..., 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. cerevisiae... artifacts sometimes associated with plasmid-based overexpression. Tim Formosa, at the University of Utah...Addgene distributes several ready-to-use, modular plasmids, combining fluorescent tags, epitope tags, protease...interest. These fluorescently tagged genes are expressed under native conditions and allow scientists to...FPs that are codon optimized specifically for expression in yeast. These tagging vectors are based on ...). Additionally, Dr. Formosa has deposited six plasmids with a multiple cloning site in place of the epitope...Jürg Bähler have deposited a large collection of plasmids for genome modification in yeast developed by ...

... is now complete! pAAV-CAG-GFP (plasmid 37825) and pAAV-hSyn-EGFP (plasmid 50465) are now available as...directs strong, ubiquitous expression. This is a Cre-dependent switch that expresses nuclear mCherry in the...entire AAV inventory. Our new AAVs include: EGFP-expressing AAV for serotype testing Calcium sensors and...testing AAV, which are small (20 ul) samples of EGFP-expressing AAV packaged in various serotypes. This collection...pAAV-hSyn-DIO-EGFP (50457-AAV5): The Synapsin promoter directs broad, neuronal expression. AAV pCAG-FLEX-EGFP-WPRE...pCAG-FLEX-EGFP-WPRE (51502-AAV5): The CAG promoter directs ubiquitous expression. pAAV-mDlx-NLS-mRuby2 (99130...directs expression in forebrain GABAergic interneurons. pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP (112677...

...specimens, such as whole mouse brains expressing green fluorescent protein (GFP) (6).   The Dodt and Frank Bradke...cranial nerve afferents labelled with a AAV8 expressing eGFP under the human ubiquitin C promoter. Anatomical...subpopulation of neurons are brightly labeled with GFP (e.g. Thy1-YFP-H mice).  However, we recently developed...compared to transgenic animals (17).  These viral plasmids are available through Addgene.  The combination...University of Miami Miller School of Medicine. Find plasmids from the Lee Lab here. Pantelis Tsoulfas is an...University of Miami Miller School of Medicine. Find plasmids from the Tsoulfas lab here.     References: H...

... size of 12–15 kDa Ease of cloning and expression from plasmids, due to only needing one open reading ...highlighted back in 2015, where sdAbs were used to create GFP scaffolds for transcriptional activation.  sdAbs ... because sdAbs are so amenable to recombinant expression (and because maintaining a llama facility is ...interesting work done developing transgenic mice that express camelid variable domains (aka nanomice), which ...domains of heavy chain only antibodies) (B) Nanomice express hybrid heavy-chain only antibodies that are the...Membrane Proteins More resources from addgene.org  Plasmids encoding nanobodies at Addgene Sybody Generation...

...accelerate your research.   Why distribute virus? Plasmid distribution is booming, but, over the years, many...list of what’s available or suggest additional plasmids with the Viral Service Suggestion Form. Lentivirus... to knockout a gene, replace it, modulate its expression, or perform a genome-wide screen, we’ve got viruses...also have lentivirus for delivering control shRNAs, GFP, and rtTA from the Tet-on system. The first two can...experiments, while rtTA can be used to activate expression of a gene under the control of the tet operator...page and includes western blots as well as other expression data. If you have any questions about the Viral...Uses and Overview AAV: A Versatile Tool for Gene Expression in Mammals Additional Resources on Addgene.org...

... single protein is expressed, with the intent of purification, it will be expressed along with all other...other cellular proteins within the expression system (bacteria, yeast, mammalian, etc.). To individually...about that protein that no other protein in the expression system has. This is where affinity tags come ...peptides which do not naturally exist in most expression systems that can be added to the N or C terminus...on: The protein you are trying to purify The expression system If you want the tag to remain on your ...MBP tag, although large, can actually increase expression and solubility of the tagged protein (Fox and... (FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry, etc.). Fluorescent tags are primarily used...

...Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent Protein Guide...associated publication for the specifics of your chosen plasmid. Types of biosensors When choosing a biosensor...biosensors as an example. Many calcium biosensors include GFP fused to calmodulin (CaM) and the chicken myosin ...translocation. Targeting your biosensor Before expressing a biosensor in eukaryotic cells, think about ...about the best place for the biosensor to be expressed. Are you looking at an intracellular or extracellular...

...Description Expression Additional considerations CMV General expression mRNA Strong mammalian expression promoter...lower expression than U6. May have better expression in neuronal cells. U6 small RNA expression shRNA...promoter, which is necessary for getting the plasmid express our gene of interest. This element is responsible...some cell types. EF1a General expression mRNA Strong mammalian expression from human elongation factor ...give consistent expression regardless of cell type or physiology. SV40 General expression mRNA Mammalian...  Commonly used in expression systems for Drosophila. Polyhedrin General expression mRNA  Strong insect...Constitutive  Commonly used in expression systems for insect cells. CaMKIIa Gene expression for optogenetics mRNA...

...selection. Expression is driven by a fairly strong EF1alpha promoter. We have verified expression of correctly-sized...2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins in human...the viral protease, Nsp5. Our set also includes a GFP construct as a control. For easier portability, all...Western Blot for 25 constructs, and confirmed expression via mass spec for two of the remaining viral ...going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC and... has been inundated with requests to share our plasmids with other researchers. We have been excited to...Gordon) have been quite busy shipping and sending plasmids to all who request them -- in locations all over...

...high-copy plasmid-borne chromoproteins lost color overnight in liquid culture, though expressing the chromoproteins...for allelic exchange Figure 3: pTOX plasmids expressing amilCP and tsPurple genes. Image: Lazarus...coli BioBrick plasmids (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue...contrast, the widely used green fluorescent protein (GFP) gives a bright green fluorescence when exposed to...at the cells on the plate. For example, if your plasmid contains a gene encoding a blue chromoprotein, ...colonies to determine which cells have taken up the plasmid. Of course, other follow up methods like sequencing...gfasPurple) (Figure 2). Figure 2: Chromoprotein expression from bacteria on agar plate. Image: Liljeruhm...

...Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent Protein Guide...Representative images of HEK293T cells and neurons expressing nLightG or nLightR before/after application of...ΔF/F0 heatmaps. White insets indicate surface expression of the indicators over white dashed lines. Scale...contains circularly permuted green fluorescent protein (GFP), and nLightR uses a red fluorescent protein, mApple...accounted for in your experimental design. Find nLight plasmids here! Next steps While nLightG and nLightR greatly...

...drive high protein expression in vivo. Expression plasmids are often lost without plasmid addiction systems...cells to retain plasmids - if a cell does not inherit plasmid due to improper plasmid partitioning, it...unstabilized plasmids were retained in less than 10% of cells while plasmids containing the plasmid addiction...ensure that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction ...only survive if they retain the plasmid. Since their discovery, plasmid addiction systems have been used...found that if a mini-F plasmid carried a specific 700 bp segment of DNA, the plasmid was propagated better...selection in one plasmid, they were able to create an efficient cloning tool. The plasmid pKSCTMF contains...

...stem cells transduced with iTOP resulting in GFP expression. Image courtesy of Diego D'Astolfo. CRISPR... mutations and gene expression changes elsewhere in the genome. Second, expression is by nature transient...control could enter cultured cells and activate expression of a luciferase reporter. CPP was not required...causes a cell to take up, and continued protein expression would require multiple transduction events. For... to binary systems in which transient protein expression has a long-lasting effect, such as Cre- or Cas9...10.1016/j.cell.2015.03.028. PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters...

...colleagues built a Molecular Expressions collection including some 3,300 plasmids along with image galleries...Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection.  mEmerald plasmids and many more! ... resources to go with them. This collection of plasmids is available in an easily searchable format on...Fluorescent Protein Collection webpage. These plasmids are divided into the following categories: Over...list of cell markers all tagged with mEmerald. Plasmids to monitor protein-protein interactions through...Specialty constructs for biosensing. Over 300 plasmid backbones spanning a rainbow of fluorescent proteins...proposition, explained Michelle Baird, who has created plasmids as a technician in the Davidson lab for the last...

...lines expressing it. The Church lab fixed this issue by creating a new dCas9-VPR expression plasmid that...transfer vector for in-vivo expression and plasmids optimized for dCas9-VPR expression in drosophila and yeast...Cas9 nickase, and Cas9 activator expressing plasmids that can co-express 1-8 gRNAs. Ordon et al., 2016 tested...Neurosci 2016. PubMed PMID: 26950006 Plasmid ID Plasmid Name Plasmid Type 74309 pcDNA3.0-TRPV4-p2A-ferritin-p2A-mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...features and applications of a partiular plasmid or set of plasmids. We hope that these articles make it ... you to find and use the plasmids you need. You can find all the hot plasmids from 2016 below. With over...

...transforming both fusion plasmids, or mating two single haploid yeast fusion plasmid transformants into a ...Addgene Browse Our Yeast Expression Vectors Find Bacterial Two Hybrid Plasmids Read our Blog Post About...activation. Vary the expression levels of bait and prey proteins - overexpression can lead to false interactions...During this period, proteins were successfully expressed as fusions that retain their individual activities...in this case a reporter gene (e.g. luciferase or GFP), regardless of whether it interacts with a transcriptional...see Figure 2a) (4). These fusions are then co-expressed in a yeast strain that also contains a reporter...bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI’s interact, this...

...Addgene.org Find plasmids from the Bethune Lab Find Plasmids from the Roux Lab Find Plasmids for FRET ...labeling, BirA* fusion proteins are expressed in cells via plasmids or by the creation of stable cell lines... enriched relative to controls using Split-BioID-GFP and BioID run on six unrelated proteins. One caveat... et al saw different levels of fusion protein expression and biotinylation depending on which terminus... requires 6-24 hours for tetracycline induced expression of the BirA* fusion protein and saturation of... al allowed 24 hours for tetracycline induced expression and biotinylation of interacting proteins. This...Interested in using Split-BioID with your research? Plasmids are available from Addgene! References Schopp...

...drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes...step as simple as heat-shocking to induce the expression of a Cre recombinase contained within the knock-in...homology arms to knock in large epitopes, such as GFP (Paix et al., 2014). A further development of this...efficiencies, obtaining as many as 100 independent GFP knock-ins from a single injected animal.  PAM sequence.... elegans. These modular toolkits build single plasmid targeting vectors that encode the gRNA transcript... C. elegans, visit Addgene’s C. elegans CRISPR plasmids page. Future perspectives It is interesting to...using modified Cas9 proteins to regulate gene expression (CRISPRi/CRISPRa), visualize specific genomic...

...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  ...all applications. Addgene’s plasmid collection includes over 10,000 plasmids with a FLAG tag (!), but did...escape. Combining Cas9-PAGE with lentiviral sgRNA expression yielded editing of 80–90% in human cell lines..., Myc and SV40 Nuclear Localization Signals, and GFP. B) Workflow to assess Cas9-PAGE editing with viral... al. 2023 with permission.   Find Cas-PAGE plasmids here!   Zhang, Z., et al. (2023). Efficient engineering...structured illumination microscopy in HeLa cells expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm...inspired and informed this section.  Find mStayGold plasmids at Addgene here!   Ando, R., et al. (2023). StayGold...

...to be expressed. These permit exquisite control of gene expression and tracing of gene expression across... basal expression. Co-expression of repressor QS along with QF leads to intermediate expression from QUAS...the Church lab uses this system to control expression of GFP.)  To turn this inducible system into a repressible...transgene permits directed expression of that gene in cells that also express GAL4. Usage of this system...UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter with...basal state, and co-expression of the inducer QF can increase QUAS driven expression by ~3,300-fold in ... Shen. “Controlling gene expression with the Q repressible binary expression system in Caenorhabditis ...

...interest: Plasmid Color Expression Description pPROEX Aqua Cyan Bacterial Expresses Aquamarine with...Cyan Mammalian Expresses mammalian optimized Aquamarine mCerulean N1 Cyan Mammalian Express a gene of interest...Cyan Mammalian Expresses mammalian optimized CyPet pCyPet-His Cyan Bacterial Expresses CyPet with C-terminal... Mammalian Expresses mammalian optimized YPet pYPet-His Yellow Bacterial Expresses YPet with C-terminal...Mammalian Express a gene of interest fused to the C-terminus of SYFP2 Clover Green Mammalian Expresses Clover...Clover (a GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange Mammalian Express a gene of interest...used pairs to measure FRET. The table below lists plasmids that can be used to create your choice of fluorescent...

...plant plasmid resources at Addgene  Choosing an expression system Choosing an appropriate expression vector....org Find plant plasmids Visit our plant resource page Deposit arabidopsis plasmids with Addgene ...this method include: Cloning and transforming a plasmid into the bacterium Agrobacterium tumeficans - a...options, but here are a couple of my favorites. Plasmids from the Tsuyoshi Nakagawa lab are binary gateway...terminally tagged fusion proteins with fluorescent (YFP, GFP, CFP, RFP, etc.) or affinity tags (HA, FLAG). There...prefer Golden Gate assembly, there are a series of plasmids from the Dinneny lab that additionally allow for...for visual selection of transformants based on expression of RFP in the seed (Emami, Yee and Dinneny, 2013...

...Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection. Cellular expression Whether BiFC constructs... independent of PPIs. Overexpression can be assessed by comparing the expression levels between BiFC constructs...because it relies on a single BiFC pair. Find BiFC plasmids for your experiment at Addgene! How to Design ... steps: fusion protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These...constructs are expressed transiently by transfection or stably impacts the success of BiFC experiments...this can also be perturbed by supraphysiological expression levels. BiFC quantification This is where BiFC...cannot be used as negative controls because their expression level, stability, and subcellular localization...

...David Goldstein’s lab, will use GFP-ATP1A3, FLAG-ATP1A3, and ATP1B3 plasmids to explore the functional differences...studying four of those rare diseases will receive plasmids from Addgene worth a total of $5000.  Lowe Syndrome...involving the primary cilia. They’ll put a variety of plasmids to work to explore the effect of various OCRL1...there. This will help us acquire a lot of those plasmids so that we can try many different things in parallel...ATP1A3. The gene encodes a sodium-potassium pump expressed specifically in neurons of the brain. McSweeney...mutations. More specifically, they’ve proposed to use plasmids with modified nickase Cas9 protein in order to...

...fluorescent protein timers When FP timers are first expressed, they predominantly fluoresce one color but slowly...timers (whether as part of protein fusions or expressed independently) originally fluoresce green or blue...displays predictable kinetics over 14 hours of expression in C. elegans embryos. However, this initial ...ideal FP timers: 1. Ratiometric determination of expression time The ratio of mature to immature fluorescence...fluorescence from the FP timer is dependent on the total expression time but independent of protein concentration...Calibration curves correlating this ratio to the total expression time can therefore be used to determine how long... long a pool of FP timers has been expressed, either in whole cells or as a pool of fusion proteins localized...

...Proteins Control Protein Expression with Cre-lox Control Protein Activity with GFP Resources at Addgene.... kinase inhibitors, psMEK1 and ERK KTR-mRuby2 expressing cells were incubated with an inhibitor of choice...feedback. 3. Studying signaling pathways in vivo Expressing photoswitchable kinases in vivo not only allows...used in other model systems, but they need to be expressed in a tissue or organism that blue and violet light...photoswitchable kinases in my experiments? You can find the plasmids from Zhou et al here. In addition, it could be...Addgene.org Find KTR reporter plasmids Find photoactivatable fluorescent proteins in empty backbones in the...

...multiplexing; expressing 5 gRNAs simultaneously produces comparable gene knockdown to single gRNA expression. Although... a superfolder GFP fusion to stabilize the protein in mammalian cells. LwaCas13a-msfGFP can mediate cleavage...tracking, modulating splicing, and regulating expression of specifically targeted RNAs. Finally (and perhaps...at this finding through analyses of global RNA expression, cell stress, and RNA size distribution. When...27256883. PubMed Central PMCID: PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky,...) (2016):270-273. PubMed PMID: 27669025. Find plasmids from this paper at Addgene.  3. Gootenberg, Jonathan...28408723. PubMed Central PMCID: PMC5526198. Find plasmids from this paper at Addgene. 4. East-Seletsky,...

...recombination, RFP is expressed. Cre can mediate one of two deletions to allow YFP or M-CFP expression; these deletions...inversion. The Brainbow-2.1 construct can express one of four colors (n-GFP, RFP, YFP or M-CFP.) The construct...research? Check out Brainbow plasmids available from Addgene! Find Brainbow Plasmids @Addgene: Brainbow 1.0...1.0, 1.1, 2.1 plasmids Brainbow 3.0, 3.1, 3.2 plasmids References: Transgenic strategies for combinatorial...editing may be the hot new way to manipulate gene expression, but other gene manipulation systems remain valuable...ways to spatially and temporally control gene expression, especially in in vivo models, and new Cre-lox... of Cre-lox. Check out our previous blog post, Plasmids 101: Cre-lox, if you need a quick primer on how...

... be degraded, leading to reduced eGFP fluorescence. Cells expressing the library are then sorted by fluorescence-activated...CUSPER bioreporter such that fluorescent proteins expression is induced prior to inoculation with IPTG. In...control of promoters that control cell cycle gene expression.  Additional Resources on the Addgene Blog ...fluorescent protein topics page Get the basics on GFP Find microbiology blog posts Resources on Addgene.org...Addgene.org Find fluorescent protein biosensors Browse Plasmids from the Tsien lab Browse the Michael Davidson...variant library is monitored by fusing the variants to eGFP followed by an internal ribosomal entry site and...a variant has wild-type levels of abundance, the eGFP will be stable and fluoresce. But if the variant...

...assays to select both over- and under-expressing clones. If GFP-encoding cDNA is co-transfected at a favorable... Pages Browse Fluorescent Protein Plasmids Find Mammalian Expression Vectors ...and PCR, depending on the level of endogenous expression. We highly recommend the use of an electronic...ampules each of 4 clones with different levels of expression of our gene of interest. Advantages of the agarose...isolate individual cell clones with different expression characteristics from a population of transfected...Time CRISPR User) The Challenges of Cell Culture Plasmids 101: Mammalian Vectors Resources on Addgene.org...the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also...