...sites are eliminated by digestion/ligation and do not appear in the final construct. The destination vector...Type IIS enzyme and ligase. Although the original destination vector + insert may spontaneously religate...ligated following a heat inactivation step. If your genes of interest or destination vector contain multiple...correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless cloning...complementary overhangs that direct assembly of the final ligation product. As shown below, a fragment with...overhangs. Entry DNA overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification...ligation can be done in one 30-minute reaction. The destination vector and entry vector(s) are placed in a single...

... or under certain stimuli. Cre recombinase catalyzes the recombination of DNA sequences located between.../c, and 129. Conversely, outbred strains were originally derived from random crosses between mice of uncharacterized...Outbred strains of mice are useful for comparing dominant and recessive traits and their impact on diseases...or into embryonic stem cells. Non-homologous recombination incorporates this DNA randomly into the mouse...knockin strains are generated via homologous recombination. A targeting vector is electroporated into embryonic... ends to the breakpoint promotes homologous recombination of the targeting vector. CRISPR/Cas9-based systems... of small molecules that promote homologous recombination or the use of long single-stranded DNA have ...

...intrinsic bacterial recombination pathway, referred to as recA-independent recombination, joins together ...modifications requires multiple recombination events to assemble the final plasmid. At least 5 fragments...assembly can be achieved in vivo using a bacterial recombination pathway that is present even in common lab cloning... The pathway is ubiquitous, with successful recombination reported in all laboratory E. coli strains tested...Garcia-Nafria et al., 2016) uses the bacterial recombination pathway to allow any cloning procedure to be...strain: when asking the cell to perform multiple recombination events, all linear fragments need to get into...For simple cloning requiring just one or two recombination events, homemade competent cells of 107 CFU/...

...ligated into Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt...Originally published Jan 28, 2016 and last updated Sep 10, 2020 by Jennifer Tsang. CRISPR makes it easy...ordered assembly of multiple components into a destination vector. The first step in CRISPR/Cas9 Golden ...together to allow for ordered assembly into a destination vector that expresses Cas9. This is illustrated...drive ordered assembly into a Cas9-containing destination vector. Note: depending on which method you use...fewer than four gRNAs, you’ll clone in a polyT-termination sequence for each unused promoter. This step ...to generate all of the overhangs needed for the final ligation step. Plasmids are then digested using ...

...delivery By Cythina Arokiaraj (Gradinaru lab) Another useful viral tool deposited by the Gradinaru lab, AAV1...recommends these recombinant antibodies for use in western blots, though the original parent clone (M2...primate brains Endothelial-specific AAV delivery Recombinant anti-FLAG tag antibodies Peptide-assisted genome...mitochondrial membranes imaged with structured illumination microscopy in HeLa cells expressing COX8a-mStayGold... photostability and brightness compared to the original, though its maturation is even faster. Published...maintaining the bright and stable fluorescence of the original (Ivorra-Molla et al., 2023). They also named their...Hridaya Patel (Addgene co-op) Researchers in the Gradinaru lab have developed an engineered adeno-associated...

...though the data is heavily dominated by the U.S. (42%), Europe (23%), and China (14%). Spikes in plasmid...instabilities in the China data, so we recommend interpreting the model results for China with more uncertainty...entered lockdown for weeks to months, pausing or terminating employment for a significant fraction of the ...encountered COVID-19 at different times. Most notably, China was affected by COVID-19 earlier than the rest of...hierarchical model: Because the U.S., Europe, and China account for over 75% of all of our plasmid orders...COVID-19 event windows as 01/15/20 -- 03/15/20 for China and 03/11/20 -- 05/19/20 (the last date in our dataset...day across regions, with Europe most impacted and China least impacted. The inferred weekend effect is, ...

...SLIC), eliminated many of LIC’s constraints by relying on the power of homologous recombination. SLIC ...reading Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Li MZ, Elledge...only does this system not use site-specific recombination, it also doesn’t require a ligation step. Based...Based on the robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized... incomplete plasmid looks like a homologous recombination intermediate to bacteria, so it can be transformed...optionally boosts efficiency with the homologous recombination protein RecA. (It’s effective with as little... branch); or by incomplete PCR, which skips the final extension step to retain products with single-stranded...

...of extensive sequences used for cloning into the final translated products. Negative selection markers ...expression vectors additionally contain various combinations of protein tags (His, Avi, and GFP) as well ...blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen." Scientific...production of a downstream peptide, the transcription terminators in this series are just the tool you need. The...this portion of the BIOFAB Plasmid Set contain terminators of varying strengths centered within GFP/RFP ...characterized in Cambray et al., 2013. These terminators can be used to precisely tune your polycistronic... for a researcher to quickly generate N- and C-terminal epitope tag fusions to a protein of interest. ...

... lambda sutures and subsequent determination of the proper coordinates of your brain region of interest...activation and the kinetics of opsin activity/inactivity. Dr. Deisseroth has also generously made many... as the vector core at the University of North Carolina) that offer his constructs. Importantly, control...open-reading frame (DIO). Upon coexpression with cre recombinase (either from a mouse line or viral co-injection...specificity dependent on the promoter of the cre recombinase while still taking advantage of the robust expression...identical for all of these surgeries. Identify coordinates through a rodent brain atlas or research papers...prior to using the virus. You may need to adjust coordinates based on your own method of identifying bregma...

...Agrobacterium binary vectors Vectors designed for use in Agrobacterium are termed binary vectors because...depositor Indu Maiti has created a new and versatile binary Ti vector for both transient and stable gene expression...and E. coli. Like plasmids used in other systems, binary vectors are cloned and prepped in E. coli. However...co-cultivation with the target plant species. Thus, binary vectors contain two origins of replication, one...transferred to the plant, similar to viral vectors’ terminal repeats. It’s a lot of information to fit into...into a single plasmid, so most first-generation binary vectors are quite large (>10 kb.) Since transformation...scientists want to transfer large genes, smaller binary vectors with a larger cloning capacity would be...

...the brain of Cre-expressing mice to trigger recombination and expression of a transgene (Lin et al., 2018...with precisely truncated attP sites to control recombination of the gene construct. A predictable number ...effector or retain the stop cassette following recombination. Image from Isaacman-Beck et al., 2019. Sparse... Sparse Predictive Activity through Recombinase Competition (SPARC), a genetic tool that allows for the... downstream gene expression. attP and attB, recombination sites. These sites are target sequences of PhiC31...where the stop cassette is removed during the recombination step, the effector is expressed. In cells where...expressed and the cells are not labeled. PhiC31, a recombinase. It specifically integrates an attP site into...

... 69090 pMJ915 MBP (N terminal); 6xHis (N terminal); 2xNLS (C terminal) 47327 pET-28b-Cas9-... 6xHis (C terminal); NLS (C terminal) 62933 pET-Cas9-NLS-6xHis 6xHis (C terminal); NLS (C terminal...62934 pET-NLS-Cas9-6xHis 6xHis (C terminal); NLS (N terminal)   Easi-CRISPR efficiency and delivery...marker, likely by breeding to a mouse line with a recombinase. If it turned out your chimeric mice did not ... a standard dsDNA repair template is used. The original design for creating a conditional allele, or floxed...that the T7 promoter will not be included in the final ssDNA template. Figure 2: Overview of ivTRT...stop codon!  Figure 3: How to design a C-terminal insertion with Easi-CRISPR. Potential guide sequences...

...natural cytidine deaminases, they evolved the adenine deaminase ADAR2DD to deaminate cytidine and used... dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase. Three rounds of rational ...Originally published Nov 30, 2017 and updated Jul 31, 2020. Cas13 enzymes are quickly becoming major ...editor: a Cas13 enzyme fused to an RNA adenosine deaminase (ADAR). Such a system would convert adenine to...protein, they examined ADAR1 and ADAR2, which deaminate adenosine to inosine in RNA, creating a functional...functional A->G change. They fused ADAR deaminase domains (ADARDD) to dPspCas13b, but observed low RNA editing...the full ADAR2DD to further optimize cytidine deaminase activity. While RESCUE can perform C-to-U edits...

...of an AAV plasmid. Inverted terminal repeat (ITR) sequence Inverted terminal repeats (ITRs) are what makes...in them.  Lox sites Lox sites, in combination with the Cre recombinase, are used to spatially and temporally...loss, it’s best to propagate AAV constructs in recombination-deficient bacterial strains, such as Stbl3s....FLEX or DIO. Lox sites are recognized by the recombinase Cre. Cre first flips the sequence between the...excise the sequence between the second pair. The final result is a permanently flipped transgene. By restricting...the end of the AAV genome, just upstream of the final ITR.  polyA  The polyadenylation or polyA signal...the end of the AAV genome, just upstream of the final ITR and downstream of the WPRE. TL;DR? Watch our...

...base editor (ACBE), they inserted the deaminase into the C-terminal domain of Cas9, which narrowed the editing...bystander edits. Their final tool, ACBE-Q (for an N108Q mutation in the deaminase), produced an average... (AAG). When adenine base editors (ABEs) were originally being developed for A → G edits, researchers ... MPG to an ABE would allow it to act on newly-deaminated inosines. Pro tip! The nucleoside inosine consists...further development. Figure 2: Mechanisms of deaminase-based adenine transversion editors. Red arrows...processes. Created with BioRender.com. The first deaminase-free base editor allows guanine transversions ...! All base editors up to this point relied on deaminase enzymes, either to directly accomplish the base...

...wtGFP is illuminated with UV-violet light (~400 nm), the chromophore photoconverts predominantly to the ...activates and inactivates Dronpa is not ideal, since this means Dronpa is quickly inactivated and less photons...PA-GFP to display almost no fluorescence when illuminated with 488nm light. However, when UV-violet light... to a 100-fold increase in fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists...derived from DsRed and mRFP, respectively. When illuminated with violet light, both variants are photoconverted...but then further exposure to blue light also inactivates the chromophore, leading to a non-fluorescent...find a version of Dronpa that doesn't become inactivated as quickly. Padron is a monomeric, reversible...

... “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector cells...interleukin (IL)-12 and interferon gamma (IFNγ) - and eliminate cancer cells. 2. Equilibrium - In a process analogous...in bacteria, the tumor cells that escape the elimination stage acquire genetic alterations and alter cell-surface...In contrast to the acute immune responses that eliminate early stage cancer cells, chronic inflammatory...tolerance-producing antigens enunciated above. Finally, signals released from the microenvironment help...cells, and often cannot eradicate metastasis or eliminate the probability of a relapse. Chemical and radiation...to aide chemotherapy and result in successful elimination of certain tumors. However, antibody stability...

...mRNA Surveillance Mechanism That Eliminates Transcripts Lacking Termination Codons. Science 295:2258–2261...generally accepted that some form of homologous recombination (HR) occurs via this region of overlap, but ...single over-sized vector, but not always.  The combination of serotype and tissue/cell type could have a...fragments are likely non-functional, they could be dominant-negative and/or toxic. A potential solution is...repairs Overview: The sequential homologous recombination approach delivers large donor templates using...Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting. Front Neurosci 11: . https://doi.org...Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger...

...detailed instructions on how to generate the final recombinant adenoviral plasmid construct, please see Addgene...bacterial strain not prone to recombination for amplification. The final purified plasmid stock should...candidates for applications in oncolysis and vaccination!) In the 65 years since its discovery, researchers...linear double-stranded DNA of ~36kb, with inverted terminal repeats (ITR) present at both ends.   ...to immune response against AdV. Importantly, recombination-competent adenovirus (RCA) can be generated ...packaging cell line is transferred into the AdV by recombination. It is therefore recommended to test for the...more complex and the presence of helper virus contaminants have slowed the use of this system. New and ...

...bringing the C-terminal and N-terminal Cre fragments together and permitting recombinase activity. Opto-Cre-Vvd... of bacterial gene expression By Meghan Rego Recombinases are enzymes that recognize, cut, and alter specific...translocation or cassette exchange. By utilizing recombinases to manipulate genomes, scientists can control...expression of their genes of interest. While recombinases are powerful gene expression tools in and of...Cells expressing an RFP reporter plasmid for recombinase activity were exposed to blue light. Cre-Vvd ...substantially improved activity as compared to other recombinase/photodimer pairs. Created with BioRender.com ...24390935/ Hypercompact genome editor CRISPR-Casɸ By Melina Fan The Doudna Lab adds to the CRISPR-Cas toolbox...