...Fusions; Coding Prefix alleviates RBS-CDS issues. Prohibited Restriction Sites: EcoRI, XbaI, SpeI, PstI...genome. Prohibited Restriction Sites: EcoRI, SpeI, NheI, PstI Restriction Sites to Avoid: PvuII, XhoI, AvrII...assembly standards – combinations of restriction sites which preserve a standard prefix and suffix sequence... are added. Therefore, the same reaction which ligates a promoter into an empty backbone can be repeated...restriction enzymes used for assembly are the prohibited sites and must be omitted from the coding sequence of ...backbone. For some standards, there are additional sites which should be avoided whenever possible. Other...PstI Restriction Sites to Avoid: NotI For more info, visit iGEM: BioBrick (RFC 10) Back to Top BioBrick...

...2013). We also have an older system using two cassettes to express the CRISPR RNA array and tracrRNA separately...guide RNA: These plasmids contain two expression cassettes, a human codon-optimized SpCas9 or SpCas9n, and...tracrRNA: This plasmid contains three expression cassettes. To target a given site, the plasmid can be digested...loops at tetraloop and stemloop - Contains BbsI sites for insertion of guide sequences Addgene plasmids... and EF1a-zeo resistance marker. Contains BsmBI sites for insertion of guide sequences Zhang Lab SAM Cloning...guide RNA: These plasmids contain two expression cassettes, a human codon-optimized SaCas9 driven by either...AAV plasmid for sgRNA cloning. GFP-KASH fusion facilitates FACS sorting of cells and nuclei. Return to top...

...D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, Gersbach CA. Nat Biotechnol . 33(5):510-7. PMID:...have additional sites throughout the genome where partial homology exists. These sites are called off-...partial homology to additional sites throughout the genome. These sites are called off-targets and should...efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. 2015....modifications to Cas9. As discussed previously, Cas9 generates double-strand breaks (DSBs) through the combined...mutant of SpCas9, retains one nuclease domain and generates a DNA nick rather than a DSB. Thus, two nickases...preventing strand separation and cutting at off-target sites. Similarly, SpCas9-HF1 lowers off-target editing...

...increasing on-target editing rates and decreasing off-target editing rates. Advances in this area include...offering flexibility and easy multiplexing/scaling. Prokaryotes have long utilized CRISPR as a powerful defensive...elucidated until 2007. Scientists hypothesized that prokaryotes used CRISPR as part of an adaptive immune system...would bind. (5) After the complex binds, Cas9 separates the double stranded DNA target and cleaves both...seen in human patients, protein tags, or loxP/FRT sites, among others. Homology-directed repair (HDR) allows...packaged in AAV. Cas9 orthologs also have distinct PAM sites that do not cross react, allowing multiple Cas9s...engineered mutant Cas enzymes with alternative PAM sites . Cpf1 , another CRISPR nuclease, recognizes a 5...

...response has developed, the antibodies are collected, tested against the antigen, packaged, and sold. This is...immortalized B cells). The genetic reorganization that creates variety in the antibody allowing for their extreme...class therefore has a valency, or number of binding sites available per antibody unit, which is typically ...multiplied by two, as each antibody has two binding sites. Isotype Description Configuration and Valency Subclasses...Monomer with a valency of 2 IgE Protects against parasites and is responsible for driving allergic reactions...in the lab. Check for and record any expiration dates. Buffers Buffers are a concern for conjugating and...reaction in the presence of several different substrates. The luminescence produced by the reaction can...

... mTORC1 integrates these signals to determine the nutritional state of the cell and regulates the switch...signaling of mTOR, a key metabolic regulator, correlates with poor prognosis in many cancers. Cancer...anabolic to catabolic processes. Active mTORC1 promotes protein and lipid synthesis, and increases energy...Enhanced signaling through either mTORC1 or mTORC2 correlates with poor prognosis in many cancers, including... cancer. Loss of the tumor suppressor p53 also promotes aberrant mTORC1 activity, and multiple familial...mTORC1. The hyperactivation of mTORC1 in cancer promotes the conditions needed for uncontrolled cell growth... which appears to signal upstream of Akt, also promotes cell survival and proliferation. mTOR Pathway ...

...nuclease sites, this tool from the Joung Lab has been expanded to identify potential DNA target sites for ...access tool from Synthego allows you to determine rates of CRISPR editing at a given locus. To use the tool...sequence, functional noncoding elements and off-target sites. This algorithm allows for the quantification of... splice site mutations. The CRISPResso suite accommodates single or pooled amplicon deep sequencing, WGS...Church Lab : a tool that identifies putative target sites for S. pyogenes Cas9, S. thermophilus Cas9, or Cpf...probability of off-target effects. Cas-Designer integrates information from the Kim Lab's Cas-OFFinder and...online predictor Identifies candidate sgRNA target sites by off-target quality. Validated for gene inactivation...

...ΔA all TAG sites changes to UAG, RF1 function removed George Church 48999 C321 all TAG sites changes to...all TAG sites changes to UAG, RF1 function removed Farren Isaacs 87359 C321.∆A.opt all TAG sites changes...to UAG George Church 49018 C321.ΔA.exp all TAG sites changes to UAG, RF1 function removed, MutS restored...mutation rate George Church 68306 C321.ΔA all TAG sites changes to UAG, RF1 function removed, deletion of...Strain 192872. Jesse Rinehart 69493 MCJ.559 TAG sites, RF1 function removed, genomic deletions for improved...incorporation Michael Jewett 69495 rEc.13.delA 13 amber sites changed to TAA, RF1 function removed Michael Jewett.... George Church 98564 C321.Ub-UAG-sfGFP all TAG sites changes to UAG, RF1 function removed, with Ubiquitin-UAG-sfGFP...

...Phosphopeptide Library ). The phosphorylation sites (“phosphosites”) consist of a central phosphoserine site...expressing recombinant phosphoproteins. These attributes make rEcoli XpS ( 192872 ) and pSerOTS-C1* ( ...site-specific incorporation of phosphoserine in >36,000 phosphosites by tandem mass spectrometry (Barber et al, Nat...synthesized using supD tRNA ( 68307 ) to generate the phosphosites with serine instead of phosphoserine (i.e. a... can then be used to identify candidate human substrates of kinases of interest by mass spectrometry in...library ( 188536 ) into 10 subpools of ~11,000 phosphosites ( 188526 ; 188527 ; 188528 ; 188529 ; 188530...precise knowledge of phosphorylated positions for testing phosphoproteomics methods, proteomics search algorithms...

...system for eukaryotes. At the time, bacterial systems from E. coli presented the best candidates for inducible...were tested for functionality in mammalian cells. The lac system with the IPTG inducer was tested first...induction. In 1992, Manfred Gossen and Hermann Bujard tested the tet system in a mammalian cell system (HeLa...these tetO sequences and the activation domain promotes expression. In the presence of tetracycline, tetracycline...26803 pEnt L1L3 EF1a-tTA-2 Contains Gateway L1L3 sites and EF1α promoter driving tTA; contains insulator...cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. ...

...functionally distinct interneurons in mice, non-human primates and humans. Nat Neurosci. 2020 Dec;23(12):1629...Plasmids Rylan Larsen Calcium Calcium sensor in astrocytes (GCaMP6f), cytosolic Conditions and constraints...413-29. Baljit Khakh Calcium Calcium sensor in astrocytes (GCaMP6f), membrane-tethered Khakh Lab pZac2.1...), diacylglycerol and STIM1 signalling system regulates directed cell migration. Nat Cell Biol. 2014 Feb...ER-targeted CEPIA1er ER-mitochondria tethering by PDZD8 regulates Ca(2+) dynamics in mammalian neurons. Science.... Mg(2+) Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation. Curr Biol...the adenosine sensor GRAB_Ado1.0 in neurons or astrocytes, (Constitutive or Cre-dependent) Li Lab GRAB_...

...near-matches to reduce risk of cleavage away from target sites (off-target effects). Ensure that the guide sequences...primers upstream and downstream of the sgRNA cleavage sites (“deletion band”; Figures 1 - 2 ). In the absence...sorted cells individually into 96-well round-bottom plates using sorter or by using limiting dilution at 30...conditions for each primer pair designed based on testing the bulk sorted cells. Run samples on 2% agarose... per well for a final volume of 150 μl. This facilitates screening by allowing a multichannel pipette ...100 μl each into two separate 96-well flat-bottom plates. Keep one plate to allow for clones to grow and... into DH5α E. coli cells and plate onto LB agar plates with the relevant antibiotic. Select multiple colonies...

...including: how closely homologous the off-target sites are compared to the on-target site, the specific...immediately adjacent to the nearly-homologous target sites. The mere presence of additional PAM sequences should...selection tool that should be helpful in minimizing or testing off-target DSB. Is it possible to target a single...summarize HR template vector design guidelines? A few notes below are considerations for designing HR donor....with PX330 plasmid, clone in your target guides, test run them and then select the best guide to co-transfect...site in the donor. For example, choosing target sites that span the knock-in gene. For making mutations...As a successful insertion will destroy the BbsI sites, a double digest should discriminate between positive...

...function of zebrafish astrocytes. Knock-in tagging - Michel Bagnat lab. Targeting cassettes encoding fluorescent...Zebrafish Society (IZFS) - An organization that promotes and advocates for zebrafish research in the international...Morsch Lab. A vertebrate model to study ALS-linked aggregates in real time. Live imaging of astroctyes - Kelly...Resources Zebrafish Genome Project - Access the latest reference sequence assembly for the zebrafish genome...

...Institute Identifying Altered Epigenetic States and Drivers in Intestinal Carcinoid and Pancreatic Neuroendocrine... Cancer Institute Finding the Causes of Small Intestinal Neuroendocrine Tumors Ramesh Shivdasani Dana-...Dana-Farber Cancer Institute Epigenetic Regulators of Intestinal Endocrine Cells and Carcinoid Tumors Qiao Zhou...Zhou Harvard University Epigenetic Regulators of Intestinal Endocrine Cells and Carcinoid Tumors Bradley ...

...simultaneously with dual labeling when used with two substrates Can be used in live or fixed cells, and for internal...protein tags based on the acyl carrier protein. Substrates are derivatives of Coenzyme A (CoA). These tags... Please see the note below for information on substrates and synthases needed for use of these tags. Plasmids...product portfolio, including the CoA-derivative substrates and the ACP and SFP Synthases. Users may still... their own fluorescent or non-fluorescent CoA substrates by purchasing coenzyme A disodium salt from a...

...nuclease operates as a dimer, cutting in the spacer region between two distinct ZF target sites. (B) A ...species almost as easily as correcting a typo in the latest draft of your dissertation. Well, that will probably...corresponding zinc fingers, potential targetable sites in your average genome are every 500 bp. TAL effectors... a T), but they still have potential targetable sites approximately every 35bp. Researchers are still ...

...pyogenes GFP Vakoc U6>sgRNA(F+E) 59986 Other/Ciona intestinalis BsaI none S. pyogenes Christiaen p(U6a-BsaI-...vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette...the expression of multipe gRNAs. Csy4 cleavable cassettes; requires expression of Csy4 (eg Plasmid 53369...gRNA module vectors, multiple gRNA expression cassettes can be assembled using Golden Gate cloning or ...polycistronic glycine tRNA-gRNA genes (PTGs) and then PTG cassettes are assembled into a Cas9 expressing vector. Yang...to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR...sgRNAs Zebrafish Plasmids for assembly of gRNA cassettes using Golden Gate ligation into Tol2-based pGGDestTol2LC...

...sequences and gRNA target sites. The first deposited PITCh plasmids were tested by fusing EGFP-2A-PuroR ...plasmids in this CRISPR-Cas tagging system were tested by tagging transcription factors with FLAG in human...plasmids in the following articles provide PCR templates for amplification of the tag (eg GFP, Flag, YFP...elegans . This system uses a 10-day protocol, generates “clean” homozygous mutants with no co-integrated...

...Cell type-biased: Astrocytes - AAV-PHP.AX with GRE or transgenic animal Oligodendrocytes - AAV-PHP.eB with...digital PCR, in vitro and (when possible) in vivo testing, and full sequencing of the final viral vector ...LY6A-nonpermissive mouse strains. **AAV1-X1 has not been tested in rats. Abbreviations: AAV, adeno-associated virus...regulatory element; NHP, non-human primate. Image courtesy of the Gradinaru Lab and the Caltech CLOVER Center...