pcDNA6.2/N-EmGFP/GW/CAT ，pcDNA6.2/N-YFP/GW/CAT荧光表达载体gateway载体-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

pcDNA6.2/N-EmGFP-DEST，pcDNA6.2/N-YFP-DEST荧光表达载体gateway技术目的载体

pcDNA6.2/C-EmGFP-DEST

pcDNA6.2/C-YFP-DEST

pcDNA6.2/N-EmGFP/GW/CAT

pcDNA6.2/N-YFP/GW/CAT

pcDNA6.2/C-EmGFP/GW/CAT

pcDNA6.2/C-YFP/GW/CAT

BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心www.biovector.net

分别带有EmGFP绿色荧光和YFP黄色荧光，CMV启动子，V5标签，Amp抗性，Blasticidin杀稻瘟菌素细胞筛选标记。

Vivid Colors™ pcDNA™6.2/EmGFPand YFP-DEST Gateway® VectorsGateway®-adapted destination vectors forexpression of N- and C-terminal EmGFP andYFP fusion proteins in mammalian cells

pcDNA™6.2/EmGFP and YFP-DEST vectorscombine the ease and flexibility of Gateway® recombinationbased cloning with the brightness of Emerald GreenFluorescent Protein (EmGFP) and Yellow FluorescentProtein (YFP) derived from Aequorea victoria GFP.Users can easily make an EmGFP or YFP N- or C-terminallytagged mammalian expression clone by performing an LRrecombination reaction between a Gateway® entry vectorcontaining the gene of choice and a Vivid Colors™pcDNA™6.2/EmGFP or YFP-DEST Gateway® Vector. Aftertransfection of the expression clone into mammalian cells,the fluorescent-tagged protein of interest can be identifiedby fluorescence detection methods for localizationexperiments. The protein of interest can also be analyzed byWestern blot.The Vivid Colors™ pcDNA™6.2/EmGFP or YFP-DESTGateway® Vectors are supplied with either an N- orC-terminal tagged EmGFP or YFP/GW/CAT plasmid thatserves as a control for transfection efficiency of theexpression clone into the target cells, as well as a control forexpression of the gene of interest.

Features of theVectorsThe Vivid Colors™ pcDNA™6.2/EmGFP or YFP-DESTGateway® Vectors contain the following elements:• Human cytomegalovirus immediate-early (CMV)promoter/enhancer for high-level expression in a widerange of mammalian cells• Two recombination sites, attR1 and attR2, downstreamof the CMV promoter for recombinational cloning of thegene of interest from an entry clone• Emerald Green Fluorescent Protein (EmGFP) or YellowFluorescent Protein (YFP) derived from Aequorea victoriaGFP for N- or C-terminal fusion to the gene of interestfor fluorescent detection• The V5 epitope tag for detection of recombinant proteinusing Anti-V5 antibodies (optional on N-terminalfusion vectors only)• CAT gene located between the two attR sites forcounterselection• The ccdB gene located between the two attR sites fornegative selection• The Herpes Simplex Virus thymidine kinasepolyadenylation signal for proper termination andprocessing of the recombinant transcript• f1 intergenic region for production of single-strandDNA in F plasmid-containing E. coli• SV40 early promoter and origin for expression of theBlasticidin resistance gene and stable propagation of theplasmid in mammalian hosts expressing the SV40 largeT antigen• Blasticidin resistance gene for selection of stable celllines• The pUC origin for high copy replication andmaintenance of the plasmid in E.coli• The ampicillin (bla) resistance gene for selection inE. coli

EmGFP and YFP

The EmGFP and YFP variants have been described in apublished review (Tsien, 1998) and are summarized in thetable below. The amino acid mutations are represented bythe single letter abbreviation for the amino acid in theconsensus GFP sequence, followed by the codon numberand the single letter amino acid abbreviation for thesubstituted amino acid.Fluorescent Protein GFP Mutations*EmGFP S65T, S72A, N149K, M153T, I167TYFP S65G, S72A, K79R, T203Y*Mutations listed are as described in the literature. When examiningthe actual sequence, the vector codon numbering starts at the firstamino acid after the initiation methionine of the fluorescent protein,so that mutations appear to be increased by one position. Forexample, the S65T mutation actually occurs in codon 66 of EmGFP.EmGFP andYFPFluorescenceThe fluorescent proteins from the Vivid Colors™pcDNA™6.2/EmGFP and YFP-DEST vectors have thefollowing excitation and emission wavelengths, as publishedin the literature (Tsien, 1998):Fluorescent Protein Excitation (nm) Emission (nm)EmGFP 487 509YFP 514 527

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