3.1 Material
3.1.5 Plasmids
Table 3.3: Plasmids used in this study.
Plasmid Description Source/
Reference ER33854 cDNA encoding for full length EcR-RB in the
pFlc-1 vector.
DGRC BDGP Gold collection pGex4T1 Vector for IPTG-inducible expression of
N-terminal GST-tagged proteins in E.coli. GE Healthcare pGex4T1 dMi-2
aa 2-690
Vector for IPTG-inducible expression of N-terminal GST-tagged dMi-2 amino acids 2-690 in E.coli.
(Murawska, 2011)
pGex4T1 dLint1 aa 1-301
Vector for IPTG-inducible expression of N-terminal GST-tagged C-terminus of dLint-1 in E.coli.
(Meier, 2012)
pING14A Vector for in vitro transcription containing an SP6 promoter.
(Hagemeier et al., 1993)
pING14A-EcR Encodes full length EcR under the control of
an SP6 promoter for in vitro transcription. (Amrhein, 2012)
pFastBac1
Baculovirus transfer vector for recombinant protein expression in Sf9 cells. (Bac-to-Bac Baculovirus Expression System)
Life Technologies
pVL1392
Baculovirus transfer vector for recombinant protein expression in Sf9 cells. (Bac-N-Blue Baculovirus Expression System)
Life Technologies
45 pFastBacDual
Mi2-FLAG
Encodes full length Mi-2 C-terminally FLAG tagged, for generation of recombinant baculovirus.
Ulla Kopiniak
Table 3.4: Plasmids generated in this study.
Plasmid Description pING14A EcR
aa 1-227
Encodes the activation function 1 (AF1) of EcR under the control of an SP6 promoter for in vitro transcription, cloned with XbaI and SmaI using oligos EcR-AF1-Xba_fwd and EcR-AF1-Sma_rev
pING14A EcR aa 228-431
Encodes the DNA-binding domain (DBD) of EcR under the control of an SP6 promoter for in vitro transcription, cloned with XbaI and SmaI using oligos Xba_fwd and EcR-DBD-Sma_rev.
pING14A EcR aa 432-655
Encodes the activation function 2 (AF2) of EcR under the control of an SP6 promoter for in vitro transcription, cloned with XbaI and SmaI using oligos EcR-AF2-Xba_fwd and EcR-AF2-Sma_rev.
pING14A EcR aa 656-878
Encodes the C-terminus of EcR under the control of an SP6 promoter for in vitro transcription, cloned with XbaI and SmaI using oligos EcR-CTerm-Xba_fwd and EcR-CTerm-Sma_rev.
pVL1392 EcR Encodes the full length EcR for generation of recombinant baculovirus, cloned with NotI and XbaI using oligos EcR_NotI_fwd and EcR_Xba_rev.
pFastBac FLAG-EcR
Encodes the full length EcR, N-terminally FLAG-tagged, for generation of recombinant baculovirus, derived from pFastBac by site directed mutagenesis using oligos FastBac_EcR_FLAG_fwd and FastBac_EcR_FLAG_rev.
pFastBac FLAG-EcR
aa 1-227
Encodes the activation function 1 (AF1) of EcR, N-terminally FLAG-tagged, for generation of recombinant baculovirus, cloned with BamHI and NotI using oligos EcRAF1_FLAG_fwd and EcRAF1_rev.
pFastBac FLAG-EcR
aa 228-431
Encodes the DNA-binding domain (DBD) of EcR, N-terminally FLAG-tagged, for generation of recombinant baculovirus, cloned with BamHI and NotI using oligos EcRDBD_FLAG_fwd and EcRDBD_rev.
46 pFastBac
FLAG-EcR
aa 432-655
Encodes the activation function 2 (AF2) of EcR, N-terminally FLAG-tagged, for generation of recombinant baculovirus, cloned with BamHI and NotI using oligos EcRAF2_FLAG_fwd and EcRAF2_rev.
pFastBac FLAG-EcR
aa 656-878
Encodes the C-terminus of EcR, N-terminally FLAG-tagged, for generation of recombinant baculovirus, cloned with BamHI and NotI using oligos EcRCT_FLAG_fwd and EcRCT_rev.
pFastBac HA-EcR
aa 1-227
Encodes the activation function 1 (AF1) of EcR, N-terminally HA-tagged, for generation of recombinant baculovirus, cloned with BamHI and NotI using oligos EcRAF1_HA_fwd and EcRAF1_rev.
pFastBac HA-EcR
aa 432-655
Encodes the activation function 2 (AF2) of EcR, N-terminally HA-tagged, for generation of recombinant baculovirus, cloned with BamHI and NotI using oligos EcRAF2_HA_fwd and EcRAF2_rev.
pGex4T1 Mi-2 aa 691-1271
Contains N-terminal GST fusion of Mi-2 ATPase domain for IPTG-inducible expression in E.coli, cloned with SmaI and NotI using oligos ATPase_Sma_fwd and ATPase_Not_rev.
pGex4T1 Mi-2 aa 691-1271 LFHAA
Contains N-terminal GST fusion of Mi-2 ATPase domain with a mutation of the LFHLL motif to LFHAA for IPTG-inducible
expression in E.coli, derived from pGex4T1 Mi-2 aa 691-1271 by site-directed mutagenesis using oligos LXXAA_ATPase_fwd and LXXAA_ATPase_rev
pGex4T1 Mi-2 aa 691-965
Contains N-terminal GST fusion of Mi-2 ATPase domain Fragment 1 for IPTG-inducible expression in E.coli, cloned with BamHI and NotI using oligos ATPase_F1_Bam_fwd and ATPase_F1_Not_rev
pGex4T1 Mi-2 aa 966-1183
Contains N-terminal GST fusion of Mi-2 ATPase domain Fragment 2 for IPTG-inducible expression in E.coli, cloned with BamHI and NotI using oligos ATPase_F2_Bam_fwd and ATPase_F2_Not_rev
pGex4T1 Mi-2 aa 1184-1271
Contains N-terminal GST fusion of Mi-2 ATPase domain Fragment 3 for IPTG-inducible expression in E.coli, cloned with BamHI and NotI using oligos ATPase_F3_Bam_fwd and ATPase_F3_Not_rev
47 pGex4T1 Mi-2
aa 1272-1982
Contains N-terminal GST fusion of Mi-2 C-terminal domain for IPTG-inducible expression in E.coli, cloned with SmaI and NotI using Cterm_Sma_fwd and Cterm_Not_rev.
pGex4T1 Chd1 aa 505-1067
Contains N-terminal GST fusion of Chd1 ATPase domain for IPTG-inducible expression in E.coli, cloned with XhoI and NotI using oligos Chd1_Xho_fwd and Chd1_Not_rev.
pGex4T1 Chd3 aa 232-804
Contains N-terminal GST fusion of Chd3 ATPase domain for IPTG-inducible expression in E.coli, cloned with EcoRI and NotI using oligos Chd3_Eco_fwd and Chd3_Not_rev.
pGex4T1 ISWI aa 99-611
Contains N-terminal GST fusion of ISWI ATPase domain for IPTG-inducible expression in E.coli, cloned with BamHI and NotI using oligos ISWI_Bam_fwd and ISWI_Not_rev.
pFastBacDual Mi2-FLAG LFHAA
Encodes full length Mi-2, C-terminally FLAG-tagged with a mutation of the LFHLL motif to LFHAA, for generation of
recombinant baculovirus. Derived from pFastBacDual Mi-2-FLAG by site directed mutagenesis using oligos LXXAA_ATPase_fwd and LXXAA_ATPase_rev.