The nucleic acid data:
IRESite Id: 119 Version: 11
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-01-24 00:00:00
IRESite record type:
  negative_control_plasmid_with_promoter_and_without_putative_IRES
The shape of the nucleic acid molecule translated:
  linear
The quality of the mRNA/+RNA sequence:
  our_best_guess
The mRNA/+RNA description: 
Putative spliced bicistronic renilla-firefly luciferase mRNA molecule with empty, 28nt long intercistronic
region. The chimeric intron in the mRNA sequence used here is spliced out as would be expected. The sequence
ends at its 3'-end right after the poly(A) signal from bovine growth hormone (BGH) mRNA and thus the 3'-UTR
might be slightly wrong (it is unknown what region of the BGH signal could was transcribed and where the
poly(A) tail was added).
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:
  reverse_engineered_sequence_and_should_match_experiment_except_3UTR
The name of the plasmid:
pRL-FL
The name of the promoter used to express this mRNA:
  CMV
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
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pRL-FL.jpg
A promoter reported in cDNA corresponding to IRES sequence:
  not tested
The total number of notable open-reading frames (ORFs):
  2
Notable Open-Reading Frames (ORFs; protein coding regions) in the mRNA/+RNA sequence:
ORF
ORF position:   1
Version: 1 Last change: 2006-05-03 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
RLuc
The description of the protein encoded in this ORF:
Renilla 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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  190-1125
ORF
ORF position:   2
Version: 4 Last change: 2006-10-30 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FLuc
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1154-2806
Remarks:
pRL-FL was created by first mutating pRL-HL (IRESite Id:185 and 186; S. Lemmon and created by K. Honda) to
introduce a SacII site immediately upstream from Fluc. This construct was then dual-digested with NotI/SacII,
blunted with T4 DNAPol filled 4 recessive bases on one end and removed 2 bases using its 3'-5' exo- activity,
and blunt-ligated. Thus the length of the intercistronic region is 28bp instead of 27bp as described in the
article. [Sherril et al. (2004)]

van Eden et al. (2004) actually mention introduction of SacII site 17nt upstream of FLuc, treatment by
T4 ligase and blunt-end ligation to create pRL-FL. But the position was actually -19 to -14.

The in vitro transcript was used in experiment shown Sherrill et al. (2004) in Fig. 2 and also
in works of van Eden et al. (2004) from the same lab.
Citations:
Sherrill K. W., Byrd M. P., Van Eden M. E., Lloyd R. E. (2004) BCL-2 translation is mediated via internal ribosome entry during cell stress. J. Biol. Chem. 279(28):29066-29074
Van Eden M. E., Byrd M. P., Sherrill K. W., Lloyd R. E. (2004) Translation of cellular inhibitor of apoptosis protein 1 (c-IAP1) mRNA is IRES mediated and regulated during cell stress. RNA. 10(3):469-481
Last change to the database: 2015-04-16 16:45:23 GMT+1