IRESite record type: negative_control_plasmid_with_promoter_and_without_putative_IRES
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.
The mRNA/+RNA sequence represented in the +DNA notation:
Credibility of mRNA sequence: only_fragment_published_or_from_author_and_the_rest_is_a_guess
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): 7752-8471
Remarks:
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.
However, this empty vector sequence we received from Chris has an extra G in the sequence acgcGgt (a typo
destroying the MluI site in the intercistronic region between env and GFP?).
This vector gives 9% expression (the background) compared to 100% when same vector with EMCV IRES is used
(Figure 2).