...also contains a CAAX sequence to bind the mRNA to the plasma membrane; this sequence prevents diffusion ...requires inserting multiple hairpin sequences into the coding sequence and 3’ UTR. Before beginning experiments...they are bound. These regions are subsequently sequenced and aligned to the genome to determine genome-...dynamics at the single molecule level. The coding sequence of Yan et al.’s reporter contains 24 copies of...

...recognizes the DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The energy from...blue colonies that do not need to be picked and sequenced for potential positive clones. Once you introduce...restriction sites on the ends) and amplify your sequence of interest with Taq polymerase using your favorite...of your insert by PCR, restriction digest, or sequencing. Pro tips Do not add 5’ phosphates to your PCR...virus DNA topoisomerase I in Escherichia coli is sequence specific."  Proc Natl Acad Sci U S A.  1991 Nov...

...form a clean DNA break, Cas3 locates its target sequence and initiates single stranded DNA degradation....increase cutting efficiency and diversify PAM sequences of Cas12a to further optimize it for therapeutic...sensitive to even a single mismatch in the target sequence Similar to Cas12, it is also significantly smaller...non-complementary ssDNA when engaged by its target sequence. Why would you want to give it a go? The high ...indiscriminately cleaves ssRNA when engaged at its RNA target sequence. The RNA-targeting ability of Cas13 makes it perfect... facts – PAM requirement: protospacer flanking sequence – A, U, or C. Best for: targeting single stranded... facts – PAM requirement: protospacer flanking sequence – none. Best for: targeting single stranded RNA...

... are theoretically 256 distinct flanking sequences, sequences that differ by only one base may result ... that they cleave outside of their recognition sequence, creating four base flanking overhangs. Since ...these overhangs are not part of the recognition sequence, they can be customized to direct assembly of ...specify assembly order, so fragments with 5’ or 3’ sequence homology cannot be assembled using this method...appropriate oligonucleotides specifying a gRNA target sequence into a Cas9-containing plasmid such as pX330. ...Golden Gate cloning Golden Gate cloning is not 100% sequence-independent: to avoid undesired digestion, the...

...depend more on the individual sequence(s) than on how well that sequence fits into the five rules. By ...three or four base-pair sequences, and exclude or include specific overhang sequences, allowing it to be used...specific to your DNA sequence, SplitSet is the tool for you. Once you input a sequence, SplitSet finds optimal...you’d like the tool to use to identify fusion sequences for either a circular or linear assembly. If you...even more specificity, you can exclude specific sequences or set a narrow search window for a site—or sites—that...

...biological process. High quality genome sequence A high-quality sequence assembly of the zebrafish genome was... will help clarify their roles in humans. The sequenced genome also permits “whole-genome/transcriptome...as the field of genetics progresses and genome sequences become available, new hypotheses about specific...organs during embryo development. Single cell sequencing-based lineage tracing Recently, gene expression... embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). Several different...such as CRISPR/Cas9, Tol2, and the high-quality sequenced zebrafish genome. One study profiled 38,731 cells...produced, rather than novel, abnormal cell types. Sequencing-based lineage tracing can help elucidate the ...

...custom CRISPR antimicrobials can target specific sequences in a single virulent bacterial species, or even...2014, Citorik et al. and Bikard et al. developed sequence-specific, antimicrobial, plasmid-based, CRISPR... cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik...targeting either genomic (blue) or plasmid-based sequences (red). Treating a mixed population, represented...targets bacteria with the given genomic or plasmid sequence from a mixed population without affecting the ...the other bacteria. Targeting a genomic sequence results in cell death. Plasmid targeting may result in ...Citorik, Robert J., Mimee, Mark, and Timothy K. Lu. “Sequence-specific antimicrobials using efficiently-delivered...

...plasmid depletion assay to discover FnCpf1’s PAM sequence requirements. Cpf1’s preferred PAM is 5’-TTN, ...in both genomic location and GC-content. After sequencing and searching for cellular RNAs important for...post-HDR, repair templates should remove the PAM sequence. Comparing Cpf1 and Cas9 on-target and off-target...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...

...Timer protein fused to the mitochondrial targeting sequence of cytochrome c oxidase subunit VIII, so that ...exchange and re-use of existing elements. DNA sequences for a desired component are cloned into Golden...channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal transcriptomes, as well as further...for CRISPR gRNA validation Selecting a gRNA sequence that effectively targets your gene/region of interest...their gRNAs. The first step is to clone the target sequence in between two fragments of EGFP in pCAG-EGxxFP...The EGFP fragments contain 482bp of overlapping sequence that direct Homologous Recombination (HR) or Single... Cas9. If the gRNA effectively cuts the target sequence, the plasmid undergoes HR or SSA to reconstitute...

...expressed from a plasmid or from an integrated sequence in a stable cell line. The genes encode the heavy...recombinant antibodies, you first need to know the sequence. Scientists are getting better and better at identifying...mass spectrometry to determine the amino acid sequences of the antibodies and then synthesize the genes...be converted into recombinant form through DNA sequencing of the line and subsequent cloning of the antibody...Hybridomas are much tougher to ship, often lack sequence data, and may genetically drift over time. Antibodies...research tools, it is shocking that the plasmids and sequences for antibodies are not shared. For instance, through...Complete De Novo Assembly of Monoclonal Antibody Sequences. Sci Rep 6: . https://doi.org/10.1038/srep31730...