pEntry-CMV-MCS-3×FLAG-EF1a-CopGFP-Kan - Basic Information

Recommended Medium	Yeast extract: 5.0 g, Peptone: 10.0 g, NaCl: 10.0 g, pH: 7.0 (25°C)
Growth Conditions	37°C; 18-24 h; Aerobic
Storage Conditions	2-8°C
Procedure	① Dissolution: Add 20 μL of sterile water to the plasmid dry powder to dissolve it, and let it stand at room temperature for 1 min; ② Mixing: Mix 200 μL of competent cells with 5-10 μL of plasmid DNA, and place on ice for 30 min; ③ Heat-shock introduction: Let stand at 42°C for 90 s; ④ Membrane pore contraction: Ice bath for 2 min; ⑤ Recovery culture: Add 800 μL of liquid Recommended Medium to each tube, and culture on a shaker at 37°C for 1 h (150 r/min); ⑥ Selection culture: Spread an appropriate volume (100 μL) of recovered cells onto LB plates with the corresponding antibiotic. Let the plates stand upright for 30 min (make sure the agar surface is completely dry), then invert and culture for 12-16 h until colonies appear; ⑦ Extraction: Pick a single monoclonal colony into LB liquid Recommended Medium with the corresponding antibiotic, culture with shaking for 12-16 h, and extract the plasmid as needed for the experiment.
Reference Application	pEntry-CMV-MCS-3×FLAG-EF1a-CopGFP-Kan is an entry vector for the Gateway cloning system in mammalian cells. It uses the CMV promoter to drive the fusion expression of the target gene with a 3×FLAG tag, while the CopGFP green fluorescent protein and kanamycin resistance are independently expressed under the EF1a promoter for labeling and selection.
Sharing Method	Public welfare sharing