...The ready-made PITCh plasmids available from Addgene are perfect for expressing GFP from a given promoter...into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about ... An overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology... the GFP-Puro will be inserted just upstream of a stop codon. One potential concern is if the GFP-Puro...Guide Pages Find CRISPR Plasmids for Your Experiments Deposit Your New Plasmid Tools with Us  ... knock-ins, some labs have used NHEJ for whole plasmid integration; however, since NHEJ is error-prone...detailed protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of...

.... An expression plasmid encoding the ORF to be expressed Finally, the researcher needs a plasmid encoding...Find Synthetic Biology Plasmids Find Bacterial Expression Systems Find Plasmids for Protein Purification...host strain for protein production, and an expression plasmid encoding the open reading frame (ORF) of ... Two expression systems have been verified for use by the Rinehart lab: pCRT7 NT TOPO E17TAG GFP and pGEX...serine) population of protein using the same expression plasmid. This is a particularly important control...Alternatively, you can express the completely wild type protein from the same plasmid.  Upon verifying that...Sep-OTSλ plasmid - Addgene # 68292 C321.ΔA bacterial strain – Addgene #68306 pCRT7 NT TOPO E17TAG GFP plasmid...

...Knockdown the expression of Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene... used for plasmid-based experiments. What are control plasmids? Generally, control plasmids help to ensure...as the expression level of Gene X in cells treated with Plasmid A is similar to the expression level of...control may be a plasmid that constitutively expresses a reporter protein (e.g., GFP or luciferase) that...Figure 1, an internal control, such as the GFP-expressing Plasmid B, could demonstrate whether the cells ...follow an example of a plasmid-based experiment, delineate its requisite control plasmids, and discuss why ...proper control plasmids would address and answer these questions. Types of control plasmids Part of planning...

... have 10 HA or 10 MYC tags built into a GFP scaffold. Some GFP nanobodies  stain poorly when tagged with...weak signals of fluorescent protein (i.e. GFP, RFP) Detecting GFP reconstitution across synaptic partners... Resources on Addgene.org More nanobody expression plasmids can be found here Watch this video to learn...Monster because they recognize this GFP scaffold itself. Fortunately the GFP1 HA Spaghetti Monster RANbody doesn... a reporter and a nanobody that’s encoded on a plasmid. A nanobody is a ~150 amino acid, single domain... sequences that targeted the following antigens: GFP, RFP,  the histone protein H2A2B, and the actin-binding...could be used to make a RANbody. RANbodies are expressed in 293T cells and are secreted from cells due ...

... of GFP, it's variants, and their relevant mutations, check out Marcy's post from last week on GFP. Additionally... a few hours. For example, superfolder GFP (sfGFP) and mNeonGFP can fold in <10min at 37°C, mCherry takes...are PA-GFP, Dendra2, and the mEOS proteins. Some proteins are reversibly switchable (e.g. rsEGFP, Dreiklang...affected by the temperature. For instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most...bioscience. ilovegfp – a site with very comprehensive data sheets on many FP variants Plasmids 101: Green... the discovery of the Green Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria....of these FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing FPs to ...

...native expression levels. However, it is advisable to first express the fusion protein from a plasmid-based...the limitations is the size of GFP: ~240 amino acids or about 28 kDa. GFP and its homologues have a beta-barrel...instance, Lifeact-mTurquoise2 (Plasmid #36201) or mScarlet-tubulin (Plasmid #85045). We often use restriction...and shape matters The green fluorescent protein (GFP) from Aequorea victoria and its variants are genetically...interference is known as steric hindrance. For instance, GFP may occlude catalytic sites or obstruct binding sites...post-translational modifications. Despite these potential issues, GFP has been used successfully in countless fusion proteins... an inert light emitting module, any tendency of GFP to homodimerize should be avoided because it will...

...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...digestion of single molecules in real-time. Find plasmids expressing tTALE-FP Shin et al., Sci. Rep. 2019. https...protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry. In comparison...need. If you'd ever like to write about a recent plasmid deposit please sign up here.  Human whole genome...the stop cassette is removed, the effector is expressed. By truncating one of the recombination sites .... https://doi.org/10.1101/788679 A destabilized GFP reporter for calcineurin activity in yeast Article...

...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 Podcast!  MarathonRT...at Yale University recently deposited a plasmid for expressing a novel reverse transcriptase (RT), MarathonRT... Their toolkit includes a set of plasmids each containing an anti-GFP nanobody fused with a different ...with GFP, YFP, mCerulean, or other related fluorophores. Find all of the nanobody toolkit plasmids here...these lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9) fused to APEX2 is expressed from a Tet/Shield1...

...amazing plasmid technologies developed by our community of depositors. With over 40,000 plasmids avaliable...amazing new plasmid tools that have come through our doors in 2015. Do you have a favorite new plasmid tool ... mediated genome modification and gene expression. The plasmids associated with this article can be obtained...system plasmids are available through Addgene and are already very popular. Use these plasmids to increase... of the integrases is supplied on its own expression plasmid or in various combinations, while the cognate...bacterial expression vector which gives close to 100% cloning efficiency and gene expression. The pUCXKT...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins...

...of the new plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  ...matured considerably faster, with expression levels comparable to GFP during early embryogenesis (Figure...selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after identifying successful knockouts... transformed with the sgRNA cassette on a plasmid expressing mCherry and resistance selection marker HygR... or want to swap out the constant domains, the plasmids for the heavy chain and light chain are also available...difficult to study in vitro, requiring either expressing it with additional cofactors or using significantly...BY license. This construct can be easily expressed and purified from E. coli, retains important functional...

...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...

...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...

...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...

...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. ...