IRESite record type: plasmid_with_promoter_and_putative_IRES_translationally_characterized
The shape of the nucleic acid molecule translated: linear
The quality of the mRNA/+RNA sequence: end-to-end_full-length_mRNA
The mRNA/+RNA description:
Bicistronic RNA encoding B2 and NS´proteins as the first and the second cistron, respectively, with mutated
CSFV IRES inserted between them. Capped T7 promoter-derived transcript has been transcribed in vitro.
The mRNA/+RNA sequence represented in the +DNA notation:
Credibility of mRNA sequence: end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the promoter used to express this mRNA: T7
Description of the plasmid (facultative for promoter-less plasmid records): pXLCSFV1-442.NS325UC357GA is a derivative of pXL10S. CSFV IRES corresponding to 1-442 bp of viral genomic
RNA and carrying mutations at positions 325 and 357 has been inserted between the two cistrons using
SalI and SnaBI enzymes. The pXLJ0S contains T7 and SP6 promoter sites and is typically used in in vitro
transcription/translation assays. Bicistronic RNA, which contains B2 and NS cistrons (GI:214094, GI:21693177),
is transcribed by T7 polymerase after vector linearization by EcoRI.
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?): not tested
The in vivo produced heterogeneous transcripts occur due to alternative splicing: not tested
A promoter reported in cDNA corresponding to IRES sequence: not tested
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid: CSFV
The description of the protein encoded in this ORF: Truncated form of influenza NS1 protein which is N-terminally fused with 3 amino acids originating from
polylinker and with N-terminal part of viral NPro protein.
The translational frameshift (ribosome slippage) involved: 0
The ribosome read-through involved: no
The alternative forms of this protein occur by the alternative initiation of translation: no
The ORF absolute position (the base range includes START and STOP codons or their equivalents): 1761-2516
Remarks:
IRESite notes about verification of plasmid sequence:
The sequence of pXLCSFV1-442.NS325UC357GA (pXLJ0S respectively) was derived from the pXLJCon sequence which
has been experimentally determined by IRESite curators (please see the IRESite entry 329). The sequence of
pXLJ0S differs from pXLJCon in a creation of SnaBI restriction site at position 4202 of plasmid sequence. A
mutation in vector backbone at position 2808 is indicated in pXLCSFV1-442.NS325UC357GA.gb file provided by
IRESite. This mutation has been detected by sequencing of pXLJCon, thus it is not clear if it is present in
pXLJ0S series as well.
The IRES absolute position (the range includes START and STOP codons or their equivalents): 1389-1830
How IRES boundaries were determined: experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF: 172
3'-end of IRES relative to last base of the STOP codon of the upstream ORF: 613
5'-end of IRES relative to first base of the START codon of the downstream ORF: -372
3'-end of IRES relative to first base of the START codon of the downstream ORF: 69
The sequence of IRES region aligned to its secondary structure (if available):
Remarks:
A compensatory mutation CU to GA at position 357 restores base pairing in stem 2 containing mutation AG to UC
at position 325 and recovers translational activity of the IRES.