Part:BBa_K3338011

CMV-EGFP-MagA-P2A-hGLuc

Usage and Biology

The composite part described here exhibits an EGFP-MagA-P2A-hGLuc-cassette under the control of a CMV-enhancer/promoter for expression in mammalian cells. It was used to determine the expression of MagA and hGLuc at the same time from one promoter.

Cloning

Theoretical Part Design

In order to test whether HeLa cells are able to simultaneously express MagA and hGLuc using a P2A-peptide, MagA and hGLuc were cloned into the pEGFP-C2 vector interspaced with P2A. In the resulting plasmid MagA is encoded with a N-terminal EGFP-tag making MagA detection very easy. The CMV promoter ensures constitutive and high expression rates mimicking the fully activated sensor.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2553
  Illegal BsaI.rc site found at 1885
  Illegal BsaI.rc site found at 2434
  Illegal SapI site found at 1575
  

Cloning

The plasmid pEGFP-C2 (BBa_K3338020) was linearized with EcoRI and SalI. The MagA, P2A und hGLuc fragments were PCR amplified using the primers shown in table 1.

Table1: Primers used to design the fragments.

Primer name	Sequence
MagA_fw	TACAAGTCCGGCCGGACTCAGATCTCGAGCTCAAGCTTCGccatggacctgcatcatcc
MagA_rv	agcaggctgaagttagtagcgattccagtgccaggtccg
P2A_fw	ccggacctggcactggaatcgctactaacttcagcctgctgaag
P2A_rv	aacagaactttgactcccataggtccagggttctcctcc
hGLuc_fw	tggaggagaaccctggacctatgggagtcaaagttctgtttgcc
hGLuc_rv	CAGTTATCTAGATCCGGTGGATCCCGGGCCCGCGGTACCGttagtcaccaccggccccct

These primers exhibit 5’ overhangs from approximately 20 bp length, that are designed using SnapGene allowing the assembly via the NEBuilder® HiFi DNA Assembly Cloning Kit. The sequence of the part was verified by sanger sequencing.