Part:BBa_K4806005

CYP81A10V7 gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYP81A10V7 (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)^*, this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (BBa_K3002017)^* is recommended.

Constructs

Fig.1 Construct design
We designed 3 level 2 constructs containing CYP81A10V7 using the modular cloning system (MoClo).

Here are the links to the built constructs:

• 1. CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806219)
• 2. CYP2D6 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806220)
• 3. CYP2D6 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806221)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP81A10V7 coding sequence the constructs contain the AβSAP(i)-promotor (BBa_K4806013), the HA-tag (BBa_K3002017)^* or mNeonGreen (BBa_K4806006) for detection or mStop (BBa_K4806009) and the tRPL23-terminator (BBa_K3002006)^*. The resistance cassette for spectinomycin is already built in the level 2 vector 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 1685
  Illegal PstI site found at 1827
  Illegal PstI site found at 2449
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1685
  Illegal PstI site found at 1827
  Illegal PstI site found at 2449
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1685
  Illegal PstI site found at 1827
  Illegal PstI site found at 2449
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1685
  Illegal PstI site found at 1827
  Illegal PstI site found at 2449
  Illegal NgoMIV site found at 1532
  
• 1000
  COMPATIBLE WITH RFC[1000]