...highly-sensitive genetically encoded temperature indicator exploiting a temperature-responsive elastin-like polypeptide...Aug 13;11(1):16519. Takeharu Nagai Temperature Fluorescent temperature indicator B-gTEMP Intracellular Heat...You may also like... Addgene Blog: Biosensors Luciferase Plasmids Subcellular Localization Optogenetics...family Cameleon calcium sensors Bcl-2-mediated alterations in endoplasmic reticulum Ca2+ analyzed with an... cAMP levels for imaging in live cells Fourth-generation epac-based FRET sensors for cAMP feature exceptional...GMP) Green fluorescent probe for cGMP (cGull) Generation of a cGMP Indicator with an Expanded Dynamic ...Monitoring the action of redox-directed cancer therapeutics using a human peroxiredoxin-2-based probe. Nat...

...guidelines? A few notes below are considerations for designing HR donor. Generally if off-target (non-specific... PCR and surveyor. I used DNA polymerase Takara Ex Taq ™ DNA Polymerase for my genomic PCR, but couldn't...In our hands, Herculase II Fusion polymerase or Kapa Hifi Polymerase work very well. Some groups have ...You may also like... Zhang Lab CRISPR plasmids General CRISPR Plasmids Dr. Le Cong, the first author of...concentration of Cas9 and guide RNA (gRNA). These considerations only matter if the PAM sequence is immediately...additional PAM sequences should not be sufficient to generate off-target DSBs; there needs to be extensive homology...most efficient and easiest way is to co-transfect several plasmid together, with each plasmid having a targeting...

...approach, stable lines with targeted genome alterations can be generated within a month. phsp70-Cas9 and a single...BbsI-crRNA : Generates crRNA for use in combination with tracrRNA. 51434 pHD-DsRed : vector for generating dsDNA...mediated genome modifications can be efficiently generated in Drosophila and transmitted through the germline...single chiRNA plasmid can be coinjected to generate mutations via imperfectly repaired DSBs. Coinjection...controlled by the regulatory sequences of an RNA polymerase III transcribed U6 snRNA. In both vectors, the...following template: example : Resources: Protocol for generating gRNAs 106.5 KB flyCRISPR website contains additional...96Ab promoter. 51019 pDsRed-attP : Vector for generating dsDNA donors for homology-directed repair to ...

...capable of controlling eukaryotic gene expression. Several endogenous promoters had been identified that responded... seven repeats of a 19 nucleotide tetracycline operator (tetO) sequence, and is recognized by the tetracycline...viewed in Krueger et al, 2004. Some cell culture sera may contain tetracycline or its derivatives which..., or TetR On or Off PI 21916 Tet-pLKO-neo 3rd generation lentiviral plasmid for inducible expression of...article for additional selection options third-generation rtTA On Fraser 44012 pInducer20 Lentiviral vector... article for additional tookit plasmids third-generation rtTA On Elledge 19407 pTREtight2 Promoter contains...single copy in mammalian cells; Expresses firefly luciferase hairpin and GFP under pTREtight promoter None...

...the new amino acid is introduced in the media. Generally, you will need two plasmids, as depicted in the...interest: The non-canonical amino acid, which is generally introduced in the media. A new codon to be allocated...in selected amino acids within a protein, any alterations in structure or function in the protein can be...and evolve proteins for various purposes from therapeutics to biopolymers. Tips for Success Before beginning...beginning to reprogram the genetic code, there are several things to consider. If you are working off of a...tRNA pair instead. For directed evolution of the liberated EcTrpRS/tRNA pair to incorporate other Uaas (derived...

...developing embryos. zfRegeneration - A database for gene expression profiling during regeneration. Seurat - R ...(embryogenesis is complete in ~3 days), short generation time (~3 months), ease of care and breeding, ...study of cancer, inflammation and infection, regeneration, human neurological disorders, microbiota studies...CRISPR Please refer to Addgene's CRISPR Guide for a general introduction to CRISPR technology or the zebrafish... Vivo Lineage Tracing during Development and Regeneration - Periklis Pantazis Lab Multibow: digital spectral...abundance, size, and localization in high-throughput. MAZERATI platform - Leonard Zon Lab. A platform to rapidly...Zebrafish Tol2 Gateway-compatible Kit for Neurodegenerative Disorders - Nicholas Cole Lab. A kit consisting...

...expression when cloning in E. coli . This alteration enables the generation of a single vector containing Cre ...separate DNA molecules, a translocation event is generated at the loxP sites. For a more thorough introduction...Drosophila. Read Addgene's Blog on Cre alternatives from Gerald Rubin's lab. Cre-containing Plasmids Scientists... Cre-EGFP fusion in a promoterless vector with several available restriction sites none Mammalian Sauer... region including the polyadenylation site and several introns tet-hCMV Mammalian Sauer 12168 pMB80 (R26...hsp70 Insect Rubin 32606 Ttr:Cre Cre expressed in visceral endoderm MCK Mammalian Hadjantonakis 33340 LGmCreER...backbone)-pEFS-Rluc-2A-Cre-WPRE-hGHpA-ITR Cre, luciferase, and sgRNA expression EFS AAV Zhang 60229 AAV...

...estrogen receptor 1 DKFZp686N23123, ER, ESR, ESRA, Era, NR3A1 ESR2 estrogen receptor 2 (ER beta) ER-BETA...MGC3886 ERAP1 endoplasmic reticulum aminopeptidase 1 A-LAP, ALAP, APPILS, ARTS-1, ARTS1, ERAAP, ERAAP1, KIAA0525...disulfide isomerase family A, member 2 PDA2, PDI, PDIP, PDIR PDIA3 protein disulfide isomerase family A...KGFLP1 keratinocyte growth factor-like protein 1 MGC125746, MGC125747, MGC126891 KGFLP2 keratinocyte growth... TDGF1 teratocarcinoma-derived growth factor 1 CR, CRGF, CRIPTO, Cripto-1 TDGF3 teratocarcinoma-derived...systems. Upon infection, the immune system triggers several processes to activate cells to recognize and get...the dominant defense strategy during the first several days following exposure to a pathogen. The innate...

...suitable for generating SARS-CoV-2 spike pseudotyped virus. It also lists several luciferase and fluorescent...mammalian sequences linked to SARS-CoV-2 infection. General Tools : Plasmids expressing Cas12a/Cas13a, Reverse...top Plasmids Encoding Mammalian Genes or Inserts Several mammalian genes have been identified as having ... our Immunology Resource Guide . Return to top General Plasmid Tools SHERLOCK and DETECTR - Simple, portable...window) reverse transcriptase, protease, and Bst polymerase constructs now available from Andrea Pauli's ...nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2. Huo, et al. Nat Struct Mol Biol. 2020...Diagnostic Toolkit Enzymes. (unpublished) Taq polymerase and MMLV reverse transcriptase expression plasmids...

... a 5'LTR and should be packaged using the 2nd generation packaging system. Didier Trono Cre-regulated ...Description Tet on or off PI 11643 pLVCT-tTR-KRAB 2nd generation; Transgene (CAG promoter) ‐ OR ‐ shRNA (H1 promoter...and Didier Trono 11779 pLVCT-rtTR-KRAB-2SM2 2nd generation; Transgene (CAG promoter) ‐ OR ‐ shRNA (H1 promoter...Aebischer and Didier Trono 11644 pLVET-tTR-KRAB 2nd generation; Transgene (hEF-1alpha promoter) ‐ OR ‐ shRNA...Aebischer and Didier Trono 11642 pLVPT-tTR-KRAB 2nd generation; Transgene (hPGK promoter) ‐ OR ‐ shRNA (H1 promoter...and Didier Trono 11652 pLVPT-rtTR-KRAB-2SM2 2nd generation; Transgene (hPGK promoter) ‐ OR ‐ shRNA (H1 promoter...Aebischer and Didier Trono 11651 pLVUT-tTR-KRAB 2nd generation; Transgene (hUbiquitin promoter) ‐ OR ‐ shRNA...

...plant expression vectors containing several cassettes and generate multi-construct transgenic plants -...addition to individual plasmids, Addgene carries several useful toolkits and plasmid collections for use...includes plasmids containing promoters that drive general and species-specific gene expression (see the column...CRISPR Please refer to Addgene's CRISPR Guide for a general introduction to CRISPR technology or the Plant ...selection and multiplex CRISPR/Cas9 assembly for fast generation of multiple Arabidopsis mutants - Niko Geldner...MoClo Plant Parts II and Infrastructure Kit for hierarchical assembly of plant multigene constructs - Johannes...publications are created. Papers from the plant biology literature are discussed with one of the authors. The podcast...

...Expression mEmerald 487 509 39 6 Monomer (A206K) mEmerald-N1 - Mammalian Expression mEmerald-C1 - Mammalian...Expression mPA-Emerald 487 509 - Monomer (A206K) mPA-Emerald-N1 - Mammalian Expression mPA-Emerald-C1 - Mammalian...HisD-rsTagRFP - Bacterial Expression rsEmerald 491 508 rsEmerald-pBAD - Bacterial Expression rsGFP1 493...Expression Species . The content for this page was generated with the help of Erik Snapp . Empty Backbones ...Mammalian Expression mEmerald-pBAD - Bacterial Expression EGFP 488 507 34 6 Prone to dimerization pcDNA3-...

...full-text of the publication here . Article Citation Generation of Genomic Deletions in Mammalian Cell Lines ... incubation time allows for sufficient cell proliferation for screening genomic DNA (gDNA) for the intended... and 7.1 ). The bulk cells have sufficiently proliferated when the concentration exceeds ~100,000 cells...80% confluence. The clones have sufficiently proliferated once macroscopically visible with ~2 mm diameter...pellets in 50 μl of DNA extraction solution. NOTE: Generally ~100,000 cells are used for DNA extraction, although... band” in separate reactions. NOTE: Numerous polymerases may be used for step 6.3 . Run samples in a thermocycler...biallelic deletion clones with a proofreading polymerase and clone the amplicons ( e.g., with a PCR cloning...

...accessory plasmids available from Addgene for generating your own specific TALE plasmids.... TALENs assembled by Golden Gate. Validated in several species. Takashi Yamamoto Additional Plasmids for... Gate TALEN kit (Bogdanove/Voytas) in order to generate TALORs (TAL Orthongal Repressors) that can be ...Grunwald pCS2TAL3-DD and pCS2TALE3-RR are next generation TALEN backbone vectors designed for use with ...segments (136AA and 63AA, respectfully). This next generation architecture has been shown to increase mutation... C63AA) and showed robust cleavage activity in several later studies. pCAG-T7-TALEN(Sangamo)-Destination.... In addition, a T7 promoter was included for generating TALEN mRNAs through in vitro transcription as...

... regulator controlling cell growth and proliferation. mTOR, a serine/threonine kinase, is found ...mutations in negative regulators of mTORC1. The hyperactivation of mTORC1 in cancer promotes the conditions...conditions needed for uncontrolled cell growth and proliferation, including an abundant supply of macromolecules...energy, as well as the presence of both pro-proliferative and anti-apoptotic signals. mTORC2, which appears...upstream of Akt, also promotes cell survival and proliferation. mTOR Pathway Plasmids Click on a name to find...specific relationship. The content for this page was generated with the help of David Sabatini . mTOR Pathway...specific relationship. The content for this page was generated with the help of David Sabatini . Return to top...

...Institute for Cell Science has deposited plasmids to generate fluorescently tagged proteins of key cellular ... stem cell (hiPSC) lines. The plasmids used to generate these lines have been deposited here with Addgene...With these plasmids researchers will be able to generate fluorescently tagged proteins of key cellular ...Microtubules 101786 ST6GAL1-mEGFP AICSDP-26 mEGFP Sialyltransferase 1 Golgi 109122 NPM1-mEGFP AICSDP-50 mEGFP ...164500 POLR2A-mEGFP AICSDP-117 mEGFP RPB1 RNA polymerase II 168798 TERF2-mEGFP AICSDP-77 mEGFP Telomeric...CTCF-binding sites 170818 PCNA-mEGFP AICSDP-116 mEGFP Proliferating cell nuclear antigen (PCNA) DNA replication ...Protocols The Allen Institute for Cell Science has several protocol videos available that may be helpful for...

... activator-like (TAL) effectors, scientists in several biological disciplines are coming closer to this...fingers recognize triplets and the FokI nuclease operates as a dimer, cutting in the spacer region between...triplet has a corresponding zinc finger, and interactions between zinc fingers within an array can reduce...zinc fingers, potential targetable sites in your average genome are every 500 bp. TAL effectors have some...history. Dr. Keith Joung’s lab at Massachusetts General Hospital also recently released a TALEN kit through... Zinc Finger Consortium with Dr. Voytas, has considerable experience in the DNA targeting field. A third...specific gene or your anxiously investigating gene therapy options for a rare disease in humans, TAL effectors...

...to LacZ, plus luciferase-2A-Cre recombinase 60226 : sgRNA cloning backbone with luciferase-2A-Cre recombinase...For cloning information, please view: Zhang Lab General Cloning Protocol 237.2 KB SpCas9 Plasmids The CRISPR...sgRNAs targeting KRAS , p53 , and LKB1 , plus luciferase-2A-Cre recombinase, and Kras G12D HDR template...of a single AAV vector ( AAV-KPL ) in the lung generated loss-of-function mutations in p53 and Lkb1 , as...plasmid contains two expression cassettes, Renilla luciferase-2A-Cre recombinase and sgRNAs targeting the mouse...plasmid contains two expression cassettes, Renilla luciferase-2A-Cre recombinase and an sgRNA targeted to LacZ...plasmid contains two expression cassettes, Renilla luciferase-2A-Cre recombinase and an sgRNA backbone for ...

...regions retaining molecular interactions and identify molecules interacting with those regions. Locus-specific...Lab web page . Figure 1: iChIP general scheme Figure 2: enChIP general scheme Fujii Lab Plasmids Individual...reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line.../dsx023. PubMed . Identification of physical interactions between genomic regions by enChIP-Seq. Fujita...specific genomic regions retaining molecular interactions by the iChIP system using recombinant exogenous...

...Resources Overview of Viral Production Viruses are generated using standard methods that have been optimized...optimized for each specific virus in order to generate high quality preparations. After production, all virus...the University of Pennsylvania Vector Core. In general, titering is performed by the facility that produced...suggests that titers obtained using ddPCR are generally 2–3 fold higher than those achieved using the ...us . Full sequencing of the Viral Genome Next-generation sequencing is performed on viral genomes isolated...preparations are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging system developed ...dilutions of the lentiviral vector, harvesting cells several days later, and isolating genomic DNA. This DNA...