LOCUS Exported 7719 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Multisite gateway vector for expression of nCas9n with bicistronic expression of mKate2. ACCESSION . VERSION . KEYWORDS pME-nCas9n-P2A-mKate2-pA SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7719) TITLE Parton Lab Plasmids for in vivo cell biology in the zebrafish REFERENCE 2 (bases 1 to 7719) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7719 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 1..100 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" regulatory 101..110 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 119..139 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 146..4246 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes MGAS15252 Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKK NLIGALLFGSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLADSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQIYNQLFEENPINASRVDAKAILSARLSKSRRL ENLIAQLPGEKRNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNSEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGAYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRGMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGHSLHEQIANLAGSPAIKKGILQTVKIVDELVKVMGH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" RBS 4159..4167 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" CDS 4262..4282 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4298..4354 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 4373..5068 /codon_start=1 /product="monomeric far-red fluorescent protein (Shcherbo et al., 2009)" /label=mKate2 /note="mammalian codon-optimized" /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC DLPSKLGHR" polyA_signal 5190..5271 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5227..5246) /label=SV40pA-R /note="SV40 polyA, reverse primer" protein_bind complement(5276..5375) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(5392..5411) /label=T7 /note="T7 promoter, forward primer" promoter complement(5393..5411) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5416..5432) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5416..5432) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5534..5553) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 5545..6354 /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind complement(5612..5631) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 6501..7089 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6990..7009 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7243..7260 /label=L4440 /note="L4440 vector, forward primer" terminator 7419..7446 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 7538..7624 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 7673..7695 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7687..7704 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7688..7704 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" ORIGIN 1 caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat tgatgagcaa 61 tgctttttta taatgccaac tttgtacaaa aaagcaggct gccaccatgg cttctccacc 121 taagaagaag agaaaggtgg gaagcatgga taagaagtat agcatcggcc tggatattgg 181 aactaactcc gtgggttggg cagtgattac agacgactac aaggtcccta gcaagaaatt 241 taaggtgctg ggtaacaccg acaggcacag catcaagaaa aatctgattg gagccctgct 301 gttcggttct ggagagactg ccgaagcaac acgcctgaaa agaacagcaa gaaggcgcta 361 taccagaagg aagaatagaa tctgttacct gcaggagatt ttctctaacg aaatggctaa 421 ggtggacgat tcattctttc ataggctgga ggaaagtttc ctggtcgagg aagataagaa 481 acacgagcgc catcctatct ttggaaacat tgtggacgag gtcgcctatc acgaaaaata 541 cccaaccatc tatcatctgc gcaagaaact ggctgactct actgataaag ccgacctgag 601 actgatctat ctggctctgg cccacatgat taagttcagg ggtcattttc tgatcgaggg 661 cgatctgaac cccgacaatt ccgatgtgga caagctgttc atccagctgg tccagattta 721 caatcagctg tttgaggaaa accctattaa tgcttccaga gtggacgcaa aagctatcct 781 gtcagccagg ctgtccaagt cacgcagact ggagaacctg attgcacagc tgcccggaga 841 aaagaggaac ggtctgtttg gaaatctgat cgctctgagt ctgggcctga ctcctaactt 901 caaaagcaat tttgatctgg ctgaggacgc caaactgcag ctgtcaaagg acacatatga 961 cgatgacctg gataacctgc tggcacagat cggagatcag tacgctgacc tgttcctggc 1021 tgccaaaaat ctgtccgacg caatcctgct gtcagatatt ctgagagtga acagcgagat 1081 tacaaaagct cctctgagtg ccagcatgat caagagatat gacgagcacc atcaggatct 1141 gaccctgctg aaggctctgg tcaggcagca gctgccagag aagtacaagg aaattttctt 1201 tgatcagtcc aagaacggct acgccggtta tatcgacgga ggcgcatcac aggaggaatt 1261 ctacaagttt atcaaaccta ttctggagaa gatggacgga actgaggaac tgctggtgaa 1321 actgaataga gaggacctgc tgaggaagca gcgcacattt gataacggtt ccatcccaca 1381 ccagattcat ctgggagagc tgcacgctat cctgaggcgc caggaagact tctacccctt 1441 tctgaaagat aaccgcgaga agatcgaaaa aattctgacc ttcagaatcc cttactatgt 1501 gggtccactg gctcgcggaa acagcagatt tgcctggatg actcgcaaat ccgaggaaac 1561 cattactcct tggaacttcg aggaagtggt cgataagggc gcctctgcac agtccttcat 1621 cgagagaatg actaattttg acaaaaacct gcccaatgag aaagtgctgc ctaagcactc 1681 cctgctgtac gagtatttca ctgtctataa cgaactgaca aaggtgaaat acgtcaccga 1741 gggcatgaga aagccagcct tcctgtcagg agagcagaag aaagcaatcg tggatctgct 1801 gtttaaaacc aacaggaaag tgactgtcaa gcagctgaag gaggactact tcaagaaaat 1861 tgaatgcttc gattccgtgg agatcagcgg agtcgaagac agatttaacg caagcctggg 1921 cgcttaccac gatctgctga agatcattaa ggataaagac ttcctggaca acgaggaaaa 1981 tgaggatatc ctggaagaca ttgtgctgac actgaccctg tttgaggaca gaggaatgat 2041 cgaggaaaga ctgaaaacct atgctcatct gttcgatgac aaggtgatga aacagctgaa 2101 gagaaggcgc tacactggct ggggtagact gagcaggaag ctgatcaacg gcattaggga 2161 taaacagtca ggaaagacaa tcctggactt tctgaaaagt gatggcttcg ccaaccgcaa 2221 ttttatgcag ctgattcacg atgacagtct gaccttcaaa gaggacatcc agaaggctca 2281 ggtgtctgga cagggccact ccctgcatga gcagattgca aacctggctg gaagcccagc 2341 catcaagaaa ggcattctgc agacagtgaa aatcgtcgat gagctggtga aagtcatggg 2401 ccataagccc gaaaacatcg tgattgagat ggctcgcgaa aatcagacaa cccagaaggg 2461 tcagaagaac agtagagaga ggatgaaaag aatcgaggaa ggcattaagg agctgggtag 2521 ccagatcctg aaagagcacc cagtggaaaa cacacagctg cagaatgaga agctgtatct 2581 gtactatctg cagaatggaa gagatatgta cgtggaccag gagctggata ttaacaggct 2641 gtctgattac gacgtggatc atatcgtccc ccagagtttc atcaaagatg acagcattga 2701 caacaaggtg ctgaccaggt ccgacaaaaa cagaggaaaa tcagataatg tccctagtga 2761 ggaagtggtc aagaaaatga agaactactg gagacagctg ctgaatgcca aactgatcac 2821 tcagaggaag tttgataacc tgacaaaagc agagcgcggt ggactgtcag aactggacaa 2881 agctggattc atcaagaggc agctggtgga aacacgccag atcactaaac acgtcgcaca 2941 gattctggat agtcgcatga acacaaagta cgatgagaat gacaaactga tcagagaagt 3001 gaaggtcatt accctgaaga gtaaactggt cagcgacttt aggaaagatt tccagtttta 3061 taaggtccgc gagattaaca attatcacca tgcccatgac gcatacctga acgccgtggt 3121 cggtaccgca ctgatcaaga aatacccaaa actggagagc gaattcgtgt acggagacta 3181 taaggtgtac gatgtcagaa aaatgatcgc caagtccgag caggaaattg gaaaagctac 3241 tgccaagtat ttcttttact caaacatcat gaatttcttt aagacagaga tcaccctggc 3301 caatggagaa atccgcaaaa ggcccctgat tgagacaaac ggagagacag gcgaaatcgt 3361 gtgggacaaa ggcagagatt ttgcaaccgt gaggaaggtc ctgagcatgc ctcaagtgaa 3421 tatcgtcaag aaaactgagg tgcagacagg cggtttctca aaagaaagta ttctgccaaa 3481 acgcaactct gataagctga tcgctagaaa gaaagactgg gaccctaaga agtatggagg 3541 ctttgactct cccactgtgg catactccgt cctggtggtc gctaaggtgg agaagggcaa 3601 aagcaagaaa ctgaaatctg tcaaggagct gctgggtatc acaattatgg agagaagctc 3661 tttcgagaag aacccaatcg attttctgga ggccaaaggt tataaggaag tgaagaaaga 3721 cctgatcatt aaactgccca agtacagtct gtttgagctg gaaaacggca ggaaacgcat 3781 gctggcaagc gctggagagc tgcagaaagg caatgaactg gccctgcctt ctaagtacgt 3841 gaacttcctg tatctggcaa gccactacga gaagctgaaa ggatctccag aggataacga 3901 acagaaacag ctgtttgtgg agcagcacaa gcattatctg gacgagatca ttgaacagat 3961 tagcgagttc tctaaaagag tgatcctggc cgacgcaaat ctggataagg tcctgtctgc 4021 ttacaacaaa cacagagata agcccatcag ggagcaggcc gaaaatatca ttcatctgtt 4081 cactctgaca aacctgggcg cacctgcagc tttcaagtac ttcgacacta caatcgatag 4141 aaagaggtac acctccacta aggaggtgct ggacgctaca ctgatccatc agagtattac 4201 cggcctgtac gaaacaagga ttgacctgtc tcagctgggt ggcgactcac cggtgagatc 4261 tcctaagaag aagagaaagg tgcctagggg cagcggcgcc accaacttca gcctgctgaa 4321 gcaggccggc gacgtggagg agaaccccgg ccccgccccc ggcagcctcg aggtgagcga 4381 gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtga acaaccacca 4441 cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga ccatgagaat 4501 caaggcggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta ccagcttcat 4561 gtacggcagc aaaaccttca tcaaccacac ccagggcatc cccgacttct ttaagcagtc 4621 cttccccgag ggcttcacat gggagagagt caccacatac gaagacgggg gcgtgctgac 4681 cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca agatcagagg 4741 ggtgaacttc ccatccaacg gccctgtgat gcagaagaaa acactcggct gggaggcctc 4801 caccgagacc ctgtaccccg ctgacggcgg cctggaaggc agagccgaca tggccctgaa 4861 gctcgtgggc gggggccacc tgatctgcaa cttgaagacc acatacagat ccaagaaacc 4921 cgctaagaac ctcaagatgc ccggcgtcta ctatgtggac agaagactgg aaagaatcaa 4981 ggaggccgac aaagagacct acgtcgagca gcacgaggtg gctgtggcca gatactgcga 5041 cctccctagc aaactggggc acagatgagc ggccgcgact ctagatcata atcagccata 5101 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga 5161 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca 5221 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cgtcgaccca 5281 gctttcttgt acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa 5341 caggtcacta tcagtcaaaa taaaatcatt atttgccatc cagctgatat cccctatagt 5401 gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc tcaaaatctc 5461 tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact gtctgcttac 5521 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc gaggccgcga 5581 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg 5641 caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga gttgtttctg 5701 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg 5761 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca 5821 tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga agaatatcct 5881 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt 5941 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca 6001 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct 6061 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc 6121 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt 6181 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac 6241 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat 6301 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaatcagaa 6361 ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga cggcgcaagc 6421 tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc agaccccgta 6481 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 6541 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 6601 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 6661 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 6721 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 6781 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 6841 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 6901 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6961 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 7021 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 7081 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 7141 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 7201 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 7261 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 7321 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 7381 acgcgtaccg ctagccagga agagtttgta gaaacgcaaa aaggccatcc gtcaggatgg 7441 ccttctgctt agtttgatgc ctggcagttt atggcgggcg tcctgcccgc caccctccgg 7501 gccgttgctt cacaacgttc aaatccgctc ccggcggatt tgtcctactc aggagagcgt 7561 tcaccgacaa acaacagata aaacgaaagg cccagtcttc cgactgagcc tttcgtttta 7621 tttgatgcct ggcagttccc tactctcgcg ttaacgctag catggatgtt ttcccagtca 7681 cgacgttgta aaacgacggc cagtcttaag ctcgggccc //