...expression ~ ~ ~ ~ ✓   ✓ SV40 and c-Myc nuclear localization sequences Improve translocation..., the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance... initial nick site (to avoid creating a double strand break), increasing editing efficiencies 2-3-fold... Figure 1: An overview of prime editing. Created with BioRender.com. How does it work? First...ones in Addgene’s repository later in the post. Advantages of prime editing Less constrained by PAM sequence... between the template and primer binding site, increase processivity, or improve thermostability. What...fold. However, the process of double nicking increases indel formation slightly. Designing the sgRNA with...

..., modular plasmids, combining fluorescent tags, epitope tags, protease sites, and selection markers. These...fluorophore tagging vectors for your single or multi-color imaging experiments. Interested in epitope tags? Others...have genome tagging systems enabled you to advance your research? Do you have a favorite tagging system that...Researchers have long taken advantage of this natural process to integrate protein tags into the genomes of S...multiple cloning site in place of the epitope tag for creation of your own unique protein fusions. This collection...promoter substitution), and tagging at either the N- or C-terminus (3xHA, 13xMyc, GST, or GFP). Longtine ...especially useful in protein complex studies where tagging of multiple protein products is desired, as multiple...

...transgene expression. Which tag should I use? When deciding on the appropriate tag, the experimental design...our studies suggest that non-fluorescent tags may be advantageous over fluorescent proteins when the fluorescence... methods, these proteins are routinely fused to ‘tag’ or ‘label’ proteins to facilitate its detection....detection. Fluorescent proteins are most widely used tags because they emit endogenous fluorescence that allow...protein (GFP) and mCherry, two of the most common tags currently used, are approximately 240 amino acids...most of them were still cytoplasmic. Use of ‘small’ tags to identify proteins Given the limited expression...monkey neurons, we next hypothesized that a different tag fused to the hM4Di might improve the transport of...

...Activity Cre-dependent Flp-dependent Cre and Flp-dependent Cre, Flp, and VCre-dependent Constitutive (non-cre-dependent...Fluorophore/Tag Green Red None Other Cre-dependent color switch Brainbow constructs Spaghetti monster tags Activity... not fluoresce) Cre dependent 1 Looger 45185 AAV-EF1a-BbTagBY EF1a TagBFP and EYFP Cre dependent 9 Sanes...(non-cre-dependent) Clear Filters ID Name Promoter Fluorophore/Tag Activity Serotype PI 37825 AAV-CAG-...EF1a EYFP Cre dependent 1, 2, 5, 9, rg* Deisseroth 28306 pAAV-FLEX-tdTomato CAG tdTomato Cre dependent...nls-EGFP Cre dependent 1, 2, 5, 8, 9, rg* Harvey 131002 pAAV-Ef1a-DIO mScarlet EF1a mScarlet Cre dependent... hSyn EGFP Cre dependent 1, 2, 5, 8, rg* Roth 50459 pAAV-hSyn-DIO-mCherry hSyn mCherry Cre dependent 1...

... well as additional useful tags, including 3xFLAG epitopes, TEV and BioTag, which can be used in protein... protein effectivity), and a fluorescent protein tag for visualization (Fig. 1b). While the probes target...ultimately leading to the degradation of the AID-tagged protein. To quantify protein degradation the Ward...44 amino acids auxin inducible degron sequence) tagged proteins. Degradation of your protein interest ...Gibson and Saptrap cloning systems to generate AID* tagged CRISPR knock-in repair constructs. These repair...repair constructs can be used to integrate the tagged AID* into genomic sites of interests via CRISPR techniques...engineering plasmid tools in about a week. Taking advantage of Golden Gate cloning, the Haynes lab’s Protocols.io...

... Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs Empty gRNA Vectors...Knockout Library 171531 Knockout Human Li 3rd ~6 9,274 MYC-CRISPR Library 173195 Knockout Human Dzikiewicz-Krawczyk...In a CRISPR screening experiment, target cells are treated with the pooled library to create a population...mutant cells that are then screened for a phenotype of interest. Screening experiments using a pooled ...1000000074 (Puromycin) Activation Human Zhang 3rd 3 70,290 SAM v1 - 3 plasmid system 1000000075 (Puromycin) Activation...CRISPR pooled libraries for large-scale functional screens. Available for knockout, activation, inhibition...Blog Posts CRISPR is a useful tool for genetic screening experiments, due to the relative ease of designing...

...additionally contain various combinations of protein tags (His, Avi, and GFP) as well as protease sites to...researcher to quickly generate N- and C-terminal epitope tag fusions to a protein of interest. The plasmids used...folding and/or protease sites to remove the epitope tags. Different plasmids in the kit can be used to purify...2015). He has also constructed a library of eGFP-tagged human ORF clones to allow testing and comparison...conveniently performed through the N-terminal eGFP tag carried on all constructs. The Cell-Free Protein ...greatly reduced costs per plasmid. We generally create kits to allow you to easily use a set of plasmids...markers built into all plasmids in the kit help decrease false positives observed in the cloning process...

...The permanent labelling of cells using cre-lox is advantageous when compared to photoactivable or photoconvertible...cells according to whether or not they have been tagged with a fluorescent protein or dye. It separates... distinguishing cell populations that have been tagged with different fluorescent proteins or dyes. 3....useful tool for marking and tracking cells is the the Cre/lox recombination system. A researcher can flank ...expression is turned on or off in cells expressing Cre. The result of a system like this is a mosaic of ...progenitor cell migrating to the epithelium). At this stage, only cells from the immune system show wide scale... In the past 15 years efforts have been made to create transgenic mice expressing fluorescent protein ...

...terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or affinity tags (HA, FLAG... plant selection markers (basta, hygromycin, kananamycin, tunicamycin) (Nakagawa et al., 2007). Alternatively...new inflorescences begin to grow. This will also increase the number of inflorescences overall, which may...them again.  This won’t hurt them, and it will increase the chances of tDNA integration. Selecting Arabidopsis... seeds too densely during selection: this will increase background growth of non-transformants and make...Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs in Transgenic Analysis of...

...tissue-specific cell types: Cre-lox recombination. What is Cre-lox? The Cre-lox system is a technology ...marker, Cre can be used to easily perform this eviction. Regulated Cre expression – placing Cre downstream...constructed. Additionally, Cre-containing adenovirus (Ad-Cre) or AAV (AAV-pgk-Cre) has been used to successfully...generated at the loxP sites. How can I use Cre-lox? The Cre/lox system is a well-established research ...prevent gene expression in the absence of Cre. In the presence of Cre, the stop codon is excised, and gene ...facilitate the use of Cre-lox technology, transgenic mice have been constructed that express Cre under a variety...analogous to Cre and loxP, respectively. The FLP-FRT technology can be an effective alternative to Cre-lox, and...

...Vectors Read our Blog Post About Tagging Yeast Genes ... Scientists took advantage of these discoveries to create a new system for screening protein-protein interactions... developed in Saccharomyces cerevisiae in 1989 and are still used extensively to screen for molecular ...identifying prey proteins after screening. It is also possible to screen libraries of bait and prey against...single-pass two-hybrid screening (6). See the Tips section for more on how to increase your rate of interactions...interactions when screening! Tips for two-hybrid screening Minimizing false positives: Run replicates of ...protein interact and enable intein splicing (5). Screening with yeast two hybrid systems While initial studies...

... expressing fluorescent proteins will be used to tag individual strains of bacteria. Their interactions...Understanding drug resistance mechanisms of Mycobacterium tuberculosis Dr. Patricia Sheen, Universidad...interested in understanding the mechanisms behind Mycobacterium tuberculosis resistance to pyrazinamide (PZA...methods and improved therapies for PZA-resistant Mycobacteria tuberculosis. Investigation of multispecies ...such as pACBSR-hyg and pFLP-hyg will be used to create gene deletions of important unknown regulatory ...sense contaminants and emit light as a response. To create this bacterial sensor, he will construct plasmids...

...DNA glycosylase inhibitor with a degradation tag (LVA tag) to achieve specific point mutations in E. coli...plasmid contains all of the desired gRNAs. This increases the chance that all the edits you want to make... cleave outside of their recognition sequence, creating flanking overhangs. These overhangs can be customized... ideal number. First you generate four unique kanamycin-resistant plasmids, each containing a different...construct, you clone all but one of your gRNAs into spectinomycin-resistant plasmids pX330S-2 to pX330S-(last ...Expression Systems (MuLE) Kit: This kit can be used to create lentiviral vectors expressing wt humanized Cas9...multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors like AAV. In addition...

...promoters, terminators, solubility and purification tags, connectors, cDNAs, and more to find the perfect...constitutive and inducible promoters, linkers, and tags makes EcoFlex a great option for those interested... a larger library of parts but don’t need to add tags to your proteins, then the CIDAR Kit is an excellent... technology has been used to optimize protein secretion and expression in the biotech workhorse yeast ...Engineering disease resistance through gene stacking, increasing production of a protein of interest, and improving...genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization, and more....can be invaluable for more complex editing and screening experiments in organisms that don’t yet have these...

...Photoactivatable GFP tagging cassettes for protein‐tracking studies in the budding yeast Saccharomyces cerevisiae...FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization...fusion, etc). For example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) ... by performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry (E26V...photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't ...an FP-chimeric protein may be at any one of many stages of synthesis and degradation. For this reason it...photoconverts predominantly to the anionic form, causing increased fluorescence when excited with 488nm blue light...

...perfect empty backbone, made sure to add the right tags to your gene, and may have even put a fluorescent...recombination, increases plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability...mtl1 DH10B Streptomycin MC1061 derivative. General cloning and storage, blue/white screening, leucine auxotroph...) TOP10 Streptomycin MC1061 derivative. General cloning and storage, blue/white screening. F- mcrA Δ...that make major improvements such as those that increase plasmid yield and/or DNA quality. Table 1 below...sensitive. dnaJ Mutation in a chaperonin gene Increases the stability of certain expressed proteins ...methylate DNA lac Lac operon mutations Blue/white screening of clones   lacIq lac repressor overproduced...

... al. shows just that - using MMEJ to insert FLAG tags into the genome of the pathogenic fungus Aspergillus...of the gene it follows. For increased versatility, it would be advantageous to adapt PITCh to insert genes... deletions or homology-directed repair (HDR) to create precise genome edits. But have you heard of another...depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh...PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without the need for...of press. However, this process accounts for a percentage of the mutations seen with TALENs and CRISPR.... between the microhomologies.MMEJ has certain advantages over HDR. Some species don’t have good HDR systems...

...chapters on genome engineering, cloning, and plasmid tags, addtional content added to the "Plasmids that Glow...resistant plasmid, pSC101, and a newly developed kanamycin resistant plasmid, pSC102, with EcoRI and selected...Plasmids 101 eBook! In keeping with our goal of creating a one-stop reference guide for plasmids we've ...

...found in Botman et al., 2019 . FP Tagging Yeast tagging vectors to create fusions of proteins of interest...Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast. 2004 Jun;21(8):661-...plasmids to overexpress various fluorescent markers and tag your proteins of interest with fluorescent proteins...Materials Fluorescent Protein Plasmids & Resources FP Tagging FP Overexpression Resources This comprehensive ...allows for constitutive overexpression of FPs and tagging of genes of interest with FPs in yeast. Characterization...TCGATGAATTCGAGCTCG–3' ID Plasmid Selectable Marker Tags Publication FP Overexpression Plasmids for constitutive...Improved blue, green, and red fluorescent protein tagging vectors for S. cerevisiae. PLoS One. 2013 Jul 2...

...uracil DNA glycosylase inhibitor and degradation tag on the fusion protein to limit the activity of the... lab created two “pCRISPomyces” systems for use in Streptomyces (Cobb et al., 2015). pCRISPomyces-1 includes...other advantages including shorter plasmid curing time. pCRISPomyces for editing in Streptomyces  Streptomyces...array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays...proteotoxic when used in Mycobacterial species, Sarah Fortune’s lab screened 11 other Cas9 orthologs to...for transcriptional silencing Mycobacterium tuberculosis and Mycobacterium smegmatis. In addition to their...Addgene depositor Luciano Marraffini’s lab took advantage of this lethality to design the first synthetic...