Skip to main content
Addgene
Open Menu

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

In Category

We narrowed to 430 results for: sequence

Showing: 61 - 80 of 430 results

  1. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
    Published
    Aug. 6, 2024, 1:15 p.m.
    ...interest: The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration...the transfer plasmid sequences into the host genome. Typically, it is the sequences between and including...Leukemia Virus) or MSCV (Murine Stem Cell Virus) sequences. Packaging genes (viral Gag-Pol): Gag is a structural...virus post‐transcriptional regulatory element; sequence that stimulates the expression of transgenes via...the 5' and 3' ends of the provirus). Contains sequences necessary for activation of viral genomic RNA ...the addition of a poly A tract just after the R sequence. This post was written and updated by multiple...

  2. Plasmids 101: How to Verify Your Plasmid Using a Restriction Digest Analysis

    Type
    Blog Post
    Published
    Aug. 28, 2014, 3:34 p.m.
    ...expected sequence. Here at Addgene, we use NGS-based quality control to confirm the sequence of all the...cleaving sequences which are similar, but not identical, to their defined recognition sequence. Most enzyme...successfully even if you don't have the full plasmid sequence. Once you have purified plasmid DNA, this method...NCBI for YGOI to find the particular reference sequence if necessary. The example plasmid on the right... dna sequencing results...

  3. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    Published
    March 28, 2018, 6:52 p.m.
    ...CRISPR enzymes require a short PAM sequence adjacent to the targeted sequence at the locus of interest. SpCas9...engineered alternative Cas9s binding distinct PAM sequences. Now, Hu et al., working in David Liu’s lab, have...PAM-interacting domain. However, these alternative PAM sequences are longer and often more complex than NGG, resulting... for and interest in enzymes with shorter PAM sequences and/or broader PAM flexibility. Creating xCas9...plasmid library contained all 64 possible NNN PAM sequences adjacent to a protospacer. If ω-dCas9 could recognize...permitting phage propagation and keeping that dCas9 sequence in the pool. According to this logic, ω-dCas9 ...off-target effects across 5 gRNAs with NGG PAM sequences were actually lower for xCas9 3.6/3.7 than for...

  4. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

    Type
    Blog Post
    Published
    Oct. 19, 2017, 1:54 p.m.
    ...adding a nuclear localization sequence (SV40 Large T nuclear localization sequence) and expressing it from ... of the DNA sequence of interest can lead to poor expression of genes within that sequence and should ...between target sites of the same sequence in FLEx vectors, the DNA sequence that should be inverted is flanked...differ in their sequence. Recombination between the two target sites with identical sequence leads to inversion...when it was discovered that it is not the exact sequence of the asymmetric target site core but its 8 bp...inversion of the flanked DNA sequence and the interior target sites. In a second step, the two compatible...

  5. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    Published
    April 28, 2016, 2:30 p.m.
    ..., a SSR will invert the DNA sequence between the sites. If a DNA sequence is floxed in the same orientation...the BFP coding sequence in the sense orientation, followed by the mCherry coding sequence in the antisense...reporter DNA sequence (such as GFP) in the sense orientation, followed by coding sequence of the gene ...loxP sites, while FLP binds FRT sites. When a DNA sequence is flanked by target sites (floxed) in opposing...loxP and lox511 sites after the inverted GeneR sequence in the opposing orientation, you have created ...

  6. Channelrhodopsins with improved light sensitivity for minimally-invasive optogenetics

    Type
    Blog Post
    Published
    Nov. 3, 2020, 2:15 p.m.
    ...recombinant channelrhodopsin sequences. The model first removed sequences from the library predicted to...reducing the library to 1,161 sequences. Thirty of these remaining sequences were selected for characterization...to have improved photocurrent properties: The sequence and structures that determine membrane expression...model was trained to predict if a channelrhodopsin sequence would result in a functional channelrhodopsin,...library of ~120,000 predicted channelrhodopsin sequences would have photocurrent properties best suited...for less-invasive optogenetics experiments. The sequences in this library were designed using structural...

  7. Design Tips for Prime Editing

    Type
    Blog Post
    Published
    Jan. 23, 2025, 2:15 p.m.
    ...scaffold sequence into the genome, ensure it is not homologous to the target genomic sequence (Chen et...experimental design, especially the design of the RNA sequences you’ll use in prime editing. We thought we’d collect...both the genomic location to be edited and the sequence of the edit, so its design changes for every new... prime editing application. And optimizing its sequence is important, as prime editing efficiency is affected...content are most likely to be successful, although sequences with G/C content outside this range can still ... more important to test different lengths and sequences, as unintended secondary structures in your pegRNA...Created with BioRender.com. What to edit The sequence you choose to install can influence the success...

  8. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    Published
    July 2, 2020, 12:40 p.m.
    ...They found that DNA sequences targeted by the gRNAs were the top enriched sequences in the experiment and...supporting roles. Enhancers are cis regulatory DNA sequences that, when bound by transcription factors, can...looping with the photo “developed” by analyzing DNA sequencing data. While “pictures” generated with current...chimeric DNA fragments by PCR or next-generation sequencing. Figure 1: Overview of the key steps of... DNA fragments can be detected by PCR or deep sequencing. A drawback of the original CAPTURE method...gRNA expression. Additionally, CAPTURE 3C-based sequencing requires a large number of cells (~5x107). Together...BioTAP-tag is a 69 aa long biotinylation targeting sequence that is recognized by biotin ligases normally ...

  9. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    Published
    Nov. 12, 2020, 2:15 p.m.
    ...anticodon,  a three nucleotide sequence complementary to the mRNA sequence. The 3’ end of  the tRNA binds...the amino acid that corresponds to the anticodon sequence. That means there are 61 possible tRNAs. Cells...and thus protein expression without altering the sequence of the protein. As DNA synthesis has become quite...scientists to re-synthesize DNA with the most optimized sequence. Unfortunately, it is not always easy to identify... certain systems, synonymous changes to a gene sequence can cause unexpected detrimental results to the...could remove information encoded in the primary sequence of a particular gene which could affect translation...available online to both determine and synthesize DNA sequences for optimal expression in your system of choice...

  10. Kiran Musunuru on the Newest TALEN Genome-Editing System

    Type
    Blog Post
    Published
    Dec. 10, 2013, 3:13 p.m.
    ...desired sequences. TALENs are much more reliable. If you design them for a particular sequence, there ...can be programmed to bind to essentially any DNA sequence desired. This technology allows you to create ...do you use this kit to target a particular DNA sequence? Musunuru: The kit includes 832 plasmids containing...DNA-binding domain to recognize any desired 15 base pair sequence in the genome in rapid fashion. The two remaining...there is a much better chance they will bind that sequence. That’s the revolutionary aspect: TALENs take a...: The power here is that you could potentially sequence a large number of individuals and find several...

  11. CRISPR-Cas14: a family of small DNA-targeting enzymes enabling high-fidelity SNP genotyping

    Type
    Blog Post
    Published
    Nov. 29, 2018, 1:53 p.m.
    ...cleave a targeted sequence of ssDNA. Unlike Cas9, Cas14 does not require a PAM sequence. In addition to ... types of nucleic acids and required a certain sequence specificity in the middle of the gRNA to be activated...mutations. By providing a gRNA specific of a viral sequences, for instance, the enzymes are activated and can...Fluorescent signal indicates the presence of this viral sequence in the assay. By adding Cas14 to the mix, you ...

  12. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    Published
    Aug. 29, 2024, 1:15 p.m.
    ... and then plasmid sequencing if you can. To quote Doench, “Whole plasmid sequencing is just part of doing...enzyme that cleaves outside of its recognition sequence. This means that each component can be designed...? The Doench lab tested the Fragmid system by sequencing 60 individual assemblies created over five months...82 of the correctly banded clones for plasmid sequencing and found 98% of the plasmids were perfectly ...confirm that at least one plasmid was the correct sequence for every individual assembly. What destination...051 (for stable fly cell lines; attL and attR sequences for genome integration by phiC31) Drosophilia...pRDB_053 (for transgenic flies; gypsy insulator sequences; white + selectable marker; attB site for genome...

  13. Genome-wide Screening Using CRISPR

    Type
    Blog Post
    Published
    Aug. 18, 2015, 2:30 p.m.
    ...next-generation sequencing (E). Considerations and tips for successful screens Next-generation sequencing - CRISPR...guide RNA (gRNA). The ~20 nucleotide targeting sequence within the gRNA is defined by the user, and it...immediately 5’ to a protospacer adjacent motif (PAM) sequence. Co-delivery of wild-type Cas9 and a gRNA generates...screening due to the broad range of potential target sequences and ease of generating gRNA-containing plasmids...generate lentivirus. Be sure to use next-generation sequencing to check the quality of your amplification. If...by a unique barcode on each plasmid. As such, sequencing CRISPR libraries after amplification and after... a screen requires the use of next-generation sequencing. Representation - Most libraries contain 3-6 ...

  14. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    Published
    July 28, 2016, 2:30 p.m.
    ...machines (RSMs), which take on a particular DNA-sequence configuration (state) based on the identity and...RSMs work? The state of an RSM is defined by the sequence within a prescribed region of DNA termed the “...straightforward way to read the state of an RSM is to sequence the register. In our paper (1), we allowed individual... register region and sent the PCR product for sequencing.  A less straightforward, but more population-wide...of state interrogation mentioned above (Sanger sequencing, qPCR, and fluorescent reporter-based) each come...with their advantages and disadvantages. Sanger sequencing is simple and the most accurate method as it ...the state of a register. However, each Sanger sequencing reaction can only be performed on one register...

  15. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    Published
    Nov. 1, 2016, 2:30 p.m.
    ...and amplification of a recombinant DNA without sequence alteration. Molecular cloning techniques are some... DNA polymerase, the construction of specific sequences for base pair overhangs, insert and vector purification...products, nor does it require the use of specific sequences. Read on to learn how easy this process is and.... The primers used to amplify the insert have sequences specific to the insert with an additional 15-17...with the appropriately sized product, purify and sequence the plasmid to confirm no mutations were introduced...FastCloning: a highly simplified, purification-free, sequence-and ligation-independent PCR cloning method." ...

  16. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

    Type
    Blog Post
    Published
    June 27, 2018, 1:16 p.m.
    ...transcription of RNA such as long-read cDNA sequencing and RNA sequencing of complex heterogeneous RNA samples...functional element (tyrosine sulfation consensus sequence, fluorophore, or APEX2), allowing for quantitative...Caspex’ construct is guided by a pre-defined sgRNA sequence to a specific genomic locus and labels the proteins...radius of approximately 20 nm of a specific DNA sequence in only 1 min. Using these lentiviral plasmids...Shield1 inducible CMV promoter and targeted to a sequence of interest with an sgRNA expressed from a separate...

  17. Behind-the-scenes of the Isolation of the Thermostable IgnaviCas9 From a Yellowstone Hot Spring

    Type
    Blog Post
    Published
    Nov. 12, 2019, 2 p.m.
    ...that remained unexplored. Using new single-cell sequencing approaches, the lab was able to eliminate the...cultures to study these microbes. From 16S rRNA sequencing, hot springs were known to be diversity hotspots... characterized a few samples by 16S rRNA gene sequencing which showed the presence of many “dark matter...samples, and years later, several whole genome sequencing projects were launched in the Quake Lab. Identifying...mini-metagenomics method using microfluidic parallelization to sequence this highly complex hot spring sample. The sample...separated into smaller samples of 5-10 cells each for sequencing, allowing for lower complexity with higher throughput...

  18. CRISPR 101: Multiplex Expression of gRNAs

    Type
    Blog Post
    Published
    Sept. 10, 2020, 11:45 a.m.
    ...Deleting a large region of the genome by removing the sequence between two target sites. Modifying multiple genes... oligonucleotides for your chosen gRNA target sequences and clone them in just as you would for a single...enzymes, which cleave outside of their recognition sequence, creating flanking overhangs. These overhangs ...the oligonucleotides specifying each gRNA target sequence into distinct expression vectors using the enzyme...the promoter-gRNA construct, but with different sequences adjacent to the sites. When digested with the ...Type IIS enzyme, the unique flanking overhang sequences can link together to allow for ordered assembly...schematic below.   Figure 2: gRNA target sequences (colored rectangles) are cloned into various plasmids...

  19. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...primers that contain the appropriate homologous sequences. NEB recommends an overlap of 15-40 bp, with a... insert both the gene of interest and the tag sequences into the vector in one step without scars as depicted...might be anticipated to occur within a terminator sequence), since this would directly compete with the required...assembling multiple fragments in one step with no scar sequences remaining in the final product. The long overlapping...fragments as compared to the smaller overlapping sequences left with a restriction digest. A study in 2013...around this is to use a combination of standard sequences with overlap regions, such as in the case of MODAL...overlap-directed methods (Casini 2014). In this case linker sequences are added before the homology regions, which allows...

  20. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    Published
    May 5, 2020, 1:15 p.m.
    ... the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity gets activated. This unleashes... of interest. Cas13 uses a complementary crRNA sequence to bind to the sample and upon binding, gets activated...trans cleavage reaction that cleaves a nearby RNA sequence that contains a fluorophore that is quenched when... Unlike Cas12a, Cas13a does not require strict sequence preferences at the target site, while Cas12a requires...SHERLOCK diagnostic test, this test varies in the sequence of steps and the Cas protein used. Here, all the...to one strand of the reverse-transcribed target sequence that are present close to the primer recognition...binds to the complementary region in the target sequence and this activity results in the trans cleavage...
Showing: 61 - 80 of 430 results