...an autonomously replicating sequence (ARS), a partial centromere sequence (CEN), both, or neither, depending... an autonomously replicating sequence (ARS) a partial centromere sequence (CEN) both or neither ...depending...molecular biologist has seen before, showing how DNA sequences are translated into amino acids. And I was astonished... Yeast shuttle vectors have these replication sequences for their yeast targets as well as a bacterial...incorporated into the host cell’s genome, so no special sequence is needed to retain the vector as cells divide...

...cognate tRNA abundance Repetitive sequences Restriction sites Sequences prone to create secondary structures...Addgene now have full sequence data, we recommend directly analyzing gene sequences for codon optimization...think it’s easy to come up with a workable DNA sequence to encode your peptide of interest and produce... the possible codons at random and expect your sequence to express well in any organism. So what are the...1990). Controlling gene expression through gene sequence - Proteins that are encoded by codons with low...

... signal sequence (gene start) and a transcription termination/polyadenylation signal sequence (gene end...viral sequences (e.g. promoters) can be studied by changing the minigenome cis-acting sequences and monitoring...approximately 19 kb in length. It contains short promoter sequences at its 3’ and 5’ ends that are crucial cis-acting... by the EBOV-specific gene start and gene end sequences. Thus, the minigenome contains all cis-acting ... under control of a T7 RNA polymerase promoter sequence. Thus, T7 RNA polymerase also needs to be present...

... efficient and precise tools for altering DNA sequences. These first base editors were both deaminases... things base editors really need from Cas9 are sequence specificity and the R-loop structure. In order...requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent motif (PAM) on... types, although efficacy varied among target sequences. The researchers hypothesized that the CGBE edit...different deaminase and UNG enzymes had different sequence preferences, which they defined thoroughly. Plus...outcomes of their various CGBEs at different target sequences and in different cell types, available at www....target loci (between 4.0% to 29.2% of studied sequences) (Huang et al., 2024). BE4max, a CBE, generated...

...hybridoma antibody sequences that have been cloned into plasmids. Because their sequences are known and encoded...non-antigen binding mouse sequence as possible with human antibody sequences.  Additional considerations...chimeric antibodies will inevitably retain some sequence from their parental species which can be bound...

...viruses. Evolving to survive Although the CRISPR sequences were initially discovered in E. coli in 1987 (... (1), the concept that these clustered repeat sequences operated as a safeguard against bacteriophages... 30bp in length and paste them into the CRISPR sequence. Separate Cas proteins then express and process...using slightly different Cas proteins and PAM sequences. A total of eight evolutionarily conserved CRISPR... generating a single point mutation in the PAM sequence, preventing the Cas nucleases from re-identifying...

...codons that alter the nucleotide sequence but not the amino acid sequence of the translated protein.  These...however, one of its biggest limitations is that sequence modifications can only be made at restriction ...and deletion of selectable and non-selectable sequences, point mutations or other small base pair changes...recombination of the substrate with the target DNA sequence. This means cloning occurs in vivo, as compared...made by PCR using primers that amplify the DNA sequence of interest and flank it with 50 base pairs of...nucleotides long (20 nucleotides that anneal to the DNA sequence of interest and 50 nucleotides of homology to ...desired alteration(s) located in the center of the sequence. Since lambda red has a higher recombination frequency...

...mismatch between the original sequence on one strand and the edited sequence on the other strand. To guide...transcriptase with a sequence optimized for human codons; additional nuclear localization sequences; and two mutations.... After nicking by Cas9, the homologous pegRNA sequence hybridizes to the target site, and the reverse...Advantages of prime editing Less constrained by PAM sequence location The prime editor extends the reach of...the PE2 enzyme. PEmax: Optimizing prime editor sequence to improve expression and activity In addition...  ✓ SV40 and c-Myc nuclear localization sequences Improve translocation to the nucleus ~ ~...prime editing with a recombinase to install large sequences (up to 10 kb) into the genome (Pandey et al., ...

...the gene of interest. This gives you loads of sequences to select from - in many cases several exons’ ...sgRNA), provided it meets two conditions:  1) The sequence is unique compared to the rest of the genome.  ...upstream of a Protospacer Adjacent Motif (PAM), the sequence NGG.  If these conditions apply to your experimental...feasible for your experiment, ensure your sgRNA sequence has little homology to other regions of the genome...polymerizes DNA onto the target strand based on the sequence of the pegRNA. Image from David Liu.   Activation... sgRNA vectors and design your sgRNA targeting sequence using one of the many freely available sgRNA design... cells, it is time to confirm that your target sequence has been modified!  The exact protocol may vary...

...simultaneously  With increasing sequencing power, scientists are able to quickly sequence an individual’s genome... develop a method that measures the effects of sequence variants on abundance simultaneously called VAMP-seq..., for Variant Abundance by Massively Parallel sequencing. In VAMP-seq, the abundance of a protein variant...Genomic DNA from each bin are then extracted and sequenced. Chiasson is currently using VAMP-seq to study...

...different synthetic peptides corresponding to linear sequences within the middle of the human c-Myc protein or...chicken homologs. By comparing the homologous sequences in these species, they found that the N-terminal... as a recombinant antibody. Researchers first sequenced the variable domains of Myc1-9E10 in the late ...affinity for the epitope, a phenomenon known as sequence context sensitivity (Schüchner, et al. 2020). ...Myc-tag antibodies appeared to be less sensitive to sequence context.    So, do Schüchner, et al.’s results...variable epitope recognition dependent on neighboring sequence context. Sci Signal 13. https://doi.org/10.1126...

...orthologs, Cpf1 efficiency varies widely with gRNA sequence. Both groups used multiple methods to examine ...single and double mismatches throughout the 23-base sequence. Double mismatches ablated Cpf1 activity, except... they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is also sensitive to single...23. Accordingly, the 3’ end of the gRNA target sequence does not have an essential function in Cpf1-mediated...4-6 base deletions at the 3’ end of the target sequence. Cpf1’s strength may lie in its low off-target...post-HDR, repair templates should remove the PAM sequence. Many research and clinical applications require...

...discrepancies between what we sequenced in house and the submitted sequences were a concern to the plasmid... currently working on the QC/QA team as a DNA Sequencing Intern. The application process When I first...approachable.     Fig. 1: Examples of DNA sequencing QC work    In addition to my responsibilities...

...majority of information on each cancer clinical sequencing report includes rare variants that haven’t been...corresponding to cancer variants found in genome sequencing studies (as well as reference cDNA clones mapping...scientific community through Addgene. These include 634 sequence confirmed mutant cDNA clones corresponding to ... worked with many collaborators to aggregate a sequence-confirmed series of clones corresponding to known...when rare variants are discovered in clinical sequencing studies. At the Broad, my colleague Cory Johannessen...

...such as this "CRISPR 101" post. Next-generation sequencing technologies are the gold standard but they remain...assays can be designed to detect either a donor sequence integrated through homology-directed repair (HDR...designed to perfectly complement the wild type sequence and is not capable of binding a mutated target... screening tool, it still requires target DNA sequencing to confirm the exact nature of your mutations...great description of the plethora of available sequencing options can be found here. Good luck with your...

...knock-in long sequences with reduced risks of unwanted mutations in mammalian cells. Long sequences traditionally...M-CREATE) dCas9-SSAP with reduced error rate for long sequence knock-ins Visual barcodes for live cell clonal...  dCas9-SSAP with reduced error rate for long sequence knock-ins by: Lucie Wilson Le Cong’s lab has developed...SSAP will make the editing of kilobase-scaled sequences more efficient and less risky.   Fig. 3...

...clonal dynamics over time using next generation sequencing (NGS). The library was developed when Carrie ...population of cells with specifc, easily read sequences that can be used to measure their clonal abundance...barcoded clone can then be monitored over time by sequencing the barcodes in the population (all barcodes ...primers). The abundance of a particular barcode sequence then corresponds to the abundance of a specific...clone; lineage tracing is performed via barcode sequencing. Using the ClonTracer library, Bhang et al. found...

... a rapid and highly efficient way to move DNA sequences into multiple vector systems. With the appropriate...scientists can easily clone in their desired DNA sequences. Gateway technology relies on the two reactions...add attB sites to either end of the KRAS coding sequence. If you choose this strategy, it’s important to...protein expression elements (ribosome recognition sequences, start codon, stop codons, reading frame considerations...verify the integrity of your expression clone via sequencing or restriction digest! Then, you can transform...Once you generate the entry clone with your DNA sequence of interest, you can move this DNA fragment across...

... cells. Cas13 enzymes also don’t require a PAM sequence at the target locus, making them more flexible...given single base protospacer flanking site (PFS) sequence, but orthologs like LwaCas13a do not require a... target RNA from bases 12-26 of a 30 nt target sequence. For the editing portion of the protein, they ...designated this system REPAIRv1. Using next-generation sequencing, they confirmed A->I editing and found that 50...target effects observed in whole-transcriptome RNA-sequencing, the authors performed rational mutagenesis and...RESCUE further expands the RNA editing toolkit and sequences that can be edited.   As discussed above, the ...

...mariner), as a method of tagmentation for next-gen sequencing pipelines (e.g. using Tn5), and as knock-in tools...A CRISPR RNA (crRNA) comprising a 32 nt spacer sequence specifying the target site. Four proteins (TniQ...interest flanked by ~50-150 bp transposon end sequences (R and L ends).  Figure 1: (A) Components.... Genomic targets require an upstream 2-bp PAM sequence for recognition by QCascade; although 5’-CC-3’...complexes survey the genome and bind target DNA sequences using RNA-DNA base pairing. Separately, TnsABC...overnight Screen colonies phenotypically or by PCR/sequencing While CRISPR-transposons are powerful and simple...