...from its own promoter. But that doesn’t mean you have to clone and transfect multiple promoter-gRNA constructs...arrays (e.g. for spacers A, B, and C, you could obtain arrays A, B, C, AB, AC, BC, and ABC). Nielsen Lab...can I express more than one gRNA from a single promoter using a plasmid like pX330? Unfortunately, the...of pX330, contains humanized wtCas9 and two U6 promoters. To use this plasmid, you simply order oligonucleotides...contain Type IIS restriction sites flanking the promoter-gRNA construct, but with different sequences adjacent...contain Type IIS restriction sites that flank the promoter-gRNA constructs. When these plasmids are digested...downstream of the 7SK, human U6, mouse U6, or human H1 promoters. If you express fewer than four gRNAs, you’ll ...

...pCMVTNT PINK1 N-myc PINK1 Myc CMV Parkinson's Mark Cookson 13314 pCMVTNT PINK1 C-myc PINK1 Myc CMV Parkinson's...Ultra-Exon1Q23-Myc-A HTT Myc UbC Huntington's Baoji Xu 110487 Ultra-Exon1Q145-Myc-A HTT Myc UbC Huntington's...pUltra-Exon1Q23-Myc-B HTT Myc UbC Huntington's Baoji Xu 110489 pUltra-Exon1Q145-Myc-B HTT Myc UbC Huntington's...Strep-Lrrk2-Myc LRRK2 Myc, Strep CMV Parkinson's Maik Hintze 161583 pPuro3.1(+)_Strep-Lrrk2-Myc LRRK2 Myc, Strep...LRRK2-WT LRRK2 His, Myc CMV Parkinson's Ted Dawson 17612 pRK5-Myc-Parkin PRKN Myc CMV Parkinson's Ted ... pGW1-Myc-DJ1-WT PARK7 V5 CMV Parkinson's Mark Cookson 29349 pGW1-Myc-DJ1-L166P/K130R PARK7 Myc CMV Parkinson's...D1994S)-Myc LRRK2 Myc, Strep CMV Parkinson's Maik Hintze 161585 pPuro3.1(+)_Strep-Lrrk2(G2019S)-Myc LRRK2...

...system is tight temporal regulation. Promoter-regulated Cre: The promoter region defines the areas in which...active promoter like CAG, or expressed only in a subset of cells under a more specific promoter (e.g. ...HIV-TAT - promotes cellular uptake of recombinant Cre Bacterial Rajewsky 13775 pCAG-Cre Cre-Myc CAG Mammalian...Cepko 13779 pRho-Cre Cre-Myc rhodopsin Mammalian Cepko 13780 pNrl-Cre Cre-Myc, Expressed in rod photorecetor... PBAD promoter Bacterial Richmond 112614 pVHC Venus and Cre-ERT2 with MCS for inserting promoter none ...inserting promoter none Mammalian Heller 112616 pmTHC TFP and Cre-ERT2 with MCS for inserting promoter none...respectively) and placed under the control of different promoters. Expression of both N and CCre in the same cell...

...screening of human promoters, the Zhang Lab found a short fragment of human IBA1 promoter (hIBA1a, 466 bp...small RNA-binding exonuclease protection factor) promotes editing (Yan, J et al., 2024). They developed ... expression from ssAAV5s with hIBA1 or hIBA1a promoters in mice. B) Quantifications showing high microglia...a CC-BY-NC-ND license.  Integrating hIBA1a promoter into AAV vectors, either alone or in combination...specificity of GFP expression for the indicated ssAAV5s. C) Quantifications showing high microglia transduction... microglia-targeting AAV plasmids here! Serrano, C., et al. (2023). Simple and Highly Specific Targeting... vectors for CROPseq-multi using selection by Puromycin, Zeocin®, or nuclear expression of BFP2.   ...

...El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer, C., Ellerbrok, H., Kamuzinzi, R., Ledent...contains a reporter gene cloned downstream of a promoter containing the target sequence of the DBD. The...as the “prey”) in close enough proximity to the promoter of the reporter gene to activate expression (Figure...organisms, including bacteria, alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the ... prey libraries can be screened using both N- and C-terminal fusions of these proteins. This way both ...Two hybrid systems were developed in Saccharomyces cerevisiae in 1989 and are still used extensively ...

...reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus, and selection markers. Need ...components, such as your CRISPR enzyme or Pol 2 promoter. The options available in each class, or module...Griffith, A. L., Szegletes, Z. M., Wen, B., Kraus, C., Miller, N. W., Steger, R. J., Escude Velasco, B....Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration by spontaneous...

...limited by the lack of well characterized inducible promoters. Far removed from the bio-industry’s vision of... the same dCas9 protein. By using an inducible promoter to modulate dCas9 expression, multiple scRNA transcriptional...on designing and using RNA scaffolds: Delebecque, C. J. et al. Designing and using RNA scaffolds to assemble... various metabolites in Escherichia coli or Saccharomyces cerevisiae with few techniques that are easily...

... by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope tags to the C-termini of Met...grow in the absence of methionine. Since the GAL1 promoter controls MET/Met gene expression, complementation...methionine synthesis (5). The budding yeast, Saccharomyces. cerevisiae, makes a great reference organism... or not proteins from the fission yeast, Schizosaccharomyces pombe, have the same function as their counterparts...yeast for budding geneticists: A primer on the Saccharomyces cerevisiae model system. Genetics 197, 33-48...yeast for young geneticists: A primer on the Schizosaccharomyces pombe model system. Genetics 201, 402-423...Surdin-Kerjan, Y. Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 61, 503-532...

...02708-5 Hrit J, Goodrich L, Li C, et al. (2018) OGT binds a conserved C-terminal domain of TET1 to regulate...functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase...89–92. https://doi.org/10.1038/301089a0. Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking...https://doi.org/10.7554/eLife.34870.  Kaas GA, Zhong C, Eason DE, Ross DL, Vachhani RV, Ming G-L, King JR...lentiviral backbones expressing blasticidin or puromycin resistance genes.  It’s that simple! Good luck...

...either the Thy1 or CAG promoter, while Brainbow AAV is under control of the EF1a promoter. Livet et al., ...insert modules (plant promoter, N-terminal tag, coding sequence of the gene of interest, C-terminal tag, plant...known as SpyLigase and is a protein domain that promotes the formation of an isopeptide bond between 2 ...insert fragments of DNA containing basic parts (promoters, UTRs, coding sequences, terminators, etc) into... creating a single transcriptional unit (Ex: a promoter, 5’UTR, coding region, and terminator). Next, ...the mitochondrial targeting sequence of cytochrome c oxidase subunit VIII, so that they can be imported...pCoofy series of plasmids contain a variety of N- and C-terminal tags (including His, S-tag, OneStrep, CBP...

...inhibitor response 1 (TIR1) expressed under a desired promoter with a specific spatiotemporal expression pattern...interactions New auxin inducible degron system for C. elegans New CRISPR plasmids New items from the viral...tool-kit for the auxin inducible degron (AID) system in C. elegans By Alyssa Cecchetelli The auxin inducible... degron (AID) system has been extensively used in C. elegans since 2015 to conditionally degrade proteins...repression, and editing with a single Cas9-VPR in Saccharomyces cerevisiae. To control Cas9 activity and influence...

...optimum of 30 °C and is therefore inefficient in mammalian cells (usually grown at 37 °C). Smart molecular...localization sequence) and expressing it from a CAGGs promoter. However FLPe is still less efficient than Cre...identification of FLPe which has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance... receptor (ER) ligand binding domain (LDB) to the C-terminus of FLP or Cre (pCAG-CreERT2 #14797). These...Sadowski. 1985. “The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently...

...Loh, P. C., & Wong, H. L. (2023). Development of constitutive and IPTG-inducible integron promoter-based...resistance cassettes (Prior et al. 2010), inducible promoters with different degrees of stringency and inducibility...Gluconacetobacter xylinus, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus...calcoaceticus, Actinobacillus pleuropneumoniae, Actinomyces naeslundii, A. viscosus, Aerobacter aerogenes...Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum, M. smegmatis, Paracoccus denitrificans...spheroides, Salmonella spp., Serratia marcescens, Streptomyces lividans, Synechococcus spp., Thiobacillus ferrooxidans...

...overexpression (by promoter substitution), and tagging at either the N- or C-terminus (3xHA, 13xMyc, GST, or GFP...

...enough to prevent confluency but high enough to promote cell growth. Overgrown cultures stimulate quiescence... for less than a day, lentivirus can be kept at 4°C. For long-term storage, viral preps should be divided...divided into single-use aliquots, and stored at -80°C. Some reports suggest rapidly freezing virus in a ... labs and hinders experiments is mycoplasma contamination. Mycoplasma contamination has been shown to ...routinely test for mycoplasma contamination. There are several commercially available mycoplasma detection kits...larger microbes such as yeast, fungi, or bacteria, mycoplasma can be extremely hard to detect with levels reaching...cells per ml before the media becomes cloudy. Mycoplasma compete with host cells for nutrients and can...

... vectors within this kit contain a variety of promoters to facilitate gene expression in bacteria, yeast...plasmids within it contain a variety of different promoters followed by leader sequences (encoding small leader...different genes was predictable based solely on the promoters and Shine-Delgarno sequences used in this so-called... easy for a researcher to quickly generate N- and C-terminal epitope tag fusions to a protein of interest...et al. "Adaptation of Enterococcus faecalis to daptomycin reveals an ordered progression to resistance....

... glutaGRO. Store at 4 °C. Pro-Tips Different brands and lots of FBS can promote or inhibit transfection...it can be inactivated in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health...obtaining accurate titers. Check the cells for mycoplasma (Link opens in a new window) regularly Do not... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

...researchers test many different combinations of promoters, terminators, solubility and purification tags...2016). The variety of constitutive and inducible promoters, linkers, and tags makes EcoFlex a great option...been used to examine the effects of different promoters, terminators, enhancers, and cross-cassette interactions... et al., 2019). Many plasmid elements such as promoters or fluorophores are interchangeable across organisms...doi.org/10.1371/journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit and Parts Library...Toolkit for Extensive and Flexible Engineering of Saccharomyces Cerevisiae.” ACS Synthetic Biology, vol. 12,...

...with pLKO.1 C. Cloning shRNA oligos into pLKO.1 C.1 Recommended materials C.2 Annealing oligos C.3 Digesting...Virus may be stored at 4°C for a few days, but should be frozen at -20°C or -80°C for long-term storage...., R is a purine (A,G), and Y is a pyrimidine (C,U). G-C content should be 36-52%. Sense 3’ end should ...annealed oligo from step C.2 20 ng digested pLKO.1 TRC-cloning vector from step C.3 (If you were unable ...Store at 4°C. m. Add 5 mL of fresh media containing antibiotics to the cells and incubate at 37°C, 5% CO ...at 37°C, 5% CO 2 overnight. Day 2: b. The target cells should be approximately 80-90% confluent. c. Dilute...solution is stable at 4°C for up to one year. The powder form of polybrene is stable at 4°C for several years...

...inducible promoters capable of controlling eukaryotic gene expression. Several endogenous promoters had been...Tetracycline-dependent promoters are developed by placing a TRE upstream of a minimal promoter. Tetracycline Off....CMV.tTA Adenoviral; CMV promoter; see article for neuronal-specific promoter and enhancer options tTA...for Tet transregulators. Krueger C, Pfleiderer K, Hillen W, Berens C. Biotechniques . 2004 Oct;37(4):546... tetracycline, expression from a tet-inducible promoter is reduced. To use tetracycline as a regulator...these tetO sequences and the activation domain promotes expression. In the presence of tetracycline, tetracycline...are: Plasmid containing a tetracycline-dependent promoter upstream of your gene of interest tTA expression...