...time. Creating a modular, selection-based gene tagging approach Multiple features of C. elegans have made... between a fluorescent protein (FP) and a 3xFLAG tag (see construct schematic below.) After self-excision...intron, and only the fluorescent protein and 3xFLAG-tag sequences are inserted into the genome. Self-excising...template plasmid to create fluorescent and 3xFLAG-tag fusion proteins. The repair template and Cas9-sgRNA...Dickinson et al illustrating the steps to fluorescently tag a C. elegans protein. The his-72 locus is shown as...little hands-on labor and could facilitate the tagging of every protein in the C. elegans genome. Although... gene tagging...

..., 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...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...multiple selection markers can ensure that all desired tags have been integrated. Simply design your amplification...

...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...

...system for protein tagging. We spoke with Mendenhall and Myers about their new CRISPR tagging system, which...www.addgene.org/crispr/tagging. Addgene: What motivated you to adapt CRISPR for use in protein tagging? Myers: Our...epitope tag onto the transcription factors. By the way, you can actually use this for tagging any protein... resistance to neomycin so that you can select the population of cells with the tagged transcription factor...factors. Our groups got together to adapt CRISPR for tagging experiments and it’s already looking like it’s ...correctly, the second plasmid inserts a small epitope tag called FLAG at the end of the transcription factor...of advice for people who may want to apply this tagging system to their own work? Mendenhall: We’ve made...

...electron microscopy (EM) tag providing superior subcellular localization of tagged proteins compared to traditional...contain a variety of N- and C-terminal tags (including His, S-tag, OneStrep, CBP, Trx, GST, Halo, MBP, ...promoter, N-terminal tag, coding sequence of the gene of interest, C-terminal tag, plant terminator, and...of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated the use of the SpyLigase...expression levels. APEX2 is a monomeric, 27 kD genetic tag which can be fused to proteins of interest for EM...same animal. Flipbow additionally incorporates SUMO tags in the FP sequence for separation from Cre-based...levels to be directly compared among the different tags To clone a sequence or gene of interest into the...

... 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...

...make it possible to insert, or knock-in, any gene, tag, or mutated exon into the genome. For this purpose...promoter responds. You can also use this method to tag an endogenous protein with GFP, as seen in blue flame...where recombination has occurred. Neomycin, puromycin, and hygromycin resistance genes are commonly used...identical molecules of DNA. Gene targeting takes advantage of this natural process to replace a targeted ...insert a resistance gene. The vector contains a neomycin resistance gene (NeoR) flanked by homology arms...remove a resistance gene. In this construct, the neomycin resistance gene is floxed so that it can be removed...the sequence of GFP is cloned together with the Neomycin resistance (NeoR) gene between exon 1 and 3 of...

...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 ...et al. "Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance....

...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...

... Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled Libraries gRNAs...Knockout Library 171531 Knockout Human Li 3rd ~6 9,274 MYC-CRISPR Library 173195 Knockout Human Dzikiewicz-Krawczyk...1000000074 (Puromycin) Activation Human Zhang 3rd 3 70,290 SAM v1 - 3 plasmid system 1000000075 (Puromycin) Activation...Library 159391 Knockout Mouse Moffat 3rd 4 182 Mycobacterium tuberculosis CRISPRi Library (RLC12) 163954 ...Inhibition M. tuberculosis Rock NA Varies 96,700 Mycobacterium smegmatis CRISPRi Library (RLC11) 163955 Inhibition...

... cloning procedure adds both V5 and His6 epitope tags to the C-termini of Met proteins, which can be used...methionine synthesis (5). The budding yeast, Saccharomyces. cerevisiae, makes a great reference organism... or not proteins from the fission yeast, Schizosaccharomyces pombe, have the same function as their counterparts...yeast for budding geneticists: A primer on the Saccharomyces cerevisiae model system. Genetics 197, 33-48...yeast for young geneticists: A primer on the Schizosaccharomyces pombe model system. Genetics 201, 402-423...Surdin-Kerjan, Y. Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 61, 503-532.... et al. Functional characterization of the Saccharomyces cerevisiae genome by gene deletion and parallel...

... 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 ...

...John Chodera's Lab - Get high expression of His-tagged human kinases in bactera. Listen to Our Segment...this kit to complement unused auxotrophies in Saccharomyces strains.  Listen to Our Segment on The Yeast...

...DNA glycosylase inhibitor with a degradation tag (LVA tag) to achieve specific point mutations in E. coli... 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 ...multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors like AAV. In addition...monocot and dicot plants, with a choice of either hygromycin or Basta selection. Yang Lab Single Transcript.... Yin, Linlin, et al. “Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs...

...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....progenitor cell migrating to the epithelium). At this stage, only cells from the immune system show wide scale...like EGFP Yellow-orange fluorescent proteins like TagRFP, tdTomato, DsRed, the mKate series, or tdKatushka2...permanent labelling of cells using cre-lox is advantageous when compared to photoactivable or photoconvertible...during the development and disintegration of mycobacterial granulomas." Immunity 28.2 (2008): 271-284. ...presentation and T cell effector function in mycobacterial granulomas." Immunity 34.5 (2011): 807-819. ...

...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) ...an FP-chimeric protein may be at any one of many stages of synthesis and degradation. For this reason it...PA-FPs by performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry...negative” photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't...

...perfect empty backbone, made sure to add the right tags to your gene, and may have even put a fluorescent...subunit S12 of 30S ribosome Confers resistance to streptomycin Tn10   Confers resistance to tetracycline ...galK rpsL (StrR) endA1 nupG fhuA::IS2 DB3.1 Streptomycin  HB101 derivative. For propagating plasmids ...lacY1 proA2 rpsL20(Smr) xyl5 Δleu mtl1 DH10B Streptomycin MC1061 derivative. General cloning and storage...lacZYA-argF)U169, hsdR17(rK- mK+), λ– HB101 Streptomycin Hybrid of E. coli K12 and E. coli B (mostly ...proAB+ lacIq lacZΔM15] hsdR17(rK-mK+) JM110  Streptomycin  For storing plasmids that should not be Dam...proAB+ lacIq lacZΔM15] hsdR17(rK-mK+)  MC1061 Streptomycin Parent of DH10B/TOP10 and derived strains, common...

... al. shows just that - using MMEJ to insert FLAG tags into the genome of the pathogenic fungus Aspergillus...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...- and locus-specific gRNAs Step 4: Select for puromycin resistant cells Step 5: PCR amplify and sequence...cassette into the FBL locus. Upon sequencing of puromycin resistant clones, they found that 80% and 50% ...follows. For increased versatility, it would be advantageous to adapt PITCh to insert genes into AAVS1, the...using the nuclease Cpf1. Since Cpf1 cuts in a staggered pattern, it is thought to be ideal for HDR-independent...

...vector with the puromycin resistance gene 63591 3xFN-Tel-TAL/pMXs-neo Expresses a 3xFLAG-tagged TAL protein...Fujii, 2013). In iChIP, specific genomic regions tagged with the recognition sequences of an exogenous ...Fig. 1). In enChIP, specific genomic regions are tagged with engineered DNA-binding molecules such as TAL... 63589 3xFN-Tel-TAL/pCMV-7.1 Expresses a 3xFLAG-tagged TAL protein recognizing a telomere repeat 63590...63590 3xFN-Tel-TAL/pMXs-puro Expresses a 3xFLAG-tagged TAL protein recognizing a telomere repeat. A retroviral...systems using different CRISPR orthologues and epitope tags. Fujita T, Yuno M, Fujii H. BMC Res Notes. 2018 ...repeat. A retroviral expression vector with the neomycin resistance gene 64325 3xFLAG-dCas9/p-bacteria ...

...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...