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The genuine presence of the identified exon trapping sequences (ETS) in the bovine transcriptome was verified by multi-tissue expression analysis using RT-PCR.
The nested protein trap pigP constructs were made by inserting P element-based protein trapping sequences into a unique HpaI site in the piggyBac vector p3E1.2, which has an intact piggyBac element (Fraser et al., 1995).
Mining a genomic interval comprising about 1 Mb for transcribed sequences using this technique, we identified a total of 92 unique exon trapping sequences.
Using the method of exon trapping, 92 unique exon trapping sequences (ETS) were discovered in a chromosomal region of poor gene coverage.
Thus, a total of 396 putative exon trapping sequences (ETS) with a size varying from 35 to 349 bp were identified.
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Efforts aimed at mapping the paths of the nascent peptide through the ribosome have focused mainly on trapping sequence-specific peptides known to interact directly with the exit tunnel.
Primers were designed for unique exon trapped sequences (ETS) exceeding a size of 60 bp (see Additional file 1) and tested for successful PCR amplification on a panel of genomic DNA from their parent BAC clones to verify the genomic origin of the trapped sequences.
Repeated attempts to identify these other linked chromosome-4 insertions by our cloning techniques and 5'RACE for trapped sequences in bone marrow extracts were unsuccessful in identifying the origin of this tissue-specific gene-trap tTA expression.
The chromosomal localization of exons on BTA6 could be inferred from physical mapping of the BACs carrying the trapped sequences by FISH or by in silico sequence similarity mapping of the corresponding BAC end sequences on the genome sequence assembly of BTA6 in combination with our high-resolution RH map [ 9].
For this gene-targeting model, the trapping vector contains a splice acceptor site along with the neomycin coding sequence and a polyadenylation sequence, and thus when inserted into the Glut1 locus, the upstream Glut1 sequence is spliced to the trapped sequence forming a truncated mRNA.
I appreciate that the "real" Ghostbusters embarked on their paranormal business venture to turn a profit, but it felt unusual to me, as a kid playing this game in a bedroom-above-a-garage, to have to count dollars and cents instead of ploughing through zap-and-trap sequences without considering the cost of using these unlicensed nuclear accelerators.
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