The nucleic acid data:
IRESite Id: 490 Version: 0
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš
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:
  our_best_guess
The mRNA/+RNA description: 
Putative in vitro ARCA (Ambion) capped T7 run-off transcript of pR-deltaEMCV-F with -400 to -1 insert of
putative LINE-1 ORF1 IRES linearized at XbaI site
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:
pR-deltaEMCV-L1_ORF1_-400-1-F
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:
L1spa
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Mus musculus
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:
pR-deltaEMCV-L1_ORF1_-400-1-F.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):
  12-947
ORF
ORF position:   2
Version: 0
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:
  not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1870-3522
Remarks:
In preparing for submission of this IRESite record, it was found that there are 3 PCR errors (A->G @ 1481,
C->T @ 1649, del @ 1784) and a nucleotide difference between the PCR template and the GenBank database (T->G @
1592).




Overview of the mutants shown in 7B:

mL1-ORF2-138-1-mut1
2804-2941

This tested sequence is identical to "mL1-ORF2-138-1" except for 3 single point mutations [2820(G->C),
2836(A->C), 2838(G->C)] introduced to disrupt a predicted stem-loop.


mL1-ORF2-138-1-mut2
2804-2941

This tested sequence is identical to "mL1-ORF2-138-1" except for 6 single point mutations [2812(C-G),
2815(T-G), 2820(G-C), 2833(C-G), 2836(A-C), 2838(G-C)] introduced to disrupt and then restore a predicted
stem-loop.


mL1-ORF2-138-1-mut32804-2941

This tested sequence is identical to "mL1-ORF2-138-1" except for 5 single point mutations [2810(A-T),
2820(G-C), 2836(A-C), 2838(G-C), 2840(T-A)] introduced to disrupt a predicted stem-loop and avoid introducing
a small ORF.


mL1-ORF2-138-1-mut4
2804-2941

This tested sequence is identical to "mL1-ORF2-138-1" except for 8 single point mutations [2810(A-T),
2812(C-G), 2815(T-G), 2820(G-C), 2833(C-G), 2836(A-C), 2838(G-C), 2840(T-A)] introduced to disrupt/restore a
predicted stem-loop and avoid introducing a small ORF.
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
IRESs:
IRES:
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  LINE1-ORF1-400-1
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1468-1867
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  521
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  920
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -402
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):


Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
The translation experiments:
Translation results:
IRESite Translation Id: 542
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:
Mus musculus ltk-
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  165.000
Name of IRES used as the positive control:
  CrPV_IGR_IRES
Name of the plasmid used as the positive control.
pR-deltaEMCV-CrPV-F
Name of the plasmid used as the negative control.
pR-deltaEMCV-F
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  6.800
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_with_polyA_tail
Remarks:
Fig. 4
Citations:
Li P. W., Li J., Timmerman S. L., Krushel L. A., Martin S. L. (2006) The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic. Acids Res. 34(3):853-864
Last change to the database: 2015-04-16 16:45:23 GMT+1