The nucleic acid data:
IRESite Id: 72 Version: 7
Originaly submitted by: Václav Vopálenský Submission date: 2005-09-20 00:00:00
Reviewed by: Václav Vopálenský Last change: 2008-07-03 12:54:42
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 spliced in vivo SV40 promoter-derived transcript produced from bicistronic plasmid pRL which
comprises renilla and firefly luciferases as the first and the second cistron, respectively and the 5' UTR (nt
8 to nt 300) plus first 27 bp from human n-myc mRNA.
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:


Warning: mRNA sequence when devoid of trailing 'A's is still not a substring of the plasmid sequence. Is it because an intron is spliced out? Stay calm then. :-)
Credibility of mRNA sequence:
  end-to-end_sequence_reverse_engineered_and_should_match_experiment
The name of the plasmid:
pRNF
The name of the promoter used to express this mRNA:
  SV40
The in vivo produced transcripts are heterogeneous (due to any of promoter?/splicing?/cleavage?/breakage?):
  no
The in vivo produced heterogeneous transcripts occur due to alternative splicing:
  no
A promoter reported in cDNA corresponding to IRES sequence:
  no
Summary of possible issues when IRES cDNA is experimentally transcribed in vivo:
Summary of experiments studying integrity of the in vivo transcripts in a particular host:
Integrity (uniformity) of mRNA tested using Northern-blot:
not_tested
Integrity (uniformity) of mRNA tested using RNase protection:
homogeneous_population_of_molecules_confirmed
Integrity (uniformity) of mRNA tested using 5'-RACE:
not_tested
Integrity (uniformity) of mRNA tested using primer extension :
not_tested
Integrity (uniformity) of mRNA tested using RT-PCR:
not_tested
Integrity (uniformity) of mRNA tested using real-time quantitative polymerase chain reaction (rtqPCR):
not_tested
Integrity (uniformity) of mRNA tested using RNAi:
not_tested
Integrity (uniformity) of mRNA tested using S1 nuclease mapping:
not_tested
Cryptic promoter presence was confirmed by expression from a promoter-less plasmid:
not_tested
Cryptic promoter presence was confirmed in an experimental setup involving inducible promoter:
not_tested
Integrity (uniformity) of mRNA molecules or possible promoter presence expressed in vivo was tested using another method, please specify in Remarks:
not_tested
The organism used:
Homo sapiens HeLa (ATCC CCL-2)
The abbreviated name of the donor gene or virus from which this IRES was excised and inserted into the plasmid:
n-myc
The origin of IRES in the plasmid:
  nuclear
The donor organism of the IRES segment:
Homo sapiens SH-SY5Y (ATCC CRL-2266)
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:
pRNF.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: Václav Vopálenský
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1-936
ORF
ORF position:   2
Version: 0
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The abbreviated name of this ORF/gene:
FFluc
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:
  no
The ORF absolute position (the base range includes START and STOP codons or their equivalents):
  1306-2958
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
IRESs:
IRES:
Version: 6 Last change: 2008-07-03 12:54:42
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The IRES name:
  n-myc
The functional status of IRES:
  functional
The IRES absolute position (the range includes START and STOP codons or their equivalents):
  989-1308
How IRES boundaries were determined:
experimentally_determined
5'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  53
3'-end of IRES relative to last base of the STOP codon of the upstream ORF:
  372
5'-end of IRES relative to first base of the START codon of the downstream ORF:
  -317
3'-end of IRES relative to first base of the START codon of the downstream ORF:
  2
The sequence of IRES region aligned to its secondary structure (if available):


Remarks:
n-myc 5' UTR (293 nt) and open reading frame (27 nt)
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
The translation experiments:
Translation results:
IRESite Translation Id: 55
Version: 6 Last change: 2008-07-06 13:36:51
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HeLa (ATCC CCL-2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  145.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
IRESite Id of the plasmid used as positive control.
  25
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
446
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
Remarks:
As a positive control was used HeLa cell line transfected by vector pRMF (alias pRmycF; formerly pGL3Rutr),
which contains full-length 5' UTR of c-myc P2 transcript inserted between renilla and firefly luciferase (see
IRESite Id #25).

As a negative control was used HeLa cell line transfected by control vector pRF (formerly pGL3R) without
insertion.

Data from Figure 3.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Translation results:
IRESite Translation Id: 56
Version: 4 Last change: 2008-07-06 13:14:49
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens MCF7 (ATCC HTB-22)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  175.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
IRESite Id of the plasmid used as positive control.
  25
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
446
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
Remarks:
As a positive control was used MCF7 cell line transfected by vector pRMF (alias pRmycF; formerly pGL3Rutr),
which contains full-length 5' UTR of c-myc P2 transcript inserted between renilla and firefly luciferase.

Data from Figure 3.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Translation results:
IRESite Translation Id: 57
Version: 4 Last change: 2008-07-06 13:14:49
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HEK 293T/17 (ATCC CRL-11268)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  375.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
IRESite Id of the plasmid used as positive control.
  25
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
446
The effect of 5'-cap analogs on translation:
no
Rapamycin affects translation:
no
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
As a positive control was used HEK293 cell line transfected by vector pRMF (alias pRmycF; formerly pGL3Rutr),
which contains full-length 5' UTR of c-myc P2 transcript inserted between renilla and firefly luciferase.

Data from Figure 3.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Translation results:
IRESite Translation Id: 58
Version: 4 Last change: 2008-07-06 13:14:49
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Mus musculus Neuro-2a [N2a] (ATCC CCL-131)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  540.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
IRESite Id of the plasmid used as positive control.
  25
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
446
The effect of 5'-cap analogs on translation:
no
Rapamycin affects translation:
no
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
As a positive control was used NB2a cell line transfected by vector pRMF (alias pRmycF; formerly pGL3Rutr),
which contains full-length 5' UTR of c-myc P2 transcript inserted between renilla and firefly luciferase.

Data from Figure 3.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Translation results:
IRESite Translation Id: 59
Version: 4 Last change: 2008-07-06 13:14:49
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens NTERA-2 cl.D1 [NT2/D1] (ATCC CRL-1973)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  383.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
IRESite Id of the plasmid used as positive control.
  25
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
446
The effect of 5'-cap analogs on translation:
no
Rapamycin affects translation:
no
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
As a positive control was used NT2 cell line transfected by vector pRMF (alias pRmycF; formerly pGL3Rutr),
which contains full-length 5' UTR of c-myc P2 transcript inserted between renilla and firefly luciferase.

ATCC number of used cell line - CRL-1973; Designation - NTERA-2 cl.D1, NT2/D1

Data from Figure 3.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Translation results:
IRESite Translation Id: 60
Version: 4 Last change: 2008-07-06 13:14:49
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens SH-SY5Y (ATCC CRL-2266)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  670.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
IRESite Id of the plasmid used as positive control.
  25
The relative translation efficiency in % of the positive control:
  100.000
The size (length) of intercistronic region in the positive control:
446
The effect of 5'-cap analogs on translation:
no
Rapamycin affects translation:
no
Type of RNA subject to translation:
  endogenous_nuclear_RNA_Pol_II_transcript
Remarks:
As a positive control was used SH-SY5Y cell line transfected by vector pRMF (alias pRmycF; formerly pGL3Rutr),
which contains full-length 5' UTR of c-myc P2 transcript inserted between renilla and firefly luciferase.

Data from Figure 3.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Translation results:
IRESite Translation Id: 498
Version: 1 Last change: 2008-07-06 14:32:07
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HeLa (ATCC CCL-2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  132.000
Name of IRES used as the positive control:
  c-myc
Name of the plasmid used as the positive control.
pRMF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  25
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  1.500
The size (length) of intercistronic region in the positive control:
446
The size (length) of intercistronic region in the negative control:
67
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
Remarks:
Translational data are derived from Fig. 1B.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Translation results:
IRESite Translation Id: 499
Version: 1 Last change: 2008-07-06 14:32:07
Originaly submitted by: Václav Vopálenský Reviewed by: Václav Vopálenský
The translation method used to study IRES function:
in vivo
The organism used for translation:
Homo sapiens HeLa (ATCC CCL-2)
The temperature (in degrees of Celsia):
37
The relative translation efficiency in % of this IRES:
  315.000
Name of IRES used as the positive control:
  EMCV-R
Name of the plasmid used as the positive control.
pREMCVF
Name of the plasmid used as the negative control.
pRF
IRESite Id of the plasmid used as positive control.
  349
IRESite Id of the plasmid used as negative control.
  184
The relative translation efficiency in % of the positive control:
  100.000
The relative translation efficiency in % of the negative control:
  1.700
The size (length) of intercistronic region in the positive control:
619
The size (length) of intercistronic region in the negative control:
67
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
Remarks:
Translational data are derived from Fig. 1B.
Citations:
Jopling C. L., Willis A. E. (2001) N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells. Oncogene. 20(21):2664-2670
Last change to the database: 2015-04-16 16:45:23 GMT+1