The nucleic acid data:
IRESite Id: 217 Version: 4
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-09-09 00:00:00
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: 
In vitro bicistronic T7 transcript containing renilla and firefly luciferases separated by LEF1 IRES
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 plasmid:
pRSTF-LEF1_1.2
The name of the promoter used to express this mRNA:
  T7
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:
LEF1
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens
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:
pRSTF-LEF1_1.2.jpg
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: 0
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:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  12-968
ORF
ORF position:   2
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
FLuc-fusion
The description of the protein encoded in this ORF:
Firefly luciferase with 5 additional N-terminal aminoacid residues.
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):
  2263-3930
Remarks:
Authentic RNA pol II promoter was confirmed between bases +280 and +640 relative to the main start site of
transcription. Comment from J. Jimenez: Our original studies of the LEF1 5'UTR were based on characterizing
a third promoter in the 5'UTR that is responsible for generating a 3.0 kb transcript. Because we had evidence
of an authentic promoter, we performed N. blot analysis. We did also attempt RT-PCR, but had technical
problems due to the high GC content of the 5'UTR.

Possibility of aberrant splicing of the plasmids with LEF-1 was therefore ruled out only by the Northern-blot
technique (Fig. 2C and 3B).
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
IRESs:
IRES:
Version: 3 Last change: 2007-09-06 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LEF1
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1091-2268
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  123
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  1300
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -1172
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  5
The sequence of IRES region aligned to its secondary structure (if available):


Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
The translation experiments:
Translation results:
IRESite Translation Id: 195
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  6.160
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.210
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_without_cap_without_polyA_tail
Remarks:
Fig. 4A
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Translation results:
IRESite Translation Id: 196
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  8.490
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.390
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_without_cap_without_polyA_tail
Remarks:
Fig. 4B, measured values provided by J. Jimenez.
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Translation results:
IRESite Translation Id: 197
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  6.610
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  6.060
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_with_GpppG_cap_without_polyA_tail
Remarks:
Fig. 4B, measured values provided by J. Jimenez.
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Translation results:
IRESite Translation Id: 198
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  2.470
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  2.810
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_with_GpppG_cap_without_polyA_tail
Remarks:
Fig. 4C, measured values provided by J. Jimenez. Cells in this experiment did not co-express PV2A protease.
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Translation results:
IRESite Translation Id: 199
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Cercopithecus aethiops COS-1 (ATCC CRL-1650)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  0.440
Name of IRES used as the positive control:
  CVB3
Name of the plasmid used as the positive control.
pRSTF-CVB3
Name of the plasmid used as the negative control.
pRSTF
IRESite Id of the plasmid used as positive control.
  210
IRESite Id of the plasmid used as negative control.
  212
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.090
The size (length) of intercistronic region in the positive control:
819
The size (length) of intercistronic region in the negative control:
131
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  exogenous_RNA_with_GpppG_cap_without_polyA_tail
Remarks:
Fig. 4C, measured values provided by J. Jimenez. Cells in this experiment did co-express PV2A protease.
Citations:
Jimenez J., Jang G. M., Semler B. L., Waterman M. L. (2005) An internal ribosome entry site mediates translation of lymphoid enhancer factor-1. RNA. 11(9):1385-1399
Last change to the database: 2015-04-16 16:45:23 GMT+1