peTurboGFP-dest1 is a mammalian expression vector encoding destabilized variant of the green fluorescent protein TurboGFP (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.

peTurboGFP-dest1 carries synthetic version of the TurboGFP-dest1 gene which 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.

peTurboGFP-dest1 vector can be used to express TurboGFP-dest1 in eukaryotic (mammalian) cells. For example it can be used as a positive control with a peTurboGFP-PRL-dest1 promoterless vector (Cat.# FP523). The vector can be also used to generate destabilized TurboGFP-tagged fusion proteins. Multiple cloning site (MCS) is located upstream of TurboGFP-dest1 coding sequence.

The vector backbone contains immediate early promoter of cytomegalovirus (P_{CMV IE}) for protein expression, 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.

A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a fusion to the TurboGFP-dest1 N-terminus when inserted in the same reading frame as TurboGFP and no in-frame stop codons are present. TurboGFP-dest1-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express TurboGFP-dest1 when transfected into eukaryotic (mammalian) cells.

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.

peTurboGFP-dest1 vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of TurboGFP-dest1 or its fusions in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].

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.