Plasmid 1834: pRET.IS.IRES-EGFP N2

• Gene/insert name: None
• Alt name: retroviral RET construct
• Alt name: removable exon trap
• Alt name: Type I vector
• Vector backbone: pRET
  (Search Vector Database)
• Vector type: Mammalian Expression, Retroviral, Cre/Lox
• 5' sequencing primer: na
• Bacterial resistance(s) Ampicillin
• Growth strain(s) DH5alpha
• Growth temperature (℃): 37
• High or low copy: High Copy
• Selectable markers: Neomycin
• Sequence: View sequences (1)
• Map: View map
• Principal Investigator: Philip Leder
• Terms and Licenses: MTA

Comments: 

See scanned map.
This is the original RET-EGFP vector. The highest virus titer that can be obtained with this construct is ~4x10^4 cfu/ml: the mRNA instability signal seems to block transcription of the virus genome at least to some extent. There are two versions, N1 and N2. Version N1 contains two XbaI sites and can be used for transfection after isolation of the XbaI-XbaI fragment. However, N1 contains an additional cryptic poly A signal between EGFP and GHpA, and its orientation is the same as that of virus transcription (from right to left in the figure). This means that N1 can not be used for the virus production: a full-length virus RNA is not produced efficiently. In contrast, N2 does not have such a cryptic poly A signal and is suitable for virus production. However, N2 contains an additional XbaI site between EGFP and GHpA, making it very difficult to isolate all the essential components of the vector on a single XbaI-XbaI fragment. Because of a weak NEO promoter, the number of G418-resistant colonies obtained after transfection (N1) or infection (N2) is fewer than that generated by other RET vectors with a strong NEO promoter. (Leder #C-1011)

Article: RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells. Ishida et al (Nucleic Acids Res 1999 Dec 15;27(24):e35. PubMed)