The nucleic acid data:
IRESite Id: 377 Version: 0
Originaly submitted by: Václav Vopálenský
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:
  end-to-end_full-length_mRNA
The mRNA/+RNA description: 
mRNA produced by bicistronic plasmid pRG-HCV1rc which comprises DsRED2 and EGFP genes as the first and the
second cistron, respectively. The reverse complement sequence of full-length 5'-UTR of HCV genomic RNA
together with the first 45 nt of a viral polyprotein gene was inserted into the intercistronic region of the
reporter pRG.
The sequence ends at its 3'-end right after the poly(A) signal from SV40 mRNA and thus the 3'-UTR might be
slightly wrong.
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 plasmid:
pRG-HCV1rc
The name of the promoter used to express this mRNA:
  CMV_IE
Description of the plasmid (facultative for promoter-less plasmid records):
Plasmid containing the full-length 5'-UTR of HCV genomic RNA together with the first 45 nt of a viral polyprotein gene inserted between DsRED2 and EGFP reporter genes in the revesrse orientation.
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:
HCV1a
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Hepatitis C virus type 1a
The DNA sequence of the plasmid in (+) orientation annotated by its secondary structure:


Plasmid sequence verified (partially/completely) by IRESite (more details in Remarks):
  plasmid sequence confirmed by IRESite curators by restriction analysis + parts by PCR + sequencing
GenBank formatted file with annotated plasmid sequence hyperlinked from vector image map:
pRG-HCV1rc.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: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
DsRED2
The description of the protein encoded in this ORF:
red fluorescent protein (DsRed2 version, Clontech)
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):
  31-726
ORF
ORF position:   2
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
EGFP
The description of the protein encoded in this ORF:
red-shifted variant of wild-type GFP optimized for brighter fluorescence and higher expression in mammalian cells (Clontech)
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):
  1156-1875
Remarks:
IRESite notes about verification of plasmid sequence:
The complete sequence of inverted HCV IRES region was determined by sequencing using DsRed1-C Sequencing
Primer (Clontech; #6483-1; 5'-AGCTGGACATCACCTCCCACAACG-3').
Citations:
Masek T., Vopalensky V., Horvath O., Vortelova L., Feketova Z., Pospisek M. (2007) Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast. J. Gen. Virol. 88(Pt 7):1992-2002
IRESs:
IRES:
Version: 1 Last change: 2008-06-11 10:42:05
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The IRES name:
  HCV_type_1a_rc
The functional status of IRES:
  defective
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  752-1136
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  26
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  410
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -404
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  -20
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
HCV IRES represents the reverse complement sequence of the full-length 5'-UTR of HCV RNA (type 1a) and of the
first 15 codons of the viral polyprotein.
Citations:
Masek T., Vopalensky V., Horvath O., Vortelova L., Feketova Z., Pospisek M. (2007) Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast. J. Gen. Virol. 88(Pt 7):1992-2002
The translation experiments:
Translation results:
IRESite Translation Id: 446
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens CCL-13 [ Chang Liver]
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  0.500
Name of IRES used as the positive control:
  HCV_type_1a
Name of the plasmid used as the positive control.
pRGIRES
Name of the plasmid used as the negative control.
pRG-L270
IRESite Id of the plasmid used as positive control.
  375
IRESite Id of the plasmid used as negative control.
  373
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0.300
The size (length) of intercistronic region in the positive control:
439
The size (length) of intercistronic region in the negative control:
304
The effect of 5'-cap analogs on translation:
not tested
Rapamycin affects translation:
not tested
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Citations:
Masek T., Vopalensky V., Horvath O., Vortelova L., Feketova Z., Pospisek M. (2007) Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast. J. Gen. Virol. 88(Pt 7):1992-2002
Last change to the database: 2015-04-16 16:45:23 GMT+1