4. Material & Methods
4.1 Material
4.1.3 Plasmids
# Name Gene Marker Origin Cloning procedure K1 pBS empty backbone
plasmid Amp Biochemistry III
K97 pBS1539 TAP::URA3 Amp/URA3 CellZome Puig et al. (2001)
K375 pT11 rDNA Amp Biochemistry III
K1224 pYM15 TAP::KANMX Amp Biochemistry III Janke et al. (2004)
K1837 pCM182-LEU2 empty backbone
plasmid Amp/LEU2 Biochemistry III LEU2 marker replaces TRP1 in pCM182 (Bellí et al., 1998; Garí et al., 1997)
p96 pYM15-TAP-
URA TAP::URA3 with S2-S3 primer Amp This study TAP cassette from pBS1539 was inserted in pYM15 using oligonucleotides #3883 and #3884
p199 ptCMS1 POL5, tetOFF Amp/LEU2 This study PCR product obtained with oligonucleotides o86-o87 ligated into pCM182-LEU2 digested with BamHI- NotI
p201 pCMS16 POL5 Amp This study [BamHI-NotI]-
fragment from ptCMS1 subcloned in pBS
p202 ptDLS5 FLAG-RPA12DE,
tetOFF Amp/LEU2 Dominik Strobel pCM182-LEU2 plasmid containing FLAG epitope
p203 ptCMS2 FLAG-POL5,
tetOFF Amp/LEU2 This study [BamHI-NotI]-fragment from ptCMS1 subcloned in ptDLS5
p229 pCMS19 pol5-DCt120;
new name: pol5-
D902P
Amp This study PCR product obtained with oligonucleotides o131-o132 cloned in pCMS16 using AQUA cloning protocol
p230 pCMS20 pol5-DCt252;
new name: pol5-
D770P
Amp This study PCR product obtained with oligonucleotides o131-o133 cloned in pCMS16 using AQUA cloning protocol
p231 pCMS21 pol5-DCt343;
new name: pol5-
D679L
Amp This study PCR product obtained with oligonucleotides o131-o134 cloned in pCMS16 using AQUA cloning protocol
# Name Gene Marker Origin Cloning procedure p232 pCMS22 pol5-DNt137;
new name: pol5-
DN137
Amp This study PCR product obtained with oligonucleotides o135-o138 cloned in pCMS16 using AQUA cloning protocol
p233 pCMS23 pol5-DNt296;
new name: pol5-
DN296
Amp This study PCR product obtained with oligonucleotides o136-o138 cloned in pCMS16 using AQUA cloning protocol
p234 pCMS24 pol5-DNt658;
new name: pol5-
DN658
Amp This study PCR product obtained with oligonucleotides o137-o138 cloned in pCMS16 using AQUA cloning protocol p239 ptCMS7 FLAG-pol5- DCt120, tetOFF; new name: FLAG-pol5- D902P
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS19 subcloned in ptCMS2 p240 ptCMS8 FLAG-pol5- DCt252, tetOFF; new name: FLAG-pol5- D770P
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS20 subcloned in ptCMS2 p241 ptCMS9 FLAG-pol5- DCt343, tetOFF; new name: FLAG-pol5- D679L
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS21 subcloned in ptCMS2 p242 ptCMS10 FLAG-pol5- DNt137, tetOFF; new name: FLAG-pol5- DN137
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS22 subcloned in ptCMS2 p243 ptCMS11 FLAG-pol5- DNt296, tetOFF; new name: FLAG-pol5- DN296
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS23 subcloned in ptCMS2 p244 ptCMS12 FLAG-pol5- DNt658, tetOFF; new name: FLAG-pol5- DN658
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS24 subcloned in ptCMS2 p262 ptAMW3 FLAG-pol5- DCt30, tetOFF; new name: FLAG-pol5- D993W
Amp/LEU2 Anna Weigert
p266 pgCMS1 POL5 Amp/URA3 Jorge Perez- Fernandez
p267 pgCMS2 GFP-pol5-
DCt343; new
name: GFP-pol5-
D679L
Amp/URA3 Jorge Perez- Fernandez
# Name Gene Marker Origin Cloning procedure p268 pgCMS3 GFP-pol5-
DNt658; new
name: GFP-pol5-
DN658
Amp/URA3 Jorge Perez- Fernandez
p269 pgCMS4 GFP-pol5-
DNt678; new
name: GFP-pol5-
DN677
Amp/URA3 Jorge Perez- Fernandez
p274 pExJPF4 FLAG-POL5-
shine-HIS Kan Jorge Fernandez Perez-
p279 pExJPF4-CTD FLAG-pol5-CTD-
HIS Kan This study [NdeI]-fragment from pExJPF4 ligated to pExJPF4-CTD
p282 pExJPF4-NTD FLAG-pol5-NTD-
HIS Kan This study [Xho]-fragment from pExJPF4 ligated to pExJPF4-NTD p283 ptCMS15 FLAG-pol5- DNt678, tetOFF; new name: FLAG-pol5- DN677
Amp/LEU2 This study [BamHI-NotI]-
fragment from pgCMS4 subcloned in ptCMS2 p286 ptJPF3 FLAG-pol5- DCt70, tetOFF; new name: FLAG-pol5- D952A
Amp/LEU2 Jorge Perez- Fernandez p287 ptAMW4 FLAG-pol5- DNt37, tetOFF; new name: FLAG-pol5- DN37
Amp/LEU2 Anna Weigert
p288 ptAMW5 FLAG-pol5-
DNt81, tetOFF;
new name: FLAG-pol5-
DN81
Amp/LEU2 Anna Weigert
p300 ptCMS17 FLAG-pol5-
DN38Int,
tetOFF; new name: FLAG-
pol5-D639H
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS25 subcloned in ptCMS2
p313 pCMS25 pol5-DN38Int;
new name: pol5-
D639H
Amp This study PCR product obtained with oligonucleotides o131-o249 cloned in pCMS16 using AQUA cloning protocol
p314 pCMS26 pol5-DN75Int;
new name: pol5-
D602T
Amp This study PCR product obtained with oligonucleotides o131-o250 cloned in pCMS16 using AQUA cloning protocol
p315 pCMS27 pol5-DC50Int;
new name: pol5-
DN727
Amp This study PCR product obtained with oligonucleotides o138-o251 cloned in pCMS16 using AQUA cloning protocol
# Name Gene Marker Origin Cloning procedure p316 pCMS28 pol5-DC92Int;
new name: pol5-
DN769
Amp This study PCR product obtained with oligonucleotides o138-o252 cloned in pCMS16 using AQUA cloning protocol p317 ptCMS19 FLAG-pol5- DN75Int, tetOFF; new name: FLAG- pol5-D602T
Amp/LEU This study [BamHI-NotI]-
fragment from pCMS26 subcloned in ptCMS2 p318 ptCMS20 FLAG-pol5- DC50Int, tetOFF; new name: FLAG-pol5- DN727
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS27 subcloned in ptCMS2 p319 ptCMS21 FLAG-pol5- DC92Int, tetOFF; new name: FLAG-pol5- DN769
Amp/LEU2 This study [BamHI-NotI]-
fragment from pCMS28 subcloned in ptCMS2
p324 ptJPF4 FLAG-pol5-
D481R, tetOFF Amp/LEU2 Jorge Fernandez Perez-
p325 ptJPF5 FLAG-pol5-
5xAla, tetOFF Amp/LEU2 Jorge Fernandez Perez-
p327 pgJPF2 GFP_woFlag Amp/URA3 Jorge Perez- Fernandez
p344 ptJPF8 FLAG-pol5-nls3,
tetOFF Amp/LEU2 Jorge Fernandez Perez-
p345 ptJPF9 FLAG-pol5-Dnls,
tetOFF Amp/LEU2 Jorge Fernandez Perez-
p346 ptJPF10 FLAG-pol5-
DLoop, tetOFF Amp/LEU2 Jorge Fernandez Perez-
Table 2: Plasmids used in this study.
Plasmids marked with “K” belong to the department of Biochemistry III at the University of Regensburg. Plasmids marked with “p” belong to the collection of the UTP lab.