rAAV-hSyn-NES-His-CaMPARI2-F391W-Flag-HA-Myc-WPRE-SV40 polyA

Promoter Details

hSyn, human synapsin promoter, mature neuron-specific promoter, 448 bp.

Functional Gene Details

CaMPARI2.0: EricR Schreiter's team first tested the purified CaMPARI2.0 protein and found that its green and red fluorescence intensities were 60% and 130% higher than the first-generation CaMPARI protein, respectively, and the calcium dissociation rate was also increased several times, making it more sensitive. What is even more exciting is that under calcium-free conditions, the photoconversion rate (PC) of CaMPARI2.0 is significantly lower than that of its predecessor, which means that CaMPARI2.0 will show higher contrast when calcium ion concentrations are different. Using the "super chameleon" CaMPARI2.0 protein, the activities of nerves in a wide range of brain areas in complex behaviors can be studied. CaMPARI2 may have important and widespread application value in the study of brain functional atlas and connectomics. CaMPARI2 CaMPARI2-F391W (phenylalanine at position 391 is mutated to tryptophan) CaMPARI2-L398T NES: nuclear export signals, nuclear export signal sequences, only distributed outside the nucleus.

CaMPARI2.0: EricR Schreiter's team first tested the purified CaMPARI2.0 protein and found that its green and red fluorescence intensities were 60% and 130% higher than the first-generation CaMPARI protein, respectively, and the calcium dissociation rate was also increased several times, making it more sensitive. What is even more exciting is that under calcium-free conditions, the photoconversion rate (PC) of CaMPARI2.0 is significantly lower than that of its predecessor, which means that CaMPARI2.0 will show higher contrast when calcium ion concentrations are different. Using the "super chameleon" CaMPARI2.0 protein, the activities of nerves in a wide range of brain areas in complex behaviors can be studied. CaMPARI2 may have important and widespread application value in the study of brain functional atlas and connectomics. CaMPARI2 CaMPARI2-F391W CaMPARI2-L398T

Fluorescent Tag

Flag、HA、His、Myc

Application

Measuring neuronal activity using genetically encoded calcium indicators (GECIs) has become a widely used method in neuroscience. Calcium imaging with GECIs is often used to measure neural activity in the complete nervous system, such as detecting the activity of a large number of neuronal cell bodies, or tracking the dynamics of neuronal subcells (such as axons, dendrites, and single synapses). Calcium imaging system is a type of optogenetic technology that uses calcium ion indicators that can sense Ca2+ concentration to detect Ca2+ concentration changes in cells or tissues and convert calcium concentration changes into fluorescent signals, thereby converting cell electrical activity into recordable light signals.

Tag Details

His: A tag protein composed of 6 histidine residues, connected to the C-terminal or N-terminal of the target protein. It has a relatively small molecular weight, low immunogenicity, hydrophilicity and variability in the presence of detergents and other additives. Therefore, under natural and denaturing conditions, polyhistidine tags are recognized as the most widely used affinity tags for a series of protein purification purposes. Recombinant proteins can be detected and purified without the use of protein-specific antibodies or probes, and the presence of anti-His tag antibodies for the detection of His-tagged proteins makes 6xHis tags more common and popular. The main uses include: affinity purification of proteins, detection of target proteins, and protein interactions. Myc: A tag protein composed of N-EQKLISEEDL-C10 amino acid residues, which can be placed at the C-terminal or N-terminal of the target protein. However, the low pH elution conditions of Myc recombinant proteins tend to reduce the activity of the protein. Therefore, the Myc tag system is widely used for detection but rarely for purification. Flag: The Flag-tagged 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. HA: The HA tag protein sequence is YPYDVPDYA, which is derived from the hemagglutinin surface epitope of influenza virus. It has 9 amino acids and has little impact on the spatial structure of the foreign target protein. It is easy to construct a tag protein fused to the N-terminus or C-terminus. Easy to detect with Anti-HA antibody detection and ELISA.

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.