pcDNA6.2/nTC-Tag-DEST

价格:2500元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Invitrogen
载体名称: pcDNA6.2/nTC-Tag-DEST
质粒类型: 哺乳动物表达载体;cDNA表达载体;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: 6796 bp
5' 测序引物及序列: T7 Forward: 5’-TAATACGACTCACTATAGGG-3’
3' 测序引物及序列: TK polyA Reverse: 5’-CTTCCGTGTTTCAGTTAGC-3’
载体标签: V5 Epitope(N-端);TC tag(N-端)
载体抗性: 氨苄青霉素、氯霉素(仅空载体)
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/cTC-Tag-DEST载体是cDNA的表达与克隆载体;
CMV启动子驱动目的基因的过表达 ;
TC Tag 即 tetracysteine 基序是一段六氨基酸残基的肽段 (Cys-Cys-Pro-Gly-Cys-Cys);
对应的检测试剂为FlAsH-EDT2和ReAsH-EDT2,既可以进行体内检测又可以在胶内直接检测,高效灵敏。
产品目录号: T34563
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

pcDNA6.2-nTC-Tag-DEST载体图谱



pcDNA6.2-nTC-Tag-DEST 多克隆位点

pcDNA6.2-nTC-Tag-DEST特征位点



pcDNA6.2-nTC-Tag-DEST 载体特征1
pcDNA6.2-nTC-Tag-DEST 载体特征2

载体简介

pcDNA6.2/nTC-Tag-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
 TC-Tag for C-terminal (pcDNA6.2/cTC-Tag-DEST) or N-terminal (pcDNA6.2/nTC-Tag-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 For a map of pcDNA6.2/cTC-Tag-DEST and pcDNA6.2/nTC-Tag-DEST, refer to pages 20 and 22, respectively. 

Tetracysteine 基序
Both the FlAsH-EDT2 and ReAsH-EDT2 reagents bind a tetracysteine motif consisting of Cys-Cys-Xaa-Xaa-Cys-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 TC-Tag. In the TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kit, 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.

Tetracysteine标记技术的优点:
The TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kit uses biarsenical labeling reagents to bind and detect proteins containing a tetracysteine motif (i.e. TC-Tag).2 Using the TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits for fluorescence labeling of recombinant proteins provides the following advantages:
 Small size of the TC-Tag (6 amino acids, 585 Da) is less likely to interfere with the structure or biological activity of the protein of interest
 FlAsH-EDT2 and ReAsH-EDT2 labeling reagents are membrane-permeable and readily cross the cell membrane, allowing labeling and detection of recombinant proteins in live mammalian cells
 FlAsH-EDT2 and ReAsH-EDT2 labeling reagents bind the TC-Tag with high specificity and high affinity (nanomolar or lower dissociation constant), allowing targeted labeling of the protein of interest5
 FlAsH-EDT2 and ReAsH-EDT2 labeling reagents become strongly fluorescent (green and red, respectively) only upon binding the TC-Tag, allowing specific detection of TC-tagged proteins
 FlAsH-EDT2 and ReAsH-EDT2 labeling reagents can be applied sequentially on the same sample, allowing temporal detection of protein turnover and trafficking.
 ReAsH-EDT2 labeling reagent can be used for both fluorescence-based microscopy and electron microscopy.
 FlAsH-EDT2 labeling reagent provides a superior alternative to yellowfluorescent protein (YFP) when coupled with cyan-fluorescent protein (CFP) for FRET-based cellular analysis.



Gateway技术
The Gateway Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda1 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/ cTC-Tag-DEST or pcDNA6.2/nTC-Tag-DEST).
3. Transfect your expression clone into the cell line of choice for transient or stable expression of your gene of interest.
For more information on Gateway, refer to the Gateway Technology with Clonase II manual. 



检测试剂盒及使用方法

The TC-FlAsH TC-ReAsH II In-Cell Tetracysteine Tag Detection Kit consists of two major components:
 The tetracysteine TC-Tag (Cys-Cys-Pro-Gly-Cys-Cys) in the pcDNA6.2/TCTag-DEST vector. When fused to a gene of interest, the TC-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, FlAsH-EDT2 or ReAsH-EDT2, which becomes fluorescent upon binding to recombinant proteins containing the TC-Tag. The FlAsH-EDT2 or ReAsH-EDT2 labeling reagents are supplied pre-complexed to the dithiol EDT (1,2-ethanedithiol) which stabilizes and solubilizes the biarsenic reagents. 

检测TC-Tag融合蛋白
Introduction Once you have transfected your expression clone into mammalian cells, you may:
 Detect protein expression and localization in live cells by fluorescence microscopy using the TC-FlAsH or TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits. For detailed guidelines and protocols, refer to the TCFlAsH or TC-ReAsH II In-Cell Tetracysteine Tag Detection Kits instruction manual.
 Detect protein expression directly in polyacrylamide gels using the Lumio Green Detection Kit.

胶内检测
For sensitive and specific in-gel detection of TC-Tagged fusion proteins, we recommend the Lumio Green Detection Kit available from Invitrogen (LC6090). The Lumio Green Detection Kit enables immediate visualization of TC-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 (LC5928) allows you to easily visualize molecular weight ranges of proteins labeled with Lumio Green Detection Reagent.


Western Blotting You may detect expression of your recombinant fusion protein using the Anti-V5 Antibody (R960-25), Anti-V5-HRP Antibody (R961-25), or Anti-V5-AP Antibody (R962-25) available from Invitrogen. You may use any method of  choice to prepare your mammalian cell lysates for Western blot analysis. 

We recommend the following guidelines:
 If you plan to analyze your samples using the Lumio Green Detection Kit in addition to Western blotting, you will need to prepare your samples using lysis buffer. Lysates containing standard Laemmli SDS-PAGE sample buffer will not be suitable for in-gel detection with the Lumio Green Detection Kit. Refer to the Lumio Green Detection Kit manual for a protocol to prepare cell lysates that are compatible with both in-gel detection and Western blot analysis.
 For cells transfected with the pcDNA6.2/nTC-Tag-p64 positive control vector, you will need to prepare lysates using RIPA or SDS-PAGE sample buffer to adequately release p64 from the nucleoli. If you are preparing samples using lysis buffer, you may sonicate your samples to release p64.
 To detect p64 (human c-myc) expression, you may use any of the Anti-V5 Antibodies or the Anti-myc Antibodies available from Invitrogen.

Note: The c-myc gene encodes a protein with an expected molecular weight of 48 kDa, however, the native protein actually runs at a range of 55–64 kDa on an SDS-PAGE gel.

载体序列

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT
AAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACA
ACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCG
ATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC
ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC
ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC
AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA
TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC
AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG
CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA
CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGTTAAGCTGCAC
CATGGCTGGTGGCTGTTGTCCTGGCTGTTGCGGTGGCGGCAAGCTGGGTAAGCCTATCCCTAACCCTCTC
CTCGGTCTCGATTCTACGAGTGCTGTTATCACAAGTTTGTACAAAAAAGCTGAACGAGAAACGTAAAATG
ATATAAATATCAATATATTAAATTAGATTTTGCATAAAAAACAGACTACATAATACTGTAAAACACAACA
TATCCAGTCACTATGGCGGCCGCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATAATG
TGTGGATTTTGAGTTAGGATCCGGCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCAC
TGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCT
CAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGC
ACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGC
AATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACT
GAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAG
ATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTC
AGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCC
GTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATC
ATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCA
GGGCGGGGCGTAAACGCGTGGATCCGGCTTACTAAAAGCCAGATAACAGTATGCGTATTTGCGCGCACCG
GTGCTAGCGTATACCCGAAGTATGTCAAAAAGAGGTGTGCTATGAAGCAGCGTATTACAGTGACAGTTGA
CAGCGACAGCTATCAGTTGCTCAAGGCATATATGATGTCAATATCTCCGGTCTGGTAAGCACAACCATGC
AGAATGAAGCCCGTCGTCTGCGTGCCGAACGCTGGAAAGCGGAAAATCAGGAAGGGATGGCTGAGGTCGC
CCGGTTTATTGAAATGAACGGCTCTTTTGCTGACGAGAACAGGGACTGGTGAAATGCAGTTTAAGGTTTA
CACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGG
CGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGG
TGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTAT
CGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGG
GGAATATAAATGTCAGGCTCCGTTATACACAGCCAGTCTGCAGGTCGACCATAGTGACTGGATATGTTGT
GTTTTACAGTATTATGTAGTCTGTTTTTTATGCAAAATCTAATTTAATATATTGATATTTATATCATTTT
ACGTTTCTCGTTCAGCTTTCTTGTACAAAGTGGTGATAACACCGGTTAGTAATGAGTTTAAACGGGGGAG
GCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAAT
AAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGAT
ACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTT
CGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCTG
GGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGC
AGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA
CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACG
GCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT
TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA
ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGA
GCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCC
CAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTC
CCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCC
CTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTT
TTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTT
TGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTG
TTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGC
CAAGCCTTTGTCTCAAGAAGAATCCACCCTCATTGAAAGAGCAACGGCTACAATCAACAGCATCCCCATC
TCTGAAGACTACAGCGTCGCCAGCGCAGCTCTCTCTAGCGACGGCCGCATCTTCACTGGTGTCAATGTAT
ATCATTTTACTGGGGGACCTTGTGCAGAACTCGTGGTGCTGGGCACTGCTGCTGCTGCGGCAGCTGGCAA
CCTGACTTGTATCGTCGCGATCGGAAATGAGAACAGGGGCATCTTGAGCCCCTGCGGACGGTGCCGACAG
GTGCTTCTCGATCTGCATCCTGGGATCAAAGCCATAGTGAAGGACAGTGATGGACAGCCGACGGCAGTTG
GGATTCGTGAATTGCTGCCCTCTGGTTATGTGTGGGAGGGCTAAGCACTTCGTGGCCGAGGAGCAGGACT
GACACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCG
GGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTT
ATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCAC
TGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAG
CTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC
AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTG
CGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACG
CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTC
GTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATA
ACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC
GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAAC
CCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCC
TGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG
TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC
GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGG
CAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG
GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA
AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGC
AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTG
GAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA
AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCT
TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGT
GTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGC
TCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAA
CTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAG
TTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC
AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT
TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCA
TAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC
TGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATA
GCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCT
GTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC
GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT
GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATA
CATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCT
GACGTC

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