■ 基本信息
啟動子: | CMV |
復制子: | pUC |
終止子: | SV40 poly(A) signal |
質粒分類: | 哺乳系列質粒；哺乳熒光質粒；哺乳紅色質粒 |
質粒大小: | 4715bp |
質粒標簽: | N-DsRed2 |
原核抗性: | Kan |
真核抗性: | G418 |
克隆菌株: | DH5a |
培養條件: | 37℃，5%CO2 |
表達宿主: | 293T等哺乳細胞 |
誘導方式: | 無需誘導，瞬時表達 |
5'測序引物: | CMV-F（CGCAAATGGGCGGTAGGCGTG） |
3'測序引物:
| SV40-polyA-R（GAAATTTGTGATGCTATTGC）
|
備注: | 哺乳細胞線粒體紅色熒光定位質粒 |
■ 質粒屬性
質粒宿主: | 哺乳細胞 |
質粒用途: | 定位 |
片段類型: |
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片段物種: |
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原核抗性: | Kan |
真核抗性: | G418
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熒光標記: |
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■ 質粒簡介
pDsRed2-Mito是一個哺乳細胞線粒體定位質粒，含有密碼子優化過的紅色熒光蛋白基因DsRed2和人細胞色素c氧化酶（Mito）亞基VIII的線粒體靶向序列。
pDsRed2-Mito is a mammalian expression vector that encodes a fusion of Discosoma sp. Red fluorescent protein (DsRed2; 1, 2) and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (Mito; 3, 4). The Mito sequence is fused to the 5'-end of DsRed2, a human codon-optimized DsRed variant that is engineered for faster maturation and lower nonspecific aggregation (1, 5). The Mito sequence targets the Mito-DsRed2 fusion protein to the host cell’s mitochondria.
To drive expression of Mito-DsRed2, this vector contains the immmediate early promoter of cytomegalovirus (PCMV IE ). SV40 polyadenylation signals downstream of the DsRed2 gene direct proper processing of the 3'-end of the Mito-DsRed2 mRNA. This vector also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 T-antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin resistance cassette—consisting of the SV40 early promoter (PSV40e), the neomycin/kanamycin resistance gene of Tn5 (Neor/Kanr), and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV TK poly A) gene—allow stably transfected eukaryotic cells to be selected using G418 (6). A bacterial promoter (P) upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli.
pDsRed2-Mito is designed for fluorescent labeling of mitochondria. The vector can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6). The Mito-DsRed2 fusion (excitation/emission maxima: 558 nm/583 nm) can be detected by fluorescence microscopy and by flow cytometry. Filter sets optimized for detecting DsRed by microscopy are available from Chroma Technology Corporation and Omega Optical Inc. Please see their websites (www.chroma.com and www.omegafilters.com) and the Living Colors? Vol. II User Manual, provided with this vector, for more information. To detect MitoDsRed2-expressing cells by flow cytometry, use the instrument’s argon-ion laser to excite the fluorophore at 488 nm and the FL-2 channel to detect the fluorophore’s emission at 583 nm.
■ 質粒圖譜
■ 質粒序列
質粒序列請下載：
ZK526pDsRed2-Mito線粒體定位質粒.txt
