| 质粒名称: | pcDNA3.1-ccdB-3xFLAG-V5 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 87063 |
| 存储实验室: | Susan Lindquist, Mikko Taipale |
|
载体骨架基本信息 |
|
| 载体名称: | pcDNA3.1 |
| 载体出品公司: | Invitrogen |
| 载体抗性: | 氯霉素和氨苄西林 |
| 载体类型: | 哺乳动物的表达 |
| 筛选标记: | 新霉素（G418） |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
文献引用方法 |
|
| 材料和方法部分: | pcDNA3.1-ccdB-3xFLAG-V5 was a gift from Susan Lindquist & Mikko Taipale (Addgene plasmid # 87063 ; http://n2t.net/addgene:87063 ; RRID:Addgene_87063) |
| 参考文献部分: | Quantitative analysis of HSP90-client interactions reveals principles of substrate recognition. Taipale M, Krykbaeva I, Koeva M, Kayatekin C, Westover KD, Karras GI, Lindquist S. Cell. 2012 Aug 31;150(5):987-1001. doi: 10.1016/j.cell.2012.06.047. 10.1016/j.cell.2012.06.047 PubMed 22939624 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pcDNA3.1-ccdB-3xFLAG-V5 7412 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Mammalian Gateway-compatible expression vector backbone with a
C-terminal 3xFLAG-V5 tag.
ACCESSION .
VERSION .
KEYWORDS pcDNA3.1-ccdB-3xFLAG-V5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7412)
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 7412)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7412
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(44..63)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 769..789
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 863..882
/label=T7
/note="T7 promoter, forward primer"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 969..1093
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 1118..1148
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 1202..1861
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(1268..1287)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
CDS 2203..2508
/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 2387..2406
/label=ccdB-fwd
/note="ccdB gene, forward primer"
protein_bind complement(2549..2673)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS 2675..2740
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 2759..2800
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
primer_bind complement(2844..2861)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 2850..2961
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 3110..3538
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(3197..3216)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 3407..3428
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(3547..3567)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 3552..3881
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 3732..3867
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3794..3813
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3930..4955
/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"
polyA_signal 5085..5206
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(5122..5141)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 5176..5195
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(5255..5271)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(5255..5271)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(5268..5290)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 5279..5295
/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)."
promoter complement(5303..5333)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 5348..5369
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5486..5503)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5657..6245)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(5737..5756)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(6416..7276)
/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 7039..7058
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(7277..7381)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
961 agatatcaac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat ataaatatca
1021 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
1081 tccagtcact atggcggccg cattaggcac cccaggcttt acactttatg cttccggctc
1141 gtataatgtg tggattttga gttaggatcc gtcgagattt tcaggagcta aggaagctaa
1201 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
1261 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
1321 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
1381 tcacattctt gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg
1441 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
1501 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
1561 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
1621 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
1681 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
1741 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca
1801 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
1861 aagatctgga tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
1921 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct
1981 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
2041 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
2101 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
2161 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
2221 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
2281 acgcccgggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
2341 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
2401 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
2461 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
2521 cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta
2581 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
2641 cgtttctcgt tcagctttct tgtacaaagt ggttgattac aaagaccatg acggtgatta
2701 taaagatcat gacatcgact acaaggatga cgatgacaag ggctcgggct cgacctcggg
2761 taagcctatc cctaaccctc tcctcggtct cgattctaca tgataactcg agtctagagg
2821 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
2881 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
2941 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
3001 ggtggggcag gacagcaagg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
3061 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
3121 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
3181 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
3241 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
3301 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
3361 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
3421 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
3481 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
3541 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
3601 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
3661 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
3721 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
3781 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
3841 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
3901 tatccatttt cggatctgat cagcacgtga tgaaaaagcc tgaactcacc gcgacgtctg
3961 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
4021 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
4081 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
4141 cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca
4201 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
4261 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
4321 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
4381 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
4441 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
4501 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
4561 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
4621 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
4681 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
4741 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
4801 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
4861 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
4921 gacgccccag cactcgtccg agggcaaagg aatagcacgt gctacgagat ttcgattcca
4981 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
5041 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
5101 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
5161 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
5221 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
5281 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
5341 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
5401 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
5461 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
5521 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
5581 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
5641 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
5701 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
5761 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
5821 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
5881 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
5941 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
6001 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
6061 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
6121 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
6181 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
6241 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
6301 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
6361 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
6421 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
6481 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
6541 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
6601 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
6661 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
6721 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
6781 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
6841 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
6901 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
6961 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
7021 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
7081 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
7141 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
7201 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
7261 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
7321 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
7381 gcacatttcc ccgaaaagtg ccacctgacg tc
//
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