... gRNA backbones, and constructs for endogenous tagging. Some examples of useful worm CRISPR resources,...streamlines the production of targeting vectors for tag insertion using CRISPR/Cas9 and the selection of ...Better CRISPR/Cas9 Editing Efficiency Fluorescent Tagging of Endogenous Genes with SapTrap Even more elegant...Editing in Caenorhabditis elegans. A modified approach has also been developed by the Fire laboratory and described...

...KB Fujii iChIP/enChIP to purify genomic DNA FLAG tagged dCas9 PDF 107.4 KB Goldstein Nematode: gRNA design...PDF 106.7 KB Mendenhall & Myers Mammalian: FLAG tagging endogenous proteins pFETCh_Donor ; additional HDR...finger nuclease sites, this tool from the Joung Lab has been expanded to identify potential DNA target sites...cloning in gRNA: pSimpleII-U6-tracr-U6-BsmBI-NLS-NmCas9-HA-NLS(s) PDF 47.5 KB Vosshall and Matthews CRISPR/Cas9... and quantify the efficiency of the targeted mutagenesis The amplicon sequence expected after HDR can ...

...pAAV_BiSSTe4_ChR2_mCherry AAV construct expressing mCherry-tagged channelrhodopsin-2 driven by SST interneuron-targeting...pAAV_BiPVe3_ChR2_mCherry AAV construct expressing mCherry-tagged channelrhodopsin-2 driven by PV+ basket cell-targeting...pAAV_BiPVe4_ChR2_mCherry AAV construct expressing mCherry-tagged channelrhodopsin-2 driven by chandelier cell-targeting...pAAV_BiLAMP5e3_ChR2_mCherry AAV construct expressing mCherry-tagged channelrhodopsin-2 driven by Lamp5 interneuron-...pAAV_BiVIPe4_ChR2_mCherry AAV construct expressing mCherry-tagged channelrhodopsin-2 driven by VIP interneuron-targeting...pAAV_BiCHATe27_ChR2_mCherry AAV construct expressing mCherry-tagged channelrhodopsin-2 driven by cholinergic neuron-targeting...pAAV_BiSSTe10_ChR2_mCherry AAV construct expressing mCherry-tagged channelrhodopsin-2 driven by SST interneuron-targeting...

...studies Addgene has a collection of plasmids suitable for creating individual fluorescently tagged proteins ...Bacterial Expresses Aquamarine with N-terminal His tag pAquaN1 Cyan Mammalian Expresses mammalian optimized...Cyan Bacterial Expresses CyPet with C-terminal His tag SCFP3A Cyan Mammalian Express a gene of interest ...Yellow Bacterial Expresses YPet with C-terminal His tag SYFP2 Yellow Mammalian Express a gene of interest...

...plasmids. Ginkgo Bioworks Plasmid Collection - Addgene has partnered with Ginkgo Bioworks to distribute SARS-CoV...DETECTR . Feng Zhang's lab at the Broad Institute has utilized the natural RNAse activity of the Cas13 ...UnLOCKING (SHERLOCK and SHERLOCKv2). The Broad Institute has published information and protocols for utilizing...bioRxiv 2020.04.20.052159 . Jennifer Doudna's lab has utilized Cas12a’s non-specific ssDNA degradation ...CRISPR Trans Reporter (DETECTR). Mammoth Biosciences has published information and protocols for utilizing... out this Suggest a Plasmid form. If the plasmid has been described previously we will reach out to the...Spike Receptor Binding Domain Site-Saturation Mutagenesis Libraries - Libraries of variant Spike 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... 63591 3xFN-Tel-TAL/pMXs-neo 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 ...sequence can be cloned into BbsI site. gRNA scaffold has efficient ver. 2 structure. 92220 pLenti_dCas9-2xAM...

...Includes tagging with CFP, YFP, and mRFP Tsoulfas Lab Lentiviral Plasmids - Fluorescently tag your gene...Addgene has assembled a collection of empty plasmid backbones with different fluorescent tags for you ... iRFP Gradia Lab Mammalian Plasmids - Includes tagging with mCherry, mCitrine, mCerulean Davidson Lab ...C. elegans Hamdoun Lab Plasmids - Set includes tagging with mCherry, Cerulean, Citrine, and eGFP pPD95... expression Yeast Thorn Lab Vectors - Includes tagging with a wide variety of colors, some specifically...Lindquist Lab Vectors - Gateway cloning; includes tagging with ECFP, EYFP, DsRed, Cerulean Bacteria Gradia...

...innocuous tags” (inntags) less likely to alter a protein’s properties. Why do we need new epitope tags? Short... looking to a tag a protein, inntags IT5 and IT6 are a great place to start. These tags are easy to use... about other Protein Tags in our Plasmids 101 Series. Get the Inntag Plasmids Tag endogenous proteins ...major caveat - a tag may interfere with protein localization and/or function, so each tagged protein must ...the effects of GFP fusions to inntags or traditional FLAG, HA, and Myc tags. Impressively, IT6-GFP did not...Short epitope tags like FLAG, HA, and Myc can adopt a number of different conformations and thus potentially...with IT5 and IT6 fusions. In contrast, Flag and HA tags had especially detrimental effects on cell size...

...purification tag blog!).   Applications of epitope tags   The tags A number of epitope tags exist...guidance on designing a GFP tag Addgene has you covered!   Potential tag issues Tags have a lot of major benefits...hemagglutinin. The tag has two negatively charged amino acids for an overall negative charge. This tag has been used...Addgene has you covered with monoclonal antibodies to Myc for all of these species. HA The HA tag is a ...fix your antibody woes.   What are epitope tags? Epitope tags are short peptides introduced at the N or...system. Tagging can also aid in protein purification! In this blog, though, we are focusing on tagging for...amino acids on the C terminal of the tag. Of the most common tags, FLAG is the most charged, with 5 negatively...

...feature provides HA frankenbody the ability to bind nascent HA tagged proteins.  Lastly, the HA frankenbody...proteins and tags including mCherry, HaloTag and SNAP-tag for multi-color imaging. Find the HA frankenbody...observed colocalization of GFP tagged HA frankenbody with an mCherry, 4xHA tagged histone protein (H2B) in ...mitochondrial protein tagged with spaghetti monster HA (smHA) which contains a 10xHA tag. These experiments... towards a HA epitope from the CDRs.  To visualize proteins of interest tagged with the HA epitope, the... the HA frankenbody? Another large advantage of the HA frankenbody is that it remains bound to HA epitopes...localization of single HA-tagged molecules over time.  The Stasevich lab also proved that HA frankenbody can ...

...Fluorescent reporter for serotonin (dendrite localization tag) Lin Tian 135420-AAV1 pAAV-syn-FLEX-jYCaMP1s Yellow...AAV vector for Cre-dependent expression of Venus-tagged parapinopsin driven by the CAG promoter. Used for...AAV vector for Cre-dependent expression of Venus-tagged parapinopsin driven by the Ef1a promoter. Used ...support from the BRAIN Initiative below. Support has been identified by depositors and/or when BRAIN Initiative...Viral Prep Description PI 65417-AAV8 pAAV-hSyn-dF-HA-KORD-IRES-mCitrine AAV packaging vector for hSyn ...forebrain GABAergic interneurons. The enhancer core has 100% seq conservation between mouse and human Jonathan...

...affinity tag is the polyhistidine (His) tag. Small and unlikely to affect function, His-tagged proteins...greatly alter the size of the tag.   Table 1: Common protein tags Tag Epitope Mass (kDa) Function Notes...Combo and cleavage tags Frequently, a single tag is not enough. What if you need one tag to increase solubility...to all tags, but rather a quick overview of why scientists use tags, with a few time-tested tags and fusion...to fuse an affinity tag to it. The FLAG, hemaglutinin antigen (HA), and c-myc tags have been the workhorses...keep your protein soluble.  Tags for affinity and purification An affinity tag, generally a relatively small...usually using a Ni2+ column. Like other affinity tags, a His tag can be fused to either the N- or C-terminus...

...directly to the sdAb, or by using a secondary against a tag, such as His, incorporated in the sdAb. sdAbs can... immune response to it. Once the immune response has developed, the antibodies are collected, tested against...epitopes on constant regions of other antibodies. This has useful research applications, and is covered in the...exist as monomers. Each antibody class therefore has a valency, or number of binding sites available per...of antibodies multiplied by two, as each antibody has two binding sites. Isotype Description Configuration... must be able to detect if and where an antibody has bound. Signaling molecules , proteins that emit a...to avoid confusing data. Check that your machine has appropriate lasers and filters for each fluorophore...

...RANbody is detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties...Spaghetti Monsters are highly antigenic tags that have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP...Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B Myc Tagged RFP, H2A2B Chicken IgY (Y) Labeled...discussed in more detail below. The HA tag can be stained for with an anti-HA antibody and serves as a backup... Spaghetti Monster tagged RANbodies can be detected either with a labeled anti- HA or -MYC antibody or...Gelsolin Spaghetti Monster: HA (H) or Myc (M) versions Labeled anti-HA or -Myc antibody Staining cultured...lot-to-lot variability in activity. No Genetic Tagging - Since antibodies are produced in animals instead...

...transgene expression. Which tag should I use? When deciding on the appropriate tag, the experimental design...DREADD to the plasma membrane. The haemagglutinin (HA) tag is a small 9 amino acid epitope of the haemagglutinin... big difference. Replacing the mCherry with the HA tag resulted in abundant expression of hM4Di in plasma...fluorescence is not needed, the use of HA and other short epitope tags (Kimple et al., 2013) could help improve...to reveal the hM4Di coupled to mCherry (top) or HA tag (bottom). The images show examples of dendrites...to the plasma membrane. Note that the use of the HA tag significantly improves the transport of hM4Di to...our studies suggest that non-fluorescent tags may be advantageous over fluorescent proteins when the fluorescence...

...related to embryonic stem cell use and has the major advantage that iPSCs can potentially be derived from... be used to create cell lines with endogenously-tagged gene variants or browse Addgene’s entire collection...Capecchi Replicating EBNA1 episome Human Fluorescent-tagged EBNA1-mediated expression of human Oct3/4, shp53...dosage tracking of reprogramming factors Fluorescent tagged episomals for stoichiometric induced pluripotent...development. Unlike other cell types, each stem cell has the potential to either remain a stem cell or become...can be faster, bypasses intermediate pluripotent stages, and can occur without cell division, all of which...mutation and minimize tumorigenicity, an inherent disadvantage of iPS cell technology. Browse Addgene's collection...

...degron tags. What is a degron tag and why should I use one?   Degron tags are a class of protein tag which...amenable to being tagged, e.g. tagging some proteins can affect their function, the SMASh tag self-cleaves ...). Choosing a degron tag Are all tags created equal? No! Choosing the ‘right’ tag does depend a lot on...Protein Tags Plasmids for Endogenous Gene Tagging in Human Cells Antibodies 101: Epitope Tags  ...) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation tag (dTAG) system functions...degradation. Like several other tags described here, the dTAG uses a small molecule, a tag, and the endogenous cellular...machinery to function. The dTAG is the smallest tag reviewed here, in regard to total tag size, which can also...

...strep-tag and, more recently, OLLAS (Escherichia coli OmpF linker and mouse langerin) and Sun Tag (Viswanathan...antibodies can have low affinity for these tags and even multimeric tags are frequently insufficient for detection...FP and epitope tags, researchers in Loren Looger’s lab have developed new molecular tags combining the ...antibody recognition of epitope tags. Thus they have created antigenic protein tags called “spaghetti monster...experiments with tag-specific antibodies. The most famous ones are influenza hemagglutinin (HA), myelocytomatosis...With the limitations of epitope tags and fluorescent proteins, it has been crucial to develop new tracers...provided better labels than fusions to 3 or more HA tags. 4. High-resolutions microscopy As indicated above...

...T2A-TurboGFP-PEST), and small epitope tags (HA, Myc, Strep tag II, AviTag, HaloTag, SpyTag). The Foerstemann lab developed... scarless tag insertion.   In addition to the obvious advantages SapTrap offers for tagging a single genetic...between the tag and homology arms). Digestion of the donor plasmids with SapI allows the tag, selectable... inserting a tag at multiple target sites. SapTrap vectors can be easily modified to tag hundreds of genes...insertion tagging), developed by the Hornung Lab, allows users to easily create C-terminal tag fusions ...endogenous protein tagging, please visit our website CRISPR/Cas Plasmids - Protein Tagging. Beyond simply...expression levels. CRISPR tagging systems offer users an easy and efficient way to tag endogenous proteins ...

... His-tagged protein purification work flow by nickel column.    Affinity tags vs. other tags There...different types of tags, including epitope tags (FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry...GST antibody? Addgene has you covered! His His tags, often called polyhistidine tags, typically include ...planning, affinity tags can make protein purification a cinch. Types of affinity tags, the proteins they...are all reviewed here. What are affinity tags? Affinity tags are used primarily as protein purification... common tags available. Maltose binding protein Maltose binding protein (MBP) is a 45 kDa tag which can.... The MBP tag, although large, can actually increase expression and solubility of the tagged protein (...