Difference between revisions of "Part:BBa K5142040"

Latest revision as of 12:08, 2 October 2024

FOLR1-T2A-mCherry cds

This part contains the coding sequences (cds) of human folate receptor protein FOLR1 and mCherry which are in the same reading frame and separated by a T2A sequence. The T2A fragment is a self-cleaving peptide fragment originally discovered in the Thosea asigna virus, and its sequence is EGRGSLLTCGDVEENPGP in which the cleavage occurs before the last proline. This design allow us to express FOLR1 and mCherry simultaneously but individually in the same cell and to easily recognize FOLR1 overexpressed cells which were used to verify the targeting of the click virus carrying folate.

FOLR1 cds was amplified by PCR from cDNA reverse-transcribed from mRNA of HeLa cells according to the gene sequence (Gene ID 2348).
The DNA sequence of T2A in this part is 5’-GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT-3’.
The DNA fragment of mCherry was created by PCR amplification according to the mCherry coding region of the plasmid pcDNA3.1-mCherry (GenBank ID: MH325107.1).

Since the fragment of FOLR1, T2A and mCherry should me linked tandemly in the same open reading frame without any stop codon, we used the technique of recombinational cloning to construct the FOLR1-T2A-mCherry cds and inserted it into p3×FLAG-CMV vector.