peTurboGFP-PRL-dest1 is a promoterless vector encoding destabilized variant of the green fluorescent protein TurboGFP, which can be used as in vivo reporter of promoter activity (see reporter description). To generate TurboGFP-dest1 variant, residues 422-461 of mouse ornithine decarboxylase (MODC) were fused to the TurboGFP C-terminus. This MODC region contains a PEST amino acid sequence that targets the protein for degradation and provides for rapid protein turnover [Li et al., 1998]. TurboGFP-dest1 retains fluorescent properties of the native protein and has a half-life of approximately 1-1.5 hours, as measured by fluorescence intensity of cells treated with the protein synthesis inhibitor, cycloheximide. Rapid TurboGFP-dest1 turnover allows accurate analysis of changes in gene regulation.

TurboGFP-dest1 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TurboGFP-dest1 coding sequence [Kozak, 1987]. Fragments of exons 2 and 3 and intron 2 of human beta globin gene are added in the 3’ UTR of TurboGFP-dest1 coding sequence in order to increase the protein expression level.

Multiple cloning site (MCS) is located upstream of the Kozak consensus translation initiation site and can be used to clone a promoter or a promoter/enchancer combination of interest. Without the addition of a functional promoter, this vector will not express TurboGFP-dest1.

The vector backbone contains SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

SV40 early promoter (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

Note: The plasmid DNA was isolated from dam⁺-methylated E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam^- host and make fresh DNA.

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.