Part:BBa_K5142041

FOLR2-T2A-mCherry cds

This part contains the coding sequences (cds) of human folate receptor protein FOLR2 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 allows us to express FOLR2 and mCherry simultaneously but individually in the same cell and to easily recognize FOLR2 overexpressed cells which were used to verify the targeting of the click virus carrying folate.

FOLR2 cds was amplified by PCR from cDNA reverse-transcribed from mRNA of HeLa cells according to the gene sequence (Gene ID 2350).
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 FOLR2, 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 FOLR2-T2A-mCherry cds and inserted it into p3×FLAG-CMV vector.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 1608
  Illegal EcoRI site found at 1566
  Illegal PstI site found at 691
  Illegal PstI site found at 1209
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 1566
  Illegal SpeI site found at 1609
  Illegal PstI site found at 691
  Illegal PstI site found at 1209
  Illegal PstI site found at 1623
  Illegal NotI site found at 7
  Illegal NotI site found at 619
  Illegal NotI site found at 1616
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal EcoRI site found at 1566
  Illegal BglII site found at 1578
  Illegal BamHI site found at 349
  Illegal BamHI site found at 786
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 1609
  Illegal EcoRI site found at 1566
  Illegal PstI site found at 691
  Illegal PstI site found at 1209
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal EcoRI site found at 1566
  Illegal XbaI site found at 16
  Illegal SpeI site found at 1609
  Illegal PstI site found at 691
  Illegal PstI site found at 1209
  Illegal PstI site found at 1623
  
• 1000
  COMPATIBLE WITH RFC[1000]