pcDNA6.2/C-EmGFP-DEST

價格:3000元

聯系方式:I47-825O-882O

買家導航

載體基本信息

出品公司: Invitrigen
載體名稱: pcDNA6.2/C-EmGFP-DEST
質粒類型: 哺乳動物表達載體;Gateway載體;熒光報告載體
高拷貝/低拷貝: 高拷貝
啟動子: CMV
克隆方法: Gateway
載體大小: 7711bp
5' 測序引物及序列: T7 forward:5’-TAATACGACTCACTATAGGG-3'
3' 測序引物及序列: TK polyA Reverse: 5’-CTTCCGTGTTTCAGTTAGC-3’
載體標簽: EmGFP Tag (C Term)
載體抗性: 氨芐青霉素、氯霉素(僅空載體具有此抗性)
篩選標記: Blasticidin
克隆菌株: ccdB陽性篩選 DB3.1 感受態細胞, 陰性篩選用 OmniMAX2-T1 感受態細胞
表達細胞(系): 常規細胞系,如293、Hela等。
備注:
pcDNA6.2/C-EmGFP-DEST載體是cDNA表達載體,是綠色熒光報告載體;

CMV啟動子驅動目的蛋白的過表達;

綠色熒光報告基因EmGFP是第三代EGFP;

EmGFP在真核細胞和原核細胞中都能可溶性型表達。
 
產品目錄號: V356-20
穩定性: 瞬表達 或 穩表達
組成型/誘導型: 組成型
病毒/非病毒: 非病毒

載體質粒圖譜和多克隆位點信息

pcDNA6.2-C-EmGFP-DEST 載體圖譜



pcDNA6.2-C-EmGFP-DEST 多克隆位點

pcDNA6.2-C-EmGFP-DEST 特征位點
pcDNA6.2-C-EmGFP-DEST 載體特征1
pcDNA6.2-C-EmGFP-DEST 載體特征2

載體簡介

系統介紹

The Vivid Colors Fluorescent Protein Gateway Destination Vectors allow you to quickly and easily fuse a protein of interest to the widely used and well-characterized fluorescent proteinsfrom the jellyfish Aequorea victoria (1,2) using the pcDNA 6.2 Gateway mammalian expression vector. These powerful Gateway Technology vectors contain the next-generation EGFP, Emerald Green Fluorescent Protein (EmGFP), or the popular Yellow Fluorescent Protein (YFP) for simple, non-invasive detection of recombinant protein. Both fluorescent proteins have been humanizedfor optimal mammalian expression (3). In addition, the Vivid Colors pcDNA 6.2 Fluorescent Protein Gateway Vectors offer:

 CMV promoter for high-level expression of the recombinant fluorescent fusion protein
 Options to fuse EmGFP or YFP to the N- and C-terminus of your protein

Vivid Colors pcDNA6.2/EmGFP and YFP-DEST vectors combine the ease and flexibility of Gateway recombination based cloning with the brightness of Emerald Green Fluorescent Protein (EmGFP) and Yellow Fluorescent Protein (YFP) derived from Aequorea victoria GFP. Users can easily make an EmGFP or YFP N- or C-terminally tagged mammalian expression clone by performing an LR recombination reaction between a Gateway entry vector containing the gene of choice and a Vivid Colors pcDNA6.2/EmGFP or YFP-DEST Gateway Vector. After transfection of the expression clone into mammalian cells, the fluorescent-tagged protein of interest can be identified by fluorescence detection methods for localization experiments. The protein of interest can also be analyzed by Western blot.
The Vivid Colors pcDNA6.2/EmGFP or YFP-DEST Gateway Vectors are supplied with either an N- or C-terminal tagged EmGFP or YFP/GW/CAT plasmid that serves as a control for transfection efficiency of the expression clone into the target cells, as well as a control for expression of the gene of interest.


載體特征

pcDNA6.2/EmGFP-DEST載體含有以下元件:
 Human cytomegalovirus immediate-early (CMV) promoter/enhancer for high-level expression in a wide range of mammalian cells
 Two recombination sites, attR1 and attR2, downstream of the CMV promoter for recombinational cloning of the gene of interest from an entry clone
 Emerald Green Fluorescent Protein (EmGFP) or Yellow Fluorescent Protein (YFP) derived from Aequorea victoria GFP for N- or C-terminal fusion to the gene of interest for fluorescent detection
 The V5 epitope tag for detection of recombinant protein using Anti-V5 antibodies (optional on N-terminal fusion vectors only)
 CAT gene located between the two attR sites for counterselection
 The ccdB gene located between the two attR sites for negative selection
 The Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript
 f1 intergenic region for production of single-strand DNA in F plasmid-containing E. coli
 SV40 early promoter and origin for expression of the Blasticidin resistance gene and stable propagation of the plasmid in mammalian hosts expressing the SV40 large T antigen
 Blasticidin resistance gene for selection of stable cell lines
 The pUC origin for high copy replication and maintenance of the plasmid in E.coli
 The ampicillin (bla) resistance gene for selection in E. coli


綠色熒光蛋白(GFP)

Green Fluorescent Protein (GFP) is a naturally occurring bioluminescent protein derived from the jellyfish Aequorea victoria (Shimomura et al., 1962). GFP emits fluorescence upon excitation, and the gene encoding GFP contains all of the necessary information for posttranslational synthesis of the luminescent protein. GFP is often used as a molecular beacon because it requires no species-specific cofactors for function, and the fluorescence is easily detected using fluorescence microscopy and standard filter sets. Commonly, GFP is fused to a protein of interest, and upon expression, the localization of the fusion protein can be detected in cells. GFP can also function as a reporter gene downstream of a promoter of interest.

Modifications have been made to the wild-type GFP to enhance its expression in mammalian systems. These modifications include amino acid substitutions that correspond to the codon preference for mammalian use, and mutations that increase the brightness of the fluorescence signal, resulting in “enhanced” GFP (Zhang et al., 1996).
Mutations have also arisen or have been introduced into GFP that further enhance and shift the spectral properties of GFP such that these proteins will emit fluorescent color variations (reviewed in Tsien, 1998). The Emerald GFP (EmGFP) and Yellow Fluorescent Protein (YFP) are such variants of enhanced GFP.


LR重組反應所需實驗材料:

 Purified plasmid DNA of your entry clone
(50-150 ng/μL in TE, pH 8.0)
 Vivid Colors pcDNA6.2/EmGFP or YFP-DEST Gateway Vector (150 ng/μL in TE, pH 8.0)
 LR Clonase II enzyme mix (page 34; keep at –20°C until immediately before use)
 pENTR-gus (supplied with LR Clonase II enzyme mix; use as a control for the LR reaction; 50 ng/μL)
 TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
 2 μg/μL Proteinase K solution (supplied with LR Clonase II enzyme mix; thaw and keep on ice until use)
 Appropriate competent E. coli host and growth medium for expression
 S.O.C. Medium
 LB agar plates containing 100 μg/mL ampicillin



Gateway哺乳動物表達載體選擇指南:

pDEST12.2   不帶標簽

pDEST26     
N-His標簽      G418篩選

pDEST27     N-GST標簽    G418篩選

pDEST40       C-His標簽、C-V5標簽   G418篩選

pcDNA-DEST40       C-His標簽、C-V5標簽   G418篩選
 
pcDNA-DEST47           C-綠色熒光蛋白 G418篩選

pcDNA-DEST53           N-綠色熒光蛋白 G418篩選

pcDNA3.2/V5-DEST      C-V5標簽       G418篩選

pcDNA6.2/V5-DEST      C-V5標簽        Blasticidin S篩選

pcDNA6.2/N-YFP-DEST            N-黃色熒光蛋白      Blasticidin S篩選
 
pcDNA6.2/N-EmGFP-DEST      N-綠色熒光蛋白   Blasticidin S篩選
 
pcDNA6.2/C-YFP-DEST            C-黃色熒光蛋白      Blasticidin S篩選
 
pcDNA6.2/C-EmGFP-DEST        C-綠色熒光蛋白   Blasticidin S篩選
 
pcDNA6.2/BioEase-DEST           N-生物素     Blasticidin S篩選

載體序列

TTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCG
CTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAA
TTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCC
TGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA
GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT
ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA
CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATG
CGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCA
TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGC
CCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACT
AGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGT
TAAGCTGAGCATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATAT
ATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGG
CGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTA
GGATCCGTCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTT
GATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACC
AGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGC
CTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAG
CTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGC
TCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGG
TGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTG
AGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCA
AATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGG
CTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGA
TCTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAA
TATATACTGATATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACA
GTTGACAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAAC
CATGCAGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAG
GTCGCCCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAG
GTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGC
CCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTA
CCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCC
GTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGT
TCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATAT
GTTGTGTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATC
ATTTTACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATGCTGTTAACATGGTGAGCAAGGGCGAG
GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG
TGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAA
GCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCGCCCGCTACCCC
GACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCT
TCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCG
CATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTAC
AACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCC
ACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC
CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC
GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGT
AATGATAAGTTTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTA
TGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGT
CCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTT
TTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCT
GCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCG
CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGC
TTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTA
GGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTG
GGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT
GTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATT
TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTG
TCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAG
TCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATT
AGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTC
TCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTC
CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCA
TTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGAC
AAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGG
CTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCG
CATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACT
GCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGA
GCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAG
TGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCA
CTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGG
TTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGT
TCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT
CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAT
GTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT
GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATG
AGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG
CTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGC
TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG
GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAAC
CGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGAC
GCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCT
CGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG
GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG
TGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT
AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT
GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTC
TGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAG
CGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC
TTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA
AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA
AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA
TCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG
CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG
GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCT
AGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCAC
GCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT
GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTA
TCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA
CTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTC
AATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGG
CGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGAT
CTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAA
GGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTAT
CAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGC
GCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACT
CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCG
CTGAGTAGTGCGCGAGCAAAAT

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