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" primer_bind 92..109 /label=L4440 /note="L4440 vector, forward primer" terminator 268..295 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" terminator 387..473 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind 522..544 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 536..553 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 537..553 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 569..668 /gene="mutant version of attL" /label=attL1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" regulatory 669..678 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 687..707 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 714..4814 /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 4727..4735 /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 4830..4850 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4866..4922 /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 4941..5636 /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 5758..5839 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5795..5814) /label=SV40pA-R /note="SV40 polyA, reverse primer" protein_bind complement(5844..5943) /gene="mutant version of attL" /label=attL2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" primer_bind complement(5960..5979) /label=T7 /note="T7 promoter, forward primer" promoter complement(5961..5979) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5984..6000) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5984..6000) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6102..6121) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 6113..6922 /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(6180..6199) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" rep_origin 7069..7657 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7558..7577 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 601 ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 661 agcaggctgc caccatggct tctccaccta agaagaagag aaaggtggga agcatggata 721 agaagtatag catcggcctg gatattggaa ctaactccgt gggttgggca gtgattacag 781 acgactacaa ggtccctagc aagaaattta aggtgctggg taacaccgac aggcacagca 841 tcaagaaaaa tctgattgga gccctgctgt tcggttctgg agagactgcc gaagcaacac 901 gcctgaaaag aacagcaaga aggcgctata ccagaaggaa gaatagaatc tgttacctgc 961 aggagatttt ctctaacgaa atggctaagg tggacgattc attctttcat aggctggagg 1021 aaagtttcct ggtcgaggaa gataagaaac acgagcgcca tcctatcttt ggaaacattg 1081 tggacgaggt cgcctatcac gaaaaatacc caaccatcta tcatctgcgc aagaaactgg 1141 ctgactctac tgataaagcc gacctgagac tgatctatct ggctctggcc cacatgatta 1201 agttcagggg tcattttctg atcgagggcg atctgaaccc cgacaattcc gatgtggaca 1261 agctgttcat ccagctggtc cagatttaca atcagctgtt tgaggaaaac cctattaatg 1321 cttccagagt ggacgcaaaa gctatcctgt cagccaggct gtccaagtca cgcagactgg 1381 agaacctgat tgcacagctg cccggagaaa agaggaacgg tctgtttgga aatctgatcg 1441 ctctgagtct gggcctgact cctaacttca aaagcaattt tgatctggct gaggacgcca 1501 aactgcagct gtcaaaggac acatatgacg atgacctgga taacctgctg gcacagatcg 1561 gagatcagta cgctgacctg ttcctggctg ccaaaaatct gtccgacgca atcctgctgt 1621 cagatattct gagagtgaac agcgagatta caaaagctcc tctgagtgcc agcatgatca 1681 agagatatga cgagcaccat caggatctga ccctgctgaa ggctctggtc aggcagcagc 1741 tgccagagaa gtacaaggaa attttctttg atcagtccaa gaacggctac gccggttata 1801 tcgacggagg cgcatcacag gaggaattct acaagtttat caaacctatt ctggagaaga 1861 tggacggaac tgaggaactg ctggtgaaac tgaatagaga ggacctgctg aggaagcagc 1921 gcacatttga taacggttcc atcccacacc agattcatct gggagagctg cacgctatcc 1981 tgaggcgcca ggaagacttc tacccctttc tgaaagataa ccgcgagaag atcgaaaaaa 2041 ttctgacctt cagaatccct tactatgtgg gtccactggc tcgcggaaac agcagatttg 2101 cctggatgac tcgcaaatcc gaggaaacca ttactccttg gaacttcgag gaagtggtcg 2161 ataagggcgc ctctgcacag tccttcatcg agagaatgac taattttgac aaaaacctgc 2221 ccaatgagaa agtgctgcct aagcactccc tgctgtacga gtatttcact gtctataacg 2281 aactgacaaa ggtgaaatac gtcaccgagg gcatgagaaa gccagccttc ctgtcaggag 2341 agcagaagaa agcaatcgtg gatctgctgt ttaaaaccaa caggaaagtg actgtcaagc 2401 agctgaagga ggactacttc aagaaaattg aatgcttcga ttccgtggag atcagcggag 2461 tcgaagacag atttaacgca agcctgggcg cttaccacga tctgctgaag atcattaagg 2521 ataaagactt cctggacaac gaggaaaatg aggatatcct ggaagacatt gtgctgacac 2581 tgaccctgtt tgaggacaga ggaatgatcg aggaaagact gaaaacctat gctcatctgt 2641 tcgatgacaa ggtgatgaaa cagctgaaga gaaggcgcta cactggctgg ggtagactga 2701 gcaggaagct gatcaacggc attagggata aacagtcagg aaagacaatc ctggactttc 2761 tgaaaagtga tggcttcgcc aaccgcaatt ttatgcagct gattcacgat gacagtctga 2821 ccttcaaaga ggacatccag aaggctcagg tgtctggaca gggccactcc ctgcatgagc 2881 agattgcaaa cctggctgga agcccagcca tcaagaaagg cattctgcag acagtgaaaa 2941 tcgtcgatga gctggtgaaa gtcatgggcc ataagcccga aaacatcgtg attgagatgg 3001 ctcgcgaaaa tcagacaacc cagaagggtc agaagaacag tagagagagg atgaaaagaa 3061 tcgaggaagg cattaaggag ctgggtagcc agatcctgaa agagcaccca gtggaaaaca 3121 cacagctgca gaatgagaag ctgtatctgt actatctgca gaatggaaga gatatgtacg 3181 tggaccagga gctggatatt aacaggctgt ctgattacga cgtggatcat atcgtccccc 3241 agagtttcat caaagatgac agcattgaca acaaggtgct gaccaggtcc gacaaaaaca 3301 gaggaaaatc agataatgtc cctagtgagg aagtggtcaa gaaaatgaag aactactgga 3361 gacagctgct gaatgccaaa ctgatcactc agaggaagtt tgataacctg acaaaagcag 3421 agcgcggtgg actgtcagaa ctggacaaag ctggattcat caagaggcag ctggtggaaa 3481 cacgccagat cactaaacac gtcgcacaga ttctggatag tcgcatgaac acaaagtacg 3541 atgagaatga caaactgatc agagaagtga aggtcattac cctgaagagt aaactggtca 3601 gcgactttag gaaagatttc cagttttata aggtccgcga gattaacaat tatcaccatg 3661 cccatgacgc atacctgaac gccgtggtcg gtaccgcact gatcaagaaa tacccaaaac 3721 tggagagcga attcgtgtac ggagactata aggtgtacga tgtcagaaaa atgatcgcca 3781 agtccgagca ggaaattgga aaagctactg ccaagtattt cttttactca aacatcatga 3841 atttctttaa gacagagatc accctggcca atggagaaat ccgcaaaagg cccctgattg 3901 agacaaacgg agagacaggc gaaatcgtgt gggacaaagg cagagatttt gcaaccgtga 3961 ggaaggtcct gagcatgcct caagtgaata tcgtcaagaa aactgaggtg cagacaggcg 4021 gtttctcaaa agaaagtatt ctgccaaaac gcaactctga taagctgatc gctagaaaga 4081 aagactggga ccctaagaag tatggaggct ttgactctcc cactgtggca tactccgtcc 4141 tggtggtcgc taaggtggag aagggcaaaa gcaagaaact gaaatctgtc aaggagctgc 4201 tgggtatcac aattatggag agaagctctt tcgagaagaa cccaatcgat tttctggagg 4261 ccaaaggtta taaggaagtg aagaaagacc tgatcattaa actgcccaag tacagtctgt 4321 ttgagctgga aaacggcagg aaacgcatgc tggcaagcgc tggagagctg cagaaaggca 4381 atgaactggc cctgccttct aagtacgtga acttcctgta tctggcaagc cactacgaga 4441 agctgaaagg atctccagag gataacgaac agaaacagct gtttgtggag cagcacaagc 4501 attatctgga cgagatcatt gaacagatta gcgagttctc taaaagagtg atcctggccg 4561 acgcaaatct ggataaggtc ctgtctgctt acaacaaaca cagagataag cccatcaggg 4621 agcaggccga aaatatcatt catctgttca ctctgacaaa cctgggcgca cctgcagctt 4681 tcaagtactt cgacactaca atcgatagaa agaggtacac ctccactaag gaggtgctgg 4741 acgctacact gatccatcag agtattaccg gcctgtacga aacaaggatt gacctgtctc 4801 agctgggtgg cgactcaccg gtgagatctc ctaagaagaa gagaaaggtg cctaggggca 4861 gcggcgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag aaccccggcc 4921 ccgcccccgg cagcctcgag gtgagcgagc tgattaagga gaacatgcac atgaagctgt 4981 acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc 5041 cctacgaggg cacccagacc atgagaatca aggcggtcga gggcggccct ctccccttcg 5101 ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc aaccacaccc 5161 agggcatccc cgacttcttt aagcagtcct tccccgaggg cttcacatgg gagagagtca 5221 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct 5281 gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc 5341 agaagaaaac actcggctgg gaggcctcca ccgagaccct gtaccccgct gacggcggcc 5401 tggaaggcag agccgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact 5461 tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact 5521 atgtggacag aagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc 5581 acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac agatgagcgg 5641 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa 5701 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac 5761 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 5821 aaagcatttt tttcactgcg tcgacccagc tttcttgtac aaagttggca ttataagaaa 5881 gcattgctta tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat 5941 ttgccatcca gctgatatcc cctatagtga gtcgtattac atggtcatag ctgtttcctg 6001 gcagctctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaataatatc 6061 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca 6121 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg 6181 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg 6241 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt 6301 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa 6361 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac 6421 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc 6481 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg 6541 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga 6601 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact 6661 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat 6721 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg 6781 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa 6841 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt 6901 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat 6961 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc 7021 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7201 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 7321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 7381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 7441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 7501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 7561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 7621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 7681 acggttcctg gccttttgct ggccttttgc tcacatgtt //