... cells. Flexible tagging: Choose untagged or add a versatile N- or C-terminal HA tag. Selectable and stable...Key elements (promoter, tag, polyA site) are flanked by unique restriction sites so you can swap sequences...plasmid set, our bacterial plasmid set for expressing tagged proteins, and stay tuned for mammalian expression...known regulators at the FOS gene promoter and the MYC locus (Figure 5). Figure 5: TurboCas protein...streptavidin immunoprecipitation (IP), and tandem mass tag (TMT) mass spectrometry (MS) in HCT116 cells (colorectal...lentiviral supernatant and selected with puromycin. Puromycin-resistant cells were fixed and labeled with... stable: A puromycin resistance gene makes stable cell line creation a breeze. Modular by design: Key ...

...dedifferentiation stage involves overexpression of four reprogramming factors- OCT4, SOX2, KLF4, and C-MYC - that...More (due to additional pluripotency stage) Less Safety Less (C-MYC, a known oncogene is one of the reprogramming...lineage, without dedifferentiating into a pluripotent stage. Cellular reprogramming The reversal of a differentiated...reprogramming [1]. The process can be divided into two stages: Dedifferentiation - Conversion of adult somatic...differentiated somatic cell to revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate...reprogrammed to iPSCs successfully [10-12]. The next stage of reprogramming consists of redifferentiation of...

...epitopes tags such as HA and MYC in vivo. The authors found that fusions to one particular tag, the IT6... for epitope tagging: Inntags When fused to your protein of interest (POI), epitope tags give you the...production of a new series of epitope tags, inntags (innocuous tags), that they believe will have fewer ...expressed either without a tag or as multiple fusions with different N- and C-terminal tags. Additionally, by ...Unfortunately, while there are many types of epitope tags (eg. HA, MYC, Flag), they can be deleterious and cause ...expression of fusions between the POI and a TAP tag (a tag with two epitopes that can be used to sequentially...the POI-tag fusion from a plasmid or from a random genomic location than to express the tagged protein...

...of four embryonic genes- OCT4, SOX2, KLF4, and C-MYC. iPSCs were first generated from mouse fibroblasts...therapeutic purposes. Both types have their advantages and disadvantages. Here is a table listing some of them... system which combined the advantages of both while limiting disadvantages led to the creation of induced...cells found in the zygote through to the morula stage are totipotent [2-4]. Pluripotency: When a cell ... Induced Pluripotent Stem Cells: Problems and Advantages when Applying them in Regenerative Medicine. ...

... you casually mention “Haery et al., showed that MYC expression was increased…” when discussing the review...retreat to the woods for months at a time, so take advantage of whatever time you have. If you’re like me, ...

...Roux lab has deposited HA tagged BioID2 for N-terminal fusions and Myc tagged BioID2 for C-terminal fusions...for an individual tagging event. Commonly used sequences, including fluorescent tags, a floxed Cbr-unc...proteins are tagged with Protein A-Renilla luciferase and the “prey” proteins are tagged with Firefly ...technology. To streamline CRISPR/Cas9-based genetic tag insertion in the C. elegans genome, Erik Jorgensen...119 selectable marker, and “connectors” that link tags to the targeted gene are supplied to the reaction...editing in worms to the point that high-throughput tagging projects can be performed. Schwartz, et al. Genetics...lentiviral transfer vector. Each gene has been FLAG-tagged for convenient experimental use. The plasmids were...

...Epitope tag antibody collection grows By Ashley Waldron We are excited to highlight the Anti-V5 tag antibody... as a tag since 1992 (Hanke et al., 1992), and has proven itself a versatile option for tagging proteins...immunodeficiency virus p27 using tag-specific monoclonal antibody and tag-linked antigen. J Gen Virol., ...Figure 3: Expression of fluorophore-tagged GOLG-4 through eight stages of embryonic development. Representative...find in this post: Anti-V5 and other new epitope tag antibodies from IPI New tool for structural studies...studies of E3 ligase cereblon Optimized mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene...Innovation (IPI), part of a new collection of epitope tag antibodies available through Addgene. Anti-V5 [IPI-SV5...

... a unique tag into a genomic site of choice via the mutational abilities of PolI3m. This tag would allow...result in a spectinomycin resistant phenotype in bacteria. While there are many known spectinomycin resistance... for mutagenesis, a feature which they used to generate bacteria resistant to both spectinomycin and streptomycin...directed mutagenesis to generate specific mutations in your plasmids Use REPLACER Mutagenesis to mutate...Mutagenesis is a tool that both evolution and molecular biologists use to tinker with DNA. Making changes...region. EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley...potential applications. Challenges of existing mutagenesis methods Several continuous diversification methods...

...protein tags can be used to facilitate the visualization of specific proteins in cells. Tagged protein...containing AHA to a fluorescent alkyne tag. The newly synthesized and tagged proteins can then be observed with...the knock-in of fluorescent tags into the gene locus of interest. An advantage of this alternative is the...here, there is the question of whether or not the tags influence behavior. A conference and a breakthrough... known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich et al. Nature Neuroscience, ...protein of interest that also had incorporated the AHA tag after pulse labeling of a determined period of time...used for cell labeling Check out other protein tagging strategies Additional Resources on Addgene.org...

... SpCas9 is also fused to a 3X FLAG tag at its N-terminus. This tag can be used to test for SpCas9 expression...Alternative forms of this vector contain GFP-tagged and un-tagged versions of SpCas9 and further variants ...SpCas9 is fused to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables...causes the ribosome to translate Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This...predecessor, which had a point mutation in the puromycin resistance gene that made it less effective in...

...expression ~ ~ ~ ~ ✓   ✓ SV40 and c-Myc nuclear localization sequences Improve translocation...ones in Addgene’s repository later in the post. Advantages of prime editing Less constrained by PAM sequence...

...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. Figure...template plasmid to create fluorescent and 3xFLAG-tag fusion proteins. The repair template and Cas9-sgRNA.... (2015) 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...