... Since researchers can generate almost any DNA sequence in the way that they like, the design and engineering...mNeonGreen, mScarlet(-I), and mCherry. Keep the linker sequence short In the early days of GFP applications, many...site and therefore consist of random amino acid sequences or (ii) designed to form an inert, unstructured...and protein of interest (POI). To indicate the sequence, people often use terminology like “C-terminal...interest usually outweigh potential negative consequences of the tag. Many thanks to our guest blogger...

...N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence (-CaaX). Both modifications...modifications are disrupted when GFP is fused to these sequences. When N- and C-terminal fusions are not tolerated...alternative is to insert the GFP into the coding sequence of the protein of interest. One of the reasons... reaction requires molecular oxygen and as a consequence GFP remains non-fluorescent under anaerobic conditions...

...segments, each with two fluorescent protein coding sequences in opposite orientations. Cre-mediated excision...the system, the fluorescent protein whose coding sequence is closest to the promoter will be expressed. ...used are fluorescently distinct, their protein sequences are highly homologous, preventing design of antibodies...three with low levels of both fluorescence and sequence overlap (coral mOrange2, jellyfish EGFP, and sea...

...MG1655    Wild type K-12 strain; first published sequence of an E. coli K-12 strain. F- λ- ilvG- rfb-50 ...and retroviral vectors or cloning or repeated sequences with the potential to recombine.  F' proA+B+ lacIq...and retroviral vectors or cloning or repeated sequences with the potential to recombine.  F- endA1 glnV44...and retroviral vectors or cloning or repeated sequences with the potential to recombine. This strain is...E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase mutation (GATC) Preparing...

...phosphorylatable residues and the bNLS and NES sequences, which have previously been characterized. See...more details on the design of the bNLS and NES sequences. Disadvantages of KTRs KTRs also have some disadvantages...

...strange breaks at target sites 5' of the 5'NGG3' PAM sequence as directed by a gRNA. 2A-Puro In this plasmid...into which you can clone your 20 nucleotide guide sequence of choice (for generation of a gRNA that will ...

...in place of SpCas9. gRNA A gRNA targeting a DNA sequence of interest is expressed in 293T cells and loaded... HDR allows for the precise insertion of a DNA sequence ranging from a single nucleotide change to large...utilize HDR, a donor template containing the desired sequence flank by homology arms must be delivered to cells...

...approaches: single-cell RNA-sequencing to identify neuronal class and deep sequencing of neurons from each class...of a GAL-4 driver that can bind to activation sequences upstream of genes to stimulate expression (Wang...

...In the O'Connor lab, we recognized that genome sequencing projects were generating huge amounts of data...available. Then, staff or students clone similar sequences from a less-well studied organism into overexpression...experiments We used TOPO cloning to insert the coding sequences for ten different MET/Met genes (Figure 1) from...

...strategies: Strategy #1 (ProA): Create promoters with sequences upstream of the start codon of retinal cell-type...Create promoters using phylogenetically conserved sequences that are upstream of the transcription initiation... factor binding sites interspaced with random sequences.   Strategy #4 (ProD): Create promoters with the...

...that recognize, cut, and alter specific target sequences in DNA resulting in a variety of outcomes including... requirement. For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where B is G, T, or C). The authors...scientists seeking a wider range of targetable genomic sequences could benefit from CRISPR-Casɸ. Pausch et al....

... RNA sensors that can detect virtually any RNA sequence (Green et al, 2014). The second was an in vitro...laboratory. The detection threshold for these RNA sequences (3 nM), however, was quite high and above what...and prototyping pipeline that can take pathogen sequence information from public databases to the production...

...expression that do not involve any changes to DNA sequence (Epigenetics literally means ‘on top of the genome...with reduced life-span, but no changes in the DNA sequence could be found. Angelman syndrome and Prader–Willi...methyltransferase identification, and bisulfite sequencing. However none of these techniques can be used...

...Weinstein RNA aptamers, which are nucleic acid sequences that fluoresce upon binding a small molecule, ...From considering protein size and shape, linker sequences, and fusion location, there are many choices to...

...editing experiment. I was in my office and opened a sequence file. Then I saw it, the chromatogram showing ... signs of a mutation introduced by CRISPR. The sequence started clean and then, about 100 bps into the...the run, it got “dirty”, with multiple sequence traces overlapping each other! When the count was done,... a reporter gene, or replacement of the murine sequence with a human gene. Advantages of using CRISPR ...

... universal sgRNA that targets the donor vector sequence. Homology arms that flank the donor plasmid cargo...essential genes and phage host factors, and identify sequence-specific toxicity of Cas9. Read the paper in ...

...CRISPRi cassette flanked by left and right Tn7 end sequences. Donor bacteria with a chromosomal copy of the...recipient bacteria due to site selection by TnsD, a sequence-specific DNA binding protein. Selection removes...DNA (purple) flanked by left and right Tn7 end sequences (green) integrates downstream of the glmS gene...

... capacity AdV) are devoid of all viral coding sequences and retain only the ITRs and packaging signal ...analyzed by endonuclease restriction digest or fully sequenced to confirm its integrity. Initial production (... quantity and primers specific for a viral DNA sequence. *Pro-Tip*: Ad5 reference material should be used...

...The gRNA conventionally contains a 20-nucleotide sequence that is complementary to the target site, or protospacer...several popular CRISPR-based tools for altering DNA sequence or regulating gene expression.     References ...to Our Interview with Alex Chavez Learn about Sequencing Options for CRISPR Genotyping Cas9 Activators...

...exploration into the effects of ancestry and germline sequence variation in cancer cell lines took them down ...indicating loci where SNPs reside on sgRNA target sequences for sgRNAs that target ancestry-associated genes...frequency (x-axis) which SNPs map to thetargeting sequences of guides across 8 CRISPR libraries. Samples are...