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A Helitron (BmHel-8) was randomly selected for insertion validation, and primers (BmHel-8-S: 5′-ATTGTCAGTGGTATCGTTGCTCC-3′, BmHel-8-A: 5′-TAAGGGAATACAATAGAGCCGTG-3′) were designed based on the flanking insertion sites.
Although the 4995 mutation results in a single amino acid change and thus may be susceptible to reversion, the mutant leucine codon selected for insertion into rDEN2Δ30-4995 would require two nucleotide changes to revert to a serine codon.
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Based on this, four predicted loops, named βB-βC, βD-βE, βF-βG and βH-βI, and the N- and C-termini were selected for insertions respectively fusions (Fig. 2).
Alternatively, selection cassettes can be introduced into the knockin construct to select for insertion.
TAGIT is a modified Tn5 transposon that uses KanR to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo.
Cells were selected for retrovirus insertion and once they reached senescence (corresponding to approximately passage 3), mRNA was extracted (P3+ras).
Ideally, the space between the first and second or the second and third tracheal rings should be selected for the insertion of the tracheostomy tube.
Strains carrying a plasmid with either WT-Pep4p, DPM-Pep4p, PRO-Pep4p or the vector control were transformed with and selected for stable insertion of pTN9 HindIII fragment (confirmed by PCR).
Nonetheless, some CIS pairs do break from this trend; for instance, insertions mutating Notch1 in the presence of an existing Rai17 insertion (p value = 0.001) appear to be more strongly selected for than insertions mutating Rai17 in the presence of an existing Notch1 insertion (0.0356).
Patients were initially selected for radiological catheter insertion if they did not have major cardiovascular or respiratory disease, significant bleeding abnormalities or a requirement for intense anticoagulation.
Independent T2 lines were selected for single locus insertions and eight independent PEG2::PEG2 lines (in peg2/− osd1/− background) were used for pollinations onto Col wild-type plants.
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