...enable this color switching, TRICK requires the following components: a GFP protein that binds to the coding... a reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter...Plasmid 64542 or 64543) Before translation, both GFP and RFP are bound to the reporter, causing it to ...appear yellow. The act of translation pushes NLS-PCP-GFP off of the mRNA, leaving only a red FP bound to the... contains a nuclear localization signal, NLS-PCP-GFP then returns to the nucleus to bind cognate untranslated...verified that translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known...Oskar protein levels were negatively correlated with GFP intensity, showing that TRICK is an accurate readout...

...containing the gene of interest (pTet-IRES-EGFP, pPRIME-Tet-GFP-FF3) and one with either tTA or rtTA (...be separated by an internal ribosome entry site (IRES) and expressed under the same promoter. Alternatively...viral titre to the number of cells transduced by a GFP encoded virus, and the reverse transcriptase activity...in terms of RCL generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for...that there is often  background activity and it requires continuous administration of Dox to repress transgene...One drawback of the Tet system is that it often requires the delivery of two distinct expression vectors...

...soCoChR-GFP Syn CoChR (soma-targeted) GFP Constitutive 9 Boyden 107712 pAAV-hSynapsin-FLEX-soCoChR-GFP Syn...pAAV-hsyn-Jaws-KGC-GFP-ER2 Syn Jaws GFP Constitutive 5, 8, rg* Boyden 84445 pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] CAG...* Deisseroth 58880 pAAV-Syn-ChR2(H134R)-GFP Syn ChR2/H134R GFP Constitutive 8, rg* Boyden 100054 pAAV....Constitutive 9 Harvey 59170 pAAV-Syn-Chronos-GFP Syn Chronos GFP Constitutive 1, 5, rg* Boyden 59171 pAAV-...Constitutive 8 Deisseroth 22222 AAV-FLEX-Arch-GFP CAG Arch GFP Cre dependent 1, 9 Boyden 28305 pAAV-FLEX-...dependent 5 Boyden 99039 pAAV-CamKII-ArchT-GFP (PV2527) CaMKII ArchT GFP Constitutive 1, 5, 9, rg* Boyden 137148...EF1a/nEF Synapsin E2 regulatory element Fluorophore GFP Red-wavelength fluorescent protein Yellow-wavelength...

... 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...Monster because they recognize this GFP scaffold itself. Fortunately the GFP1 HA Spaghetti Monster RANbody doesn...than HRP-RANbody staining Requires secondary antibody staining HA Tagged GFP, RFP, H2A2B Myc Tagged RFP...sensitive than HRP-RANbody staining Requires secondary antibody staining GFP, RFP, H2A2B   Are you ready... sequences that targeted the following antigens: GFP, RFP,  the histone protein H2A2B, and the actin-binding...recognize the scaffold and is useful for detecting GFP. Spaghetti Monster tagged RANbodies can be detected...

...engineered BFP variant that converts to GFP after a base editing event. GFP positive cells can be sorted and ... their hypothesis with GFP fluorescence. To do this they generated a silent GFP construct, that contains...the histidine to a tyrosine generating a GFP variant (GFPY66) that has a shifted emission spectra. Thus...lab was inspired by work that used an integrated GFP reporter that is converted into BFP (blue fluorescent...engineered a BFP variant that undergoes conversion to GFP after being targeted by a cytidine deaminase base... a successful base editing event would result in GFP fluorescence that can be visualized and sorted via...results in shift in emission spectra from BFP to GFP. Image from Standage-Beier et al., 2019. The team...

...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...base FP (for example, EBFP2(1-10) can be used with GFP(11)). Some of the articles linked below only provide...Blue EBFP2 EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with... Kamiyama Cerulean Cerulean(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with...background signal (especially for some older split-GFP constructs or certain cell environments). Also consider... variant of mScarlet Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of...

... the GFP-Puro will be inserted just upstream of a stop codon. One potential concern is if the GFP-Puro...Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about a month...detailed protocol for MMEJ-mediated knock-in of a GFP-Puro cassette into a given locus, just upstream of...microhomology to the insertion locus flanking the GFP-Puro cassette. Three double stranded breaks are necessary...necessary for knock-in: one on either side of the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies...microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the locus (see figure below). ... overview of PITCh. The PITCh plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology ...

... 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...on many FPs (particularly those derived from GFP) requires oxygen. Therefore, these FPs cannot be used ...affected by the temperature. For instance, enhanced GFP (EGFP) was optimized for 37°C, and is therefore most... the discovery of the Green Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria....below (or emission wavelenght range).  By mutating GFP, the variants blue FP (BFP), cyan FP (CFP), and yellow...

...collection is now complete! pAAV-CAG-GFP (plasmid 37825) and pAAV-hSyn-EGFP (plasmid 50465) are now available...DreO-bGHpA (50363-AAV5) Flp vectors pAAV-EF1a-mCherry-IRES-Flpo (55634-AAV1) pAAV-EF1a-Flpo (55637-AAV1) Viral...our entire AAV inventory. Our new AAVs include: EGFP-expressing AAV for serotype testing Calcium sensors... testing AAV, which are small (20 ul) samples of EGFP-expressing AAV packaged in various serotypes. This...recently released these controls: pAAV-hSyn-DIO-EGFP (50457-AAV5): The Synapsin promoter directs broad...broad, neuronal expression. AAV pCAG-FLEX-EGFP-WPRE (51502-AAV5): The CAG promoter directs ubiquitous expression...interneurons. pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP (112677-AAV5 and 112677-AAVrg): The EF1a promoter...

...P2A-linked accessory genes gvpNJKFGWV marked with Emerald GFP. These plasmids can be used directly in transient...gating for the top quartile (or higher) of BFP and GFP producing cells. After FACS, return the cells to ...Simply cotransfect a mixture of 1248 ng of CMV-gvpA-IRES-BFP plasmid and 954 ng CMV-gvpNJKFGWV plasmid into...

...reported was PA-GFP. PA-GFP was derived from Jellyfish (Aequorea victoria) GFP (wtGFP) by mutating Threonine...mutation in PA-GFP generates a chromophore population that is more neutral, causing PA-GFP to display almost...example, by engineering PA-GFP tagged to a mitochondrial matrix protein (mito-PAGFP) the Youle Lab was able...the neutral to the anionic form, which allows PA-GFP to exhibit up to a 100-fold increase in fluorescence...when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent PA-FPs...in vivo imaging models perfer the PA-RFPs over PA-GFP. Another class of irreversible PA-FPs are the photoconvertible...Fluorescence) Brightness Excitation/ Emission (nm) PA-GFP Monomer UV-Violet Dark/ Green ~200 Medium 504/ 517...

...recombinase. If GFP is under control of an endogenous promoter, you can use expression GFP to track cells...tag an endogenous protein with GFP, as seen in blue flame plasmid OCT4-eGFP-PGK-Puro from the Jaenisch lab...In order to both knockout a given gene and insert GFP into the genome, we’d create a plasmid similar to...to the one shown below, where the sequence of GFP is cloned together with the Neomycin resistance (NeoR... 3 of the targeted gene. Upon recombination, the GFP/NeoR cassette is inserted in place of exon 2. Thus...gene is disrupted (knocked out) but the inserted GFP is expressed (knocked in). As seen in the above example...knockout/knock-in targeting vector designed to insert GFP into a given locus.   Conditional knockouts ...

...the viral protease, Nsp5. Our set also includes a GFP construct as a control. For easier portability, all...vectors if you need to. Additionally, they have an IRES-Puro marker to facilitate stable cell clone selection...

...overlap. Figure 4 compares the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore...to fuse them with a fluorescent reporter, such as GFP or mCherry, through genetic modification. As your... you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies together in...while wavelengths over 700 nm make up the infrared (IR) spectrum. When photons are absorbed by matter, they...vector also encodes for eGFP, which serves as a transduction reporter. Note that eGFP is not fused to CD45...-Cy7. Next comes our transduction reporter, eGFP. As eGFP emits fluorescence by itself and does not need...cytometer which channel to use to detect eGFP. As you saw in Figure 4, eGFP strongly overlaps with FITC, hence...

...pSpCas9(BB)). The 2A-GFP allows for screening of transfected cells. Find pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR...pSpCas9n(BB)-2A-GFP (PX461) - Similar to pSpCas9(BB)-2A-Puro (PX458), pSpCas9n(BB)-2A-GFP (PX461) expresses...requested plasmid of 2017. Find pMD2.G. pSpCas9(BB)-2A-GFP (PX458) - This plasmid from the Zhang lab expresses...nick, rather than cleave DNA. Find pSpCas9n(BB)-2A-GFP (PX461). pSpCas9(BB)-2A-Puro (PX459) - This mammalian...lab that contains the gag and pol genes. It also requires pRSV-Rev and the envelope expressing plasmid pMD2...

...dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP, and...and GFP shRNA. Mice carrying the R26DsRedR; CRUSH-GFP; and Nestin-Cre alleles showed efficient GFP knockdown...identified a family of GFP proteins in the cephalochordate Branchiostoma floridae, named bfloGFPs. They went on...and demonstrate that they can successfully create GFP fusion proteins with a variety of genes across the...enabled scientists to engineer a wide variety of GFPs with diversity in fluorescence brightness, intensity...Cas9 gene editing. Dr. Masahito Ikawa has created a GFP reporter plasmid for scientists to validate the efficacy...plasmid undergoes HR or SSA to reconstitute functional GFP, and the cells will turn green. Mashiko et al.,...

...array plus a GFP tag. Four weeks post-infection, there was strong expression of Cpf1 and GFP in the targeted...4 targets when an array of 4 crRNAs, Cpf1, and a GFP tag were transfected on a single plasmid. This is...~75% of neurons were co-transduced with Cpf1 and GFP.  Of these neurons, ~15% had indels at all 3 targeted...transduction One vector expresses Cas9, eGFP, and up to 4 gRNAs Each gRNA requires its own promoter   Kabadi et ...crRNA/gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5'...vector expresses Cas9 and up to 7 gRNAs Each gRNA requires its own promoter Sakuma et al   Multiplexed...gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9 Nissim et alTsai et al...

...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 ...Fluorescent Proteins Control Protein Activity with GFP Resources on Asddgene.org Find Fluorescent Protein...of these orthologs is key: since each ortholog requires a different PAM sequence, a gRNA designed for ...color is specified by PAM sequence, each color requires a different Cas9 ortholog, and the target sequence...in combination with gene editing. CRISPRainbow requires very short, 11-mer gRNAs, which do not induce ...

...MSCV-IRES-EGFP IRES Retroviral pMSCV-pBabeMCS-IRES-RFP IRES Retroviral pMSCV-IRES-YFP II IRES Retroviral...pCMMP-MCS-IRES-Puro IRES Retroviral pEF1a-IRES-Neo IRES Mammalian MSCV-IRES-Luciferase IRES Retroviral...plasmids for research purposes are described below. IRES Elements Translation in eukaryotes usually begins... element called an Internal Ribosome Entry Site (IRES) to allow for initiation of translation from an ...mRNA. In the figure above, you can see that the IRES element acts as another ribosome recruitment site...co-expression of two proteins from a single mRNA. IRES was originally discovered in poliovirus RNA, where...in eukaryotic cells.1,2 Since then, a variety of IRES sequences have been discovered - many from viruses...

...Rabies and Neuronal Tracing References Wickersham IR, Finke S, Conzelmann KK, Callaway EM. 2007a. Retrograde...Nat Methods. 4(1):47-9. PMID: 17179932 Wickersham IR, Lyon DC, Barnard RJ, Mori T, Finke S, Conzelmann...Description Serotype PI 52473 pAAV-synP-FLEX-splitTVA-EGFP-B19G Can be used to complement deletion-mutant rabies...virus AAV1 Wickersham 100798 pAAV-syn-FLEX-splitTVA-EGFP-tTA These AAV together can be used to complement...