...please improve your plasmid maps!" After thoughtful design, vetting, and tweaking, we’re excited to announce...identifies plasmid features that differ by only a few nucleotides, such as the fluorescent proteins EGFP and mEGFP... software. In fact, SnapGene’s feature library includes nearly 3x as many features as could be detected...greater than our previous mapping software and now includes Type IIS restriction enzymes that are commonly...the gene, the name of its gene product, and a description of its function. You can also click one location...enzymes that cut the given sequence. The table includes enzyme names, the locations where the enzymes ...features detected in the plasmid sequence. The table includes feature names, locations, sizes, colors used to...

...have pre-designed plasmids for these systems! Ion sensors One common biosensor class is designed to monitor...all have specific sensors designed for them. For example, this class includes tension monitors associated...interest for many disciplines! Most of the sensors designed for calcium generate a fluorescent output, which... fluorescence in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium.... Physiological factors Unlike the previously described sensors, this class measures either factors as...to use them, you’ll need to use the vectors (or redesign the tools). It’s a good thing, though! Viral delivery...

...discussed methods. As described above, you design two primers, each containing the desired mutations, to amplify...make a point mutation, you design two oligonucleotide primers with the desired mutation, then use those ... figure above shows guidelines for primer design; a desired insertion should be present in both primers...vitro recombination. Across all modifications described in Trehan et al., REPLACR-mutagenesis’ median ...

...coveted plasmid with no substantial delays. Design, design, design Before diving into the experimental work...experimental work. Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate... to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least 24 bp with the template...from a large genome, you can recheck your primer design or optimize your primers or PCR conditions. Purifying...second and more detailed look at your experimental design. Full lawn of cells. The antibiotic in your plate...complementary to standard sequencing primers. If not, design primers to sequence your insert. When you get your...

... model to help them construct “designer” channelrhodopsins with desired characteristics. The model was...Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more light sensitive...constrained and poorly understood. It’s hard to design new channelrhodopsins with high-throughput proteins...Using machine-learning to overcome channelrhodopsin design challenges To overcome these challenges, the Arnold...experiments. The sequences in this library were designed using structural information about parental channelrhodopsins...existing channelrhodopsins CoChR and/or ChR2(H134R). Designer channelrhodopsins paired with a systemic AAV allow...Find our chemogenetics and optogenetics science guides ...

...utilizing chemogenetics (Designer Receptors Exclusively Activated By Designer Drugs, DREADDs) and optogenetics...beg the question: is axonal transport desirable or not desirable? If diffuse gene expression is tolerated...examining the use of DREADDs (Designer Receptors Exclusively Activated By Designer Drugs) to determine the ...to their Biosafety Level-2 or 2+. Please Note: Despite the fact that AAVs themselves are not pathogenic... CNS tropism (4). If axonal transport is not desirable, transport can be reduced by injecting a smaller...miRNAs), short hairpin RNAs (shRNAs), antisense oligonucleotides (ASOs), zinc finger nucleases, and many others...

...Marraffini’s lab took advantage of this lethality to design the first synthetic bacterial CRISPR system in ...gRNA cassette for plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE...citrea scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering...Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces...editing efficiency, perhaps due to its condensed design. For both systems, custom spacers/gRNAs are easily...et al., 2013), the Qi lab employed a gRNA-based design more familiar to those using CRISPR in eukaryotes...plasmid tools that can be used in this way. As described in the above CRISPRi section, the Marraffini Lab...

...that requires modest gene activation. Synergistic Activation Mediator (SAM) Description SAM uses specially...-component system dependent on gRNA design (SAM) or peptide design (SunTag). This streamlines its delivery...genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation can occur through fusing dCas9...requires a relatively simple construct, it exhibits modest levels of gene activation, with some genes experiencing...VPR and SunTag) (Chavez et al., 2016). SunTag Description Rather than using a single copy of VP64 per each...expressed consistently throughout cells. VPR Description VPR fuses a tripartite complex with dCas9 to ...

...expression in the retina.  Four approaches for designing synthetic promoters  To build a library of synthetic...synthetic promoters, the Roska lab used four different design strategies: Strategy #1 (ProA): Create promoters...promoters with the help of epigenetic analysis and designed using stretches of DNA that were transcriptionally...mice. Figure 1: The four strategies used to design synthetic promoters. Legend: TSS: transcription...class of cells  Of the four different promoter designs, promoters from strategy #4 had the highest rate...here.  Applications for synthetic promoter AAVs Besides restricting AAV transgene expression to particular...research for gene therapy. The Roska lab hopes to design improved versions of their current promoters, as...

...DREADDs DREADDs DREADDS, or designer receptors exclusively activated by designer drugs, are G protein-coupled...Relationship Studies for Designer Receptors Exclusively Activated by Designer Drugs. ACS Chemical Neuroscience...Chemogenetics has become a popular tool in the field as it provides a noninvasive method for manipulating cell activity...Roth's and Takafumi Minamimoto's lab called deschloroclozapine (DCZ) shows promise as a CNO alternative ... Gomez J, Ramirez M, Hu X, Galvan A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics...Conditional expression and signaling of a specifically designed Gi-coupled receptor in transgenic mice. Nature...

... human gut flora. Guides targeted essential and non-essential genes with two guides per gene. The library...expressing guides that target essential genes were less represented while bacteria containing guides targeting... Jason Peters, Oren Rosenberg, and Carol Gross provides a streamlined suite of tools for using CRISPRi...existing bacterial CRISPRi methods. First, its modular design allows for customization with strain-specific promoters...s take a look at the essential gene folA which encodes the target for the antibiotic trimethoprim. When... modular nature allows for the customization of guides, promoters, selection markers, and different versions...

...This post was contributed by Patrick Miller-Rhodes from the University of Rochester Medical Center.  ...plasmids for your experiment at Addgene! How to Design a BiFC Experiment Much like FRET, BiFC experiments...quantification (Kerppola, 2006, 2013). These are described in more detail below: Fusion protein construction...each PIP and empirically test which combination provides the best fluorescent signal. As for linker sequences...single FP. Your control construct may generate a modest level of fluorescence due to the random association...lentiviral expression. For more detail on how to design BiFC experiments, detailed troubleshooting tips...Many thanks to our guest blogger Patrick Miller-Rhodes from the University of Rochester Medical Center...

...harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps...knock-in. Then choose which Cas enzyme to use and design a gRNA to where you would like to introduce the...sequence to introduce, which is done through the design and use of a donor DNA molecule. Your donor molecule...strand break by homologous recombination.    Designing donors for HDR The basis of the HDR pathway relies...used as well. Below are some considerations for designing donor DNA for successful HDR events. CRISPR cut...need to be used due to synthesis limits of oligonucleotides. These donors traditionally have larger homology...or part of the guide’s recognition sequence is destroyed via mutation. By introducing a mutation in either...

...of screenshots to highlight changes to both the design and functionality of searches. A better way to ...posts, protocols, and more. Selecting crispr in Guides will return links including our CRISPR guide.  ... isn’t new to Addgene’s plasmid search. The new design of the plasmid search page moves the filters from...Lentiviral’ which eliminates most of the plasmids designed for other uses. I could look through these resulting... information about the subject, link to related guides and protocols, and provide easy to sort tables ...to the CRISPR Resources page: The page links to guides, our CRISPR eBook, plasmid kits, and curated pages...by a convenient table of plasmids specifically designed for this purpose: Still stumped? Ask an Addgenie...

...and some of the most popular commercial strains designed to avoid them. Why do I need an expression strain...protease: Strains where this is completely deleted (designated lon or Δlon) similary reduce proteolysis of the...which can prevent the culture from reaching the desired density within a reasonable time-frame. For these...and pLysE—the difference being that the latter provides tighter control of basal expression. What if I...don't see protein overexpression? The strains described above should generate sufficient expression levels...you’ve tried a common strain and don’t get the desired level (or any) protein expression? Low expression...is accomplished by growing your culture to the desired density at 37°C and reducing the temperature or...

...episodic memory, a special type of memory which includes autobiographical events (see Jin & Maren, 2015...neurons make to other distant brain areas. It is desirable for tracing compounds to outline neurons in the...applying small electric currents via inserted electrodes) may be applied, but these methods can have a...Nevertheless, the use of these tracers over the past few decades was essential to build up our current understanding...connections (Lo & Anderson, 2011). In the past two decades, several virus species have been modified and used...single synaptic step, thus preventing further widespread expansion of RABV. This improvement resulted ...out our retrograde AAV blog post. An additional desirable property for network visualization would be the...

...subtilis and Pichia pastoris pCri also includes plasmids designed for protein expression in systems other...construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed...plasmids are divided into two sets, each of which includes plasmids with different varieties of tags, both...other eukaryotes better than E. coli. Plasmids designed for these systems may include a signal peptide...can improve disulfide bond formation. pCri also includes a special feature for difficult-to-purify membrane...membrane proteins. pCri vector pCri-13a, designed for use in B. subtilis, contains the MISTIC (membrane-...

...protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated...integrated. Simply design your amplification primers with the desired targeting homology—in frame, of ...These tagging vectors are based on previously described pFA6a-link vectors and include a Kan, SpHIS5, ...former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide variety...3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. ...

...see: Uhlen et al., 2016). In this post, we will describe the approaches that can be used to determine selectivity...intended application — here, flow cytometry — is the desired target, using one or more of the approaches outlined...interpreting your data therefore necessitates careful design of RNAi sequences, confirmation of RNA knockdown...the target at different levels, it is possible to design experiments where the performance of the antibody...We find that cell tracker dyes are helpful in designing such panels, so that pre-stained cell lines can...pass type-1 membrane protein), which has been designated CD365 in the most recent HLDA workshop (HLDA10...recognized by either antibody was not shared. Tables describe that the antibodies both recognized the target...

...the virus doesn’t completely disappear; it just hides in latently infected cells. To truly cure HIV-1,... dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 ...Why can’t the body mount an immune response to destroy these reservoirs? Essentially, the immune system...enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target the 5’ long terminal ...prevent reinfection of those cells. Kaminski et al. designed gRNAs to target the HIV-1 5’ and 3’ LTRs and expressed...could be used to combat this problem, just as ART includes multiple drugs to lower the odds of developing...strategy may be a better option than direct cleavage. Despite the potential difficulties in translating these...