pcDNA3.1 Plasmid Map: Key Elements for Mammalian Expression

The pcDNA3.1 plasmid family (Invitrogen/Thermo Fisher) is a widely used mammalian expression vector. Key elements include:

Core Features

• CMV promoter: Strong constitutive promoter for high-level expression in mammalian cells (not T7—T7 is for in vitro transcription).
• Multiple cloning site (MCS): Flanked by restriction sites for directional cloning.
• BGH polyadenylation signal: Bovine growth hormone polyA for mRNA stability and nuclear export.
• SV40 origin: For episomal replication in cells expressing SV40 large T antigen (e.g., HEK293T).
• f1 origin: Single-stranded DNA production for sequencing/mutagenesis.
• Neomycin/kanamycin resistance (Neo^R/Kan^R): G418 selection in mammalian cells; kanamycin for bacterial selection.
• Ampicillin resistance (Amp^R): Bacterial selection (β-lactamase).
• ColE1 origin: High-copy replication in E. coli.
• T7 promoter: Upstream of MCS for in vitro transcription (IVT) applications.

Version Differences

• pcDNA3.1(+): CMV → MCS → BGH polyA in forward orientation.
• pcDNA3.1(-): Reverse orientation (useful for certain cloning strategies).
• pcDNA3.1/V5-His, /myc-His, /HA: Include epitope tags and His₆ for detection/purification.
• pcDNA3.1/CT-GFP, /NT-GFP: GFP fusion variants.

Applications

Transient/transient transfection, stable cell line generation, protein expression, functional studies, antibody production. High copy number in E. coli facilitates plasmid prep.

Note: The original description incorrectly listed T7 as the mammalian promoter. CMV is the mammalian promoter; T7 is for IVT.