Table 4 Plasmids available for transgene expression using the one-promoter system
PlasmidaPromoterbUpstream GenecSelectable Markerd
pMO449eHSP70A/RBCS2CrVENUS-3FLAGAPHVIII
pMO470eHSP70A/RBCS2CrVENUS-3FLAGAPHVIII
pMO471eHSP70A/RBCS2CrVENUS-3FLAGAPHVIII
pMO488HSP70A/RBCS2CrVENUSAPHVIII
pMO490HSP70A/RBCS2sfGFP-3FLAGAPHVIII
pMO519HSP70A/RBCS2CrmCHERRYAPHVIII
pMO520HSP70A/RBCS2CrmCHERRY-3FLAGAPHVIII
pMO507PSADCrVENUS-3FLAGAPHVIII
pMO508TUB2CrVENUS-3FLAGAPHVIII
pMO561fTUB2CrVENUS-3FLAGAPHVIII
  • a Available plasmids with one-promoter expression constructs of the general structure shown in Figure 1B.

  • b Except as indicated in note f, all promoters are followed by the first three codons and intron of RBCS2.

  • c All genes indicated have been codon optimized for Chlamydomonas. These genes can be replaced in toto by HpaI–StuI fragments containing other GOIs whose expression/overexpression is desired. Fluorescence tagging of a gene product of interest can be achieved by inserting the coding sequence, in frame, at the HpaI site (C-terminal tagging) or the StuI site (N-terminal tagging).

  • d Other selectable markers could be inserted as NdeI–BamHI fragments. To date, however, we have not had success with selectable markers other than APHVIII (see text).

  • e These plasmids differ only in the short linker between CrVENUS-3FLAG and APHVIII (see Figure 2D and Table 2).

  • f Same as pMO508 except that the RBCS2 ATG start codon has been changed to TTG. This allowed expression of CrVENUS-3FLAG, presumably from its own start codon (i.e., without the additional amino acids from RBCS2).