The nucleic acid data:
IRESite Id: 178 Version: 2
Originaly submitted by: Martin Mokrejš
Reviewed by: Martin Mokrejš Last change: 2007-11-02 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: 
Bicistronic mRNA transcribed by T7 polymerase in vitro or in vivo in BT7-H cells transiently expressing T7 Pol
coding for RLuc and CAT-fusion reporter genes separated by GBV-B IRES segment with 14 nts from GBV-B (incl.
ATG).
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:
pBL-RLuc-GBB+15-CATT
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:
GBV-B
The origin of IRES in the plasmid:
  viral
The donor organism of the IRES segment:
Hepatitis GB virus B
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:
pBL-RLuc-GBB+15-CATT.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):
  32-967
ORF
ORF position:   2
Version: 1 Last change: 2006-07-13 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
CAT-fusion
The description of the protein encoded in this ORF:
Chloramphenicol acetyltransferase with N-terminal 5 aminoacid residues from GBV-B.
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):
  1468-2151
Remarks:
14 bases come from GBB polyprotein and the 15th base is g, according to Fig. 7a.

The integrity of in vitro synthesized T7 transcripts was verified by agarose gel electrophoresis. The
integrity of in vivo transcripts synthesized in BT7-H cells was dependent on the Tpsi terminator function and was
not confirmed in this work.
Citations:
Rijnbrand R., Abell G., Lemon S. M. (2000) Mutational analysis of the GB virus B internal ribosome entry site. J. Virol. 74(2):773-783
IRESs:
IRES:
Version: 1 Last change: 2006-07-16 00:00:00
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The IRES name:
  GBV-B+14
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1023-1481
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  56
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  514
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -445
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  13
The sequence of IRES region aligned to its secondary structure (if available):


Citations:
Rijnbrand R., Abell G., Lemon S. M. (2000) Mutational analysis of the GB virus B internal ribosome entry site. J. Virol. 74(2):773-783
The translation experiments:
Translation results:
IRESite Translation Id: 152
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vitro
The in vitro translation system:
rabbit reticulocytes lysate
The organism used for translation:
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  70.000
Name of IRES used as the positive control:
  GBV-B
Name of the plasmid used as the positive control.
pBL-RLuc-GBB+3-CATT
IRESite Id of the plasmid used as positive control.
  153
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
500
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. 6b
Citations:
Rijnbrand R., Abell G., Lemon S. M. (2000) Mutational analysis of the GB virus B internal ribosome entry site. J. Virol. 74(2):773-783
Translation results:
IRESite Translation Id: 153
Version: 1 Last change: 2006-07-16 00:00:00
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 BT7-H
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  12.880
Name of IRES used as the positive control:
  GBV-B
Name of the plasmid used as the positive control.
pBL-RLuc-GBB+3-CATT
IRESite Id of the plasmid used as positive control.
  153
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
500
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_cytoplasmic_uncapped_T7_transcript_without_polyA_tail
Remarks:
Fig. 6c
Citations:
Rijnbrand R., Abell G., Lemon S. M. (2000) Mutational analysis of the GB virus B internal ribosome entry site. J. Virol. 74(2):773-783
Translation results:
IRESite Translation Id: 178
Version: 0
Originaly submitted by: Martin Mokrejš Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vitro
The in vitro translation system:
rabbit reticulocytes lysate
The organism used for translation:
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  80.000
Name of IRES used as the positive control:
  GBV-B+63
Name of the plasmid used as the positive control.
pBL-RLuc-GBB+63-CATT
IRESite Id of the plasmid used as positive control.
  180
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
500
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. 7.
Citations:
Rijnbrand R., Bredenbeek P. J., Haasnoot P. C., Kieft J. S., Spaan W. J., Lemon S. M. (2001) The influence of downstream protein-coding sequence on internal ribosome entry on hepatitis C virus and other flavivirus RNAs. RNA. 7(4):585-597
Last change to the database: 2015-04-16 16:45:23 GMT+1