... CRISPR Parasites CRISPR Plasmids: Parasites Browse CRISPR Plasmids ...Eukaryotic parasites continue to cause substantial death and disease in the world. Parasites such as Plasmodium...been designed for use in parasites. CRISPR...propelled both reverse and forward genetics of these parasites. Moreover, CRISPR-dCas9 gene regulation has also...also been established for some of these parasites. Together, these tools have enabled precise genetic ...changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and ...

...for serotype testing: AAV1, AAV2, AAV5, AAV8 and AAV9... Viral Service AAV Serotype Testing AAV Viral Service...Service: Serotype Testing AAV These AAV encode fluorescent reporters and can be used to compare the tropism...Inventory . The catalog ID for the 20 µL sized testing sample is denoted with a .T suffix (for example... (for example, AAV1). AAV Vectors for Serotype Testing pAAV-CAG-GFP (Plasmid #37825) Description : Ready-to-use...

...expressing secreted recombinant proteins from human erythrocytes that was created for systematic screening of...

...serotype testing panel for the eye and brain.... Viral Service AAV University of Florida Serotype Testing Panel... Viral Service: University of Florida Serotype Testing Panel for the Eye and Brain You may also like: ...by ddPCR, in vitro and (when possible) in vivo testing, and full sequencing of the final viral vector ...injection in mice and macaque. This serotype also demonstrates enhanced transduction of cells in culture for... with improved mGluR6 promoters in rodents and primates. Mol Ther . 2016 Aug;23(8-9):680-9. PMID: 27115727...

...Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat. Methods . 2013 July. PubMed PMID...trans from pSPgRNA, the dCas9-VP64 fusion protein activates the expression of downstream genes. Multiple sgRNAs...

...TAAAGACTCCTCCACCAGccaaacctgcggccccTGCAGTGTCCAGTAGTGA tes TAL3190 & TAL3191 TTGAGGACCATGACGTGCagctggagactgaagaGAGCAAGAAAGTTGGGAA... Joung, Peterson, and Yeh labs at MGH to target sites within various endogenous zebrafish genes; many ...Gene Name TALENs Full Target Site (5' to 3'; half-sites in CAPS, spacer in lowercase) aak1 TAL3208 & TAL3209...

...GJ, Ramakrishnan C, Kozicz T, Deisseroth K, Thiele TE, McElligott ZA, Holmes A, Heisler LK, Kash TL. 2016...recombinase recognition sequences (e.g. Lox sites, FRT sites) inside of the introns (A,D). The addition..., 2014. INTRSECT INTRSECT (intronic recombinase sites enabling combinatorial targeting) is a synthetic...portion of the ORF that is sandwiched between the sites; the starting direction of these ORF fragments (...design. How-to guide for the molecular design and testing of novel INTSRECT plasmids for groups interested...targeting genes. Recombinases Addgene ID Plasmid Logic Sites and Mutations 55636 pAAV-EF1a-Cre None 55632 pAAV-Ef1a-mCherry-IRES-Cre... recombinase-dependent Addgene ID Plasmid Logic Sites and Mutations 55641 pAAV-Ef1a-fDIO EYFP Flp 55640...

...recognizes 34 bp loxP sites, and the orientation and location of the loxP sites determines how the genetic...region of DNA between the loxP sites is reversed. Deletion: If the sites face in the same direction, the...If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites. For a... that recognize specific mutant loxP sites, VloxP and SloxP sites respectively. Split Cre: Cre recombinase...injection into fertilized zygotes Mammalian Sauer 21270 pBigT Cre activates gene of interest by removing... 38042 pAAV-CA-FLEX Cre activates gene of interest, using 4 mutated lox sites AAV Uchida Cre Reporters...expression. Flp recombinase recognizes FRT sites. Flp binding to these sites can result in excision, inversion ...

...acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis. Nat Commun. 2016...and c-Myc from polycistronic cassettes KLF4 N-terminal variance modulates induced reprogramming to pluripotency...30. Zhou Astrocytes Dopaminergic Neurons Lentiviral Human Efficient conversion of astrocytes to functional...Fibroblasts Astrocytes Lentiviral Mouse Direct conversion of fibroblasts into functional astrocytes by defined...Oct4, Sox2, Klf4, and c-Myc from polycistronic cassettes. piggyBac transposition reprograms fibroblasts...To Delivery Method Species Article PI iPSCs Cardiomyocytes Lentiviral Human Robust cardiomyocyte differentiation...Sci U S A. 2012 May 29. Palecek Fibroblasts Cardiomyocytes Lentiviral Mouse Optimization of direct fibroblast...

...MS2 binding sites within this plasmid. pZE31-DsRed-ms2x2 26248 MS2 coat protein binding sites Philippe Cluzel...strain USO hisB- pyrF- rpoZ- , this promotes growth on plates lacking His and containing 3-AT. FLIParaF....microscopy. pZE31ms2 25860 MS2 coat protein binding sites Philippe Cluzel RNA localization: Contains a 2x ...appropriate cDNA has been fused to the MS2 binding sites within this plasmid. Return to Top Protein Purification...N-terminal GST tag and TEV cleavage site. Includes sites for LIC cloning, and a "stuffer" fragment that includes...mOCR, MBP, with and without SUMO or TEV cleavage sites to remove tags Scott Gradia Arabinose inducible ...MBP, GFP, with and without SUMO or TEV cleavage sites to remove tags. Scott Gradia Lactose/IPTG inducible...

...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. ...