...effects of GFP fusions to inntags or traditional FLAG, HA, and Myc tags. Impressively, IT6-GFP did not reduce...yeast cell size or viability compared to untagged GFP overexpression, while the rest of the fusions had...: 315-323. Pubmed PMID: 23435261. Resources at Addgene Read about other Protein Tags in our Plasmids 101...

...this general design scheme to generate two more OptoNBs, one against mCherry and one against GFP. Insertion...Lab monitored how AsLOV2 insertion into mCherry or GFP nanobodies affected binding of their target proteins...co-localization with a membrane-localized mCherry or GFP protein. With this system, if the OptoNB can bind....  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced ...telephone, until the message reaches the nucleus where gene expression changes lead to cell proliferation and... on Addgene.org Find nanobody expression plasmids here Learn more about optogenetics in Addgene's optogenetics...loop 1 always generated OptoNBs with dark-induced binding, but insertion into loop 6 generated both blue ...

...TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing GFP that can be...plasmid - Addgene # 68292 C321.ΔA bacterial strain – Addgene #68306 pCRT7 NT TOPO E17TAG GFP plasmid -...during optimization experiments. This version of GFP will contain a phosphoserine at amino acid 17 instead...instead of a stop codon, which will allow full length GFP to be translated), and second, through a gel shift...protein. Up to 80% p-Ser incorporation into E17TAG GFP has been achieved with the Rinehart system (6); however... - Addgene #68295 SupD plasmid – Addgene # 68307 For further technical reference and protocols for use...candidate regulatory kinase is known, it’s possible to generate non-phosphorylated recombinant protein and perform...

...nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing that GFP’s  structure...Protein Activity with GFP Resources at Addgene.org Find the Spinach Plasmids at Addgene Find Broccoli Plasmids...Resources on the Addgene Blog Tips for Using FRET in Your Experiments Learn more about GFP Controlling Protein...increase its fluorescence, essentially mimicking GFP.  Though the aptamer, called Spinach, required longer...and used in assays for over half a century; the Addgene blog has an excellent primer and tips from experts...can be created in many different ways, but the general premise is that an aptamer in its unbound state...likely to trigger immunoresponses. Nucleotides, in general, are far less immunogenic than proteins. Assays...

...among other things) gene delivery, generating pluripotent stem cells, and developing gene therapies for monogenic...and lentiviral vectors that are used for gene regulation, gene of interest expression, knockout, and screening...gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for...for cloning and expressing your gene of interest with GFP expression.  Cloning and expressing RNAi pMKO...technology CAG-GFP-IRES-CRE (Plasmid #48201) This retroviral vector from the Gage lab encodes GFP and Cre-recombinase...Experimental uses include: Cre-dependent gene expression, gene knockout, and selection marker removal. ...Expressing your gene of interest pLenti-puro (Plasmid #39481) You can clone your gene of interest into...

...detect gene copy number based on the number of fluorescent puncta observed. By labeling two genes of a ...Proteins Control Protein Activity with GFP Resources on Asddgene.org Find Fluorescent Protein Plasmids ...vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its gRNA-directed targeting...signal to noise ratio of the system, they modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly...the amount of background fluorescence from unbound GFP-dCas9. With these improvements, labeling efficiency...Chen et al. successfully labeled protein-coding genes with both intronic and exonic gRNAs. In fact, the...also monitor the spatial relationship of the two genes over time, at a range of distances from 2-75 Mbp...

...array plus a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted...Cpf1 multiplexing method, other multiplex CRISPR gene editing methods relied solely on Cas9. Overall, ...4 targets when an array of 4 crRNAs, Cpf1, and a GFP tag were transfected on a single plasmid. This is...Lastly, an AAV vector was used to multiplex edit 3-4 genes in a primary culture of mouse neurons and in vivo...~75% of neurons were co-transduced with Cpf1 and GFP.  Of these neurons, ~15% had indels at all 3 targeted...summary of multiplexing efficiency in vivo. Multiplex gene editing with CRISPR-Cpf1 is one of the latest developments...transfection, viral transduction) for simultaneous multi-gene editing with a single crRNA array. Are you interested...

...includes a set of plasmids each containing an anti-GFP nanobody fused with a different functional element...using different detection methods. Since the anti-GFP nanobody is able to bind fluorescent proteins derived...here can be used to target any protein fused with GFP, YFP, mCerulean, or other related fluorophores. Find...specifically investigated protein binding at the TERT gene, but this method enables the unbiased discovery ...cDNA sequencing and RNA sequencing of complex heterogeneous RNA samples. Furthermore, such high processivity...

...systems allow researchers to understand bacterial gene clusters that produce useful natural materials, ...capable of robust expression of high G+C content genes, as validated through monitoring the expression ...coding sequence for a green fluorescent protein (GFP). The success of the A-to-G conversion could be seen...through the success of the change in expression of GFP, for the stop codon should have changed from “TGA...cytometry is used to sort populations by mCherry and GFP expression to check for ABE’s. b) Quantification ...human mRNA binding proteins with 10 guide RNAs per gene. The lab modified the lentiviral backbone to encode...pooled library from Junjie Chen's lab targets human genes known or suspected to be involved in DNA damage ...

... to track where GFP labelled B cells were in mounted slices of these organs. These GFP labelled cells ...enabled by fluorescent proteins The optimization of GFP and its derivatives by the late Roger Tsien changed...stimulus. Using an epifluorescence microscope and GFP fusion proteins controlled by promoters specific ...the immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph... the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after infection...molecule (see figure to the right). The excitation generated by these two-photons occurs only at a chosen focal...Laser include: Blue-green fluorescent proteins like EGFP Yellow-orange fluorescent proteins like TagRFP, ...

... later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the...genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445-AAVrg) alone at 1.1E6 vg/mL, or both... Karl Deisseroth (Addgene viral prep # 55632-AAVrg). pAAV-CAG- FLEX-rc [Jaws-KGC-GFP-ER2] was a gift from... plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral helper sequences...viral vectors are tested in vitro and in vivo for gene expression and/or function. These data are sometimes...copies of the lentiviral Rev responsive element (RRE) gene and the cellular ribonuclease P/MRP 30 kDa subunit...vector genome titers by droplet digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org...

...capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph of H1.8-GFP nucleosomes collected...editing, this epigenetic strategy doesn’t induce DNA damage or permanently alter a gene product; it simply...In MagIC-cryo-EM, target proteins (expressed as a GFP fusion in this proof-of-concept study) are captured...SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines ...) 3D model of the obtained structure of the H1.8-GFP-bound nucleosome. Images adapted from Arimura et ...on Prime-editing-Assisted Site-Specific Integrase Gene Editing (PASSIGE), their previously-reported technique...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...

...lactose released activates expression of GFP from the lac operon. GFP expression can then be used to determine...(including lactose), and a lac operon controlled GFP. When the enzyme breaks down the HMO of interest,...Fatima's work and keep checking back in with the Addgene blog for future award opportunities. Xin Zhou:...when exposed to ultraviolet light. To do so, she generated fusion proteins in which Dronpa domains flanked...Proteins, or FLiPs.  Xin later optimized these first generation FLiPs by mutating Dronpa to lower its aggregation...second sensor is a fusion protein consisting of sfGFP fused to a His-tagged ABC transporter known to bind...Chemical Biology! Additional Resources on the Addgene Blog Learn More about Photosactivatable Fluorescent...

...plasmids Tagging human transcription factors with GFP Kevin White’s lab recently deposited a set of CRISPR...plasmids to tag human transcription factors with GFP. As part of the Encyclopedia of DNA Elements (ENCODE...proteins for optogenetic control of CRISPR-Cas9. By combining AcrIIA4, a Streptococcus pyogenes Cas9 inhibitor... a few of the new CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR...adapted for detecting SNPs. Learn more from this Addgene blog post Read the paper in Science Find the...the Cas14 plasmids Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR proteins that...editing is now possible. Learn more from this Addgene blog post Read the paper in Nature Methods Find...

...Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126.   Gene disruption in...with expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered...tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent reporter...through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the new mScarlet3 and...developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus, an increasingly...Here, the researchers were able to target multiple genes in M. abscessus either simultaneously or stepwise...system is induced to cause disruption of the target gene. Lastly, curing the mCherry-sgRNA plasmid leaves...

... cassette to the gene of interest. The gene to be expressed is amplified using a gene-specific forward...sgRNA-targeted gene, resulting in increased activation of endogenous transcription of the target gene. The brilliant...of downstream genes. Nihongaki et al describe the targeted activation of endogenous genes and detail the...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant*...resistance gene (missing the first two codons). Two stop codons are inserted between the truncated gene and ...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins... inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (...

...deletion of one or several of these genes permits the expression of genes farther downstream in the construct... two tandem sets of genes aligned head-to-head and the deletion of one of the gene pairs. Brainbow 2.0... from the default GFP (green) to RFP, CFP, or YFP fluorescence. In Brainbow 2.1, GFP is tagged with a ...recombination in this mouse generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic...Brainbow uses a Cre/lox system to label neurons. Genes encoding the fluorescent proteins are organized ...organized in tandem with a single promoter, but only the gene directly following the promoter is expressed. Cre-mediated... which uses Cre to trigger the inversion of two genes aligned head-to-head. Another variation, Brainbow...

...reporter gene (e.g. GFP) to identify and enrich positive cells or a selection marker to generate stable ...permanently knock out or knock-in a gene? Do you want to activate or repress gene expression? Are you trying to...separate transfer vectors. May contain reporter gene (e.g. GFP) to identify and enrich positive cells. Likely... create a fusion with a reporter protein such as GFP? All of these outcomes can be most effectively achieved...conjunction with activator or repressor domains to control gene expression. Base editors can help you make precise... for each, and potential delivery methods.   General considerations for planning your CRISPR experiment...Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts or knock-ins, or introducing mutations...

...Joachim Goedhart shares some strategies for generating GFP fusion proteins. From considering protein size... PS: they’re free. 3. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection by ... biology over the past year: CRISPR, optogenetics, chemogenetics, plasmids, fluorescent proteins, careers...proteins at Addgene. 7. Designing Your Chalk Talk for the Academic Job Interview by Erik Snapp Addgene depositor...2020! Additional resources on the Addgene Blog Read other Addgene news Find the most recent plasmid ...posts Resources at Addgene Browse the repository Find educational resources at Addgene Search for plasmids...discovery. We’ve documented many new tools on the Addgene blog and have created and updated guides on various...

...easily modified to tag hundreds of genes, generating powerful genetic screening libraries. Additional protein... to screen for the genetically modified strains that contain your newly tagged gene. Most insertions can... to generate high-throughput targeting vectors. These vectors can be used to tag endogenous genes and ...-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans.” Genetics. 202(4) (2016):1277-88....Since the discovery of GFP over 50 years ago, the growing spectrum of fluorescent proteins (FPs) has ...protein fusions is to replace the genomic copy of your gene of interest with a fluorescent protein fusion. CRISPR...repair pathway, a new user-defined DNA sequence (like GFP) can be inserted at the DSB. CRISPR has allowed scientists...