pmR-mCherry 质粒

pmR-mCherry 质粒货号：kl-zl-21001

质粒类型：	RNAi载体
启动子：	CMV
克隆方法：	多克隆位点，限制性内切酶
载体大小：	4729 bp
载体抗性：	Kanamycin (卡那霉素)
筛选标记：	Neomycin (新霉素)

Description

pmR-mCherry is a mammalian expression vector designed to constitutively express a microRNA of interest. Transfected cells can be identified by the coexpression of mCherry, a mutant fluorescent protein derived from the tetrameric Discosoma sp.red fluorescent protein, DsRed (1). Coexpression of mCherry and your microRNA of interest allows easy monitoring and/or selection of microRNA-expressing cells by fluorescence microscopy or flow cytometry. The excitation and emission maxima of the native mCherry protein are 587 nm and 610 nm, respectively.

The pmR-mCherry multiple cloning site (MCS) is positioned in the 3’UTR, downstream of the mCherry coding sequence. Expression of mCherry and microRNA precursors cloned into the MCS is driven by the constitutively active human cytomegalovirus immediate early promoter (P_{CMV IE}), located just upstream of the mCherry sequence. Both the fluorescent protein and the microRNA are expressed from a single mRNA transcript, which is cleaved by Drosha and Dicer to generate the mature microRNA.

Use

A small genomic fragment containing the precursor of the microRNA of interest must be isolated and cloned into pmR-mCherry.This is most easily accomplished by PCR amplification from genomic DNA. We recommend including 100-300 bp of genomic DNA flanking the actual microRNA precursor to ensure efficient processing by Drosha. The orientation of the cloned microRNA precursor should be the same as that of the mCherry transcript. The sequence of the microRNA precursor and flanking genomic DNA can be obtained from a number of public databases including GenBank (http：//www.ncbi.nlm.nih.gov/) and EMBL-Bank (http：//www. ebi.ac.uk/embl/). The UCSC Genome Bioinformatics Site (http：//genome.ucsc.edu/) hosts an easy-to-navigate genomic database with tracks for microRNAs. The Sanger Institute hosts miRBase, a compilation of known microRNA sequences (http：//microrna.sanger.ac.uk/).

The pmR-mCherry vector can be transfected into mammalian cells using any standard transfection method. If desired, stable transfectants can be selected using G418. Overexpressed microRNA can be detected using Clontech’s Mir-X™ miRNA qRT-PCR SYBR®Kit (Cat. Nos. 638314 and 638316). For Western analysis, the mCherry protein can be detected using either the Living Colors®DsRed Polyclonal Antibody (Cat. No. 632496) or the Monoclonal Antibody (Cat. Nos. 632392 and 632393).

Location of features

0P_{CMV IE}(human cytomegalovirus immediately early promoter)： 1–589

0mCherry (human codon optimized; 3)： 613–1332

0MCS (multiple cloning site)： 1338–1401

0SV40 early polyA+signals： 1548–1582

0P_{SV40 e}(SV40 early promoter and enhancer sequences)： 2274–2542

0Kanamycin/neomycin resistance gene： 2625–3419

0HSV TK polyA+(herpes simplex virus thymidine kinase polyadenylation signals)： 3655–3673

0pUC origin of replication： 4004–4647

Propagation in E. coli

0Suitable host strains： DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM109 or XL1-Blue.
0Selectable marker： plasmid confers resistance to kanamycin (50 μg/ml) in E. colihosts.

0E. coli replication origin： pUC

0Copy number： high

Excitation and emission maxima of mCherry

0Excitation maximum = 587 nm

0Emission maximum = 610 nm

References
1.Shaner, N. C., et al.(2004) Nature Biotech.22(12)：1567-72.
2.Gorman, C. (1985) In DNA Cloning： A Practical Approach, Vol. II.Ed. D. M. Glover (IRL Press, Oxford, U.K.) pp. 143–190.

3.Haas, J., et al.(1996)Curr. Biol.6(3)：315–324.

Note： The vector sequence was compiled from information in the sequence databases, published literature,and other sources, together with partial sequences obtained by Clontech.This vector has not been completely sequenced.
Notice to Purchaser

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