...constitutively-expressed mARG plasmids driven by CMV promoters. Labs that are new to the mARG expression are advised... that, when expressed, enable cells to be visualized under ultrasound imaging. Expression of ARGs results...in bacteria, expressed poorly at body temperature (37oC) and were too burdensome to express in situ. As...that the plasmid could be maintained in the absence of antibiotics, resulting in the plasmid pBAD-bARGSer-AxeTxe...Fig. 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram of pBAD-bARGSer-AxeTxe... gene expression in deep tissue. However, not unlike the first-generation bARGs, poor expression and acoustic...Increasing expression and decreasing genetic footprint We took a cue from the native expression patterns...

... hSyn FLEx mGFP-2A-Synaptophysin-mRuby from Liqun Luo’s lab expresses membrane tethered GFP to enhance...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...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head...indels to measure time. These plasmids with different promoters driving expression of Cas9 were used in this...vectors expressing cytoplasmic targeted tdTomato and pre-synaptic targeted synaptophysin tagged eGFP. AAV...Hutchinson Cancer Research Center developed a pooled plasmid library encompassing all possible amino acid mutations...

...neuronal expression. CAG-FLEX: CAG promoter, FLEXed for expression in Cre-expressing cells hSynap: Human Synapsin...neuronal expression. hSynap-FLEX: Synapsin-1 promoter, FLEXed for expression in Cre-expressing cells. hSynap-FLP...systems you can use to express the iGluSnFR3 variants: pRSET: Bacterial expression vector, use with IPTG...FLP-dependent expression GFAP: Glial fibrillary acidic protein (promoter). Good for Glial expression (not neurons...lab swapped eGFP for sfGFP to create SF-iGluSnFR. This improved variant has higher expression levels in ...regulation. To create iGluSnFR, the Looger lab wedged GFP between two halves of a bacterially derived periplasmic...increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR in vitro, showing ...

...because they can be expressed in bacteria, while mAbs generally require mammalian expression systems. scFvs... and insect cells, they are most often expressed from a plasmid in bacteria (Ahmed et al., 2012). One ...binding scFv that’s fused to GFP. When the multiple copies of the scFv-GFP fusion bind the SunTag scaffold...One challenge of bacterial expression is the proper formation of the disulfide bond between the VL and VH...requires an oxidizing environment to form. When expressed in the non-oxidizing cytoplasm of bacteria, scFvs...more of these approaches are often used for scFv expression in bacteria:  targeting to the oxidizing periplasm...to allow for proper disulfide bond formation.  expression in strains of bacteria that are redox mutants...

...observe expression from Agrobacterium, but not the plant. For example, we have seen strong expression of the...comparison of the GUS, LUC and GFP reporter system for gene expression studies in plants." Plant Biology...transformation Read our Plasmids 101 blog posts Resources on Addgene.org Browse our Plant Plasmids and Resources...page Find synthetic biology plant plasmids Find CRISPR plant plasmids ...way to overexpress a gene and evaluate its function. In nature Agrobacterium contains a plasmid with a... transgenic cultures or plants, our transient expression method enables us to quickly screen and investigate...reporter gene so that the transformation and gene expression can be tracked. During a transient leaf transformation...

...of the new plasmids, antibodies, and viral preps in our repository through our Hot Plasmids articles.  ...2 nucleocapsid protein (Plasmid #141391-LV [+N]) or membrane protein (Plasmid #141386-LV [+M], negative...: A) Representative confocal images showing GFP expression from ssAAV5s with hIBA1 or hIBA1a promoters...Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s. C) Quantifications...using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi ... over the original CROPseq. Find CROPseq-multi plasmids here Walton, R.T., Qin, Y., Blainey, P.C. (2024...potential improvements to the editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime...

...Similar to last year, the top requested plasmid from 2016 is an expression vector for CRISPR nuclease, SpCas9...This is a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA in...mammalian cell types. Purpose  SpCas9 This plasmid expresses Streptococcus pyogenes (Sp) Cas9 from a Cbh...this is a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA of...for sure, as is obvious from our most requested plasmid, SpCas9 is still going strong as the basis for ...experiments. So, without further ado, the most request plasmid in 2016 was... pSpCas9(BB)-2A-Puro (PX459) V2.0...sequence as directed by a gRNA. 2A-Puro In this plasmid, SpCas9 is fused to the puromycin resistance gene...

...cloning plasmids, expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and ... of scientists and plasmid lovers - serves as an introduction to plasmids. Plasmids 101 will provide you...quantity inside a cell Plasmids 101: Green Fluorescent Protein (GFP) Plasmids 101: Luciferase Monitor...be asked to design, modify, or construct a plasmid. A plasmid is a small circular piece of DNA found in... components that make up a plasmid and why each is important. Our “Plasmids 101” series designed to educate...advancing the field.   What is a plasmid? At their most basic level, plasmids are small circular pieces of...cloning site. The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell ...

...hiPSCs  EMX1  67% Mouse BM cells from GFP transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~...compared to DNA transfection of gRNA and Cas9 expression plasmids. These results are in line with previous ...infiltrate cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction... gRNA targeting a DNA sequence of interest is expressed in 293T cells and loaded into Nanoblades by association...Nanoblades, different viral envelope protein(s) are expressed to pseudotype Nanoblades. The team found that ...types they tested. Interested in using Nanoblades? Plasmids are available from Addgene. Genome editing primary...measured by their maintenance of pluripotency marker expression and response to LPS stimulation, respectively...

...Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains 1 kb homology...and tested plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with GFP. These plasmids...found within the homology areas of the plasmid to prevent the plasmid from being cut and destroyed by CRISPR...introduced donor plasmid as a repair template. This results in the insertion of the GFP tag precisely where... the tag. Created with BioRender.com. Plasmids, plasmids everywhere To find out more about gene edited...found in the Cell Catalog on our data portal. The plasmids should be functional in many different human cell...constructs below. Read on to find out how these plasmids work and to discover more resources for your genomic...

...about GFP Controlling Protein Activity with GFP Resources at Addgene.org Find the Spinach Plasmids at ...nature of GFP fluorescence to create an improved fluorescent aptamer system. Knowing that GFP’s  structure...increase its fluorescence, essentially mimicking GFP.  Though the aptamer, called Spinach, required longer...processing using broccoli reveals optimal aptamer expression strategies.” Chem Biol. 2015 May 21;22(5):649...at Addgene Find Broccoli Plasmids at Addgene View All Fluorescent Protein Resources ...

...analyte), and accumulation of ATP activated GFP expression (receptor), then the transducer could be a ...plate reader, or any other instrument which measured GFP levels and converted that signal into a readable ...genetically encoded, and luckily, we have pre-designed plasmids for these systems! Ion sensors One common biosensor...retrovirus, can either permanently integrate and/or express long term (up to several years!). Biosensors tend...biosensor to specific tissues or cell (sub)types for expression and downstream monitoring. Nervous about making... vehicle, we’ve got you covered with packaging plasmids! References and Resources References  Bhalla ... as stable systems, rather than transiently overexpressing at the time of use. Another great viral perk...

...stability of modified Ti plasmids during plant infection, as well as plasmid yield during preparation ...novel versatile gene expression vector for transient assays as well as stable expression of foreign genes ...GreenGate Plasmids Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More Plasmids 101: Origin...Agrobacterium carries a “tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into... Ti vector for both transient and stable gene expression applications in plants. This smaller, easily ...in two species: Agrobacterium and E. coli. Like plasmids used in other systems, binary vectors are cloned...It’s a lot of information to fit into a single plasmid, so most first-generation binary vectors are quite...

...as close as possible to the basal expression level, induce expression at an opportune time or go all out... tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following the expression pattern of a defined promoter...you find what you need.   More Plasmid Resources from Addgene: Plasmids 101 blog series Addgene's Molecular...for overexpressing YGOI in an isolated, controlled environment. In most cases bacterial expression vectors... in molecular biology to isolate, multiply, or express the insert they carry in the target cell. These...most important thing you need to know is your expression system or environment. The host organism will...need. You will also have to make sure that your plasmid has been incorporated into the host organism, usually...

...with some plasmids. You just need three plasmids to start making AAV:  the packaging plasmid which contains... transfer plasmid an AAV transfer plasmid. ITR sequences are 145 bases each and AAV plasmids have two ...of an AAV transfer plasmid. Parts shown in green are key elements of an AAV plasmid. Parts shown in grey... elements of an AAV plasmid First let’s talk about the key parts of an AAV plasmid. Inverted terminal ...Promoter The promoter drives the expression of the AAV transgene. On a plasmid map, promoters are upstream ... of expression. For example, CAG is a strong promoter that drives very high, ubiquitous expression, while...transfer plasmids have a promoter, but this element isn’t required when the transfer plasmid harbors non-coding...

...genome and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you...virus (MoMLV) and consisted of two plasmids: the packaging plasmid containing all the structural genes...crossover event between the vector plasmid and the packaging plasmid caused the Psi sequence to be placed... facilitates nuclear export) is expressed from a separate plasmid, and the promoter of the 5’LTR has been...(tTA, also expressed by your transfer vector) binds to the TRE and activates the expression of your gene...usually 293T cells, with your transfer plasmid and your packaging plasmids. For this, you can use the transfection...distinct expression vectors to target cells - one containing the gene of interest (pTet-IRES-EGFP, pPRIME-Tet-GFP-FF3...

...Addgene-Seeding Labs plasmid grant is a collaborative endeavor providing plasmids to researchers in developing...the light‐inducible bacterial expression system pDawn to clone and express newly identified proteins and...project the expression of isolated bacterial proteins can be achieved using a bacterial expression system ...system such as the pET GFP LIC cloning vector and the pOKD plasmids. The recombinant Cry proteins will then...pQE-30-HyPer3 (2) which he will co-express together with a plasmid encoding glycine oxidase. The goal ...Addgene-Seeding Labs Plasmid Grant Check Out Previous Addgene-Seeding Labs Plasmid Grant Awardees Listen...CRISPR for plants Bacterial Expression Systems Learn about Global Plasmid Distribution ...

...  Gersbach Lab multiplexing plasmids: This plasmid set allows you to express 2-4 gRNAs, with four being...containing plasmids. Each destination vector contains GFP, enabling you to select cells with high GFP expression...Gibson Assembly Multiplexing Plasmids: These plasmids can be used to express up to 8 gRNAs after Golden ...Golden Gate Assembly Multiplex Plasmids: These plasmids allow expression of 2-5 gRNAs in zebrafish. Custom...Why use multiplexed gRNAs? By expressing multiple gRNAs on the same plasmid, you’ll make sure that each ...receive is: can I express more than one gRNA from a single promoter using a plasmid like pX330? Unfortunately... you’re working in Drosophila, a two-gRNA expressing plasmid is available from the Bullock lab, and gRNAs...

...) in a plasmid backbone. A separate plasmid contains the transposase gene (red) for expressing the transposase....12137. PubMed. Find the Plasmids at Addgene Kowarz Lab plasmids Izsvak Lab plasmids  ...). The original plasmid is empty following the removal of the transposon; the plasmid is then degraded...constitutive expression (pSBbi derivatives) and one set of vectors for inducible expression (pSBtet derivatives...transposase enzyme.2.) The transposase enzyme is expressed (red stars) and binds the inverted repeats (green...transposition; and 2) a transposon containing the gene-expression cassette that can translocate within the genome...derivatives). The constitutive and inducible expression vectors were developed into 16 variants with different...

...blot showing GFP expression in cells that have been transfected with a plasmid encoding GFP. Actin, a common...used for. Addgene’s Antibody Plasmid Collection includes plasmids that express antibodies, nanobodies, and...such as parasites or microbes, or to proteins expressed on the surface of cells that have been infected...the protein out of solution, or isolate cells expressing this protein. Read on to learn more about antibodies...context of the immune system, each isotype is expressed at different times of the immune response and ...immunoglobulin genes, recombinant antibodies are plasmid-based. Their sequences can be optimized for specificity...Additionally, the choice of vector can impact the expression level of an antibody (Ayyar et al., 2017). Single-chain...

...their genomes. Unlike plasmids used for expression in yeast and bacteria, plasmids used for stable transfections...Your Perfect Plasmid Backbone Read More: Plasmids 101: Antibiotic Resistance Genes Plasmids 101: Origin...Although plasmids do not naturally exist in mammals, scientists can still reap the benefits of plasmid-based...straightforward and the resultant cells can either express the plasmid DNA transiently (similar to bacteria) or ...reporter is unique to the cell, is expressed from the plasmid, and can be assayed conveniently. A direct..., it is sufficient for the transfected plasmid to be expressed transiently. Since the DNA introduced in...components expressed in trans within the cell for effective replication. Cell lines expressing the Epstein–Barr...

...Should I Use? Plasmids 101: Green Fluorescent Protein (GFP) Fluorescent Protein Guide Expression Vectors from...localization, or protease protection. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins...box of tags and fusion proteins to aid in the expression and purification of recombinant proteins. Tags...stability activity, can be readily purified using plasmids found in this paper (available at Addgene).   ...   plasmids 101...Biochemists and molecular biologists who need to overexpress and purify proteins can face any number of technical... some of the hurdles to overcome in order to overexpress a recombinant protein? It is not generally in...

.... pBLADEONLY: This plasmid contains only BLADE and is based on the pTRC99a plasmid. It does not include...promoter. If you already have a pBAD33-based plasmid expressing your gene of interest, all you have to do...used a strain transformed with pBLADE expressing superfolder GFP instead of mCherry under the PBAD promoter...-inducible AraC dimers in E. coli) that turns expression on and off with blue light.   Inspired by @UriAlonWeizmann...AraC and other promoter systems, head over to our Plasmids 101: Inducible Promoters article.) Modifying AraC...wild-type AraC, it still reached the level of expression needed for most applications.   Figure...reporter downstream of the PBAD promoter. To use this plasmid with your gene of interest, you would replace mCherry...

... into the brain of Cre-expressing mice to trigger recombination and expression of a transgene (Lin et .... GAL4 expressed in the fly binds to the UAS binding motif, driving downstream gene expression. attP and...effector is expressed. In cells where the stop cassette remains, the effector is not expressed and the cells...components and expression is controlled by the upstream recombination outcome. Find SPARC plasmids at Addgene...controlling gene expression in flies Resources on Addgene.org Find fluorescent protein plasmids at Addgene...the system or using chemical inducers of gene expression (del Valle Rodríguez et al., 2011).  Genetics... construct. A predictable number of cells will express the effector or retain the stop cassette following...

...phosphatidic acid probe GFP-PASS. B) Time series of HEK 293T cells expressing plasma membrane-targeted... of katanin to that anchor. So, you’ll need to express both the EB3N-VVDfast-iLID anchor module and the...module, which are also available as lentiviral plasmids if you want to make a stable cell line. This design...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with your...Opto-RhoGEF design and activity. B) OptoTIAM (magenta) expressed in a monolayer of endothelial cells, showing cell...either CRY2 or CIB1 to your protein of interest and express the other separately, then use blue light to generate...developing embryo is accessible for injection of plasmids and other materials.    Figure 3: Optogenetic...

...Find the plasmids from this publication at Addgene. Multichromatic control of gene expression in Escherichia...these two systems can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive... by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust. Synthetic...engineer new pathways with sophisticated gene expression controls. Most cell types do not respond to low-intensity...via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly...light-responsive system is spread across three plasmids, with the response regulator encoded in the genome...et al. condensed the system to two streamlined plasmids. The utility of these TCSs was limited by a few...

... blot for the myc-tagged protein GFP-smFP-myc expressed from Plasmid 98926. See the Applications section...of the new plasmids, antibodies, and viral preps in the repository through our hot plasmids articles. ...Think we missed something hot? You can pitch a Hot Plasmids section (antibodies and viral preps welcome!) ... you can also find several other constructs in plasmid form. Fig. 1: Summary of the approach and...JEDI-2p viral preps here (and here!)Find JEDI-2p plasmids here!  Liu, Zhuohe et al. Sustained deep-tissue...B) Cortical slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential...voltage traces of action potentials in a HcKCR1-expressing mouse neuron without (top) and with (bottom) ...

... FRET tools, double dimerization GFPs, and circular permutation GFPs. Hopefully we will have new tools...Plant Kit makes it really easy to clone genes for expression in plant cells. Sylvester and I chose the 96 ...that are not used so much but have comparable expression levels to things people do use a lot. The problem...susceptible to a lot of pathogens. It’s also easy to express genes in, which makes it a good laboratory workhorse...simultaneous complementation, with a knockout and expression construct on the same piece of DNA. We do a lot...Quick, Versatile Plant Transgenesis with GreenGate Plasmids  ...

... (ARG) plasmids to better monitor gene expression in vivo using ultrasound. These ARG plasmids contain...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... recent plasmid deposit please sign up here.      Listen to this episode of the Hot Plasmids podcast ...pXPR_053. They expressed this construct in bone marrow stem cells from Cas9-expressing mice, and used ...responses in C. elegans. Find the split BoTN/B plasmids at Addgene. Liu Q et al., Neuron. 2019. PubMed... microbe travels through a host organism, the expression of some genes can change depending on the location...

...without mCherry and GFP fluorescent controls. Alternatively, you can order individual plasmids from the library...Addgene.org Plasmid Pooled Libraries Pooled library amplification Zhang Lab CRISPR Plasmids The Human TFome...factors (TFs) act as the gate keepers for gene expression, turning transcription on and off by binding ...binding proximal to their target genes. Since gene expression patterns determine everything from stem cell differentiation...(aka the MORF Collection) and an accompanying expression atlas, including over 3,500 human TF isoforms...TF library, with each cell receiving only 1 TF expression construct. Cells were cultured following a differentiation...from Joung et al.)    This atlas linked TF expression to differentiation outcomes as well as global...