■ 基本信息
启动子: | CMV |
复制子: | pUC |
终止子: | SV40 poly(A) signal |
质粒分类: | 哺乳系列质粒；哺乳荧光质粒；哺乳红色质粒 |
质粒大小: | 4715bp |
质粒标签: | N-DsRed2 |
原核抗性: | Kan |
真核抗性: | G418 |
克隆菌株: | DH5a |
培养条件: | 37℃，5%CO2 |
表达宿主: | 293T等哺乳细胞 |
诱导方式: | 无需诱导，瞬时表达 |
5'测序引物: | CMV-F（CGCAAATGGGCGGTAGGCGTG） |
3'测序引物:
| SV40-polyA-R（GAAATTTGTGATGCTATTGC）
|
备注: | 哺乳细胞线粒体红色荧光定位质粒 |
■ 质粒属性
质粒宿主: | 哺乳细胞 |
质粒用途: | 定位 |
片段类型: |
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片段物种: |
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原核抗性: | Kan |
真核抗性: | G418
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荧光标记: |
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■ 质粒简介
pDsRed2-Mito是一个哺乳细胞线粒体定位质粒，含有密码子优化过的红色荧光蛋白基因DsRed2和人细胞色素c氧化酶（Mito）亚基VIII的线粒体靶向序列。
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.
■ 质粒图谱
■ 质粒序列
质粒序列请下载：
ZK526pDsRed2-Mito线粒体定位质粒.txt
