The nucleic acid data:
IRESite Id: 376 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-HCV1 which comprises DsRED2 and EGFP genes as the first and the
second cistron, respectively. The full-length 5'-UTR of HCV genomic RNA together with the first 45 nt of a
viral polyprotein gene was inserted in frame into the intercistronic region of the reporter pRG vector, thus
making the N-terminal fusion of the first 15 amino acids residues from the HCV polyprotein with the EGFP
protein. The authentic initiation 342AUG codon of HCV was substituted for 342UUG.
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-HCV1A/UUG
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. The authentic initiation 342ATG codon of HCV was substituted for 342TTG.
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:
  viral
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-HCV1A/UUG.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):
  1166-1885
Remarks:
IRESite notes about verification of plasmid sequence:
The complete sequence of 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: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Martin Mokrejš
The IRES name:
  HCV_type_1a_A/UUG
The functional status of IRES:
  defective
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  762-1146
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  36
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  420
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_type_1a_A/UUG IRES represents the full-length 5'-UTR of HCV RNA (type 1a) and the first 15 codons of viral
polyprotein. The authentic initiation codon of HCV polyprotein was mutated to UUG.
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: 445
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:
  30.000
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_III_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