...easy enough to contemplate DNA in terms of its sequencing, in vivo studies should always take DNA’s chromatin...expression pattern and an auxin inducible degron (AID) sequence attached to your protein of interest. TIR1 interacts...of auxin, this TIR1 complex binds to the degron sequence ultimately leading to the degradation of the AID-tagged...(minimal 44 amino acids auxin inducible degron sequence) tagged proteins. Degradation of your protein ...

...adding long-read sequencing capabilities to complement our next-generation sequencing, setting new standards... We’re also building a portal to easily share sequences and characterization data for over 150,000 plasmids...

...containing the modifying sequence flanked by approximately 50 base pairs of sequence homologous to the chromosomal... typically contain both a protein modification sequence and a selectable gene product for isolation of...

... target sequence) in my HR template an issue? Yes. If you have intact protospacer+PAM sequence within ... to mutate the PAM 'NGG' sequence in the HR template. Since the PAM sequence is required for successful...Nature Protocol article . Should I add the PAM sequence to the oligo when cloning my target spacers into... backbone? There is no need to add the NGG PAM sequence. Because the 'NGG' of the PAM is used to select...overhang. When designing oligos for cloning my target sequence into a backbone that uses the human U6 promoter...to add a G nucleotide to the start of my target sequence? The human U6 promoter prefers a 'G' at the transcription...adds it when they order the oligo. If your spacer sequence starts with a 'G', you naturally have one and ...

...target have high sequence identity to the spacer sequence and contain a PAM sequence just downstream of...CRISPR plasmids for targeting RNA sequences, which include the enzymes C2c2/Cas13a and Cas13b. CRISPR...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...

... reaction for traditional restriction cloning. Sequence and Ligation Independent Cloning (SLIC) could ...Invitrogen and based on lambda phage integration sequences, relies on site-specific recombination reactions...components and define their final order through sequence homology. Similarly, Golden Gate assembly allows...FastCloning: a highly simplified, purification-free, sequence-and ligation-independent PCR cloning method." ...

... the exact original sequence, it can restore the original protein-coding sequence using a recoding strategy...synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both methods...; a subsequent drive can restore the wild-type sequence (save for the residual sgRNAs and possibly cas9...unpredictable ecological effects. Because the consequences of such a mistake would necessarily extend far...

... 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...

...the guide sequences consist of a 20-mer (“protospacer sequence”) upstream of an “NGG” sequence (“protospacer...reverse complement of each guide sequence. Example reverse complement sequences of the Pim1 sgRNA from Table...representative sequencing primer; other flanking primers may be utilized. Choose a sequence-verified colony...tools 1 . Use these tools to help identify guide sequences that minimize identical genomic matches or near-matches.... NOTE: In this example, sgRNA-A’s protospacer sequence and PAM happen to fall on the bottom (Crick) strand... strand while sgRNA-B’s protospacer sequence and PAM fall on the top (Watson) strand ( Figure 2A ). However... independent of orientation of the protospacer sequence/PAM relative to the top or bottom strand. Determine...

...immunoprecipitation followed by high-throughput sequencing, ChIP-Seq, is the go-to method for mapping where...noise, reducing the read depth necessary when sequencing and lowering the amount of tissue needed per ...subsequent library preparation and high-throughput sequencing. Improvements to the CUT&RUN method and using...

...Transposons are sequences of DNA that can move around in a genome. In a laboratory setting, transposons...the tranposon to integrate. Additionally, the TA sequence is duplicated near the gene of interest insertion...between a greater number of extinct transposon sequences and then testing various combinations of changes...

...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 ...

...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...

...?  scFv sequences have traditionally been cloned by amplifying the VLand VH antibody sequences of hybridomas...semi-synthetic or entirely synthetic VL and VH sequences. Phage display also allows for screening scFv ...antibodies. To do this, antibody variable domain sequences are expressed as scFvs and screened with in vitro...assays to select for strong binders. Then the sequences for the variable domains of the selected scFvs...

... generating insertions or deletions in the DNA sequence. This same approach is effective to target short...very small deletions and insertions in the DNA sequence can completely disrupt expression of the transcribed...Cas9 generates double-strand breaks, a new DNA sequence can be knocked-in, or introduced, into the break...off-target effects since promoter and enhancer sequences remain intact (Engreitz et al. 2019). The interactions...

...The raw data is then normalized to a reference sequence present in the same amount in all samples (usually...to run multiplex assays, allowing the reference sequence and the gene of interest to be amplified in the...for both the gene of interest and the reference sequence. At the end of the run, each droplet is read as...Complexity of the gene of interest Some gene sequences are harder to amplify and quantify than others...

... editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence targeted by the...complex, promoting its interaction with the degron sequence and ultimately its degradation. While other eukaryotes...

...use a plasmid if it didn’t include any plasmid sequence data? Of course not. Unfortunately, the vast majority... majority of antibodies are supplied with no sequence information, leading to problems with reproducibility...affinity reagent screening, and next-generation sequencing of hybridomas have opened the door for a new ...

...sgRNA) that bears complementarity to a 20bp target sequence (Qi et al. 2013). Modifying the CRISPRi system... will bind to a PAM-adjacent target nucleotide sequence then subsequently recruit a transcriptional regulator...Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell, 152...

...Design your gRNA sequence to direct the dCas9-FP fusion to a specific genomic sequence - potential target...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...dPspCas13b does not require a Protospacer Flanking Sequence (PFS), making it a very flexible editing system... For both enCHIP and CAPTURE, design your gRNA sequence to direct dCas9 to a specific locus, avoiding ...regions. Fluorophores may also be fused to gRNA sequences to produce fluorescence at a target region, as...