In fusion advantage

ef1a:DsRed vector. A ef1a:DsRed cassette generated with KOD Hot Start DNA Polymerase (Toyobo) (primers: TAA TTT AAA TAG ATC TTC GAG CAG GGG GAT CAT CTA ATC A; CTA GAT GGC CAG ATC TGC CCG GGA CTT GAT TAG GGT GAT GGT TCA CGT AGT G, T_m = 59°C, 30 cycles) from plasmid Ale237 (kind gift of Alessandro Mongera) was inserted in plasmid 587jk (kind gift of Dr. Jana Krauß) using BglII restriction sites through In-Fusion Advantage (Clontech) cloning according to manufacturer's protocol.
ef1a:DsRed; ef1a:kcnk5bwild type and mutant vectors. The ef1a promoter was amplified from plasmid Ale237 (primers: ATT AAT TCG AGC TCG GTA CCC CTC GAG CAG GGG GAT CAT CT; GAA CAA GCA AGC TGG GTA CCC CGG CCG TCG AGG AAT TCT TTG, T_m = 59°C, 30 cycles) and inserted into the pSGEM vector at the KpnI restriction site using In-Fusion Advantage (Clontech) cloning. The ef1a:kcnk5b cassette was amplified from the resulting plasmid as above (primer: AAA CCT AGG TCG AGC AGG GGG ATC ATC T; AAA CCT AGG ATG ACC ATG ATT ACG CCA AGC TAT), digested with AvrII and inserted into ef1a:DsRed vector using the SpeI restriction site.

c-Myc was amplified from an IMAGE clone (Lawrence Livermore National Laboratory, Livermore, CA) and then inserted at the BamH1 restriction site of the lentiviral inducible pLVX-Tight-Puro vector (Clontech, Mountain View, CA) by IN-Fusion Advantage (Clontech) homologous fusion. Lentiviral pLVX-Teton-Genet vector (Clontech) was used to stably express the tetracycline-controlled transactivator. The human HES6 cDNA sequence was amplified and inserted into the retroviral vector pBabe-puro (Cell Biolabs, San Diego, CA). For knock-down of HES6, annealed oligonucleotides were inserted into the lentiviral pSicoR vector (Ventura et al, 2004 (link)) with a puromycin resistance cassette. Knock-down of the AR was achieved using the lentiviral pSicoR vector with a blasticidin resistance cassette.