LOCUS Exported 9531 bp ds-DNA circular SYN 15-AUG-2017 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pVEGFR2_TEV(N)_NES_TCS(Q'L)_HA-dCas9(N)_P2A_Puro-WPRE+T262 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9531) 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..9531 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 12..810 /note="CBh" CDS 823..3177 /codon_start=1 /label=KDR /translation="MQSKVLLAVALWLCVETRAASVGLPSVSLDLPRLSIQKDILTIKA NTTLQITCRGQRDLDWLWPNNQSGSEQRVEVTECSDGLFCKTLTIPKVIGNDTGAYKCF YRETDLASVIYVYVQDYRSPFIASVSDQHGVVYITENKNKTVVIPCLGSISNLNVSLCA RYPEKRFVPDGNRISWDSKKGFTIPSYMISYAGMVFCEAKINDESYQSIMYIVVVVGYR IYDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQS GSEMKKFLSTLTIDGITRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLV EATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVI LTNPISKEKQSHVVSLVVYVPPQIGEKSLISPVDSYQYGTTQTLTCTVYAIPPPHHIHW YWQLEEECANEPSQAVSVTNPYPCEEWRSVEDFQGGNKIEVNKNQFALIEGKNKTVSTL VIQAANVSALYKCEAVNKVGRGERVISFHVTRGPEITLQPDMQPTEQESVSLWCTADRS TFENLTWYKLGPQPLPIHVGELPTPVCKNLDTLWKLNATMFSNSTNDILIMELKNASLQ DQGDYVCLAQDRKTKKRHCVVRQLTVLERVAPTITGNLENQTTSIGESIEVSCTASGNP PPQIMWFKDNETLVEDSGIVLKDGNRNLTIRRVRKEDEGLYTCQACSVLGCAKVEAFFI IEGAQEKTNLEIIILVGTAVIAMFFWLLLVII" CDS 880..3114 /product="http://www.uniprot.org/blast/?about=P35968[20-764 ]&key=Topological%20domain" /label=ExtracellularSequence misc_feature 1711 /label=A mutation from Val to Ile (from original vector) CDS 3202..3555 /codon_start=1 /label=TEV protease /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ REERICLVTTNFQT" CDS 3568..3591 /codon_start=1 /label=NES ptk2 /translation="LDLASLIL" CDS 3604..3624 /codon_start=1 /label=TCS (Q'L) /translation="ENLYFQL" CDS 3643..3669 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" CDS 3685..5286 /codon_start=1 /note="hSpCas9" /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMR" misc_feature 5287..5304 /note="(GGGGS)3 linker" CDS 5307 /codon_start=1 /label=rtTA /protein_id="rtTA-M2" /translation="" CDS 5314..5373 /codon_start=1 /label=P2A /translation="GATNFSLLKQAGDVEENPGP" CDS 5380..5973 /codon_start=1 /label=Puro /protein_id="Puro" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKG" protein_bind 6010..6537 /label=protein bind misc_feature 6555..6786 /note="bGH polyA" misc_feature 6795..6935 /label=R-ITR rep_origin 7027..7333 /direction=RIGHT /label=f1 Origin misc_feature 7852..8709 /label=AmpR rep_origin 8860..9527 /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 accggtgcca ccatgcagag caaggtgctg 841 ctggccgtcg ccctgtggct ctgcgtggag acccgggccg cctctgtggg tttgcctagt 901 gtttctcttg atctgcccag gctcagcata caaaaagaca tacttacaat taaggctaat 961 acaactcttc aaattacttg caggggacag agggacttgg actggctttg gcccaataat 1021 cagagtggca gtgagcaaag ggtggaggtg actgagtgca gcgatggcct cttctgtaag 1081 acactcacaa ttccaaaagt gatcggaaat gacactggag cctacaagtg cttctaccgg 1141 gaaactgact tggcctcggt catttatgtc tatgttcaag attacagatc tccatttatt 1201 gcttctgtta gtgaccaaca tggagtcgtg tacattactg agaacaaaaa caaaactgtg 1261 gtgattccat gtctcgggtc catttcaaat ctcaacgtgt cactttgtgc aagataccca 1321 gaaaagagat ttgttcctga tggtaacaga atttcctggg acagcaagaa gggctttact 1381 attcccagct acatgatcag ctatgctggc atggtcttct gtgaagcaaa aattaatgat 1441 gaaagttacc agtctattat gtacatagtt gtcgttgtag ggtataggat ttatgatgtg 1501 gttctgagtc cgtctcatgg aattgaacta tctgttggag aaaagcttgt cttaaattgt 1561 acagcaagaa ctgaactaaa tgtggggatt gacttcaact gggaataccc ttcttcgaag 1621 catcagcata agaaacttgt aaaccgagac ctaaaaaccc agtctgggag tgagatgaag 1681 aaatttttga gcaccttaac tatagatggt ataacccgga gtgaccaagg attgtacacc 1741 tgtgcagcat ccagtgggct gatgaccaag aagaacagca catttgtcag ggtccatgaa 1801 aaaccttttg ttgcttttgg aagtggcatg gaatctctgg tggaagccac ggtgggggag 1861 cgtgtcagaa tccctgcgaa gtaccttggt tacccacccc cagaaataaa atggtataaa 1921 aatggaatac cccttgagtc caatcacaca attaaagcgg ggcatgtact gacgattatg 1981 gaagtgagtg aaagagacac aggaaattac actgtcatcc ttaccaatcc catttcaaag 2041 gagaagcaga gccatgtggt ctctctggtt gtgtatgtcc caccccagat tggtgagaaa 2101 tctctaatct ctcctgtgga ttcctaccag tacggcacca ctcaaacgct gacatgtacg 2161 gtctatgcca ttcctccccc gcatcacatc cactggtatt ggcagttgga ggaagagtgc 2221 gccaacgagc ccagccaagc tgtctcagtg acaaacccat acccttgtga agaatggaga 2281 agtgtggagg acttccaggg aggaaataaa attgaagtta ataaaaatca atttgctcta 2341 attgaaggaa aaaacaaaac tgtaagtacc cttgttatcc aagcggcaaa tgtgtcagct 2401 ttgtacaaat gtgaagcggt caacaaagtc gggagaggag agagggtgat ctccttccac 2461 gtgaccaggg gtcctgaaat tactttgcaa cctgacatgc agcccactga gcaggagagc 2521 gtgtctttgt ggtgcactgc agacagatct acgtttgaga acctcacatg gtacaagctt 2581 ggcccacagc ctctgccaat ccatgtggga gagttgccca cacctgtttg caagaacttg 2641 gatactcttt ggaaattgaa tgccaccatg ttctctaata gcacaaatga cattttgatc 2701 atggagctta agaatgcatc cttgcaggac caaggagact atgtctgcct tgctcaagac 2761 aggaagacca agaaaagaca ttgcgtggtc aggcagctca cagtcctaga gcgtgtggca 2821 cccacgatca caggaaacct ggagaatcag acgacaagta ttggggaaag catcgaagtc 2881 tcatgcacgg catctgggaa tccccctcca cagatcatgt ggtttaaaga taatgagacc 2941 cttgtagaag actcaggcat tgtattgaag gatgggaacc ggaacctcac tatccgcaga 3001 gtgaggaagg aggacgaagg cctctacacc tgccaggcat gcagtgttct tggctgtgca 3061 aaagtggagg catttttcat aatagaaggt gcccaggaaa agacgaactt ggaaatcatt 3121 attctagtag gcacggcggt gattgccatg ttcttctggc tacttcttgt catcatcggc 3181 ggtggctcgg gtggtggctc gggagaaagc ttgtttaagg ggccgcgtga ttacaacccg 3241 atatcgagca ccatttgtca tttgacgaat gaatctgatg ggcacacaac atcgttgtat 3301 ggtattggat ttggtccctt catcattaca aacaagcact tgtttagaag aaataatgga 3361 acactgttgg tccaatcact acatggtgta ttcaaggtca agaacaccac gactttgcaa 3421 caacacctca ttgatgggag ggacatgata attattcgca tgcctaagga tttcccacca 3481 tttcctcaaa agctgaaatt tagagagcca caaagggaag agcgcatatg tcttgtgaca 3541 accaacttcc aaactggcgg tggctcgcta gatttagcta gcttaatcct cggcaagctt 3601 ggcgaaaacc tgtattttca gctaggcgga ggcagcacta gttacccata cgacgtacca 3661 gattacgctg gaggttctgg atccgacaag aagtacagca tcggcctggc catcggcacc 3721 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag 3781 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc 3841 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc 3901 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg 3961 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac 4021 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc 4081 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg 4141 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac 4201 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac 4261 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct 4321 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag 4381 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag 4441 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac 4501 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc 4561 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc 4621 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc 4681 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac 4741 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac 4801 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg 4861 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag 4921 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg 4981 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc 5041 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc 5101 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag 5161 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg 5221 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga 5281 atgagaggtg gcggtggctc gggtaccgga tctggagcca ccaatttttc tctgctgaag 5341 caggctggtg atgttgaaga aaaccctgga cctgaattca tgaccgagta caagcccacg 5401 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc 5461 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc 5521 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg 5581 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg 5641 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa 5701 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc 5761 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga 5821 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac 5881 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga 5941 ccgcgcacct ggtgcatgac ccgcaagggg taactaagta aggatcgatc caagatatcg 6001 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 6061 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 6121 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 6181 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 6241 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 6301 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac 6361 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg 6421 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 6481 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 6541 ctccccgcca attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 6601 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 6661 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 6721 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc 6781 tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 6841 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 6901 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct 6961 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 7021 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 7081 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 7141 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 7201 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 7261 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 7321 caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 7381 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 7441 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 7501 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 7561 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7621 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 7681 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 7741 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7801 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 7861 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 7921 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 7981 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 8041 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 8101 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 8161 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 8221 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 8281 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 8341 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 8401 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 8461 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 8521 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc 8581 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 8641 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 8701 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 8761 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 8821 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 8881 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 8941 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 9001 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 9061 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 9121 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 9181 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 9241 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 9301 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 9361 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 9421 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 9481 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat g //