Injection:Article Title: TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.
Article Snippet: .. The final injection plasmid was generated byGateway cloning recombining the p5E ubi vector (Mosimann et al., 2011; Addgene #27320), the pME:Cas9P2A-H2B-Halotag vector, a p3E vector containing the U6c:gRNA2switch sequence and a pDestTol1 vector (Shin et al., 2016). .. 12. ubi:Cas9-H2B-Halotag_gRNA3switch_Tol1 (Figure 1): this plasmid was cloned like (11) but recombining a p3E vector con- taining a U6c:gRNA3switch sequence obtained by gene synthesis (IDT) The gRNA3switch follows the same design as gRNA2switch (see gRNA switch optimization design and cloning) but contains a target for gRNA2 instead, and several nucleotide changes in the scaffold region to avoid repeats with gRNA2switch.
Plasmid Preparation:Article Title: TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.
Article Snippet: .. The final injection plasmid was generated byGateway cloning recombining the p5E ubi vector (Mosimann et al., 2011; Addgene #27320), the pME:Cas9P2A-H2B-Halotag vector, a p3E vector containing the U6c:gRNA2switch sequence and a pDestTol1 vector (Shin et al., 2016). .. 12. ubi:Cas9-H2B-Halotag_gRNA3switch_Tol1 (Figure 1): this plasmid was cloned like (11) but recombining a p3E vector con- taining a U6c:gRNA3switch sequence obtained by gene synthesis (IDT) The gRNA3switch follows the same design as gRNA2switch (see gRNA switch optimization design and cloning) but contains a target for gRNA2 instead, and several nucleotide changes in the scaffold region to avoid repeats with gRNA2switch.
Generated:Article Title: TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.
Article Snippet: .. The final injection plasmid was generated byGateway cloning recombining the p5E ubi vector (Mosimann et al., 2011; Addgene #27320), the pME:Cas9P2A-H2B-Halotag vector, a p3E vector containing the U6c:gRNA2switch sequence and a pDestTol1 vector (Shin et al., 2016). .. 12. ubi:Cas9-H2B-Halotag_gRNA3switch_Tol1 (Figure 1): this plasmid was cloned like (11) but recombining a p3E vector con- taining a U6c:gRNA3switch sequence obtained by gene synthesis (IDT) The gRNA3switch follows the same design as gRNA2switch (see gRNA switch optimization design and cloning) but contains a target for gRNA2 instead, and several nucleotide changes in the scaffold region to avoid repeats with gRNA2switch.
Cloning:Article Title: TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.
Article Snippet: .. The final injection plasmid was generated byGateway cloning recombining the p5E ubi vector (Mosimann et al., 2011; Addgene #27320), the pME:Cas9P2A-H2B-Halotag vector, a p3E vector containing the U6c:gRNA2switch sequence and a pDestTol1 vector (Shin et al., 2016). .. 12. ubi:Cas9-H2B-Halotag_gRNA3switch_Tol1 (Figure 1): this plasmid was cloned like (11) but recombining a p3E vector con- taining a U6c:gRNA3switch sequence obtained by gene synthesis (IDT) The gRNA3switch follows the same design as gRNA2switch (see gRNA switch optimization design and cloning) but contains a target for gRNA2 instead, and several nucleotide changes in the scaffold region to avoid repeats with gRNA2switch.
Sequencing:Article Title: TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.
Article Snippet: .. The final injection plasmid was generated byGateway cloning recombining the p5E ubi vector (Mosimann et al., 2011; Addgene #27320), the pME:Cas9P2A-H2B-Halotag vector, a p3E vector containing the U6c:gRNA2switch sequence and a pDestTol1 vector (Shin et al., 2016). .. 12. ubi:Cas9-H2B-Halotag_gRNA3switch_Tol1 (Figure 1): this plasmid was cloned like (11) but recombining a p3E vector con- taining a U6c:gRNA3switch sequence obtained by gene synthesis (IDT) The gRNA3switch follows the same design as gRNA2switch (see gRNA switch optimization design and cloning) but contains a target for gRNA2 instead, and several nucleotide changes in the scaffold region to avoid repeats with gRNA2switch.
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