rAAV-EF1a-fDIO-hChR2(H134R)-3XFlag-WPRE-hGH polyA

Promoter Details

Ef1α, strong promoter, human targeted elongation factor 1α (EF1A) gene, can be stably expressed in most types of cells, especially cells where some CMV promoters are silenced, such as stem cells, 1178bp.

Functional Gene Details

hChR2 (H134R): It is the most common form of ChR2. h refers to the coding sequence of the gene that has been optimized for the human codon table. H134R refers to the mutation of histidine at position 134 to arginine. This mutation inhibits the binding of H+ by ChR2 and increases the passage ability of Na+, thereby increasing the overall conductivity of the molecule and thereby increasing the inward current excited by light. Generally, blue light around 470nm is used for excitation.

Fluorescent Tag

Flag

Fluorescent Protein Details

EGFP: Green fluorescent protein, excitation wavelength 488nm, emission wavelength 507nm.

Application

Expressing light-sensitive ion channel proteins, such as ChR2, ChrimsonR, etc., in neurons through rAAV viral vectors can specifically excite neurons and explore the effects of light stimulation on neurons, neural circuits and animal behavior.

Regulatory Element Details

fDIO: It is an element that cooperates with Flp to control the expression of target genes. If there is no Flp recombinase, the target gene will not be expressed. Only in the presence of Flp can the target gene be expressed normally.

Tag Details

Flag: The Flag tag protein encodes a hydrophilic polypeptide of 8 amino acids (DYKDDDDK). At the same time, the Kozak sequence constructed in the vector makes the fusion protein with FLAG more efficient in eukaryotic expression systems. The target protein fused to FLAG can be directly purified by non-denaturing affinity chromatography through FLAG to purify the active fusion protein with high purification efficiency. Fusion proteins containing FLAG can also be detected and identified through methods such as Westem Blot and ELSA. FLAG fused to the N-terminus can be cleaved by enterokinase (DDDK) to obtain the specific target protein.

Notes

1. Short-term storage at 4°C; long-term storage at -80°C

2. It can be stored at -80°C for more than 6 months. Each time it is frozen and thawed, the titer will be reduced by 10%, and repeated freezing and thawing should be avoided; if the virus is stored for more than 6 months, it is recommended to re-measure the virus titer before use

3. According to the experimental needs, the virus can be appropriately diluted and divided into the volume required for each experiment.