LOCUS Exported 9363 bp ds-DNA circular SYN 16-AUG-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pCXCR4_TCS(Q'G)_NLS-HA-dCas9(C)-VP64_T2A_MCP-P65-HSF1+T283 SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 9363) 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..9363 /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..1961 /codon_start=1 /label=CXCR4 /translation="MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIY SIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYF GNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIP ALLLTIPDFIFANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCII ISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVH KWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSV STESESSSFHSS" CDS 1962..2060 /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 2061..2081 /codon_start=1 /label=efficient TEV cleavage site /translation="ENLYFQG" CDS 2082..2102 /codon_start=1 /label=SV40 NLS /translation="PKKKRKV" CDS 2121..2147 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" misc_feature 2148..2156 /label=GGS linker misc_feature 2163..2174 /note="(GGGGS)3 linker" CDS 2175..4673 /codon_start=1 /note="hSpCas9" /translation="KPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEI SGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTY AHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIH DDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPE NIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEV VKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQI LDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIME RSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALP SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLD KVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL IHQSITGLYETRIDLSQLGGD" misc_feature 4674..4706 /note="NLS" CDS 4707..4871 /codon_start=1 /note="VP64" /translation="GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDAL DDFDLDMLIN" CDS 4893..4946 /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 4959..5348 /codon_start=1 /label=MCP /translation="MASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKV TCSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCEL IVKAMQGLLKDGNPIPSAIAANSGIY" misc_feature 5400..5432 /label=NLS CDS 5439..5990 /codon_start=1 /label=P65 /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED FSSIADMDFSALLSQISSSGQ" CDS 6006..6377 /codon_start=1 /label=HSF1 /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED PTISLLTGSEPPKAKDPTVS" misc_feature 6387..6618 /note="bGH polyA" misc_feature 6627..6767 /label=R-ITR rep_origin 6859..7165 /direction=RIGHT /label=f1 Origin misc_feature 7684..8541 /label=AmpR rep_origin 8692..9359 /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 tcgatatgga ggggatatca atctacacat cagataatta cacggaagag atgggttccg 961 gcgattacga ctctatgaaa gagccgtgtt ttagagagga aaacgcgaac tttaacaaga 1021 tctttctccc caccatctac agcatcatct ttctcacagg catcgtaggg aacggcctgg 1081 tcatcctggt gatgggatac caaaagaagc tgaggtcaat gaccgacaag tataggctcc 1141 atctgtccgt ggccgacctc ctgtttgtga ttaccctgcc tttttgggca gttgacgctg 1201 tcgctaattg gtacttcggc aacttcctct gtaaggcagt gcacgttatc tacactgtga 1261 atctttatag ttccgtcttg atcttggcct ttatcagtct cgacaggtat ttggcgattg 1321 tgcacgctac caactcacaa cgacctagaa aactcctggc tgagaaggtg gtttacgttg 1381 gtgtgtggat tccagctctc ctgttgacaa taccagactt cattttcgct aatgtgagcg 1441 aggccgatga cagatacatt tgtgaccgat tttacccaaa cgatctgtgg gtagtggtat 1501 ttcagttcca acacattatg gtcgggctga tcttgcccgg cattgtcata ctgtcttgct 1561 actgcatcat tatttctaag ctgtcacact caaaaggcca ccaaaagagg aaggctctga 1621 aaacaacggt gatcctgata ctggccttct tcgcatgttg gctgccctac tatatcggca 1681 tcagcattga ctcatttata ctcctggaaa ttatcaagca gggctgcgag ttcgagaaca 1741 ccgttcataa gtggatttct ataaccgagg ccctcgcctt ctttcactgt tgtttgaatc 1801 cgattctcta cgcgtttctt ggcgccaaat ttaaaacaag cgcccaacat gcactgacat 1861 cagtgtctag ggggagctct ctgaaaatcc tctccaaggg aaaacgaggc ggacatagca 1921 gtgtcagcac tgagtccgaa tccagctcat ttcatagctc tatcgatacc ggtggacgca 1981 ccccacccag cctgggtccc caagatgagt cctgcaccac cgccagctcc tccctggcca 2041 aggacacttc atcgaccggt gagaacctgt acttccaggg ccccaagaag aagaggaagg 2101 tggggggagg cagcactagt tacccatacg acgtaccaga ttacgctgga ggttctggat 2161 ccggcggtgg ctcgaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc 2221 tgctgttcaa gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga 2281 aaatcgagtg cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc 2341 tgggcacata ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg 2401 aaaacgagga cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga 2461 tgatcgagga acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc 2521 tgaagcggcg gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc 2581 gggacaagca gtccggcaag acaatcctgg atttcctgaa gtccgacggc ttcgccaaca 2641 gaaacttcat gcagctgatc cacgacgaca gcctgacctt taaagaggac atccagaaag 2701 cccaggtgtc cggccagggc gatagcctgc acgagcacat tgccaatctg gccggcagcc 2761 ccgccattaa gaagggcatc ctgcagacag tgaaggtggt ggacgagctc gtgaaagtga 2821 tgggccggca caagcccgag aacatcgtga tcgaaatggc cagagagaac cagaccaccc 2881 agaagggaca gaagaacagc cgcgagagaa tgaagcggat cgaagagggc atcaaagagc 2941 tgggcagcca gatcctgaaa gaacaccccg tggaaaacac ccagctgcag aacgagaagc 3001 tgtacctgta ctacctgcag aatgggcggg atatgtacgt ggaccaggaa ctggacatca 3061 accggctgtc cgactacgat gtggaccata tcgtgcctca gagctttctg aaggacgact 3121 ccatcgacaa caaggtgctg accagaagcg acaaggcccg gggcaagagc gacaacgtgc 3181 cctccgaaga ggtcgtgaag aagatgaaga actactggcg gcagctgctg aacgccaagc 3241 tgattaccca gagaaagttc gacaatctga ccaaggccga gagaggcggc ctgagcgaac 3301 tggataaggc cggcttcatc aagagacagc tggtggaaac ccggcagatc acaaagcacg 3361 tggcacagat cctggactcc cggatgaaca ctaagtacga cgagaatgac aagctgatcc 3421 gggaagtgaa agtgatcacc ctgaagtcca agctggtgtc cgatttccgg aaggatttcc 3481 agttttacaa agtgcgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg 3541 ccgtcgtggg aaccgccctg atcaaaaagt accctaagct ggaaagcgag ttcgtgtacg 3601 gcgactacaa ggtgtacgac gtgcggaaga tgatcgccaa gagcgagcag gaaatcggca 3661 aggctaccgc caagtacttc ttctacagca acatcatgaa ctttttcaag accgagatta 3721 ccctggccaa cggcgagatc cggaagcggc ctctgatcga gacaaacggc gaaaccgggg 3781 agatcgtgtg ggataagggc cgggattttg ccaccgtgcg gaaagtgctg agcatgcccc 3841 aagtgaatat cgtgaaaaag accgaggtgc agacaggcgg cttcagcaaa gagtctatcc 3901 tgcccaagag gaacagcgat aagctgatcg ccagaaagaa ggactgggac cctaagaagt 3961 acggcggctt cgacagcccc accgtggcct attctgtgct ggtggtggcc aaagtggaaa 4021 agggcaagtc caagaaactg aagagtgtga aagagctgct ggggatcacc atcatggaaa 4081 gaagcagctt cgagaagaat cccatcgact ttctggaagc caagggctac aaagaagtga 4141 aaaaggacct gatcatcaag ctgcctaagt actccctgtt cgagctggaa aacggccgga 4201 agagaatgct ggcctctgcc ggcgaactgc agaagggaaa cgaactggcc ctgccctcca 4261 aatatgtgaa cttcctgtac ctggccagcc actatgagaa gctgaagggc tcccccgagg 4321 ataatgagca gaaacagctg tttgtggaac agcacaagca ctacctggac gagatcatcg 4381 agcagatcag cgagttctcc aagagagtga tcctggccga cgctaatctg gacaaagtgc 4441 tgtccgccta caacaagcac cgggataagc ccatcagaga gcaggccgag aatatcatcc 4501 acctgtttac cctgaccaat ctgggagccc ctgccgcctt caagtacttt gacaccacca 4561 tcgaccggaa gaggtacacc agcaccaaag aggtgctgga cgccaccctg atccaccaga 4621 gcatcaccgg cctgtacgag acacggatcg acctgtctca gctgggaggc gacagcccca 4681 agaagaagag aaaggtggag gccagcggac gggctgacgc attggacgat tttgatctgg 4741 atatgctggg aagtgacgcc ctcgatgatt ttgaccttga catgcttggt tcggatgccc 4801 ttgatgactt tgacctcgac atgctcggca gtgacgccct tgatgatttc gacctggaca 4861 tgctgattaa cggtaccgct agcggcagtg gagagggcag aggaagtctg ctaacatgcg 4921 gtgacgtcga ggagaatcct ggcccagtga gcaagcttat ggcttcaaac tttactcagt 4981 tcgtgctcgt ggacaatggt gggacagggg atgtgacagt ggctccttct aatttcgcta 5041 atggggtggc agagtggatc agctccaact cacggagcca ggcctacaag gtgacatgca 5101 gcgtcaggca gtctagtgcc cagaagagaa agtataccat caaggtggag gtccccaaag 5161 tggctaccca gacagtgggc ggagtcgaac tgcctgtcgc cgcttggagg tcctacctga 5221 acatggagct cactatccca attttcgcta ccaattctga ctgtgaactc atcgtgaagg 5281 caatgcaggg gctcctcaaa gacggtaatc ctatcccttc cgccatcgcc gctaactcag 5341 gtatctacag cgctggagga ggtggaagcg gaggaggagg aagcggagga ggaggtagcg 5401 gacctaagaa aaagaggaag gtggcggccg ctggatcccc ttcagggcag atcagcaacc 5461 aggccctggc tctggcccct agctccgctc cagtgctggc ccagactatg gtgccctcta 5521 gtgctatggt gcctctggcc cagccacctg ctccagcccc tgtgctgacc ccaggaccac 5581 cccagtcact gagcgctcca gtgcccaagt ctacacaggc cggcgagggg actctgagtg 5641 aagctctgct gcacctgcag ttcgacgctg atgaggacct gggagctctg ctggggaaca 5701 gcaccgatcc cggagtgttc acagatctgg cctccgtgga caactctgag tttcagcagc 5761 tgctgaatca gggcgtgtcc atgtctcata gtacagccga accaatgctg atggagtacc 5821 ccgaagccat tacccggctg gtgaccggca gccagcggcc ccccgacccc gctccaactc 5881 ccctgggaac cagcggcctg cctaatgggc tgtccggaga tgaagacttc tcaagcatcg 5941 ctgatatgga ctttagtgcc ctgctgtcac agatttcctc tagtgggcag ggaggaggtg 6001 gaagcggctt cagcgtggac accagtgccc tgctggacct gttcagcccc tcggtgaccg 6061 tgcccgacat gagcctgcct gaccttgaca gcagcctggc cagtatccaa gagctcctgt 6121 ctccccagga gccccccagg cctcccgagg cagagaacag cagcccggat tcagggaagc 6181 agctggtgca ctacacagcg cagccgctgt tcctgctgga ccccggctcc gtggacaccg 6241 ggagcaacga cctgccggtg ctgtttgagc tgggagaggg ctcctacttc tccgaagggg 6301 acggcttcgc cgaggacccc accatctccc tgctgacagg ctcggagcct cccaaagcca 6361 aggaccccac tgtctcctaa gaattcctag agctcgctga tcagcctcga ctgtgccttc 6421 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 6481 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 6541 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa 6601 tagcaggcat gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc 6661 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt 6721 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg 6781 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 6841 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 6901 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 6961 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 7021 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 7081 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 7141 ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 7201 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 7261 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 7321 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 7381 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 7441 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 7501 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 7561 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 7621 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 7681 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 7741 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 7801 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 7861 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 7921 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 7981 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 8041 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 8101 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 8161 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 8221 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 8281 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 8341 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtgga 8401 agccgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 8461 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 8521 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 8581 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 8641 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 8701 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 8761 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 8821 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 8881 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 8941 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 9001 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 9061 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 9121 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 9181 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 9241 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 9301 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 9361 atg //