pcDNA6.2/cLumio-DEST

pcDNA6.2/cLumio-DEST

载体基本信息

出品公司:	Ybscience
载体名称:	pcDNA6.2/cLumio-DEST
质粒类型:	哺乳动物表达载体；cDNA表达载体；荧光报告载体；Gateway载体
高拷贝/低拷贝:	高拷贝
克隆方法:	Gateway
启动子:	CMV
载体大小:	6809 bp
5' 测序引物及序列:	T7 Forward: 5’-TAATACGACTCACTATAGGG-3’
3' 测序引物及序列:	BGH Reverse: 5-TAGAAGGCACAGTCGAGG-3
载体标签:	V5 Epitope(N-端）, Lumio（C-端）
载体抗性:	氨苄青霉素
筛选标记:	Blasticidin
克隆菌株:	DB3.1
宿主细胞（系）:	常规细胞系，如293、Hela等
备注:	pcDNA6.2/cLumio-DEST载体是cDNA的表达与克隆载体； 表达C-端含lumio标签的融合蛋白，可以在体内或胶内直接进行检测； 与传统荧光报告 基因相比，报告蛋白更小，检测方法跟简便高效； tetracysteine Lumio tag (Cys-Cys-Pro-Gly-Cys-Cys)。
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

 

载体描述：

The Mammalian Lumio Gateway Vector Kits contain Gateway-adapted destination vectors designed for use with the Lumio Technology. The pcDNA6.2/Lumio-DEST vectors supplied with each kit facilitate in vivo fluorescence labeling and detection of recombinant proteins when used with a Lumio In-Cell Labeling Kit. 

载体特征：

The pcDNA6.2/cLumio-DEST and pcDNA6.2/nLumio-DEST vectors contain the following elements:
 Human cytomegalovirus immediate-early (CMV) promoter/enhancer for high-level expression in a wide range of mammalian cells
 Lumio tag for C-terminal (pcDNA6.2/cLumio-DEST) or N-terminal (pcDNA6.2/nLumio-DEST) fusion to the gene of interest for fluorescence detection
 Two recombination sites, attR1 and attR2, downstream of the CMV promoter for recombinational cloning of the gene of interest from an entry clone
 Chloramphenicol resistance 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 plasmidcontaining 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 resistance gene for selection in E. coli


Gateway 技术简介：

The Gateway Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (Landy, 1989) to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems. To express your gene of interest in mammalian cells using Gateway Technology, simply:
1. Clone your gene of interest into a Gateway entry vector to create an entry clone.
2. Generate an expression clone by performing an LR recombination reaction between the entry clone and a Gateway destination vector (e.g. pcDNA6.2/cLumio-DEST or pcDNA6.2/nLumio-DEST).
3. Transfect your expression clone into the cell line of choice for transient or stable expression of your gene of interest.


Lumio 技术的优势：

The Lumio System is based on the FlAsH (Fluorescein Arsenical Hairpin) technology which uses biarsenical labeling reagents to bind and detect proteins containing a tetracysteine motif (i.e. Lumio tag) (Griffin et al., 1998). Using the Lumio Technology and the Lumio In-Cell Labeling Kits for fluorescence labeling of recombinant proteins provides the following advantages:
 Small size of the Lumio tag (6 amino acids, 585 Da) is less likely to interfere with the structure or biological activity of the protein of interest
 Lumio Labeling Reagents are membrane-permeable and readily cross the cell membrane, allowing labeling and detection of recombinant proteins in live mammalian cells
 Lumio Labeling Reagents bind the Lumio tag with high specificity and high affinity (nanomolar or lower dissociation constant), allowing targeted labeling of the protein of interest
 Lumio Labeling Reagents become strongly fluorescent only upon binding the Lumio tag, allowing specific detection of Lumio-tagged proteins

Lumio 系统的组成：

The Lumio System consists of two major components:
 The tetracysteine Lumio tag (Cys-Cys-Pro-Gly-Cys-Cys) in the pcDNA6.2/Lumio-DEST vector. When fused to a gene of interest, the Lumio tag allows the expressed fusion protein to be specifically recognized by a biarsenical labeling reagent. For more information on the tetracysteine motif, see below.
 A biarsenical labeling reagent, Lumio Green or Lumio Red, which becomes fluorescent upon binding to recombinant proteins containing the Lumio tag.
The Lumio Green and Lumio Red Labeling Reagents are supplied precomplexed to the dithiol EDT (1,2-ethanedithiol) which stabilizes and solubilizes the biarsenic reagents. 

Tetracysteine Motif
The Lumio Reagents bind a tetracysteine motif consisting of Cys-Cys-Xaa-XaaCys-Cys where Cys equals cysteine and Xaa equals any amino acid other than cysteine. This motif is rarely seen in naturally occurring proteins allowing specific fluorescence labeling of recombinant proteins fused to the Lumio tag. In the Lumio System, the optimized Cys-Cys-Pro-Gly-Cys-Cys tetracysteine motif is used as this motif has been shown to have a higher affinity for and more rapid binding to biarsenic compounds as well as enhanced stability compared to other characterized motifs (Adams et al., 2002).
 
Lumio Green Detection Kit
For sensitive and specific in-gel detection of Lumio-tagged fusion proteins, we recommend the Lumio Green Detection Kit available from Invitrogen (Catalog no. LC6090). The Lumio Green Detection Kit enables immediate visualization of Lumio-tagged proteins in polyacrylamide gels using a UV transilluminator or a visible light laser-based scanner and without the need for staining or western blotting. In addition, the BenchMark Fluorescent Protein Standard (Catalog no.LC5928) allows you to easily visualize molecular weight ranges of proteins labeled with Lumio Green Detection Reagent. 


实验要点：

Generating an Entry Clone
Introduction To recombine your gene of interest into pcDNA6.2/cLumio-DEST or pcDNA6.2/nLumio-DEST, you will need an entry clone containing the gene of interest. Many entry vectors including pENTR/D-TOPO (Catalog no. K2400-20) are available from Invitrogen to facilitate generation of entry clones.

Tag-On-Demand System
The pcDNA6.2/cLumio-DEST vector is compatible with the Tag-OnDemand System which allows expression of both native and C-terminallytagged recombinant protein from the same expression construct.
The System is based on stop suppression technology originally developed by RajBhandary and colleagues (Capone et al., 1985) and consists of a recombinant adenovirus expressing a tRNAser suppressor. When an expression vector encoding a gene of interest with the TAG (amber stop) codon is transfected into mammalian cells, the stop codon will be translated as serine, allowing translation to continue and resulting in production of a C-terminally-tagged fusion protein.

If you wish to express a human or mouse gene of interest, we recommend using an Ultimate Human ORF (hORF) or Ultimate Mouse ORF (mORF) Clone available from Invitrogen. Each Ultimate ORF Clone is a fully sequenced clone provided in a Gateway entry vector that is ready-to-use in an LR recombination reaction with pcDNA6.2/cLumio-DEST. In addition, each clone contains a TAG stop codon, making it fully compatible for use in the Tag-On-Demand System. 

Points to Consider for pcDNA6.2/nLumio-DEST
pcDNA6.2/nLumio-DEST allows expression of recombinant proteins with an N-terminal peptide containing the Lumio and V5 epitope tags and contains an ATG initiation codon within the context of a Kozak consensus sequence. To include the Lumio and V5 epitope tags, your insert in the entry clone should:
 not contain a Kozak initiation sequence
 be in frame with the Lumio and V5 epitope tags after recombination
 contain a stop codon

 

载体序列

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT
AAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACA
ACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCG
ATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC
ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC
ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC
AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA
TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC
AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG
CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA
CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTGAGC
ATCAACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAAATTAG
ATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACATATCCAGTCACTATGGCGGCCGCATT
AGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATGTGTGGATTTTGAGTTAGGATCCGGCG
AGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCC
AATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCA
GCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCAC
ATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATAT
GGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGA
ATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTG
GCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCA
GTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATAC
GCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTC
GGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAAAGATCTGGATCCG
GCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCTGATTTTTGCGGTATAAGAATATATACTGA
TATGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGACAGCG
ACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGCAGAAT
GAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGCCCGGT
TTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTACACCT
ATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACG
GATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTG
CATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGG
AAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAAT
ATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGTGTTTT
ACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTTACGTT
TCTCGTTCAGCTTTCTTGTACAAAGTGGTTGATGCTGTTAACGGGAAGCCTATCCCTAACCCTCTCCTCG
GTCTCGATTCTACGCGTACCGGTGCTGGTGGCTGTTGTCCTGGCTGTTGCGGTGGCGGCTAGTAATGAGT
TTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAAT
AAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTG
GCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCC
CACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAG
ATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGT
GGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCT
TCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGAT
TTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCC
CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT
GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATT
GGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGG
TGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCA
GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAC
CATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCAT
GGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGT
GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATC
TGATCAGCACGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGA
ACTAAACCATGGCCAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAA
CAGCATCCCCATCTCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACT
GGTGTCAATGTATATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTG
CGGCAGCTGGCAACCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGG
ACGGTGCCGACAGGTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAG
CCGACGGCAGTTGGGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCC
GAGGAGCAGGACTGACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCG
GAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCA
CCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAA
GCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATAC
CGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCT
CACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA
CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAT
GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTC
GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA
GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGG
CCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCA
GAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT
CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC
ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC
CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC
TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT
TCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC
AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTT
GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT
CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC
CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC
AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT
GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACC
GCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGA
AGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTT
CGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGG
TATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAA
GCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA
TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTC
AACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAAT
ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAA
GGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTT
TACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCG
ACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTC
TCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG
AAAAGTGCCACCTGACGTC