...has been used to construct plasmids for use in model organisms such as E. coli and yeast. Because this approach...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...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...overcome this overlap, Yiyang Gong’s lab created two plasmids containing a red-shifted Ace-mScarlet, a fusion...channelrhodopsins. Find the Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al., Sci Report 2019. PubMed...

...hold the plasmid together, even if there are gaps or flaps remaining. This incomplete plasmid looks like...robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized, and rapid multi-part...cloning uses short sticky ends to insert genes into plasmid backbones, base pairing between these ~4 base overhangs... overhangs isn’t strong enough to hold a plasmid together through transformation, so the cloned vector...longer single-stranded overhangs can hold a nicked plasmid together long enough to get it into cells and let... of LIC is often limited to specially designed plasmids. Sequence- and ligation-independent cloning: A...fixing up the “damage” and producing a complete plasmid. Besides being compatible with any vector, the ...

...infections. The Cas3 enzymes target several strains of E. coli and lethally chop their genomes into many, many...ijms24021563 Resources on Addgene.org CRISPR Plasmids and Resources CRISPR 101 eBook CRISPR History ...Hussein M, Molina MA, Berkhout B, Herrera-Carrillo E. A CRISPR-Cas Cure for HIV/AIDS. Int J Mol Sci. 2023...

... demonstrated in 2005 that proteins fused with E. coli dihydrofolate reductase (eDHFR) could be labeled...resources References Bottanelli, F., Kromann, E. B., Allgeyer, E. S., Erdmann, R. S., Wood Baguley, S., Sirinakis...Allgeyer, E. S., Zhong, S., Thompson, A. D., Lowe, N., Butler, R., Bewersdorf, J., Rothman, J. E., Johnston...freezer, you’ll probably find lots of different plasmids containing the same protein of interest tagged...Addgene’s collection, so you may be able to get a plasmid ready-to-use without any cloning at all! As mentioned...Schepartz, A., Baddeley, D., Toomre, D. K., Rothman, J. E., & Bewersdorf, J. (2016). Two-colour live-cell nanoscale....chembiol.2019.01.003 Gallagher, S. S., Sable, J. E., Sheetz, M. P., & Cornish, V. W. (2009). An In Vivo...

...Standards Find Plasmids for Creating and Controlling Gene Networks Browse All SynBio Plasmids ...Standards Synthetic Photobiology: Optogentics for E. coli Browse All Synthetic Biology Blog Posts Resources...

...Addgene.org Find the Spinach Plasmids at Addgene Find Broccoli Plasmids at Addgene View All Fluorescent... fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer libraries, the Jaffrey...PubMed Central PMCID: PMC4800036. 12. Wang, Rongsheng E., et al. “Aptamer-Based Fluorescent Biosensors.” Curr...nucleotide aptamer they called Broccoli. As a product of SELEX and FACS, Broccoli is inherently more stable...new RNA-based scaffolds to contain and stabilize Broccoli cassettes, thereby extending their use into mammalian...mammalian cells. They’ve also dimerized Broccoli to further enhance its biosensor capabilities (see Fig...et al. “In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies....

...Duby, J. E., Steinbeck, B. J., McCreary, J., Pierce, S. E., Tolar, J., Meissner, T. B., Chaikof, E. L., Osborn...this evolving field! Find prime editing plasmids! Find plasmids from David Liu's lab! This post was originally...the CRISPR guide Find CRISPR plasmids by function Browse our entire plasmid collection ...prime editors with RT domains derived from the E. coli Ec48 retron RT and the S. pombe Tf1 retrotransposon...for treating genetic diseases. In our summer Hot Plasmids post, we covered the Liu lab’s first demonstration...-01025-z Doman, J. L., Pandey, S., Neugebauer, M. E., An, M., Davis, J. R., Randolph, P. B., McElroy, ...Solley, S. C., Gilbert, L. A., Chan, M. M., Bauer, D. E., Marson, A., Parsons, L. R., & Adamson, B. (2024)...

...methionine (i.e. the plasmid complements the deletion). Find the Plasmids Here Plasmid features enable multiple... that allow students to propagate the plasmids in either E. coli (ori and ampR) or S. cerevisiae (2µm ...the overexpression plasmids. Finally, students determine if the presence of the plasmid corrects any known...diverged. Tips on using the Pathways Over Time plasmids The plasmids are used as part of a semester-long research...less-well studied organism into overexpression plasmids. Students next transform deletion strains from...phenotypic abnormalities due to the deletion (i.e. the plasmid complements the deletion). If the phenotype is ...cerevisiae met deletion strains with overexpression plasmids carrying MET/Met genes from either S. pombe or...

...Chris Voigt for speaking with us! Find the Plasmids: Plasmids for multi-input CRISPR/Cas9 genetic circuits...circuits are linked to the regulatory networks within E. coli host cells. Image from Nielson and Voigt...

... best bet is the Plasmid Map: We auto-generate a plasmid map based off of the plasmid sequence and feature...name of the strain.  Most Addgene plasmids are maintained in the E. coli lab strain DH5a, but others may...colleagues. Top of the plasmid page The ‘breadcrumb’ trail at the top of any plasmid page gives the hierarchy... to the individual plasmid page. The depositing lab page contains all the plasmids from the same lab while...contains all the plasmids deposited from the same publication. Next up, we have the plasmid name: This is...other plasmids with similar names in the collection, but you can use this name to find this plasmid from...A quicker way to find this plasmid through search is... The Addgene plasmid number: This is the unique...

...recombined plasmid to overtake full-size plasmids (or for smaller plasmids to overtake larger plasmids, if the...How many unique plasmids does your library contain?), the variance of size of plasmids in your library...transformation amplification protocol is the Chong Zhang E. coli Genome-wide Inhibition Library. DNA and cell amounts...lead to issues such as recombination and loss of plasmid diversity, so Addgene strives to distribute aliquots...library (How important is it that every single plasmid in your library is present, and how will you ensure...30 to 200 times as many colonies as individual plasmids in your library. As an example, if you want 100x... your library, and your library contains 5,000 plasmids, your goal for your transformation would be to...

...express and purify His-tagged Cas9 from E. coli using a plasmid such as pET-28b-Cas9-His from Alex Schier...commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9 and contains...chosen for both Cas9 and gRNA expression       Plasmid DNA may be incorporated into the genome     Off-target...targets with similar efficiency as compared to plasmid-based expression of Cas9/gRNA and can be used for... as CMV or EF1A promoters found in many CRISPR plasmids) are not expressed. Because this method does ...fluorophore tagged dCas9 may require the use of plasmid or viral-mediated delivery. You’ve decided to...experimental systems that may not be amenable to plasmid based methods. One of the most common techniques...

...specificity and binding affinity, can be produced in E. coli, and are both soluble and easily engineered.  Monobodies...Resources Additional resources on the Addgene blog Plasmids 101: Aptamer FluorophoresAntibodies 101: scFVs...available from AddgeneBrowse our recombinant antibody plasmid collection References Grumezescu, A. M. (2018)....

...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...They generated plasmids tagged with EB3 to mark the growing tips of microtubuli and plasmids tagged with ...and dCas9-VPR targeting plasmids can be found at Addgene. Check out these plasmids for piggyBac gRNA delivery...highlights from recent CRISPR plasmids. To find all of the CRISPR plasmids available from Addgene, head... Find the plasmids at Addgene The MoClo CRISPR/Cas Toolkit for plants includes 95 plasmids consisting...Nanobodies are easily expressed in the cytosol of E. coli, however the nanobody proteins tend to get stuck...

...resistance DNA plasmids are traditionally grown in bacteria as a way to produce more of the plasmid, isolate...isolate individual plasmids, or express a gene contained in a plasmid. During the propagation and/or transformation...receive a copy of the plasmid or will lose it during division. These non-plasmid-containing bacteria can...Over-Agar Antibiotic Plating Plasmids 101 eBook Resources on the Addgene blog Plasmids 101: Antibiotic Resistance...Resistance Plasmids 101: Blue-White Screening Choosing Your Perfect Plasmid Backbone  ...Plasmids need antibiotic resistance genes so that you can specifically isolate bacteria containing your...your experiment — to grow, express, or isolate plasmids. The presence of an antibiotic resistance gene...

...resistance genes in E. coli using plasmid and phagemid delivery systems. Phagemids are plasmids containing a ...genome is lethal. If this DSB occurs on a plasmid, the plasmid will be eliminated from the bacterium, which...sequence-specific, antimicrobial, plasmid-based, CRISPR systems (Find the Citorik Plasmids here). In both cases, ...sequence results in cell death. Plasmid targeting may result in plasmid loss and antibiotic resensitization...this resistance gene being encoded by a high-copy plasmid. In the case of the genomically encoded quinolone...RNAs (crRNAs) targeting either genomic (blue) or plasmid-based sequences (red). Treating a mixed population...specifically targets bacteria with the given genomic or plasmid sequence from a mixed population without affecting...

...the transfer plasmid, a plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral...coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.   ... all plasmids used for viral production are propagated in the endA-mutated NEB Stable strain of E. coli...contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic...offline and discarded. Confirmation of the transfer plasmid Addgene uses a rigorous barcode matching system...undergoes PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed... system and PCR-based confirmation of transfer plasmids used in Addgene’s viral production. Barcoding ...

...Activity Learn more about protein tags Read about E. coli strains for protein expression Additional Resources... How to Use the Nanobody Toolbox Find Nanobody Plasmids Structural comparison As the name suggests, ... Species Antibody Subclass Nanobody Addgene Plasmid Rabbit N/A* TP897 pTP1183 (1x cysteine) pTP955...Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express your nanobody of choice ...column. bdNEDP1 protease can be generated using plasmid pDG02583. Label the Nanobodies: Purified nanobodies...Journal of Cell Biology. PubMed PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M... on Addgene.org Find more nanobody expression plasmids here Additional Resources Looking for monoclonal...

...trp High levels of gene expression Promoter from E. coli tryptophan operon Repressible Gets turned off with...Once you've gotten to know what a plasmid is in general, it's time to take a look at some of its parts...origin of replication are necessary to replicate plasmids and make sure the cells maintains it. Here, we'll...the promoter, which is necessary for getting the plasmid express our gene of interest. This element is responsible.... This means that the promoter present in your plasmid backbone must to be compatible with the type of...transcript, you will also need to make sure your plasmid has a promoter suited to working in your host organism...development-specific promoters available to scientists. Plasmids are oftentimes put to therapeutic uses, and in...

...Francisella Cpf1 (FnCpf1), they employed an E. coli plasmid depletion assay to discover FnCpf1’s PAM sequence...system." Cell (2015). PubMed PMID: 26422227. Find plasmids from this publication at Addgene. 2. Zetsche,....1 (2016): 31-34. PubMed PMID: 27918548. Find plasmids from this publication at Addgene. 3. Kim, Daesik...Biotechnol. (2016). PubMed PMID: 27272384 Find plasmids from this publication at Addgene. Kleinstiver...Biotechnol. (2016). PubMed PMID: 27347757. Find plasmids from this publication at Addgene. Makarova, Kira...PMID: 26411297. Resources at Addgene Find CRISPR plasmids at Addgene Read Addgene's CRISPR Resource Guide...

...transforming both fusion plasmids, or mating two single haploid yeast fusion plasmid transformants into a ...reporter system (e.g. the Gateway-compatible bait Y2H plasmid pEZY202  from Yu-Zhu Zhang’s lab) is an example...fusions for analyzing gene expression in Escherichia coli and yeast. Methods Enzymol. 1983;100:293-308. PubMed...Yeast Expression Vectors Find Bacterial Two Hybrid Plasmids Read our Blog Post About Yeast Vectors Read our... Blaudez, D., Sanders, D., Revuelta, J.L., Boles, E., André, D., and Frommer, W.B. K+ channel interactions...

...analyzing the plasmid and viral DNA sequences and expression profile at multiple steps from plasmid amplification...recombination persists even in recombinase deficient E. coli and under conditions used to minimize recombination...to flip back into the inverse orientation (See Plasmids 101: FLEx Vectors). While this strategy greatly...recombination that are typically employed during viral plasmid amplification (Fischer, KB. 2019). This means that...of this finding, we now include guidance on the plasmid page of DIO/FLEx AAV vectors where this occurs ... mechanism of spontaneous reversion during AAV plasmid amplification. On the left homologous pairs of ....2020.618020 Additional resources on the Addgene blog Plasmids 101: FLEx Vectors Important Considerations When...

...mutagenesis of Plasmids. A) Generation of mutagenized plasmids. PCR primers (green) amplify the plasmid template...with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are isolated from the resulting colonies...the final circular plasmid product pictured here. B) Screening of recovered plasmids for the mutation of...Smaller plasmids (~3 kb) are generally more efficiently amplified than larger constructs, but plasmids as ...DpnI) to eliminate the parent plasmid from the PCR products, the plasmid template should be isolated from...have taken up the mutagenized plasmid. Note that any residual parental plasmid (usually from incomplete DpnI...additional restriction site into your plasmid, then, when the plasmid is digested with the appropriate restriction...

...new origin of replication increases plasmid copy number in E. coli. The authors used pXPG to study the...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 ...system plasmids are available through Addgene and are already very popular. Use these plasmids to increase... can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61 beta BFP-Sec61...luciferase reporter gene plasmid pGL3 pGL3 luciferase reporter gene plasmids have been used extensively...find all of our Hot Plasmid articles on our website. We update the our Hot Plasmids articles every quarter...

...PMC4884002. 10. Boer, E. de, Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni, E., Heck, A., … Strouboulis...biotinylating proteins with BirA. You can find plasmids that contain both AviTag and BirA at Addgene! ...sequestering biotin from being absorbed. In 1940 Esmond E. Snell was able to purify the protein responsible ...). References 1. Chapman-Smith, A., & Cronan, J. E. “Molecular Biology of Biotin Attachment to Proteins...PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic biotinylation of proteins: A post-.... and R. L. H. “The Discovery of Avidin by Esmond E. Snell.” J Biol Chem 279.e5 (2004). PubMed PMID: 20353954...of the E.coli BPL, BirA. If you recall, BirA biotinylates only one lysine in the entire E.coli proteome...

...implement RSMs in E. coli with two plasmids (Figure 3 below): Input Plasmid: The input plasmid (high copy, ...inducible promoter (PPhlF).  Output Plasmid: The output plasmid (single copy, bacterial artificial chromosome...through Addgene. Their plasmid maps and GenBank files can be found on their plasmid pages. All two-input...indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160 Fig. 4A pNR220...two-input RSMs and three three-input RSMs. The plasmids used to construct these RSMs are listed in the...paper (1). These parts are also annotated on the plasmid maps and GenBank files uploaded to Addgene. We ... Visit Our SynBio Pages Check Out More Lu Lab Plasmids Find More Genome Engineering Tools  ...

... be used for. Addgene’s Antibody Plasmid Collection includes plasmids that express antibodies, nanobodies...Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp. Front...immunoglobulin genes, recombinant antibodies are plasmid-based. Their sequences can be optimized for specificity...protein (Wang et al., 2013). These proteins are also plasmid-derived. However, a disadvantage to ScFv is their...nanobodies, or synbodies, can be produced from plasmids, saving time and money. Nanobodies and synbodies...expression in cells that have been transfected with a plasmid encoding GFP. Actin, a common internal control ... as well as tools for developing and producing plasmid-based antibodies - and we’re always growing our...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...articles provide a synopsis of a plasmid or group of plasmids' functions and applications. We hope that... you to find and use the plasmids you need. You can find all the hot plasmids from 2017 below. With over...over 50,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...editing for precise genome modification. Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169...Bacterial Expression Construct SpCas9 Inhibition (E. coli) Mammalian Expression Construct SpCas9 Inhibition...

...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...about a recent plasmid deposit please sign up here.   Listen this episode of Hot Plasmids! Light controls... with broccoli aptamers or MS2 tags using the same number of fluorophores. You can find plasmids containing...Hahn's lab at the University of North Carolina (plasmids at Addgene). It consists of two domains, LOV2 ...the Reuben Harris lab has deposited a reporter plasmid that allows for rapid fluorescence-based quantification...editing complexes are more efficient than the BE3 plasmid containing rat APOBEC1. Finally, they show that...

...Browse fluorescent protein plasmids and resources See all Jaffrey lab plasmids ... cellular processes by transforming cells with plasmids encoding aptamer-tagged RNA and later introducing...aptamer downstream of a known promoter. Addgene’s Plasmids 101: Aptamer Fluorophores describes the enrichment...Nilaratanakul, Voraphoj, Debra A. Hauer, and Diane E. Griffin. "Development and characterization of sindbis... respectively. Both Red Broccoli and Orange Broccoli are derived from Broccoli, and share similar properties...properties to Broccoli despite binding to a different small molecule. Notably, like Broccoli, Red Broccoli and ...was described in the same study as Red Broccoli and Orange Broccoli, two more aptamers that bind DFHO and...

... our CRISPR guide Find CRISPR plasmids for your research Browse plasmids for base editing References ...10.1534/genetics.111.131433 Doudna JA, Charpentier E (2014) The new frontier of genome engineering with...CS, Ralston EJ, Lee AH, Amora R, Miller JC, Leung E, Meng X, Zhang L, Rebar EJ, Gregory PD, Urnov FD, ...serves as the code of life. In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty’s experiment debunked...

... tag plasmids here! Where should I place the tag and how many do I need? When designing a plasmid containing...from Addgene More Resources on the Addgene blog Plasmids 101: Protein Tags Antibodies 101: ChIP Antibodies...101: Introduction to Antibodies References Hochuli E, Bannwarth W, Döbeli H, Gentz R and Stüber D. Genetic...insulin-like growth factor I from culture medium of E.coli. Bio/Technology. 1987, 5:379-382. Bornhorst JA ...