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: 3UTR_possibly_incomplete
The mRNA/+RNA description:
Putative in vivo transcript of plasmid with chimeric MoMLV provirus driven from CMV IE promoter. The capped
and unspliced viral transcript encodes gag/gag-pol and GFP proteins separated by EMCV IRES.
The mRNA/+RNA sequence represented in the +DNA notation:
Credibility of mRNA sequence: reverse_engineered_fragment_and_the_rest_is_a_guess
The name of the promoter used to express this mRNA: CMV_IE
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: not tested
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: not_tested
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: homogeneous_population_of_molecules_confirmed
The description of the protein encoded in this ORF: green fluorescence 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: not tested
The ORF absolute position (the base range includes START and STOP codons or their equivalents): 8296-9021
Remarks:
Presence of alternative splice products was monitored functionally by evaluation of efficiency of the virus
to replicate and produce reverse-transcriptase which activity in the media was measured.
From: C. Logg
Date: Mar 5 2009
The provirus sequence is almost all derived from a clone of Moloney MLV (NC_001501). We switched the ecotropic
env gene with the amphotropic env gene from MLV 4070A (M33469), so it's a hybrid of those two MLV strains. In
MoMLV, there is a stop codon at the end of gag, which is sometimes bypassed. In these cases, a gag-pol
polyprotein is produced, and this is how pol is translated. Pol doesn't have a separate start codon. I believe
all of the sequences are correct. We've sequenced the entire virus-derived portions of the original provirus
plasmids from which all of the other provirus plasmids were constructed. For each of these individual later
plasmids, we've at least verified the IRES/UTR/control-GFP insert sequence. I am assuming that no mutations
outside that region were acquired during the cloning. Each of the provirus plasmids has either a BsiWI or MluI
at the 5' side of the IRES (just after the env stop codon) and a NotI at the 3' side of GFP (just before the
MLV 3' UTR). These are the sites we used to transfer the IRES-GFP cassettes.