LOCUS pMB66 9000 bp ds-DNA circular SYN 15-AUG-2013 DEFINITION . ACCESSION . VERSION . KEYWORDS pMB66 - Phsp-16.48 Cas9 mEGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9002) 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) FEATURES Location/Qualifiers rep_origin complement(6..461) /direction=LEFT /label="f1 ori" /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind 603..619 /label="M13 fwd" /note="common sequencing primer, one of multiple similar variants" promoter 626..644 /label="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" CDS 1048..1113 /codon_start=1 /product="three tandem FLAG epitope tags, followed by an enterokinase cleavage site" /label="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 1120..1140 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label="SV40 NLS" /translation="PKKKRKV" CDS join(1165..2205,2257..3243,3295..4356,4408..5418) /codon_start=1 /label="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(5425..5727,5781..5855,5907..6026,6078..6293) /codon_start=1 /label="mEGFP" /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLKE FVTAAGITHGMDELYK" CDS 6300..6374 /codon_start=1 /label="egl-13 NLS" /translation="MSRRRKANPTKLSENAKKLAKEVEN" 3'UTR 6384..6749 /label="tbb-2 UTR" promoter complement(6815..6833) /label="T3 promoter" /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(6854..6870) /label="M13 rev" /note="common sequencing primer, one of multiple similar variants" protein_bind 6878..6894 /bound_moiety="lac repressor encoded by lacI" /label="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(6902..6932) /label="lac promoter" /note="promoter for the E. coli lac operon" rep_origin complement(7256..7844) /direction=LEFT /label="ori" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(8015..8875) /codon_start=1 /gene="bla" /product="beta-lactamase" /label="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(8876..8980) /gene="bla" /label="AmpR promoter" promoter 660..1033 /label="Phsp-16.48" ORIGIN 1 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag 61 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac 121 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga 181 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc 241 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg 301 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 361 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 421 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct 481 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 541 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 601 ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggtaccgc 661 tggacggaaa tagtggtaaa gtgacatgat tgtagtttga agatttcaca attagagtga 721 atgttgtttg gttcggtttt gtcactgtat ttatactcat ttccaccttt ttctagaaca 781 ttcgagctgc ttcttAcaaa aggaggcgac tcacattcag aacattgaga aatagtgtgc 841 gactgaagaa acccagatac tttttcaatc tgcgtctctt tgcacctatg gggtgtattt 901 tgaaatgaat gcatctagga ccttctagaa cattctaaac ggctgcagga tacgggtata 961 taagccaatc gtgttcagag gaaaccaata cactttgttc aagtgcttac tgttcattct 1021 ctaaacttca agaactagaa aaaaatggac tacaaggacc acgacggaga ctacaaggac 1081 cacgacatcg actacaagga cgacgacgac aagatggccc caaagaagaa gcgtaaggtc 1141 ggaatccacg gagtcccagc cgccgacaag aagtactcca tcggactcga catcggaacc 1201 aactccgtcg gatgggccgt catcaccgac gagtacaagg tcccatccaa gaagttcaag 1261 gtcctcggaa acaccgaccg tcactccatc aagaagaacc tcatcggagc cctcctcttc 1321 gactccggag agaccgccga ggccacccgt ctcaagcgta ccgcccgtcg tcgttacacc 1381 cgtcgtaaga accgtatctg ctacctccaa gagatcttct ccaacgagat ggccaaggtc 1441 gacgactcct tcttccaccg tctcgaggag tccttcctcg tcgaggagga caagaagcac 1501 gagcgtcacc caatcttcgg aaacatcgtc gacgaggtcg cctaccacga gaagtaccca 1561 accatctacc acctccgtaa gaagctcgtc gactccaccg acaaggccga cctccgtctc 1621 atctacctcg ccctcgccca catgatcaag ttccgtggac acttcctcat cgagggagac 1681 ctcaacccag acaactccga cgtcgacaag ctcttcatcc aactcgtcca aacctacaac 1741 caactcttcg aggagaaccc aatcaacgcc tccggagtcg acgccaaggc catcctctcc 1801 gcccgtctct ccaagtcccg tcgtctcgag aacctcatcg cccaactccc aggagagaag 1861 aagaacggac tcttcggaaa cctcatcgcc ctctccctcg gactcacccc aaacttcaag 1921 tccaacttcg acctcgccga ggacgccaag ctccaactct ccaaggacac ctacgacgac 1981 gacctcgaca acctcctcgc ccaaatcgga gaccaatacg ccgacctctt cctcgccgcc 2041 aagaacctct ccgacgccat cctcctctcc gacatcctcc gtgtcaacac cgagatcacc 2101 aaggccccac tctccgcctc catgatcaag cgttacgacg agcaccacca agacctcacc 2161 ctcctcaagg ccctcgtccg tcaacaactc ccagagaagt acaaggtaag tttaaacata 2221 tatatactaa ctaaccctga ttatttaaat tttcaggaga tcttcttcga ccaatccaag 2281 aacggatacg ccggatacat cgacggagga gcctcccaag aggagttcta caagttcatc 2341 aagccaatcc tcgagaagat ggacggaacc gaggagctcc tcgtcaagct caaccgtgag 2401 gacctcctcc gtaagcaacg taccttcgac aacggatcca tcccacacca aatccacctc 2461 ggagagctcc acgccatcct ccgtcgtcaa gaggacttct acccattcct caaggacaac 2521 cgtgagaaga tcgagaagat cctcaccttc cgtatcccat actacgtcgg accactcgcc 2581 cgtggaaact cccgtttcgc ctggatgacc cgtaagtccg aggagaccat caccccatgg 2641 aacttcgagg aggtcgtcga caagggagcc tccgcccaat ccttcatcga gcgtatgacc 2701 aacttcgaca agaacctccc aaacgagaag gtcctcccaa agcactccct cctctacgag 2761 tacttcaccg tctacaacga gctcaccaag gtcaagtacg tcaccgaggg aatgcgtaag 2821 ccagccttcc tctccggaga gcaaaagaag gccatcgtcg acctcctctt caagaccaac 2881 cgtaaggtca ccgtcaagca actcaaggag gactacttca agaagatcga gtgcttcgac 2941 tccgtcgaga tctccggagt cgaggaccgt ttcaacgcct ccctcggaac ctaccacgac 3001 ctcctcaaga tcatcaagga caaggacttc ctcgacaacg aggagaacga ggacatcctc 3061 gaggacatcg tcctcaccct caccctcttc gaggaccgtg agatgatcga ggagcgtctc 3121 aagacctacg cccacctctt cgacgacaag gtcatgaagc aactcaagcg tcgtcgttac 3181 accggatggg gacgtctctc ccgtaagctc atcaacggaa tccgtgacaa gcaatccgga 3241 aaggtaagtt taaacagttc ggtactaact aaccatacat atttaaattt tcagaccatc 3301 ctcgacttcc tcaagtccga cggattcgcc aaccgtaact tcatgcaact catccacgac 3361 gactccctca ccttcaagga ggacatccaa aaggcccaag tctccggaca aggagactcc 3421 ctccacgagc acatcgccaa cctcgccgga tccccagcca tcaagaaggg aatcctccaa 3481 accgtcaagg tcgtcgacga gctcgtcaag gtcatgggac gtcacaagcc agagaacatc 3541 gtcatcgaga tggcccgtga gaaccaaacc acccaaaagg gacaaaagaa ctcccgtgag 3601 cgtatgaagc gtatcgagga gggaatcaag gagctcggat cccaaatcct caaggagcac 3661 ccagtcgaga acacccaact ccaaaacgag aagctctacc tctactacct ccaaaacgga 3721 cgtgacatgt acgtcgacca agagctcgac atcaaccgtc tctccgacta cgacgtcgac 3781 cacatcgtcc cacaatcctt cctcaaggac gactccatcg acaacaaggt cctcacccgt 3841 tccgacaaga accgtggaaa gtccgacaac gtcccatccg aggaggtcgt caagaagatg 3901 aagaactact ggcgtcaact cctcaacgcc aagctcatca cccaacgtaa gttcgacaac 3961 ctcaccaagg ccgagcgtgg aggactctcc gagctcgaca aggccggatt catcaagcgt 4021 caactcgtcg agacccgtca aatcaccaag cacgtcgccc aaatcctcga ctcccgtatg 4081 aacaccaagt acgacgagaa cgacaagctc atccgtgagg tcaaggtcat caccctcaag 4141 tccaagctcg tctccgactt ccgtaaggac ttccaattct acaaggtccg tgagatcaac 4201 aactaccacc acgcccacga cgcctacctc aacgccgtcg tcggaaccgc cctcatcaag 4261 aagtacccaa agctcgagtc cgagttcgtc tacggagact acaaggtcta cgacgtccgt 4321 aagatgatcg ccaagtccga gcaagagatc ggaaaggtaa gtttaaacat gattttacta 4381 actaactaat ctgatttaaa ttttcaggcc accgccaagt acttcttcta ctccaacatc 4441 atgaacttct tcaagaccga gatcaccctc gccaacggag agatccgtaa gcgtccactc 4501 atcgagacca acggagagac cggagagatc gtctgggaca agggacgtga cttcgccacc 4561 gtccgtaagg tcctctccat gccacaagtc aacatcgtca agaagaccga ggtccaaacc 4621 ggaggattct ccaaggagtc catcctccca aagcgtaact ccgacaagct catcgcccgt 4681 aagaaggact gggacccaaa gaagtacgga ggattcgact ccccaaccgt cgcctactcc 4741 gtcctcgtcg tcgccaaggt cgagaaggga aagtccaaga agctcaagtc cgtcaaggag 4801 ctcctcggaa tcaccatcat ggagcgttcc tccttcgaga agaacccaat cgacttcctc 4861 gaggccaagg gatacaagga ggtcaagaag gacctcatca tcaagctccc aaagtactcc 4921 ctcttcgagc tcgagaacgg acgtaagcgt atgctcgcct ccgccggaga gctccaaaag 4981 ggaaacgagc tcgccctccc atccaagtac gtcaacttcc tctacctcgc ctcccactac 5041 gagaagctca agggatcccc agaggacaac gagcaaaagc aactcttcgt cgagcaacac 5101 aagcactacc tcgacgagat catcgagcaa atctccgagt tctccaagcg tgtcatcctc 5161 gccgacgcca acctcgacaa ggtcctctcc gcctacaaca agcaccgtga caagccaatc 5221 cgtgagcaag ccgagaacat catccacctc ttcaccctca ccaacctcgg agccccagcc 5281 gccttcaagt acttcgacac caccatcgac cgtaagcgtt acacctccac caaggaggtc 5341 ctcgacgcca ccctcattca ccaatccatc accggactct acgagacccg tatcgacctc 5401 tcccaactcg gaggagacct gcagatgtcc aagggagagg agctcttcac cggagtcgtc 5461 ccaatcctcg tcgagctcga cggagacgtc aacggacaca agttctccgt ctcaggagag 5521 ggagagggag acgccaccta cggaaagctc accctcaagt tcatctgcac caccggaaag 5581 ctcccagtcc catggccaac cctcgtcacc accttcactt acggagtcca atgcttctcc 5641 cgttacccag accacatgaa gcgtcacgac ttcttcaagt ccgccatgcc agagggatac 5701 gtccaagagc gtaccatctt cttcaaggta agtttaaaca ttaattaata ctaactaacc 5761 ctgattattt aaattttcag gacgacggaa actacaagac ccgtgccgag gtcaagttcg 5821 agggagacac cctcgtcaac cgtatcgagc tcaaggtaag tttaaacagt tcggtactaa 5881 ctaaccatac atatttaaat tttcagggaa tcgacttcaa ggaggacgga aacatcctcg 5941 gacacaagct cgaatacaac tacaactccc acaacgtcta catcatggcc gacaagcaaa 6001 agaacggaat caaggtcaac ttcaaggtaa gtttaaacat gattttacta actaactaat 6061 ctgatttaaa ttttcagatc cgtcacaaca tcgaggacgg atctgtccaa ctcgccgacc 6121 actaccaaca aaacacccca atcggagacg gaccagtcct cctcccagac aaccactacc 6181 tctccaccca atccgccctc tccaaggacc caaacgagaa gcgtgaccac atggtcctca 6241 aggagttcgt caccgctgcc ggaatcaccc acggaatgga cgagctctac aagctgcaga 6301 tgtcccgtcg tcgtaaggcc aacccaacca agctctccga gaacgccaag aagctcgcca 6361 aggaggtcga gaactaggaa ttcatgcaag atcctttcaa gcattccctt cttctctatc 6421 actcttcttt ctttttgtca aaaaattctc tcgctaattt atttgctttt ttaatgttat 6481 tattttatga ctttttatag tcactgaaaa gtttgcatct gagtgaagtg aatgctatca 6541 aaatgtgatt ctgtctgatg tactttcaca atctctcttc aattccattt tgaagtgctt 6601 taaacccgaa aggttgagaa aaatgcgagc gctcaaatat ttgtattgtg ttcgttgagt 6661 gacccaacaa aaagaggaaa ctttattgtg ccgccaagaa aaaagtctca ttgcctaaga 6721 gaaacatatg tggttggaaa tcttccagat cgtggatcac tagtatgtgg ttggaaatct 6781 tccagatcgt ggatcagagc tccagctttt gttcccttta gtgagggtta atttcgagct 6841 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 6901 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 6961 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 7021 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 7081 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 7141 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 7201 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 7261 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 7321 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 7381 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 7441 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 7501 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 7561 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 7621 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 7681 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 7741 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 7801 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 7861 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 7921 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 7981 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 8041 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 8101 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 8161 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 8221 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 8281 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 8341 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 8401 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 8461 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 8521 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 8581 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 8641 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 8701 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 8761 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 8821 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 8881 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 8941 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc //