LOCUS Exported 9480 bp ds-DNA circular SYN 15-AUG-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pBDKRB2_TCS(Q'G)_NLS-HA-dCas9(C)-VP64_T2A_MCP-P65-HSF1+T272 SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 9480) AUTHORS Weatherall Institute TITLE Direct Submission JOURNAL Exported Tuesday, Aug 15, 2017 from SnapGene 3.3.4 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9480 /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 4824..4988 /codon_start=1 /note="VP64" /translation="GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDAL DDFDLDMLIN" CDS 5010..5063 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 5076..5465 /codon_start=1 /label=MCP /translation="MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKV TCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCEL IVKAMQGLLKDGNPIPSAIAANSGIY" misc_feature 5517..5549 /label=NLS CDS 5556..6107 /codon_start=1 /label=P65 /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED FSSIADMDFSALLSQISSSGQ" CDS 6123..6494 /codon_start=1 /label=HSF1 /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED PTISLLTGSEPPKAKDPTVS" misc_feature 6504..6735 /note="bGH polyA" misc_feature 6744..6884 /label=R-ITR rep_origin 6976..7282 /direction=RIGHT /label=f1 Origin misc_feature 7801..8658 /label=AmpR rep_origin 8809..9476 /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 agcggacggg ctgacgcatt ggacgatttt gatctggata 4861 tgctgggaag tgacgccctc gatgattttg accttgacat gcttggttcg gatgcccttg 4921 atgactttga cctcgacatg ctcggcagtg acgcccttga tgatttcgac ctggacatgc 4981 tgattaacgg taccgctagc ggcagtggag agggcagagg aagtctgcta acatgcggtg 5041 acgtcgagga gaatcctggc ccagtgagca agcttatggc ttcaaacttt actcagttcg 5101 tgctcgtgga caatggtggg acaggggatg tgacagtggc tccttctaat ttcgctaatg 5161 gggtggcaga gtggatcagc tccaactcac ggagccaggc ctacaaggtg acatgcagcg 5221 tcaggcagtc tagtgcccag aagagaaagt ataccatcaa ggtggaggtc cccaaagtgg 5281 ctacccagac agtgggcgga gtcgaactgc ctgtcgccgc ttggaggtcc tacctgaaca 5341 tggagctcac tatcccaatt ttcgctacca attctgactg tgaactcatc gtgaaggcaa 5401 tgcaggggct cctcaaagac ggtaatccta tcccttccgc catcgccgct aactcaggta 5461 tctacagcgc tggaggaggt ggaagcggag gaggaggaag cggaggagga ggtagcggac 5521 ctaagaaaaa gaggaaggtg gcggccgctg gatccccttc agggcagatc agcaaccagg 5581 ccctggctct ggcccctagc tccgctccag tgctggccca gactatggtg ccctctagtg 5641 ctatggtgcc tctggcccag ccacctgctc cagcccctgt gctgacccca ggaccacccc 5701 agtcactgag cgctccagtg cccaagtcta cacaggccgg cgaggggact ctgagtgaag 5761 ctctgctgca cctgcagttc gacgctgatg aggacctggg agctctgctg gggaacagca 5821 ccgatcccgg agtgttcaca gatctggcct ccgtggacaa ctctgagttt cagcagctgc 5881 tgaatcaggg cgtgtccatg tctcatagta cagccgaacc aatgctgatg gagtaccccg 5941 aagccattac ccggctggtg accggcagcc agcggccccc cgaccccgct ccaactcccc 6001 tgggaaccag cggcctgcct aatgggctgt ccggagatga agacttctca agcatcgctg 6061 atatggactt tagtgccctg ctgtcacaga tttcctctag tgggcaggga ggaggtggaa 6121 gcggcttcag cgtggacacc agtgccctgc tggacctgtt cagcccctcg gtgaccgtgc 6181 ccgacatgag cctgcctgac cttgacagca gcctggccag tatccaagag ctcctgtctc 6241 cccaggagcc ccccaggcct cccgaggcag agaacagcag cccggattca gggaagcagc 6301 tggtgcacta cacagcgcag ccgctgttcc tgctggaccc cggctccgtg gacaccggga 6361 gcaacgacct gccggtgctg tttgagctgg gagagggctc ctacttctcc gaaggggacg 6421 gcttcgccga ggaccccacc atctccctgc tgacaggctc ggagcctccc aaagccaagg 6481 accccactgt ctcctaagaa ttcctagagc tcgctgatca gcctcgactg tgccttctag 6541 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac 6601 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca 6661 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagagaatag 6721 caggcatgct ggggagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct 6781 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 6841 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta 6901 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac 6961 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 7021 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 7081 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 7141 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca 7201 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 7261 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa 7321 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 7381 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc 7441 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 7501 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 7561 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 7621 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 7681 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 7741 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 7801 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 7861 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 7921 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 7981 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 8041 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 8101 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 8161 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 8221 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 8281 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 8341 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 8401 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 8461 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc 8521 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 8581 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 8641 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 8701 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 8761 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 8821 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 8881 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 8941 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 9001 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 9061 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 9121 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 9181 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 9241 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 9301 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 9361 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 9421 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg //