pHYDA2-RSP3
$30.00
From Carolyn Silfow, University of Minnesota, February 2024
Vector: pBluescript II KS (+)
Origin: A 2.6 kb NheI fragment containing the RSP3 gene was isolated from the plasmid pEKrsp3.epitope and inserted into pBluescript II KS linearized with XbaI (pBluescript-RSP3 plasmid). A 6.5 kb EcoRI – HindIII fragment containing the HYDA2 gene was isolated from BAC clone 1g24 and cloned into pBluescript II KS to generate pHYDA2. The 1 kb upstream promoter sequence of HYDA2 was amplified from pHYDA2. The primers added EcoRI and BamHI sites to the 5’ and 3’ of each amplicon, respectively, for later cloning. The PCR product was cloned into the pCR-Blunt vector (Invitrogen, Carlsbad, CA). The promoter sequence was excised from the plasmid by digestion with EcoRI and BamHI and ligated into the pBluescript-RSP3 plasmid at the 5’ end of the RSP3 gene to generate pHYDA2-RSP3.
Insert: HYDA2 promoter fused to RSP3 gene (tagged with HA tag)
Comment: This reporter gene construct was transformed into strain CC-1032 to create strains CC-4510 and CC-4512.
Selection in E. coli: Ampicillin
E. coli host strain: XL-1 Blue MRF’
Sun X, Perera S, Haas N, Lefebvre PA, Silflow CD. Using an RSP3 reporter gene system to investigate molecular regulation of hydrogenase expression in Chlamydomonas reinhardtii. Algal Research. 2013 Oct; 2(4):341–351.