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The 'run-off' transcript from pJEV-1 with vector sequences at the either end of the insert was not infectious, but transcripts of the full-length PCR amplicon from pJEV-1 produced infectious virus upon transfection.
This ensures a more efficient first-strand synthesis and a shorter poly-A region at the 3' end of the insert.
Comprehensive N-terminal and C-terminal truncation libraries were generated using an exonuclease III and mung bean nuclease protocol (Fig. 1A) by cutting the pair of restriction sites at the end of the insert to be truncated.
In practical terms, this tag identification was only feasible for the 5' ESTs derived from the second library (where the tag was incorporated at the 5' end of the insert), as well as for a limited number of sequences (i.e. 5' ESTs including the tag-containing 3' end) derived from the first library (where the tag was incorporated at the 3' end of the insert).
Short sequences from each end of the insert were extracted to generate two reads.
One sequence attempt was made from each end of the insert fragment for each of the individual clones.
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The unmerged reads were typically those pairs from the longer end of the insert-size distribution.
After ethanol precipitation, PCR amplifications were performed according to the protocol instructions both with Pwht1 and Plac1 primers for the 5' end and with Pry4 and Pry1 primers for the 3' end of the inserted transgene.
In order to determine the minimal sequence requirements for termination-reinitiation in VP2 expresssion, deletions of increasing size were made from the 5' end of the inserted viral information (Figure 1b).
Variations included partial inversion of the 5' end of the inserted sequence about 20%% (36/195) of inserts where both ends were found by discordant read analysis.
Sequencing was mainly performed from the 3' end of the inserts, starting from 300 ng plasmidic DNA; some 5' sequencing was also performed for sequences belonging to abundant unigene clusters.
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