LOCUS pMB62 9255 bp ds-DNA circular SYN 15-AUG-2013 DEFINITION . ACCESSION . VERSION . KEYWORDS pMB62 - Peft-3 Cas9 mEGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9255) AUTHORS Selma Waaijers, Vincent Portegijs, Jana Kerver, Bennie B. C. G. Lemmens, Marcel Tijsterman, Sander van den Heuvel, and Mike Boxem TITLE CRISPR/Cas9-targeted mutagenesis in C. elegans JOURNAL Genetics (2013) COMMENT FEATURES Location/Qualifiers primer_bind 50..66 /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 73..91 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" promoter 127..735 /note="Peft-3" CDS 750..815 /codon_start=1 /product="three tandem FLAG epitope tags, followed by an enterokinase cleavage site" /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 822..842 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" CDS join(867..1907,1959..2945,2997..4058,4110..5120) /codon_start=1 /note="Cas9" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS join(5127..5429,5483..5557,5609..5728,5780..5995) /note="mEGFP" CDS 6002..6076 /codon_start=1 /note="egl-13 NLS" /translation="MSRRRKANPTKLSENAKKLAKEVEN" 3'UTR 6086..6428 /note="tbb-2 UTR" promoter complement(6517..6535) /note="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6556..6572) /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 6580..6596 /bound_moiety="lac repressor encoded by lacI" /note="lac operator" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(6604..6634) /note="lac promoter" /note="promoter for the E. coli lac operon" rep_origin complement(6958..7546) /direction=LEFT /note="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(7717..8577) /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(8578..8682) /gene="bla" /note="AmpR promoter" rep_origin complement(8708..9163) /direction=LEFT /note="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" ORIGIN 1 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 61 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc ccccctcgag 121 gtcgacgcac ctttggtctt ttattgtcaa cttccattgg ttcttccatt gtttctgtta 181 aattaatgaa tttttcataa aataaagaca ttatacaata taaaaatgaa gaatttattg 241 aaaataaact gccagagaga aaaagtatgc aacactcccg ccgagagtgt ttgaaatggt 301 gtacggtaca ttttcgtgct aggagttaga tgtgcaggca gcaacgagag ggggagagat 361 ttttttgggc cttgtgaaat taacgtgagt tttctggtca tctgactaat catgttggtt 421 ttttgttggt ttattttgtt tttatctttg tttttatcca gattaggaaa tttaaatttt 481 atgaatttat aatgaggtca aacattcagt cccagcgttt ttcctgttct cactgtttag 541 tcgaattttt attttaggct ttcaacaaat gttctaactg tcttatttgt gacctcactt 601 tttatatttt tttaattttt aaaaatatta gaagtttcta ggataatttt ttcgactttt 661 attctctcta ccgtccgcac tcttcttact tttaaattaa attgtttttt tttcagttgg 721 gaaacacttt gctcatctag aaaaaaatgg actacaagga ccacgacgga gactacaagg 781 accacgacat cgactacaag gacgacgacg acaagatggc cccaaagaag aagcgtaagg 841 tcggaatcca cggagtccca gccgccgaca agaagtactc catcggactc gacatcggaa 901 ccaactccgt cggatgggcc gtcatcaccg acgagtacaa ggtcccatcc aagaagttca 961 aggtcctcgg aaacaccgac cgtcactcca tcaagaagaa cctcatcgga gccctcctct 1021 tcgactccgg agagaccgcc gaggccaccc gtctcaagcg taccgcccgt cgtcgttaca 1081 cccgtcgtaa gaaccgtatc tgctacctcc aagagatctt ctccaacgag atggccaagg 1141 tcgacgactc cttcttccac cgtctcgagg agtccttcct cgtcgaggag gacaagaagc 1201 acgagcgtca cccaatcttc ggaaacatcg tcgacgaggt cgcctaccac gagaagtacc 1261 caaccatcta ccacctccgt aagaagctcg tcgactccac cgacaaggcc gacctccgtc 1321 tcatctacct cgccctcgcc cacatgatca agttccgtgg acacttcctc atcgagggag 1381 acctcaaccc agacaactcc gacgtcgaca agctcttcat ccaactcgtc caaacctaca 1441 accaactctt cgaggagaac ccaatcaacg cctccggagt cgacgccaag gccatcctct 1501 ccgcccgtct ctccaagtcc cgtcgtctcg agaacctcat cgcccaactc ccaggagaga 1561 agaagaacgg actcttcgga aacctcatcg ccctctccct cggactcacc ccaaacttca 1621 agtccaactt cgacctcgcc gaggacgcca agctccaact ctccaaggac acctacgacg 1681 acgacctcga caacctcctc gcccaaatcg gagaccaata cgccgacctc ttcctcgccg 1741 ccaagaacct ctccgacgcc atcctcctct ccgacatcct ccgtgtcaac accgagatca 1801 ccaaggcccc actctccgcc tccatgatca agcgttacga cgagcaccac caagacctca 1861 ccctcctcaa ggccctcgtc cgtcaacaac tcccagagaa gtacaaggta agtttaaaca 1921 tatatatact aactaaccct gattatttaa attttcagga gatcttcttc gaccaatcca 1981 agaacggata cgccggatac atcgacggag gagcctccca agaggagttc tacaagttca 2041 tcaagccaat cctcgagaag atggacggaa ccgaggagct cctcgtcaag ctcaaccgtg 2101 aggacctcct ccgtaagcaa cgtaccttcg acaacggatc catcccacac caaatccacc 2161 tcggagagct ccacgccatc ctccgtcgtc aagaggactt ctacccattc ctcaaggaca 2221 accgtgagaa gatcgagaag atcctcacct tccgtatccc atactacgtc ggaccactcg 2281 cccgtggaaa ctcccgtttc gcctggatga cccgtaagtc cgaggagacc atcaccccat 2341 ggaacttcga ggaggtcgtc gacaagggag cctccgccca atccttcatc gagcgtatga 2401 ccaacttcga caagaacctc ccaaacgaga aggtcctccc aaagcactcc ctcctctacg 2461 agtacttcac cgtctacaac gagctcacca aggtcaagta cgtcaccgag ggaatgcgta 2521 agccagcctt cctctccgga gagcaaaaga aggccatcgt cgacctcctc ttcaagacca 2581 accgtaaggt caccgtcaag caactcaagg aggactactt caagaagatc gagtgcttcg 2641 actccgtcga gatctccgga gtcgaggacc gtttcaacgc ctccctcgga acctaccacg 2701 acctcctcaa gatcatcaag gacaaggact tcctcgacaa cgaggagaac gaggacatcc 2761 tcgaggacat cgtcctcacc ctcaccctct tcgaggaccg tgagatgatc gaggagcgtc 2821 tcaagaccta cgcccacctc ttcgacgaca aggtcatgaa gcaactcaag cgtcgtcgtt 2881 acaccggatg gggacgtctc tcccgtaagc tcatcaacgg aatccgtgac aagcaatccg 2941 gaaaggtaag tttaaacagt tcggtactaa ctaaccatac atatttaaat tttcagacca 3001 tcctcgactt cctcaagtcc gacggattcg ccaaccgtaa cttcatgcaa ctcatccacg 3061 acgactccct caccttcaag gaggacatcc aaaaggccca agtctccgga caaggagact 3121 ccctccacga gcacatcgcc aacctcgccg gatccccagc catcaagaag ggaatcctcc 3181 aaaccgtcaa ggtcgtcgac gagctcgtca aggtcatggg acgtcacaag ccagagaaca 3241 tcgtcatcga gatggcccgt gagaaccaaa ccacccaaaa gggacaaaag aactcccgtg 3301 agcgtatgaa gcgtatcgag gagggaatca aggagctcgg atcccaaatc ctcaaggagc 3361 acccagtcga gaacacccaa ctccaaaacg agaagctcta cctctactac ctccaaaacg 3421 gacgtgacat gtacgtcgac caagagctcg acatcaaccg tctctccgac tacgacgtcg 3481 accacatcgt cccacaatcc ttcctcaagg acgactccat cgacaacaag gtcctcaccc 3541 gttccgacaa gaaccgtgga aagtccgaca acgtcccatc cgaggaggtc gtcaagaaga 3601 tgaagaacta ctggcgtcaa ctcctcaacg ccaagctcat cacccaacgt aagttcgaca 3661 acctcaccaa ggccgagcgt ggaggactct ccgagctcga caaggccgga ttcatcaagc 3721 gtcaactcgt cgagacccgt caaatcacca agcacgtcgc ccaaatcctc gactcccgta 3781 tgaacaccaa gtacgacgag aacgacaagc tcatccgtga ggtcaaggtc atcaccctca 3841 agtccaagct cgtctccgac ttccgtaagg acttccaatt ctacaaggtc cgtgagatca 3901 acaactacca ccacgcccac gacgcctacc tcaacgccgt cgtcggaacc gccctcatca 3961 agaagtaccc aaagctcgag tccgagttcg tctacggaga ctacaaggtc tacgacgtcc 4021 gtaagatgat cgccaagtcc gagcaagaga tcggaaaggt aagtttaaac atgattttac 4081 taactaacta atctgattta aattttcagg ccaccgccaa gtacttcttc tactccaaca 4141 tcatgaactt cttcaagacc gagatcaccc tcgccaacgg agagatccgt aagcgtccac 4201 tcatcgagac caacggagag accggagaga tcgtctggga caagggacgt gacttcgcca 4261 ccgtccgtaa ggtcctctcc atgccacaag tcaacatcgt caagaagacc gaggtccaaa 4321 ccggaggatt ctccaaggag tccatcctcc caaagcgtaa ctccgacaag ctcatcgccc 4381 gtaagaagga ctgggaccca aagaagtacg gaggattcga ctccccaacc gtcgcctact 4441 ccgtcctcgt cgtcgccaag gtcgagaagg gaaagtccaa gaagctcaag tccgtcaagg 4501 agctcctcgg aatcaccatc atggagcgtt cctccttcga gaagaaccca atcgacttcc 4561 tcgaggccaa gggatacaag gaggtcaaga aggacctcat catcaagctc ccaaagtact 4621 ccctcttcga gctcgagaac ggacgtaagc gtatgctcgc ctccgccgga gagctccaaa 4681 agggaaacga gctcgccctc ccatccaagt acgtcaactt cctctacctc gcctcccact 4741 acgagaagct caagggatcc ccagaggaca acgagcaaaa gcaactcttc gtcgagcaac 4801 acaagcacta cctcgacgag atcatcgagc aaatctccga gttctccaag cgtgtcatcc 4861 tcgccgacgc caacctcgac aaggtcctct ccgcctacaa caagcaccgt gacaagccaa 4921 tccgtgagca agccgagaac atcatccacc tcttcaccct caccaacctc ggagccccag 4981 ccgccttcaa gtacttcgac accaccatcg accgtaagcg ttacacctcc accaaggagg 5041 tcctcgacgc caccctcatt caccaatcca tcaccggact ctacgagacc cgtatcgacc 5101 tctcccaact cggaggagac ctgcagatgt ccaagggaga ggagctcttc accggagtcg 5161 tcccaatcct cgtcgagctc gacggagacg tcaacggaca caagttctcc gtctcaggag 5221 agggagaggg agacgccacc tacggaaagc tcaccctcaa gttcatctgc accaccggaa 5281 agctcccagt cccatggcca accctcgtca ccaccttcac ttacggagtc caatgcttct 5341 cccgttaccc agaccacatg aagcgtcacg acttcttcaa gtccgccatg ccagagggat 5401 acgtccaaga gcgtaccatc ttcttcaagg taagtttaaa cattaattaa tactaactaa 5461 ccctgattat ttaaattttc aggacgacgg aaactacaag acccgtgccg aggtcaagtt 5521 cgagggagac accctcgtca accgtatcga gctcaaggta agtttaaaca gttcggtact 5581 aactaaccat acatatttaa attttcaggg aatcgacttc aaggaggacg gaaacatcct 5641 cggacacaag ctcgaataca actacaactc ccacaacgtc tacatcatgg ccgacaagca 5701 aaagaacgga atcaaggtca acttcaaggt aagtttaaac atgattttac taactaacta 5761 atctgattta aattttcaga tccgtcacaa catcgaggac ggatctgtcc aactcgccga 5821 ccactaccaa caaaacaccc caatcggaga cggaccagtc ctcctcccag acaaccacta 5881 cctctccacc caatccgccc tctccaagga cccaaacgag aagcgtgacc acatggtcct 5941 caaggagttc gtcaccgctg ccggaatcac ccacggaatg gacgagctct acaagctgca 6001 gatgtcccgt cgtcgtaagg ccaacccaac caagctctcc gagaacgcca agaagctcgc 6061 caaggaggtc gagaactagg aattcatgca agatcctttc aagcattccc ttcttctcta 6121 tcactcttct ttctttttgt caaaaaattc tctcgctaat ttatttgctt ttttaatgtt 6181 attattttat gactttttat agtcactgaa aagtttgcat ctgagtgaag tgaatgctat 6241 caaaatgtga ttctgtctga tgtactttca caatctctct tcaattccat tttgaagtgc 6301 tttaaacccg aaaggttgag aaaaatgcga gcgctcaaat atttgtattg tgttcgttga 6361 gtgacccaac aaaaagagga aactttattg tgccgccaag aaaaaagtct cattgcctaa 6421 gagaaacata tgtggttgga aatcttccag atcgtggatc actagtatgt ggttggaaat 6481 cttccagatc gtggatcaga gctccagctt ttgttccctt tagtgagggt taatttcgag 6541 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 6601 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 6661 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 6721 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 6781 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6841 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6901 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7381 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 7501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 7561 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 7621 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 7681 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 7741 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 7801 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7861 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7921 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7981 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 8041 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 8101 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 8161 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 8221 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 8281 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 8341 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 8401 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 8461 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 8521 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 8581 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 8641 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 8701 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc 8761 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag 8821 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg 8881 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca 8941 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa 9001 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg 9061 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta 9121 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg 9181 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 9241 aaagggggat gtgct //