.... P., White, M. F., Yakunin, A. F., Garrett, R. A., Van Der Oost, J., . . . Koonin, E. V. (2015). An updated...in your lab, from knockouts to pooled library screening. Science...CRISPR Basics Small Edits Large Edits Genome-Wide Screening Cas9 Fusion Tools Cas9 Alternatives Resources ...the efficient but error-prone non-homologous end joining (NHEJ) pathway and the less efficient but high-... , and increases the likelihood that any cell containing the CRISPR plasmid will have all desired genomic...researchers to target anywhere from 2–7 genetic loci by cloning multiple gRNAs into a single plasmid — though some...coding sequence ~1 kb shorter than SpCas9 while retaining the same basic function. Its size allows it to...

...Anti-Calretinin [L122/68R] Calretinin Human Mouse IgG2a 177469 Anti-Calretinin [L122/6R] Calretinin Human...220384 Calretinin [L122/110R] Calretinin Human Mouse IgG2a 220385 Calretinin [L122/117R] Calretinin Human...220386 Calretinin [L122/132R] Calretinin Human Mouse IgG2a 220387 Calretinin [L122/133R] Calretinin Human...220388 Calretinin [L122/136R] Calretinin Human Mouse IgG2a 220389 Calretinin [L122/20R] Calretinin Human...-CALB2/calretinin [L122/68R-2b] CALB2/calretinin Human Mouse IgG2b 206642 Anti-CALB2/calretinin [L122/...-CALB2/calretinin [L122/68R-1] CALB2/calretinin Human Mouse IgG2a 206691 Anti-CALB2/calretinin [L122/6R... receptor Rat Mouse IgG2a 114484 Anti-Stonin-2 [N346/9R] Stonin-2 Human Mouse IgG2a 114485 Anti-NSD3 [...

... for Cloning 67720 Worm NotI/BamHI none S. pyogenes Colaiacovo pRB17 52527 Fly blunt end cloning none ...yes, cut N. meningitidis Thomson pSmart-Nm-sgRNA-BbsI 49157 Mammalian U6 none N. meningitidis Thomson pXCas9H840A...none N. meningitidis Pederson pLH-stsgRNA1.1 64116 Mammalian/Lentiviral U6 none N. meningitidis Pederson...none N. meningitidis Pederson pLH-stsgRNA3.1 64118 Mammalian/Lentiviral U6 none N. meningitidis Pederson...the same cell at the same time. Single plasmids containing both the gRNA and a Cas protein act as an all-in-one...or nick. Selection , such as Puromycin or EGFP Cloning enzyme used for insertion of your gRNA sequence...used with, such as S. pyogenes, S. aureus, N. meningitidis, etc. gRNA scaffolds are specific for the species...

... in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions...present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions...complement including additional nucleotides for cloning and expression purposes. NOTE: Here we discuss ...AAAC” before the guide’s reverse complement for cloning into the pX330 vector using BbsI restriction enzyme...be 24-mer oligos ( Table 4 ). Design Deletion Screening Primers Design one set of primers internal to ...primers described in step 2.1 may be omitted. CRISPR Cloning Anneal and phosphorylate oligos. Resuspend oligos...oligos into pX330 using a Golden Gate assembly cloning strategy 3 . Prepare a 50 μl reaction mix: 100 ...

... RALGDS Ral guanine nucleotide dissociation stimulator RAPGEF RAPGEF1 RAPGEF2 Rap guanine nucleotide exchange...antagonist STK STK3 STK4 Serine/threonine kinase STK11 Serine/threonine kinase 11 TBK1 TANK binding kinase...small GTPase and is a member of the G protein (guanine nucleotide binding protein) family. GTPases serve...Rho GTPase activating protein 35 ARHGEF2 Rho/Rac guanine nucleotide exchange factor 2 CCND CCND1 CCND2 CCND3...binding protein Rac) RAF AFAF BRAF RAF1 Serine/threonine kinases: A-Raf proto-oncogene B-Raf proto-oncogene...like RASGRF RASGRF1 RASGRF2 Ras protein specific guanine nucleotide releasing factor RASGRP RASGRP1 RASGRP2...member ROCK ROCK1 ROCK2 Rho associated coiled-coil containing protein kinase ROS1 ROS proto-oncogene 1, receptor...

...resistances. Comprehensive multi-cloning site compatible with standard cloning using many different restriction.... pET-HIS-Sangamo 40786 TALEN Gang Bao Gateway cloning and expression of an N-terminally His-tagged TALEN.... pET-Sangamo-His 40787 TALEN Gang Bao Gateway cloning and expression of a C-terminally His-tagged TALEN...epitope tags like His for easy western blotting or staining, and RNA transcript tags to monitor RNA localization...His tag and an MBP tag. Compatible with Gateway cloning. pDEST-periHisMBP 11086 Tac (lactose/IPTG inducible...formation or prevent toxicity. Compatible with Gateway cloning. pGTvL1-SGC 39188 pTac (lactose/IPTG inducible)... and TEV cleavage site. Includes sites for LIC cloning, and a "stuffer" fragment that includes the SacB...

...spatiotemporal serotonin release in vivo. bioRxiv 2023.05.27.542566. Yulong Li Serotonin Serotonin (5-HT) sensor...measuring serotonin dynamics. Nat Neurosci. 2021 Apr 5. Yulong Li , J. Julius Zhu Serotonin Serotonin sensor...19(2):231-241. Tommaso Patriarchi Serotonin Red and green serotonin sensors GRAB_5-HT3.0 Dual-color GRAB...Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning. Cell. 2020 Dec 12. pii: S0092-8674...-8674(20)31612-3. Lin Tian Serotonin Serotonin (5-HT) sensor psychLight Psychedelic-inspired drug discovery...: S0092-8674(21)00374-3. Lin Tian Serotonin Cilia-targeted serotonin sensor GRAB-HTR6 A serotonergic axon-cilium...calcium indicator, based on C-terminal domain of troponin C Griesbeck Lab Twitch Plasmids Oliver Griesbeck...

... Parniske. GreenGate Cloning System to create plant expression vectors containing several cassettes and...Scott Lenaghan. Joint Modular Cloning (JMC) Toolkit includes a modular cloning chassis and set of functional...as the promoter or cloning method of choice. ID Plasmid Description Promoter Cloning Method PI Return to...Protein Guide Addgene Blog: Plant Biology CRISPR Cloning Transcription Factors RNAi Empty Backbones Fluorescent...in plants. The tables below highlight plasmids spanning a wide variety of molecular biology technologies...proteins. Addgene’s collection includes plasmids containing promoters that drive general and species-specific...Vladimir Nekrasov. The MoClo-compatible zCas9i Cloning Kit includes a high efficiency intron-optimized...

...should sequence the region you are planning to modify prior to designing your gRNA, as sequence variation... into plasmids containing the gRNA scaffold using standard restriction-ligation cloning. However, the ...amplification of targeted region from DNA, subcloning into a plasmid, and screening individual clones. PCR amplification...sequence. For example, gRNA targeting sequences containing a G nucleotide at position 20 (1 bp upstream ...of the PAM) may be more efficacious than gRNAs containing a C nucleotide at the same position in spite ...design software ). Additionally, many plasmids containing validated gRNAs are now available through Addgene...the exact cloning strategy will depend on the gRNA vector you have chosen, so it is best to review the protocol...

...Adenoviral For recombining shuttle plasmids Vogelstein 16401 pAdEasy-2 Adenoviral For recombining shuttle plasmids...16402 pShuttle Shuttle For production of viruses containing transgene under a chosen promoter Vogelstein ...pShuttle-CMV Shuttle For production of viruses containing transgene under CMV promoter Vogelstein 16404...Shuttle For production of GFP-trackable viruses containing transgene under a chosen promoter Vogelstein ...Shuttle For production of GFP-trackable viruses containing transgene under CMV promoter Vogelstein 16407...Shuttle For production of mRFP-trackable viruses containing transgene under CMV Bamburg 50958 ShuttleNSE ...ShuttleNSE Shuttle For production of viruses containing transgene under Neuron-specific enolase (NSE) promoter ...

...46910 pHR-SFFV-dCas9-BFP Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS ...pHR-SFFV-dCas9-BFP-KRAB Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS,...pMSCV-LTR-dCas9-VP64-BFP Human expression vector containing MSCV LTR promoter, dCas9 that is fused to 2x ...51023 pSLQ1658-dCas9-EGFP Human expression vector containing dCas9 that is fused to 2x NLS and EGFP for CRISPR...pMSCV-LTR-dCas9-p65AD-BFP Human expression vector containing MSCV LTR promoter, dCas9 that is fused to 2x ...46914 pU6-sgGFP-NT1 Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting GFP (NT1...46917 pU6-sgCXCR4-2 Human pSico-based U6 vector containing murine U6 promoter and sgRNA targeting endogenous...

...Anthony Brown 11908 pEGFP-N1 alpha-actinin 1 Actin Filaments alpha-actinin 1 EGFP Carol Otey 27676 Cortactin-pmCherryC1... Protein PI 71367 Human Beta-catenin GFP Adherens Junctions Beta-catenin EGFP Alpha Yap 67937 mouse E-...Huttenlocher 11908 pEGFP-N1 alpha-actinin 1 Focal Adhesions alpha-actinin 1 EGFP Carol Otey 31923 pPaxillin-PSmOrange...Michael Davidson 55001 mCherry-Beta-Catenin-20 Adherens Junctions Beta-Catenin mCherry Michael Davidson 30313... resides. There are a variety of methods for determining subcellular localization. One of the most common...guide to find the appropriate backbone). Then, by examining the overlap between your fusion protein and the...the cell’s internal framework that assists in sustaining structure and function. The cytoskeleton is primarily...

...editing window specified by the gRNA. Adenine base editors convert adenine to inosine, which is replaced by...with a wide variety of Cas-containing plasmids. ID gRNA Plasmid Promoter Cloning Enzyme(s) Validated In Resistance...preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions... repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted...your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re ... based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express... same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one...

...editing window specified by the gRNA. Adenine base editors convert adenine to inosine, which is replaced by...with a wide variety of Cas-containing plasmids. ID gRNA Plasmid Promoter Cloning Enzyme(s) Validated In Resistance...preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions... repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted...your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re ... based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express... same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one...

...editing window specified by the gRNA. Adenine base editors convert adenine to inosine, which is replaced by...with a wide variety of Cas-containing plasmids. ID Plasmid Promoter Cloning Enzyme Co-expressed Cas9 Cas9...preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions... repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted...your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re ... based on factors such as selectable marker or cloning method. When using CRISPR, you will need to express... same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one...

... in one cloning step rather than two. Stephen Ekker Golden Gate TALEN 2.0 Golden Gate cloning method. ...Keith Joung Zhang Lab TALE Toolbox PCR/Golden Gate cloning method. Optimized for human expression. Feng Zhang... Assembly Kit Assembly by ligation-independent cloning (LIC). Validated in human cells. Veit Hornung Musunuru...RR) used are obligate heterodimers that require cloning of left and right TALEN monomer proteins into opposite...are mammalian expression/mRNA synthesis vectors containing unique shorter N- and C-terminal domains (N153AA...fluorescent TAL effectors to visualize subcellular positioning of target DNA sequences in living cells (TALE-mediated...TALEN kit, (ii) a lacZ fragment for blue/white-screening in E.coli , (iii) CAG promoter and Kozak sequence...

... expression, Ligation Independent Cloning (LIC) Bacteria Lac, T7, araBAD p15TV-L - His-tagged... Plasmids - More LIC bacterial cloning vectors pTD plasmid ... pACU2 - Modified pUAST vector containing both P-elements and attB site for ...GFP-tag fusions for transcription pCSDest - Easy cloning of your gene using Gateway recombination... protein. Just remember that when you are designing your plasmid you should keep your gene "in... GFP for mammalian expression pcDNA3 GFP LIC cloning vector - C-terminal GFP for mammalian... expression Puromycin Mammalian pLKO.1 - TRC cloning vector - Lentiviral shRNA expression...

...- Barbier Lab Cloning and Expression Bacteria and yeast expression vectors : FX cloning System - Geertsma...Parts Kit : Molecular cloning for E. coli - Densmore Lab EcoFlex MoClo : Modular cloning for applications ...Microbiology : Sets of plasmids for fluorescence, cloning and expression, and synthetic biology. CRISPR Resources...sets of plasmids intended for use together (e.g. cloning and expression systems) and therefore supplied ....0 Yeast Toolkit - Borodina Lab Golden GATEway cloning system - Wittbrodt Lab MultiSite Gateway Kit : ...predicted open reading frames for vaccinia virus - Bennink and Yewdell Labs Yarrowia lipolytica Golden Gate...

...to add the CACC and AAAC cloning overhang. When designing oligos for cloning my target sequence into a...published with thorough step-wise instructions on designing and using Cas9-based genome engineering tools ...Should I add the PAM sequence to the oligo when cloning my target spacers into the PX330 vector backbone...guidelines? A few notes below are considerations for designing HR donor. Generally if off-target (non-specific... the Bench FAQs How can I quickly check if the cloning of my oligo into a backbone like PX330 worked? ...Why does the PX260 plasmid use 30nt oligos for cloning but PX330 only uses 20nt? The reason for the 30nt...the construct is from our 'Chimeric RNA' design (meaning the mature processed form of crRNA is fused chimerically...

...editing window specified by the gRNA. Adenine base editors convert adenine to inosine, which is replaced by...preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions... repaired using error-prone non-homologous end joining (NHEJ). Double nicking strategies reduce unwanted...your gene of interest’s promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re ... based on factors such as selectable marker or cloning method. To isolate mammalian vectors, simply type... same cell at the same time. Single plasmids containing both the gRNA and Cas protein act as all-in-one...protein can be paired with a wide variety of Cas-containing plasmids. Do you have suggestions for other plasmids...