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: hopefully_full-length_mRNA
The mRNA/+RNA description:
Putative spliced in vitro SV40 promoter-derived transcript of pRAF plasmid.
The mRNA/+RNA sequence represented in the +DNA notation:
Warning: mRNA sequence when devoid of trailing 'A's is still not a substring of the plasmid sequence. Is it because an intron is spliced out? Stay calm then. :-)
Credibility of mRNA sequence: end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the promoter used to express this mRNA: SV40
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?): yes
The in vivo produced heterogeneous transcripts occur due to alternative splicing: yes
A promoter reported in cDNA corresponding to IRES sequence: not tested
Summary of possible issues when IRES cDNA is experimentally transcribed in vivo:
Summary of experiments studying integrity of the in vivo transcripts in a particular host:
Integrity (uniformity) of mRNA tested using Northern-blot: heterogeneous_population_of_molecules_found
Integrity (uniformity) of mRNA tested using RNase protection: not_tested
Integrity (uniformity) of mRNA tested using 5'-RACE: not_tested
Integrity (uniformity) of mRNA tested using primer extension : not_tested
Integrity (uniformity) of mRNA tested using RT-PCR: not_tested
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR): not_tested
Integrity (uniformity) of mRNA tested using RNAi: not_tested
Integrity (uniformity) of mRNA tested using S1 nuclease mapping: not_tested
Cryptic promoter presence was confirmed by expression from a promoter-less plasmid: not_tested
Cryptic promoter presence was confirmed in an experimental setup involving inducible promoter: not_tested
Integrity (uniformity) of mRNA molecules or possible promoter presence expressed in vivo was tested using another method, please specify in Remarks: not_tested
The description of the protein encoded in this ORF: Firefly luciferase
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: not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents): 1757-3409
Remarks:
The putative Apaf-1 IRES (read 5'-UTR) has been cloned in frame with the AUG codon of FLuc. Therefore, there
is missing 'ag' from the 3'-end of 5'-UTR to provide space for 'cc' bases for NcoI site (ccATGg).
Analysis of integrity of mRNA transcripts produced in vivo from this plasmid was done as a Northern-blot
analysis of poly(A)+ messages using randomly-primed probe against FLuc. Unexpected transcript of about 1.3kb
was detected in all lanes (pGL3, pGAL, pRF, pRAF).
The IRES absolute position (the range includes START and STOP codons or their equivalents): 1180-1754
How IRES boundaries were determined: experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF: 53
3'-end of IRES relative to last base of the STOP codon of the upstream ORF: 627
5'-end of IRES relative to first base of the START codon of the downstream ORF: -577
3'-end of IRES relative to first base of the START codon of the downstream ORF: -3
The sequence of IRES region aligned to its secondary structure (if available):
Remarks:
The activity of the putative IRES has also been studied in various cell lines. The HeLa, CHO-T and HepG2 cell
lines were showing about 5-17x higher FLuc activity (Fig. 7).