LOCUS Exported 9015 bp ds-DNA circular SYN 16-AUG-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pBDKRB2_TCS(Q'G)_NLS-HA-dCas9(C)T2A_DHFR_PCP_VP64+T301 SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 9015) AUTHORS Weatherall Institute TITLE Direct Submission JOURNAL Exported Wednesday, Aug 16, 2017 from SnapGene 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9015 /organism="recombinant plasmid" /mol_type="other DNA" misc_feature 12..810 /note="CBh" misc_feature 817..824 /label=konzak CDS 825..905 /codon_start=1 /product="p.363: At the 5′ end, we included a cleavable signal sequence to promote membrane localization23 and a Flag epitope tag to allow monitoring of cell-surface expression by immunohistochemistry. -- Highlighted 11 mar 2016 flag - DYKDDDDK but where is the ""K""?" /label=membrane localisation signal+ FLAG /translation="MKTIIALSYIFCLVFADYKDDDDASID" CDS 906..2078 /codon_start=1 /label=BDKBR2 /translation="MFSPWKISMFLSVREDSVPTTASFSADMLNVTLQGPTLNGTFAQS KCPQVEWLGWLNTIQPPFLWVLFVLATLENIFVLSVFCLHKSSCTVAEIYLGNLAAADL ILACGLPFWAITISNNFDWLFGETLCRVVNAIISMNLYSSICFLMLVSIDRYLALVKTM SMGRMRGVRWAKLYSLVIWGCTLLLSSPMLVFRTMKEYSDEGHNVTACVISYPSLIWEV FTNMLLNVVGFLLPLSVITFCTMQIMQVLRNNEMQKFKEIQTERRATVLVLVVLLLFII CWLPFQISTFLDTLHRLGILSSCQDERIIDVITQIASFMAYSNSCLNPLVYVIVGKRFR KKSWEVYQGVCQKGGCRSEPIQMENSMGTLRTSISVERQIHKLQDWAGSRQ" CDS 2079..2177 /codon_start=1 /label=crucial for arrestin recruitment /note="added sequence from the C terminus of the V2 vasopressin receptor (V2 tail) after each recep- tor sequence, to promote arrestin recruitment20,24–26. " /translation="IDTGGRTPPSLGPQDESCTTASSSLAKDTSSTG" CDS 2178..2198 /codon_start=1 /label=efficient TEV cleavage site /translation="ENLYFQG" CDS 2199..2219 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" CDS 2238..2264 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" misc_feature 2265..2273 /label=GGS linker misc_feature 2280..2291 /note="(GGGGS)3 linker" CDS 2292..4790 /codon_start=1 /note="hSpCas9" /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGD" misc_feature 4791..4823 /note="NLS" CDS 4836..4889 /codon_start=1 /label=T2A /note="2A peptide from Thosea asigna virus capsid protein" /translation="EGRGSLLTCGDVEENPGP" CDS 4890..5363 /codon_start=1 /gene="folA" /product="E. coli dihydrofolate reductase" /label=ecDHFR /translation="MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRH TWESIGRPLPGRKNIILSSQPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFL PKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILER" CDS 5367..5411 /codon_start=1 /label=GGGGS linker /translation="GGGGSGGGGSGGGGS" CDS 5412..5774 /codon_start=1 /label=PP7 /translation="SKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLR QNGAKTAYRVNLKLDQADVVDSGLPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLV ATSQVEDLVVNLVPLGR" CDS 5781..5825 /codon_start=1 /label=GGGGS- linker /translation="GGGGSGGGGSGGGGS" CDS 5829..5849 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 5874..6023 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" misc_feature 6039..6270 /note="bGH polyA" misc_feature 6279..6419 /label=R-ITR rep_origin 6511..6817 /direction=RIGHT /label=f1 Origin misc_feature 7336..8193 /label=AmpR rep_origin 8344..9011 /direction=RIGHT /label=pUC ori ORIGIN 1 ctagaggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg 121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg 181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc 241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc 301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt 361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg 421 cgcgcgccag gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg 481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg 541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg 601 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 661 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa 721 gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 781 cctgcctgaa atcacttttt ttcaggttgg accggtgcgc caccatgaag acgatcatcg 841 ccctgagcta catcttctgc ctggtattcg ccgactacaa ggacgatgat gacgccagca 901 tcgatatgtt ctcaccctgg aagatttcta tgttcctttc tgtgcgcgag gacagcgtgc 961 ccacaactgc gagttttagc gccgacatgc ttaacgtgac cctgcagggc cccactctca 1021 acgggacatt tgctcagtct aaatgtcctc aagtggaatg gctcggctgg ttgaacacta 1081 tccagccccc tttcctttgg gttctttttg ttctggccac gctggaaaac atctttgttc 1141 tctctgtgtt ttgcctccat aagagctcct gtacggtggc cgaaatttac ctgggtaatt 1201 tggccgcagc ggatctcatt ttggcctgtg gcctgccttt ctgggctatt actatcagca 1261 ataattttga ttggttgttt ggcgagacct tgtgtcgcgt ggtgaatgca attatcagta 1321 tgaatctgta cagctcaatc tgcttcctta tgctcgtcag cattgacagg tacctcgccc 1381 ttgtaaagac tatgagcatg ggccgcatga ggggcgtacg ctgggctaag ctctatagcc 1441 tggtgatttg gggttgcacc ctcctcctca gttcacctat gctggtcttc cgaacgatga 1501 aggagtactc tgacgagggc cacaatgtta ccgcatgcgt tatcagttac ccatctttga 1561 tttgggaggt cttcacaaac atgctgctga acgtggtggg ttttttgctc cccctttctg 1621 tgattacctt ctgtacaatg cagatcatgc aagttttgcg aaacaacgaa atgcaaaaat 1681 ttaaggagat acagaccgag cgaagggcca cagtgctggt cttggtcgtt ttgctgttgt 1741 tcattatctg ttggctgcct ttccagatca gcactttcct ggacacgttg cacagactcg 1801 gaatcctgag tagttgtcag gacgaaagga tcatcgacgt tatcactcaa atcgcgtcct 1861 tcatggcata ctccaatagc tgcttgaacc cattggtgta tgttattgtg ggaaaacggt 1921 ttaggaagaa gagctgggaa gtgtaccaag gggtgtgcca aaagggaggc tgccgatcag 1981 agccaatcca aatggaaaat agcatgggta ccctgagaac ctccattagc gtggagcgac 2041 agatccacaa gctccaggac tgggctggct ctagacagat cgataccggt ggacgcaccc 2101 cacccagcct gggtccccaa gatgagtcct gcaccaccgc cagctcctcc ctggccaagg 2161 acacttcatc gaccggtgag aacctgtact tccagggccc caagaagaag aggaaggtgg 2221 ggggaggcag cactagttac ccatacgacg taccagatta cgctggaggt tctggatccg 2281 gcggtggctc gaagcccgcc ttcctgagcg gcgagcagaa aaaggccatc gtggacctgc 2341 tgttcaagac caaccggaaa gtgaccgtga agcagctgaa agaggactac ttcaagaaaa 2401 tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga tcggttcaac gcctccctgg 2461 gcacatacca cgatctgctg aaaattatca aggacaagga cttcctggac aatgaggaaa 2521 acgaggacat tctggaagat atcgtgctga ccctgacact gtttgaggac agagagatga 2581 tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga caaagtgatg aagcagctga 2641 agcggcggag atacaccggc tggggcaggc tgagccggaa gctgatcaac ggcatccggg 2701 acaagcagtc cggcaagaca atcctggatt tcctgaagtc cgacggcttc gccaacagaa 2761 acttcatgca gctgatccac gacgacagcc tgacctttaa agaggacatc cagaaagccc 2821 aggtgtccgg ccagggcgat agcctgcacg agcacattgc caatctggcc ggcagccccg 2881 ccattaagaa gggcatcctg cagacagtga aggtggtgga cgagctcgtg aaagtgatgg 2941 gccggcacaa gcccgagaac atcgtgatcg aaatggccag agagaaccag accacccaga 3001 agggacagaa gaacagccgc gagagaatga agcggatcga agagggcatc aaagagctgg 3061 gcagccagat cctgaaagaa caccccgtgg aaaacaccca gctgcagaac gagaagctgt 3121 acctgtacta cctgcagaat gggcgggata tgtacgtgga ccaggaactg gacatcaacc 3181 ggctgtccga ctacgatgtg gaccatatcg tgcctcagag ctttctgaag gacgactcca 3241 tcgacaacaa ggtgctgacc agaagcgaca aggcccgggg caagagcgac aacgtgccct 3301 ccgaagaggt cgtgaagaag atgaagaact actggcggca gctgctgaac gccaagctga 3361 ttacccagag aaagttcgac aatctgacca aggccgagag aggcggcctg agcgaactgg 3421 ataaggccgg cttcatcaag agacagctgg tggaaacccg gcagatcaca aagcacgtgg 3481 cacagatcct ggactcccgg atgaacacta agtacgacga gaatgacaag ctgatccggg 3541 aagtgaaagt gatcaccctg aagtccaagc tggtgtccga tttccggaag gatttccagt 3601 tttacaaagt gcgcgagatc aacaactacc accacgccca cgacgcctac ctgaacgccg 3661 tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga aagcgagttc gtgtacggcg 3721 actacaaggt gtacgacgtg cggaagatga tcgccaagag cgagcaggaa atcggcaagg 3781 ctaccgccaa gtacttcttc tacagcaaca tcatgaactt tttcaagacc gagattaccc 3841 tggccaacgg cgagatccgg aagcggcctc tgatcgagac aaacggcgaa accggggaga 3901 tcgtgtggga taagggccgg gattttgcca ccgtgcggaa agtgctgagc atgccccaag 3961 tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt cagcaaagag tctatcctgc 4021 ccaagaggaa cagcgataag ctgatcgcca gaaagaagga ctgggaccct aagaagtacg 4081 gcggcttcga cagccccacc gtggcctatt ctgtgctggt ggtggccaaa gtggaaaagg 4141 gcaagtccaa gaaactgaag agtgtgaaag agctgctggg gatcaccatc atggaaagaa 4201 gcagcttcga gaagaatccc atcgactttc tggaagccaa gggctacaaa gaagtgaaaa 4261 aggacctgat catcaagctg cctaagtact ccctgttcga gctggaaaac ggccggaaga 4321 gaatgctggc ctctgccggc gaactgcaga agggaaacga actggccctg ccctccaaat 4381 atgtgaactt cctgtacctg gccagccact atgagaagct gaagggctcc cccgaggata 4441 atgagcagaa acagctgttt gtggaacagc acaagcacta cctggacgag atcatcgagc 4501 agatcagcga gttctccaag agagtgatcc tggccgacgc taatctggac aaagtgctgt 4561 ccgcctacaa caagcaccgg gataagccca tcagagagca ggccgagaat atcatccacc 4621 tgtttaccct gaccaatctg ggagcccctg ccgccttcaa gtactttgac accaccatcg 4681 accggaagag gtacaccagc accaaagagg tgctggacgc caccctgatc caccagagca 4741 tcaccggcct gtacgagaca cggatcgacc tgtctcagct gggaggcgac agccccaaga 4801 agaagagaaa ggtggaggcc agcagcggca gtggagaggg cagaggaagt ctgctaacat 4861 gcggtgacgt cgaggagaat cctggcccaa tgatcagtct gattgcggcg ttagcggtag 4921 attacgttat cggcatggaa aacgccatgc cgtggaacct gcctgccgat ctcgcctggt 4981 ttaaacgcaa caccttaaat aaacccgtga ttatgggccg ccatacctgg gaatcaatcg 5041 gtcgtccgtt gccaggacgc aaaaatatta tcctcagcag tcaaccgagt acggacgatc 5101 gcgtaacgtg ggtgaagtcg gtggatgaag ccatcgcggc gtgtggtgac gtaccagaaa 5161 tcatggtgat tggcggcggt cgcgttattg aacagttctt gccaaaagcg caaaaactgt 5221 atctgacgca tatcgacgca gaagtggaag gcgacaccca tttcccggat tacgagccgg 5281 atgactggga atcggtattc agcgaattcc acgatgctga tgcgcagaac tctcacagct 5341 attgctttga gattctggag cggcgaggtg gaggaggttc aggaggtgga ggctctggag 5401 gtggaggcag ctccaaaacc atcgttcttt cggtcggcga ggctactcgc actctgactg 5461 agatccagtc caccgcagac cgtcagatct tcgaagagaa ggtcgggcct ctggtgggtc 5521 ggctgcgcct cacggcttcg ctccgtcaaa acggagccaa gaccgcgtat cgcgtcaacc 5581 taaaactgga tcaggcggac gtcgttgatt ccggacttcc gaaagtgcgc tacactcagg 5641 tatggtcgca cgacgtgaca atcgttgcga atagcaccga ggcctcgcgc aaatcgttgt 5701 acgatttgac caagtccctc gtcgcgacct cgcaggtcga agatcttgtc gtcaaccttg 5761 tgccgctggg ccgtagcgct ggaggaggtg gaagcggagg aggaggaagc ggaggaggag 5821 gtagcggacc taagaaaaag aggaaggtgg cggccgctgg atccggacgg gctgacgcat 5881 tggacgattt tgatctggat atgctgggaa gtgacgccct cgatgatttt gaccttgaca 5941 tgcttggttc ggatgccctt gatgactttg acctcgacat gctcggcagt gacgcccttg 6001 atgatttcga cctggacatg ctgattaact aagaattcct agagctcgct gatcagcctc 6061 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 6121 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 6181 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 6241 ttgggaagag aatagcaggc atgctgggga gcggccgcag gaacccctag tgatggagtt 6301 ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg 6361 acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct gcctgcaggg 6421 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca 6481 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 6541 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 6601 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 6661 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt 6721 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 6781 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat 6841 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt 6901 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact 6961 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc 7021 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 7081 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga 7141 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 7201 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 7261 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 7321 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 7381 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 7441 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 7501 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 7561 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 7621 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 7681 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 7741 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 7801 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 7861 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 7921 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 7981 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 8041 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 8101 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 8161 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 8221 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 8281 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 8341 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 8401 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 8461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 8521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 8581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 8641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 8701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 8761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 8821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 8881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 8941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 9001 ccttttgctc acatg //