Part:BBa_K4806006

mNeonGreen-tag for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of mNeonGreen (B5). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)^*, this level 0 part leads to detection of your expressed target protein like CYP3A4 (BBa_K4806000).

Constructs

Fig.1 Construct design
We designed 4 level 2 constructs containing mStop using the modular cloning system (MoClo).

Here are the links to the built constructs:

• 1. The POR gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806213)
• 2. CYP9Q3 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806224)
• 3. CYP3A4 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806204)
• 4. CYP81A10V7 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806221)

These constructs were transformed into Chlamydomonas reinhardtii. Besides mNeonGreen the constructs contain the CAβSAP(i)-promotor (BBa_K4806013), either the POR (BBa_K4806003), CYP9Q3 (BBa_K4806004), CYP3A4 (BBa_K4806000) or the CYP81A10V7 coding sequence (BBa_K4806005) and the tRPL23-terminator (BBa_K3002006)^*. The resistance cassette for hygromycin or spectinomycin is already built in the level 2 vector pMBS810/pMBS807 we are using. The usage of this vector allows the direct assembly of level 0 parts to level 2 constructs, facilitating the cloning time (Niemeyer & Schroda, 2022).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 327
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  Illegal AgeI site found at 656
  
• 1000
  COMPATIBLE WITH RFC[1000]