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Enter a sequence to perform a BLAST-based search of Addgene plasmid sequence databases.

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We narrowed to 538 results for: sequence

Showing: 161 - 180 of 538 results

  1. Hot Plasmids and Viral Preps - September 2021

    Type
    Blog Post
    ...end-point sequencing assays.  This tool was designed by putting together the coding sequence of a mCherry...base edited cell populations without end-point sequencing assays. These plasmids use “Cas9-mediated adenosine...protein followed by a stop codon and the coding sequence for a green fluorescent protein (GFP). The success...

  2. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ... the iBARs can be detected by next generation sequencing.  In order to avoid a high false-positive rate...gRNAs and to embed these barcodes within the gRNA sequences. This allowed tracing of the performance of each...experiment by counting both the gRNAs and their iBAR sequences. Since the sgRNAiBAR library covers every annotated...stain” DNA. TALE proteins can bind specific DNA sequences which brings the attached fluorescent protein ...

  3. CRISPR Guide

    Type
    Collection
    ... be any ~20 nucleotide sequence, provided it meets two conditions: The sequence is unique compared to ...(PAM) The PAM sequence (NGG) serves as a binding signal for Cas9, but the exact sequence depends on which... target, the seed sequence (8–10 bases at the 3′ end of the gRNA targeting sequence) will begin to anneal...matters. Mismatches between the target sequence in the 3′ seed sequence inhibit target cleavage, while mismatches...determined by both the gRNA sequence and the Cas9 enzyme. Ideally, a gRNA targeting sequence will have perfect ...require PAM sequences in very close proximity to the region to be edited, making the PAM sequence (NGG) limiting...target sequence in your donor DNA with a silent mutation that does not change the amino acid sequence. HDR...

  4. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    ...recombineering is unsuccessful and the wild-type sequence persists, Cas9 will cleave the gene of interest...have inactivating mutations in the Cas9 or spacer sequence, and these rare events are easily detectable using...effective targeting either the promoter or the coding sequence and the template or non-template strand, among...population based on the presence of specific target sequences (Citorik et al., 2014). These could be genes, ...introduce cytosine-to-thymine substitutions in target sequences (Banno et al., 2018). These substitutions were...approximately a five-base pair window of the target sequence which could be adjusted by changing the sgRNA ...2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32:1146...

  5. Designer PUF Proteins for Any RNA Target

    Type
    Blog Post
    ...nucleases to transcription factors – to specific sequences of DNA has become commonplace. This ability has...networks. A comparable system for targeting specific sequences of RNA is highly desirable for extending the complexity...possible to produce a protein that binds an alternate sequence. By comparison, Abil explained, the specificity...

  6. Quick, Versatile Plant Transgenesis with GreenGate Plasmids

    Type
    Blog Post
    ...cassettes: plant promoters, N-terminal tags, coding sequences of the gene of interest, C-terminal tags, plant...endonucleases except that they cut at arbitrary sequences a defined distance away from the recognition site...and recognition are independent. “They see one sequence and cut another,” Lohmann said. “It allows us ...

  7. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...and PCR genotyping (Sanger sequencing); use primers flanking the tag sequence and expect a size-shift in...pulled-down and later coupled to high-throughput sequencing to identify genome-wide binding patterns of the...facilitates in-frame insertion of tag-encoding sequences at the intended target site. With the widespread...protein, and a donor DNA that contains the tag sequence flanked by homology arms (Figure 1). In the first...donor DNA (ssODN) that contains the tag-encoding sequence and short homology arms (60-70 bp) is added to...ID(s) as an input query, pick crRNA (guide RNA sequence targeting the gene of interest) and ssODN (tag-encoding... promoter region of both the alleles should be sequence-confirmed for "indel-free" knock-in. Another good...

  8. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...self-transcribing active regulatory region sequencing) that tests enhancer sequences outside of their endogenous chromatin..., and these sequence changes are subsequently identified through transcriptome sequencing. Importantly... due to the variable availability of enhancer sequences in different chromatin states. Alexander Stark...libraries are cloned downstream of a minimal promoter sequence. Should one of these random stretches of DNA contain...its enrichment over input levels using next gen sequencing. The STARR-seq backbone is available from Addgene... genome (the identity of the genes and target sequences can be found here). To use the library, the authors...cells and surviving “mutant” colonies were deep sequenced to identify the gRNAs that confer protection to...

  9. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    ...target any sequence of DNA. Second, NgAgo target specificity is dictated by a DNA guide sequence rather than...the views of Addgene. Addgene performs Sanger sequencing on select regions of all distributed plasmids...First, unlike Cas9, NgAgo does not require a PAM sequence for target recognition, which gives a researcher...sites in a single experiment. Also, the NGG PAM sequence might not always be available at an optimal distance...

  10. CRISPR Activators: A Comparison Between dCas9-VP64, SAM, SunTag, VPR, and More!

    Type
    Blog Post
    ...target specific sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of interest...recruits transcriptional machinery to specific sequences, causing targeted gene regulation. This can be...either initiation or elongation, depending on which sequence is targeted. Performance dCas9-VP64 activation...Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 152:...

  11. CRISPR Plasmids - Bacteria

    Type
    Collection
    ...target have high sequence identity to the spacer sequence and contain a PAM sequence just downstream of...specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell ...transcription of a specific gene. Design your gRNA sequence to direct the dCas9-activator to promoter or regulatory...promoter/enhancer or the beginning of the coding sequence. If the plasmid you’re using does not also express...they have recognized and cleaved the target RNA sequence, they adopt an enzymatically active state and ... of the sequence to be cleaved. This non-specific cleavage is thought to activate programmed cell death...immunoprecipitate FLAG-tagged Cas9. Design your gRNA sequence to direct dCas9 to a specific locus, avoiding ...

  12. Custom CRISPR Screens & the Green Listed Software

    Type
    Blog Post
    ...separated cells are subsequently sequenced by next generation sequencing to identify which gRNAs are differentially...then be separated by sorting, and subsequently sequenced to identify which signaling molecules were involved...different “Options”, including adding adapter sequences for cloning purposes.  Press run and wait for...reference library, as well as GC%, reverse compliment sequences, and more. Output_Compact: A text file that describes...name for each gRNA, as well as the suggested gRNA sequence including adapters. Output_UserInputParams: Contains... into your vector of choice using the adapter sequences that you have specified.   Run your screen! ...differentially expressed in the separate groups and, as a consequence, which genes are good candidates for being involved...

  13. Synthetic Biology - Assembly Standards Guide

    Type
    Collection
    ...additional 5' clamping sequence such as GTTTCTTC). Note that all prefix/suffix sequences are given for the ...Assembly standards define standard prefix and suffix sequences that make it easy to combine components for synthetic...sites which preserve a standard prefix and suffix sequence at either end of the assembly, even as more parts...registries, including the specific prefix and suffix sequences for each (these can be built into PCR primers ...prohibited sites and must be omitted from the coding sequence of the part and backbone. For some standards, ...Thr-Gly; Use N-parts prefix to preserve N-term sequence. Prohibited Restriction Sites: EcoRI, XbaI, NgoMIV...

  14. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ... require DNA sequence-specific binding. Patients’ HIV-1 genomes will need to be sequenced to determine...-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed that most of the HIV...sites with up to 7 mismatches to the gRNA target sequence. Kaminski et al. next tested their procedure in...second challenge for CRISPR HIV-1 therapies is sequence specificity. Whereas antiviral therapies target...resistance to CRISPR therapies through PAM or seed sequence mutations. Multiple gRNAs could be used to combat...

  15. The Advances Behind The World's First Personalized CRISPR Treatment

    Type
    Blog Post
    ... varied depending on the chromosomal location, sequence context, and cell type. Multiple labs worked to...preferences Base editors can only access a short sequence, between four and eight bases, close to the PAM...Cas9. While the wild-type enzyme requires a PAM sequence of NGG (where N represents any base), their engineered...lower efficiency — essentially allowing nearly any sequence to be targeted (Walton et al., 2020). The initial...paired with the highest-efficiency sgRNA. The PAM sequence near this site is AGC, so in a secondary screen... won’t stop here. By coincidence, KJ’s genetic sequence was able to tolerate bystander edits—unintended...

  16. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...you are dealing with a defective gene, Xm, the sequence of which is identical to the correct gene, Xw,...window ranging from 1-17 bp relative to the PAM sequence (Zong et al., 2018) (see CBE-3 in Figure 3). ...versions of SpCas9 and SaCas9, which recognize PAM sequences other than NGG (Table-1). Type Nickase Cas9...location of your target base in the protospacer sequence. If you do not find a suitable ‘NGG’ PAM at your...editor variant from Table 1 depending on the PAM sequence. Avoid targeting a ‘C’ placed immediately after... ‘G’ base because CBE is inefficient in a ‘GC’ sequence context. If this cannot be avoided, select the...

  17. 5 Great Apps for Lab Life

    Type
    Blog Post
    ...helpful aspect to this app; the user can search by sequence, category of enzyme, or name to discover all the...alphabet on the side of the screen to find the sequence for your enzyme of interest. Lastly, the Tm calculator... set up an optimal PCR depending on the primer sequence and the NEB polymerase used. Overall, while much...the NEB inventory only, the restriction enzyme sequence resource is universally useful. 4. Chemical Safety...

  18. Predicting Adverse Reactions to Monoclonal Antibody Drugs

    Type
    Blog Post
    ... origin; purity, mechanism, and stability; and sequence of biologic product, mode of administration, and...prediction of T cell epitopes based on the amino acid sequence of the antibody.  NetMHCPan is one of the more... is a former co-op and current Scientist, DNA Sequencing and QC at Addgene.  References and resources...2022). In silico Immunogenicity Assessment for Sequences Containing Unnatural Amino Acids: A Method Using...

  19. Antibodies 101: Monoclonal Antibodies

    Type
    Blog Post
    ...peptide that represents a short, unique amino acid sequence found in the protein, or, are injected with the...epitope, so it’s less likely that this amino acid sequence will be shared across other proteins. Conversely...Genetic drift refers to changes in the nucleic acid sequence of the antibody-encoding genes over time as the...hybridoma. Any genetic drive can be identified by sequencing the heavy and light chains. To circumvent any...

  20. Synthesized by Ginkgo Bioworks, Shared by Addgene: SARS-CoV-2 Plasmids for Many Expression Systems

    Type
    Blog Post
    ...Though we’ve confirmed the expected features and sequences in each plasmid, none of these constructs have...plasmids in our collection, we’ve relied on Sanger sequencing as a first pass QC. The lab team manually transforms...mixing this isolated plasmid DNA with appropriate sequencing primers, and hand deliver them to our Sanger ...plasmids we perform our standard full plasmid sequencing by NGS. Addgene’s QC scientists immediately screen...
Showing: 161 - 180 of 538 results