Recombinant plasmid pBG60 containing Sindbis virus (SINV) that allows for fast gene of interest addition with Gateway recombination.
The Sindbis virus genome was inserted into the pcDNA3.1 Zeo+ expression vector such that the transcription start site of the CMV promoter overlaps with the first viral nucleotide of the viral genome. A Hepatitis D virus (HDV) ribozyme was added to the 3’ end of the viral genomic 35bp polyA sequence. The structural proteins from the 5’ subgenomic promoter were replaced with the Blasticidin-S-deaminase (BSD) gene for replicon selection. A Gateway recombination cassette is present behind the 3’ subgenomic promoter to allow genes of interest in pDONR plasmids to be easily inserted into the replicon. Transfection of Gateway recombined versions of construct into mammalian cells produces a self-replicating Sindbis virus genome that expresses the BSD protein and genes of interest, that can be packaged into infectious virus-like particles by co-transfection with packaging plasmid pBG256 that can be used to insert the replicon into poorly or non-transfectable cells (in vitro or in vivo).
From the laboratory of Brian J. Geiss, PhD, Colorado State University.
|Gene/insert name:||Sindbis virus replicon|
|Organism:||Sindbis Virus Strain TE3'2J (AR339)|
|Grow in E. coli at 37 C:||DB3.1 or similar ccdB Survival E. Coli Cells prior to Gateway Recombination, DH5-alpha or XL10-Gold E-coli after Gateway Recombination|
|Cloning Site 5':||MluI|
|Cloning Site 3':||XhoI|
|Insert Size:||11,132 bp|
|Vector Backbone and Size:||pcDNA3.1 Zeo+|
|High or low copy:||High|
|Storage:||Room Temperature (Dried), -20C (liquid)|
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