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:
In vitro T3 run-off transcript produced from bicistronic plasmid pRML-p(A) which comprises renilla and firefly
luciferases as the first and the second cistron, respectively and the 5' UTR of human c-myc (nt from -407 to
-2) mRNA transcribed from major P2 promoter. These mRNAs are bearing a poly(A) tail.
The mRNA/+RNA sequence represented in the +DNA notation:
Credibility of mRNA sequence: end-to-end_sequence_completely_same_as_in_the_experiment
The name of the promoter used to express this mRNA: T3
Description of the plasmid (facultative for promoter-less plasmid records): Plasmid containing 5' UTR of human c-myc transcribed from major P2 promoter (nt from -407 to -2) inserted
between renilla and firefly luciferase reporter genes, with the poly(A)tract.
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: c-myc
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): 1416-3071
IRESite notes about verification of plasmid sequence:
PCR amplification of the sequence encoding bicistronic RNA using M13_reverse (CAGGAAACAGCTATGAC) and
M13_forward (GTAAAACGACGGCCAGT) primers gave one band of expected size.
Sequent sequencing of the vector part's and sequencing of the bicistronic transcription unit using
M13_reverse, M13_forward, Fluc_seq_reverse (AGGAACCAGGGCGTATCTC), Fluc_seq_forward (GTGGACGAAGTACCGAAAGG) and
Rluc_seq_rev (CTGCATGTTTTTCTGAATC) primers resulted in final sequence of supposed mRNA which was in silico
combined with backbone plasmid pBluescript-KS+ (GI: 58065) resulting in the final sequence of vector presented
here in IRESite. More detailed data are annotated in the Genbank sequence file 1343.gb provided on this page
The translation method used to study IRES function: in vitro
The in vitro translation system: HeLa cell lysate
The organism used for translation:
The temperature (in degrees of Celsia): 37
The relative translation efficiency in % of this IRES: 100.000
Name of the plasmid used as the negative control. pRML
IRESite Id of the plasmid used as negative control. 410
The relative translation efficiency in % of the negative control: 14.100
The size (length) of intercistronic region in the negative control: 423
The effect of 5'-cap analogs on translation: no
Rapamycin affects translation: not tested
Type of RNA subject to translation: exogenous_RNA_with_GpppG_cap_with_polyA_tail
Whereas the cap-dependent translation of the 5' cistron (RLuc) in the bicistronic reporter mRNAs is strongly
inhibited by adding 7mGpppG analog, the c-myc IRES-driven translation of the 3' cistron (FFLuc) is not
significantly affected. By contrast, addition of ApppG, cap analog that does not bind the translation
initiation factor eIF4E, does not significantly impair translation from either cistron.
The translation of c-myc.IRES-pA RNA is about 7-fold higher efficient than the translation of the
non-polyadenylated counterpart c-myc.IRES RNA (IRESId: 410).
Translational data are derived from Table 1.