| Products/Services Used | Details | Operation |
|---|---|---|
| Custom Vector Construction | The vector pET15b-His6-Ebp1 for expression in E. coli of N-terminally His6-tagged Ebp1 was made by inserting the appropriate DNA sequence between Nde1/BamH1 sites of pET15b (GenScript, Piscataway NJ, USA). Ebp1 cysteine substitution mutants were made (Genscript) by introducing individual surface-exposed cysteine residues at positions 22, 37, 55, 65, 94, 131, 165, 208, 221, 254, 271, 311 and 360. The transcription vectors for dicistronic DC Aichivirus IRES-containing mRNA [28] and monocistronic EMCV nt 373–1656 mRNA [41] have been described. pUC57-FMDV nt 278–891 was made by inserting FMDV O1K nt 278–891 (GenBank: X00871.1) flanked by SnaBI and NdeI sites and an upstream T7 promoter into the EcoRV site of pUC57 (Genscript). pUC57[T7-Stem-GTCV] was prepared (Genscript) by synthesizing a DNA fragment with flanking HindIII and BamH1 sites that corresponded to a T7 promoter. This DNA fragment was inserted into pUC57. This plasmid was used by GenScript to generate [GUAA-UACG], [GUAA-CCUU], [destab. Dom L], [AUG619AAA], [AUG628AAA], [AUG723AAA] and [AUG619AUC] substitution mutants and the [ΔIb] and [ΔStem 1] deletion mutants. The transcription vector pUC57[T7-Stem-XL-GTCV] was prepared (GenScript) by inserting a 1533 nt long BstEII-BamHI DNA fragment of pUC57[T7-Stem-GTCV] corresponding to the entire modified GTCV sequence. pUC57-T7-Stem-RaCV1[nt 1–1386] was prepared (GenScript) by inserting DNA corresponding to a T7 promotor and a stable hairpin (ΔG = −32.40 kcal/mol) linked to nt 1–1386 of the RaCV1 genome or a stable hairpin (ΔG = −33.40 kcal/mol) linked to RaCV1 nt 1–1538 between HindIII and BamHI sites of pUC57. The RaCV1 sequence was modified (Genscript) to generate [UAA612], [UAA612UAA720], [GAA366-8CUC]. pUC57[T7-DC RaCV1 wt] was used (Genscript) to generate [AUG603-context], [AUG603-AAG], [AUG711AAA], [AUG630], [AUG669], [J2-dest] and [J3-dest] substitution mutants and [Stem1 + AUG630], [Stem2 + AUG630] and [Stem3 + AUG630] insertion mutants. | Get A Quote |
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Caliciviruses have positive-sense RNA genomes, typically with short 5'-untranslated regions (5'UTRs) that precede the long open reading frame 1 (ORF1). Exceptionally, some avian caliciviruses have long 5'UTRs containing a picornavirus-like internal ribosomal entry site (IRES), which was likely acquired by horizontal gene transfer. Here, we identified numerous additional avian calicivirus genomes with IRESs, predominantly type 2, and determined that many of these genomes contain a ~200-300 codon-long ORF (designated ORF1*) that overlaps the 5'-terminal region of ORF1. The activity of representative type 2 IRESs from grey teal calicivirus (GTCV) and Caliciviridae sp. isolate yc-13 (RaCV1) was confirmed by in vitr... More
