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- 聯系人：Dr.Xu, Biovector NTCC Inc.
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載體基本信息
| 出品公司: | Invitrogen |
|---|---|
| 載體名稱: | pcDNA6.2/cTC-Tag-DEST |
| 質粒類型: | 哺乳動物細胞表達載體；cDNA表達載體；熒光報告載體；Gateway載體 |
| 高拷貝/低拷貝: | 高拷貝 |
| 克隆方法: | Gateway |
| 啟動子: | CMV |
| 載體大小: | 6809 bp |
| 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: | V5 Epitope(C-端）；TC tag(C-端） |
| 載體抗性: | 氨芐青霉素 ，氯霉素（僅空載體） |
| 篩選標記: | Blasticidin |
| 克隆菌株: | DB3.1 |
| 宿主細胞（系）: | 常規細胞系，如293、Hela等 |
| 備注: | pcDNA6.2/cTC-Tag-DEST載體是cDNA的表達與克隆載體； CMV啟動子驅動目的基因的過表達 ； TC Tag 即 tetracysteine 基序是一段六氨基酸殘基的肽段 (Cys-Cys-Pro-Gly-Cys-Cys)； 對應的檢測試劑為FlAsH-EDT2和ReAsH-EDT2，既可以進行體內檢測又可以在膠內直接檢測，高效靈敏。 |
| 產品目錄號: | T34563 |
| 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
載體簡介
載體特征 pcDNA6.2/cTC-Tag-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 TC-Tag for C-terminal 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
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標記技術的優點：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.
檢測試劑盒及使用方法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.
檢測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.
膠內檢測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. Gateway技術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. 載體序列
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
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