pcDNA6.2/N-YFP-DEST

pcDNA6.2/N-YFP-DEST

载体基本信息

出品公司:	Ybscience
载体名称:	pcDNA6.2/N-YFP-DEST
质粒类型:	哺乳动物表达载体；Gateway载体；荧光报告载体
高拷贝/低拷贝:	高拷贝
启动子:	CMV
克隆方法:	Gateway
载体大小:	7530 bp
5' 测序引物及序列:	CMV forward 5’-CGCAAATGGGCGGTAGGCGTG-3’
3' 测序引物及序列:	TK polyA Reverse: 5’-CTTCCGTGTTTCAGTTAGC-3’
载体标签:	YFP Tag (N Term), V5 Epitope Tag(C Term）
载体抗性:	氨苄青霉素、氯霉素（仅空载体具有此抗性）
筛选标记:	Blasticidin
克隆菌株:	ccdB阳性筛选 DB3.1 感受态细胞， 阴性筛选用 OmniMAX2-T1 感受态细胞
表达细胞（系）:	常规细胞系，如293、Hela等。
备注:	pcDNA6.2/N-YFP-DEST载体是cDNA表达载体，是黄色荧光报告载体； CMV启动子驱动目的蛋白的过表达。
稳定性:	瞬表达 或 稳表达
组成型/诱导型:	组成型
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

载体简介

 

系统介绍

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 proteins from 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 humanized for 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.


载体特征

pcDNA6.2/N-YFP-DEST载体含有以下元件：
 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


绿色荧光蛋白(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重组反应所需实验材料：

 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哺乳动物表达载体选择指南：

pDEST12.2   不带标签

pDEST26     N-His标签      G418筛选

pDEST27     N-GST标签    G418筛选

pDEST40       C-His标签、C-V5标签   G418筛选

pcDNA-DEST40       C-His标签、C-V5标签   G418筛选
 
pcDNA-DEST47           C-绿色荧光蛋白 G418筛选

pcDNA-DEST53           N-绿色荧光蛋白 G418筛选

pcDNA3.2/V5-DEST      C-V5标签       G418筛选

pcDNA6.2/V5-DEST      C-V5标签        Blasticidin S筛选

pcDNA6.2/N-YFP-DEST            N-黄色荧光蛋白      Blasticidin S筛选
 
pcDNA6.2/N-EmGFP-DEST      N-绿色荧光蛋白   Blasticidin S筛选
 
pcDNA6.2/C-YFP-DEST            C-黄色荧光蛋白      Blasticidin S筛选
 
pcDNA6.2/C-EmGFP-DEST        C-绿色荧光蛋白   Blasticidin S筛选
 
pcDNA6.2/BioEase-DEST           N-生物素     Blasticidin S筛选

 

载体序列

TTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCG
CTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAA
TTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCC
TGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATA
GGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT
ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA
CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATG
CGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCA
TTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGC
CCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAAC
CGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCC
TCCGGACTCTAGAGGATCCCTACCGGTGATATCCTCGAGACCATGGTGAGCAAGGGCGAGGAGCTGTTCA
CCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA
GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG
CCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGC
GCCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGA
CGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG
AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA
ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGA
GGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG
CCCGACAACCACTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGG
TCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGCTCGAGCCC
ATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAG
ATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATT
AGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGTCG
AGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCC
AATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCA
GCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCAC
ATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATAT
GGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGA
ATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTG
GCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCA
GTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATAC
GCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTC
GGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCG
GCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGA
TATGTATACCCGAAGTATGTCAAAAAGAGGTATGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCG
ACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAAT
GAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGT
TTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGGCTGGTGAAATGCAGTTTAAGGTTTACACCT
ATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACG
GATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTG
CATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGG
AAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAAT
ATAAATGTCAGGCTCCCTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTT
ACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTT
TCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATAACGGGAAGCCTATCCCTAACCCTCTCCTCGGTCTCG
ATTCTACGCGTACCGGTTAGTAATGAGTTTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATAC
CGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTC
ATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATA
CGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAAC
GTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCT
GTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCT
AGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTA
AATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGG
GTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTT
CTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTA
TAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT
AATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAA
AGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGC
AAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCC
GCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC
TCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCC
CGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATC
GGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCC
TCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGC
TCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAA
CTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATG
AGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAA
AGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTAT
GTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACC
GCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCG
GGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAG
CAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTC
ATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGC
TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAA
AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCG
GGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC
GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA
CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC
CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG
AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTT
TCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT
CTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC
GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCG
TCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA
GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAG
TATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA
ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT
CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTT
TGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAT
CTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCG
ATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT
TACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT
AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATT
AATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTA
CAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG
AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT
AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG
TAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAG
TTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT
GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCA
CTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAG
GCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA
TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATA
AACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCC
GATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTG
CTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAAT