The nucleic acid data:
IRESite Id: 434 Version: 0
Originaly submitted by: Tomáš Mašek
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: 
Bicistronic RNA encoding B2 and NS´proteins as the first and the second cistron, respectively, with mutated
CSFV IRES inserted between them. Capped T7 promoter-derived transcript has been transcribed in vitro.
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:
pXLCSFV1-442.NS353U
The name of the promoter used to express this mRNA:
  T7
Description of the plasmid (facultative for promoter-less plasmid records):
pXLCSFV1-442.NS353U is a derivative of pXL10S. CSFV IRES corresponding to 1-442 bp of viral genomic RNA and carrying mutation at position 353 has been inserted between the two cistrons using SalI and SnaBI enzymes. The pXLJ0S contains T7 and SP6 promoter sites and is typically used in in vitro transcription/translation assays. Bicistronic RNA, which contains B2 and NS cistrons (GI:214094, GI:21693177), is transcribed by T7 polymerase after vector linearization by EcoRI.
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:
CSFV
The origin of IRES in the plasmid:
  viral
The donor organism of the IRES segment:
Classical swine fever virus Alfort/Tuebingen
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: pXLCSFV1-442.NS353U.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: Tomáš Mašek Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
B2
The description of the protein encoded in this ORF:
Xenopus laevis B2 cyclin
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  39-1217
ORF
ORF position:   2
Version: 0
Originaly submitted by: Tomáš Mašek Reviewed by: Martin Mokrejš
The abbreviated name of this ORF/gene:
NS
The description of the protein encoded in this ORF:
Truncated form of influenza NS1 protein which is N-terminally fused with 3 amino acids originating from polylinker and with N-terminal part of viral NPro protein.
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1761-2516
Remarks:
IRESite notes about verification of plasmid sequence:
The sequence of pXLCSFV1-442.NS353U(pXLJ0S respectively) was derived from the pXLJCon sequence which
has been experimentally determined by IRESite curators (please see the IRESite entry 329). The sequence of
pXLJ0S differs from pXLJCon in a creation of SnaBI restriction site at position 4202 of plasmid sequence.  A
mutation in vector backbone at position 2808 is indicated in pXLCSFV1-442.NS353U.gb file provided by
IRESite. This mutation has been detected by sequencing of pXLJCon, thus it is not clear if it is present in
pXLJ0S series as well.
Citations:
Fletcher S. P., Jackson R. J. (2002) Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5' untranslated region important for IRES function. J. Virol. 76(10):5024-5033
IRESs:
IRES:
Version: 0
Originaly submitted by: Tomáš Mašek Reviewed by: Martin Mokrejš
The IRES name:
  CSFV_1-442_353U
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  1389-1830
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  172
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  613
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -372
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  69
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
Changing A to U in pseudoknot loop at position 353 of CSFV IRES does not influence IRES activity.
Citations:
Fletcher S. P., Jackson R. J. (2002) Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5' untranslated region important for IRES function. J. Virol. 76(10):5024-5033
The translation experiments:
Translation results:
IRESite Translation Id: 481
Version: 1 Last change: 2008-07-04 13:52:40
Originaly submitted by: Tomáš Mašek 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:
  113.000
Name of IRES used as the positive control:
  CSFV_1-442
Name of the plasmid used as the positive control.
pXLCSFV1-442.NS
Name of the plasmid used as the negative control.
pXL0S
IRESite Id of the plasmid used as positive control.
  359
IRESite Id of the plasmid used as negative control.
  356
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  0
The size (length) of intercistronic region in the positive control:
543
The size (length) of intercistronic region in the negative control:
193
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:
Data from Table 2.
Citations:
Fletcher S. P., Jackson R. J. (2002) Pestivirus internal ribosome entry site (IRES) structure and function: elements in the 5' untranslated region important for IRES function. J. Virol. 76(10):5024-5033
Last change to the database: 2011-04-08 22:43:22 GMT+1