701 gaaagagtaa gaagctcaag tctgttaagg aacttctcgg aatcactatc atggaaaggt 5761 catctttcga gaagaaccca atcgatttcc tcgaggctaa gggatacaaa gaggttaaga 5821 aggatctcat catcaagctc ccaaagtact cactcttcga actcgagaac ggtagaaaga 5881 ggatgctcgc ttctgctggt gagcttcaaa agggaaacga gcttgctctc ccatctaagt 5941 acgttaactt tctttacctc gcttctcact acgagaagtt gaagggatct ccagaagata 6001 acgagcagaa gcaacttttc gttgagcagc acaagcacta cttggatgag atcatcgagc 6061 agatctctga gttctctaaa agggtgatcc tcgctgatgc aaacctcgat aaggtgttgt 6121 ctgcttacaa caagcacaga gataagccta tcagggaaca ggcagagaac atcatccatc 6181 tcttcaccct taccaacctc ggtgctcctg ctgctttcaa gtacttcgat acaaccatcg 6241 ataggaagag atacacctct accaaagaag tgctcgatgc taccctcatc catcagtcta 6301 tcactggact ctacgagact aggatcgatc tctcacagct cggtggtgat tcaagggctg 6361 atcctaagaa gaagaggaag gttggttcg //