...effects of GFP fusions to inntags or traditional FLAG, HA, and Myc tags. Impressively, IT6-GFP did not reduce...about other Protein Tags in our Plasmids 101 Series. Get the Inntag Plasmids Tag endogenous proteins with...cell size or viability compared to untagged GFP overexpression, while the rest of the fusions had some negative... in a scientist’s toolbox. As we’ve covered in Plasmids 101, tags can help you determine localization ...protein of interest, purify it, or determine its expression level without the need for a custom antibody....testing indicated that inntags IT5, IT6, and IT10 expressed well in cells and did not form undesirable aggregates...

...and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid Transduction...2019b). CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a... out a pair of genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME Inducible knockout...knockout of a single gene pXPR_068 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME ...types (lineage-based) pXPR_070 Violet-excited GFP (vex) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks...of genes sequentially pXPR_220 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME...  The X-CHIME deposit also includes pXPR_071, a plasmid that leads to higher titer lentivirus preparations...

...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...of robust expression of high G+C content genes, as validated through monitoring the expression of fluorescent...seen through the success of the change in expression of GFP, for the stop codon should have changed from... is used to sort populations by mCherry and GFP expression to check for ABE’s. b) Quantification of base-editing... CRISPR plasmids are always being added to the repository. To find all of the CRISPR plasmids available... Xiao Wang Lab has recently deposited a set of plasmids that helps identify base edited cell populations...

...of the new plasmids, antibodies, and viral preps in our repository through our Hot Plasmids articles. ...2024). In MagIC-cryo-EM, target proteins (expressed as a GFP fusion in this proof-of-concept study) are...capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph of H1.8-GFP nucleosomes collected...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 ...be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM here!   Arimura, Y., Konishi...disease phenotype in an animal model. Find PE-eVLP plasmids here! The lab’s other recent deposit builds on...

...donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression system...included with each expression system as well as suitable applications. Expression System Components ...System Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible...or electroporation. Transient expression of CRISPR components, expression decreases as RNA is degraded ...using cationic lipids. Transient expression of CRISPR components, expression decreases as gRNA and Cas9 protein... provide an overview of some CRISPR mammalian expression systems, the typical applications for each, and...gene? Do you want to activate or repress gene expression? Are you trying to create a single point mutation...

...in the Lab Plasmids 101: Screens vs. Selections Plasmids 101: Knockout /Knock-In Plasmids ...could infer that the gene which GFP was monitoring was being expressed in the same pattern. These systems...the Lab Bench Plasmids 101 e-book Additional resources on the Addgene blog Plasmids 101: Using Transposons...fundamentally the reporter construct monitors the expression of a nearby gene. The reporter gene serves as...as a proxy for the endogenous gene – its expression levels (high/low) and patterns (tissue/timing) can... way. For example, if green fluorescent protein (GFP) was the reporter gene and green cells were observed...function of a gene. For example, they eliminate the expression of the gene (by CRISPR knockout, shRNA depletion...

...upstream of GFP increased expression of GFP six-fold, but a stabilized promoter increased expression only two...this case, plasmid copy number is the input and gene expression is the output. The input, plasmid copy number...required to decouple gene expression and plasmid number. In a suite of plasmids modified from pSC101 with... gene expression also remained stable regardless of plasmid copy number. Stable gene expression whether... genes were expressed. What’s the use of stable gene expression? Constant gene expression can enable more...Researchers express genes of interest from plasmids in order to study gene function or to engineer cells...a look at how plasmids recently deposited by Chris Voigt’s lab help keep gene expression stable regardless...

...co-injection of the targeting vector and a Cas9 expression plasmid will insert the desired genetic tag and marker...as a universal donor plasmid. The CRISPaint toolkit includes several donor plasmids for protein tagging...line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging vectors (eg GFP, Flag, YFP...includes a prebuilt donor plasmid library containing several types of fluorescent (EGFP, tagRFP, mCherry) and...Since the discovery of GFP over 50 years ago, the growing spectrum of fluorescent proteins (FPs) has ...repair pathway, a new user-defined DNA sequence (like GFP) can be inserted at the DSB. CRISPR has allowed scientists...The Jorgensen Lab recently developed a modular plasmid assembly toolkit called SapTrap to improve CRISPR...

...the transfer plasmid, a plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral... later, Cre-dependent GFP expression was detected with direct fluorescence. GFP was not detected in the.... coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.   ...contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic...vectors are tested in vitro and in vivo for gene expression and/or function. These data are sometimes reported...viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445-AAVrg) alone at 1.1E6 vg/mL, ...the absence of Cre. mCherry expression alone was detected. pAAV-Ef1a-mCherry-IRES-Cre was a gift from ...

...self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP...apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11) to increase fluorescence... reporters (Zhang et al., 2019). How does FlipGFP work? GFP contains 11 β-strands and one central ɑ-helix..., and Drosophila. They treated HeLa cells expressing FlipGFP with staurosporine, which induces apoptosis...Lab used FlipGFP to track the spatiotemporal dynamics of apoptosis during embryo development. GFP fluorescence...diseases and disorders. You can find the plasmids for FlipGFP and FlipCherry at Addgene. References ...protein-protein interaction sensor based on tripartite split-GFP association." Scientific reports 3 (2013): 2854. ...

...curated collections of plasmids as kits with greatly reduced costs per plasmid. We generally create kits...: FX Cloning Kit BIOFAB Plasmid Set pCri System Cell Free Protein Expression Kit FREQ Seq PUF Assembly... of GFP and RFP fusions as their test genes, the researchers at the BIOFAB showed that expression of many...just the tool you need. The plasmids in this portion of the BIOFAB Plasmid Set contain terminators of ...your polycistronic E. coli expression constructs. Some applications of plasmids from this kit include the...Process with pCri Cell-free protein expression kit Cell-free expression systems have been improved for both...benchmarking of cell-free expression systems, and tested for product integrity, expression level, and aggregation...

...high expression, so adding this G could help with expression, though it is possible for the plasmid to ...use double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase). The concept...experiment, consider co-transfecting with GFP. This allows you to sort for GFP-positive cells and to enrich for...articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions...genome editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR genome editing experiment...concept of the double nickase system is that you can express two different chimeric gRNAs with the Cas9 nickase...backbone that uses the human U6 promoter to drive expression, is it necessary to add a G nucleotide to the...

...activates Ras. They expressed this OptoNB-SOS fusion protein in cells that expressed membrane-localized... Resources on Addgene.org Find nanobody expression plasmids here Learn more about optogenetics in Addgene's...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...more OptoNBs, one against mCherry and one against GFP. Insertion of AsLOV2 into loop 1 always generated....  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced ...until the message reaches the nucleus where gene expression changes lead to cell proliferation and differentiation...

...630 64 Luciferin ATP No Common uses for plasmids expressing luciferase Luciferase reporter assay The ...Read More: All Plasmids 101 Articles Browse plasmids that contain luciferase ...assays have since become a gold standard in gene expression analysis and a luciferase gene (one of many available...choose from) is now a common feature in reporter plasmids.  Learn How Luciferase Can Be Used In Concert ...effect of promoters or enhancer regions on gene expression can be determined by detection of the reporter...such as excitation of Green Fluorescent Protein (GFP) because of the nature of the chemical reaction compared...to using a high-intensity laser to rapidly excite GFP. As a result of the different mechanisms to produce...

...promoter is expressed. Cre-mediated deletion of one or several of these genes permits the expression of genes... from the default GFP (green) to RFP, CFP, or YFP fluorescence. In Brainbow 2.1, GFP is tagged with a ...fluorescence of the downstream OFP, GFP, or RFP. Cells that express the construct but do not undergo recombination...activate the expression of a different fluorescent protein. Therefore, each cell could express a unique combination...this mouse generates cells that randomly express either nuclear GFP, membrane CFP, cytoplasmic YFP, or cytoplasmic...copy of the transgene expressing either YFP or CFP, and cells that do not express Cre fluorescing with ...the low expression of XFP of Brainbow by incorporating a Tet-Off system to boost XFP expression. The increased...

...Protein Activity with GFP Resources on Asddgene.org Find Fluorescent Protein Plasmids Find Fluorescent Protein...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...sequences, which recruit fluorescent proteins BFP, GFP, and RFP fused to the respective hairpin binding ...hairpins generates white light. All 6 gRNAs can be expressed in a single vector, pCRISPRainbow-DONOR1, with...CRISPRainbow gRNA will permit labeling, but a newly expressed standard length gRNA would “switch” the system...24360272. PubMed Central PMCID: PMC3918502. Find plasmids from this publication at Addgene. 2. Ma, Hanhui...

...Chang-Deng Hu Green sfGFP Split super-folder GFP, our most-requested GFP(1-10) and GFP(11) Versatile protein...Commun. 2016 Bo Huang spGFP Split superpositive GFP Split-superpositive GFP reassembly is a fast, efficient...popular split-FP plasmids from our catalog to help you find the right split-FP plasmid for your experiment... vitro, and can be used for measuring protein expression or localization in cells, or even single-particle...base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles linked below only provide...below, and click the Article link to find all the plasmids from the associated paper:   Color Base FP...Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with...

...In the presence of Cre, GFP expression is lost, allowing for rescued expression of GeneR.   Sometimes...elements to turn off expression of one gene, while simultaneously turning on the expression of another! What...FLEx switch that turns BFP expression off, while turning on mCherry expression. For this FLEx switch to ...presence of Cre recombinase. This plasmid now specifically drives expression of mCherry instead of BFP. Beyond...allowing for expression of GeneR. This allows a researcher to test whether or not expressing GeneR alone...potential to express the transduced reporter gene. Generating a FLEx switch to control expression of an optogenetic...ensures Cre-dependent reporter expression, the potential for background expression of the reporter will be greatly...

...Fluorescent Protein Resources Find Other Plasmids that Glow (Luciferase Plasmids) Find Optogenetics Resources ...remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant...contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite multiple studies ...protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general... a tag with a serious drawback. The standard EGFP plasmid that used to be sold by Clontech and is in a...in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently...best solution is to use truly monomeric FPs. EGFP and other GFP family members (superfolder, Emerald, CFP...

...individually expressed proteins run in separate lanes. B: individually expressed and coexpressed proteins ...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling...editing within a single expression cassette is advantageous to Cas9’s larger expression requirements (Figure...Image from Santilli, et al. 2021 Plasmids are used to express 10, 15, 20, 30, 40, 50, 60, 80 and 100...through two polycistronic expression vectors (3570 lanes/50 mLs) coexpressing a set of either 10, 30, 50...

...antibodies (mouse, rabbit, and chicken) to this tag! GFP  GFP is more than a peptide – it’s a 238 amino acid...localization. If you need monoclonal GFP antibodies or guidance on designing a GFP tag Addgene has you covered!...endogenous WT levels of the same protein when both are expressed in the same system. Tagging can also aid in protein...acid protein that can also be used as a tag! GFP was originally derived from jellyfish (Aequorea victoria...epitope tag as well as a fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry...antibodies also exist for imaging. The obvious con of GFP is its size – which can possibly interfere with protein...Addgene blog Antibodies 101: Epitope availability Plasmids 101: Protein Tags Antibodies: Of Myc and Men ...

...S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness... There are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made...examined on a case-by-case basis. GFP is sensitive to acid The chromophore of GFP can exist in a protonated and...its acid tolerance is maintained in cells.   GFP is big GFP is a 28 kDa protein that resembles a cylinder...2013; Hilaire et al., 2017). GFP can only be used for tagging proteins GFP fusion proteins are made by ... a fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps... al., 2018). GFP is not tolerated in my fusion It is standard practice to attach the GFP to the N- or ...

... in a plasmid with loxP sites such that their expression is turned on or off in cells expressing Cre. ...immune lineage, scientists could easily track GFP expressing cells. For example, by harvesting lymph nodes... to track where GFP labelled B cells were in mounted slices of these organs. These GFP labelled cells ... depicts the infiltration of neutrophils expressing LysM-GFP (green) into a draining lymph node after ...allows the visualization of fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell...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 ...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...transduced at levels similar to that of a single AAV expressing GFP. The lab tested the base editors in vivo and...determined protein expression levels from different promoters, and inhibited gene expression using CRISPRi....need. If you'd ever like to write about a recent plasmid deposit please sign up here.        AAV delivery... Figure 1: The base editor is split into two plasmids and co-transfected into cells for reconstitution...voltage indicators, however, fall short compared to GFP-based sensors in terms of response and brightness...

...can also detect expression of toxin mRNA from Clostridium difficile. As mRNA expression cannot be identified...conjugative mobilization of broad host-range IncQ plasmids." Plasmid 62.2 (2009): 57-70. PubMed PMID: 19465049...page Deposit microbiology plasmids with Addgene Find synthetic biology plasmids ...family-RP4 conjugation system. This system can deliver plasmids to both Gram-positive and Gram-negative cells.... Wang lab further developed a modular suite of plasmids (pGT) with various replicative origins that can...selectable marker and a desired genetic payload (ex: GFP). Thus, cells can be both sorted using FACS and isolated...genera across four phyla that have taken up the plasmid through conjugation in vivo studies. However, they...

...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles. ...transcriptional activation events as bands of induced GFP-iPAK4 expressed under promoters like the tet-responsive element.... Today, it's time for another edition of Hot Plasmids!  Here's what you'll find in this post:  New...., 2023 under CC-BY 4.0 License. Find PE6 plasmids here!  Doman, J.L., Pandey, S., et al. (2023)....therapies and medical applications.  Find DIRECTED plasmids here! Strebinger, D., et al. (2023). Cell type-specific...pulse-chase experiments, and more. Find LOV-Turbo plasmids here! Next, Qin and Cheah et al. described TransitID...LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments open up new possibilities...

...pMAK464 (NBαEGFP, Addgene #140702) The pMAK461-64 plasmids were expressed in SHuffle T7 Express competent...AAV for expression in forebrain GABA-ergic interneurons. These include mDlx-GFP, hDlx-FLEX-GFP, hDlx-Flex-dTomato...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...too high expression of these markers can lead to non-specific labeling. They generated plasmids tagged ...easier to clone, express, and purify compared to antibodies. Nanobodies are easily expressed in the cytosol...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly ...

...monitor autophagy, the Pho8Δ60 mutant protein is expressed. Pho8Δ60 localizes to the cytosol in an inactive...electron microscopy analysis is time consuming. GFP-tagged Atg8p or LC3: ATG8 is part of a group of genes...with autophagosomal membranes, so tagging it with GFP allows for tracking the localization or accumulation...autophagosomes and autophagic bodies in yeast. A GFP-tagged version of LC3, the mammalian Atg8p homolog...pH-insensitive RFP variant, DsRed.T3 and a pH-sensitive GFP variant, super ecliptic pHluorin (SEP). They are ...yeast Rosella is targeted to the cytosol when expressed without a signal sequence and to the mitochondria...Fluorescent Protein Oligomerization Seeing Red: Simple GFP Photoconversion Light Sheet Fluorescence Microscopy...

...lactose released activates expression of GFP from the lac operon. GFP expression can then be used to determine...fantastic tools it’s now easy to monitor protein expression, localization, and protein-protein interactions...(including lactose), and a lac operon controlled GFP. When the enzyme breaks down the HMO of interest,...Addgene.org Find Fluorescent Protein Biosensors Browse Plasmids from the Tsien Lab Browse the Michael Davidson...second sensor is a fusion protein consisting of sfGFP fused to a His-tagged ABC transporter known to bind...

.... Various soCoChR plasmids described in this work including the soCoChR-GFP plasmid as well as several...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...write about a recent plasmid deposit please sign up here.     Listen to the Hot Plasmids episode! Expanding...in patched cells expressing wild type CoChR, these APs are absent in soCoChR-expressing cells. Finally,...further probe cellular function. The CRY2clust Plasmid can be found here. Park et al. Nat Commun. 2017...C-terminus of the protein. Unlike the diffuse neuronal expression observed with wild type CoChR, soCoChR localizes...