LOCUS Exported 7364 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION Mammalian Gateway-compatible expression vector backbone with an N-terminal 3xFLAG-V5 tag. ACCESSION . VERSION . KEYWORDS pcDNA3.1-3xFLAG-V5-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7364) AUTHORS Taipale M, Krykbaeva I, Koeva M, Kayatekin C, Westover KD, Karras GI, Lindquist S TITLE Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition. JOURNAL Cell. 2012 Aug 31;150(5):987-1001. doi: 10.1016/j.cell.2012.06.047. PUBMED 22939624 REFERENCE 2 (bases 1 to 7364) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7364 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 181..201 /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" rep_origin complement(210..638) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(320..341) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 532..551 /label=F1ori-R /note="F1 origin, reverse primer" polyA_signal 684..908 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind 897..914 /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" protein_bind 965..1089 /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(1130..1435) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(1232..1251) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(1777..2436) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 2351..2370 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(2490..2520) /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" protein_bind complement(2545..2669) /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(2667..2708) /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS complement(2727..2792) /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" primer_bind complement(2861..2885) /label=LNCX /note="Human CMV promoter, forward primer" promoter complement(2882..3085) /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind complement(2911..2931) /label=CMV-F /note="Human CMV immediate early promoter, forward primer" enhancer 3086..3465 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" primer_bind 3637..3656 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3731..3835 /gene="bla" /label=AmpR promoter CDS 3836..4696 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(4054..4073) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 4867..5455 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5356..5375 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5609..5626 /label=L4440 /note="L4440 vector, forward primer" protein_bind 5743..5764 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 5779..5809 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5817..5833 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 5822..5844 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 5841..5857 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 5841..5857 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" polyA_signal 5906..6027 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5917..5936) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5971..5990 /label=SV40pA-R /note="SV40 polyA, reverse primer" CDS complement(6157..7182) /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" promoter complement(7231..196) /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 7245..16 /label=SV40 ori /note="SV40 origin of replication" primer_bind complement(7299..7318) /label=SV40pro-F /note="SV40 promoter/origin, forward primer" ORIGIN 1 gagttagggg cgggatgggc ggagttaggg gcgggactat ggttgctgac taattgagat 61 gcatgctttg catacttctg cctgctgggg agcctgggga ctttccacac ctggttgctg 121 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac 181 accctaactg acacacattc cacagaatta attcgcgtta aatttttgtt aaatcagctc 241 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 301 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 361 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 421 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 481 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 541 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 601 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc tagagcccca 661 gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat gcctgctatt 721 gtcttcccaa tcctccccct tgctgtcctg ccccacccca ccccccagaa tagaatgaca 781 cctactcaga caatgcgatg caatttcctc attttattag gaaaggacag tgggagtggc 841 accttccagg gtcaaggaag gcacggggga ggggcaaaca acagatggct ggcaactaga 901 aggcacagtc gaggctgatc agcgggttta aacgggccct ctagactcga gttactatag 961 agcaaccact ttgtacaaga aagctgaacg agaaacgtaa aatgatataa atatcaatat 1021 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca 1081 gtcactatgg tcgacctgca gactggctgt gtataaggga gcctgacatt tatattcccc 1141 agaacatcag gttaatggcg tttttgatgt cattttcgcg gtggctgaga tcagccactt 1201 cttccccgat aacggagacc ggcacactgg ccatatcggt ggtcatcatg cgccagcttt 1261 catccccgat atgcaccacc gggtaaagtt cacgggagac tttatctgac agcagacgtg 1321 cactggccag ggggatcacc atccgtcgcc cgggcgtgtc aataatatca ctctgtacat 1381 ccacaaacag acgataacgg ctctctcttt tataggtgta aaccttaaac tgcatttcac 1441 cagcccctgt tctcgtcagc aaaagagccg ttcatttcaa taaaccgggc gacctcagcc 1501 atcccttcct gattttccgc tttccagcgt tcggcacgca gacgacgggc ttcattctgc 1561 atggttgtgc ttaccagacc ggagatattg acatcatata tgccttgagc aactgatagc 1621 tgtcgctgtc aactgtcact gtaatacgct gcttcatagc atacctcttt ttgacatact 1681 tcgggtatac atatcagtat atattcttat accgcaaaaa tcagcgcgca aatacgcata 1741 ctgttatctg gcttttagta agccggatcc agatctttac gccccgccct gccactcatc 1801 gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac aaacggcatg 1861 atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat 1921 ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa 1981 actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata 2041 ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa 2101 atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt 2161 gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg ccatacggaa 2221 ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg 2281 cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata 2341 ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat 2401 atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa 2461 tctcgacgga tcctaactca aaatccacac attatacgag ccggaagcat aaagtgtaaa 2521 gcctggggtg cctaatgcgg ccgccatagt gactggatat gttgtgtttt acagtattat 2581 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt 2641 tctcgttcag cttttttgta caaacttgta gaatcgagac cgaggagagg gttagggata 2701 ggcttacccg aggtcgagcc cgagcccttg tcatcgtcat ccttgtagtc gatgtcatga 2761 tctttataat caccgtcatg gtctttgtag tccatggctg gatcctctag agtcggtgtc 2821 ttctatggag gtcaaaacag cgtggatggc gtctccaggc gatctgacgg ttcactaaac 2881 gagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg 2941 cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat 3001 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat 3061 tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact 3121 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac 3181 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt 3241 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta 3301 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag 3361 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg 3421 accccgtaat tgattactat taataactag tcaataatca atgtcaacgc gtatatctgg 3481 cccgtacatc gcgaagcagc gcaaaacgcc taaccctaag cagattcttc atgcaattgt 3541 cggtcaagcc ttgccttgtt gtagcttaaa ttttgctcgc gcactactca gcgacctcca 3601 acacacaagc agggagcaga tactggctta actatgcggc atcagagcag attgtactga 3661 gagtgcacca taggggatcg ggagatctcc cgatccgtcg acgtcaggtg gcacttttcg 3721 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 3781 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 3841 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 3901 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 3961 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4021 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat 4081 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4141 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 4201 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 4261 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 4321 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 4381 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 4441 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 4501 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 4561 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 4621 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 4681 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 4741 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 4801 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 4861 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 4921 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 4981 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca 5041 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5101 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 5161 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagtg 5221 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 5281 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 5341 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 5401 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 5461 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 5521 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 5581 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 5641 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga 5701 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac 5761 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt 5821 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctctagct 5881 agaggtcgac ggtatacaga catgataaga tacattgatg agtttggaca aaccacaact 5941 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 6001 accattataa gctgcaataa acaagttggg gtgggcgaag aactccagca tgagatcccc 6061 gcgctggagg atcatccagc cggcgtcccg gaaaacgatt ccgaagccca acctttcata 6121 gaaggcggcg gtggaatcga aatctcgtag cacgtgctat tcctttgccc tcggacgagt 6181 gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc 6241 cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc 6301 gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata 6361 gagttggtca agaccaatgc ggagcatata cgcccggagc cgcggcgatc ctgcaagctc 6421 cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca 6481 gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat 6541 gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac 6601 gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc 6661 gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc 6721 aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg 6781 gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac 6841 cggctgcaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc 6901 acggcgggag atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg 6961 aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc 7021 ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg 7081 agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag 7141 aaacttctcg acagacgtcg cggtgagttc aggctttttc atcacgtgct gatcagatcc 7201 gaaaatggat atacaagctc ccgggagctt tttgcaaaag cctaggcctc caaaaaagcc 7261 tcctcactac ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa 7321 aaaaattagt cagccatggg gcggagaatg ggcggaactg ggcg //