Supplementary Table S1 597
Polymorphic markers used for mapping of RAA7.
598 599
Supplementary Table S2 600
Oligonucleotides used in this work.
601 602
Supplementary Figure S1 603
Sequence of Raa7 wild-type and mutants 604
605
Supplementary Figure S2.
606
Complementation of raa7 mutant with Raa7-HA 607
608
Supplementary Figure S3.
609
Expression of the RAA7-TAP construct 610
611
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764 765 766
Table 1 767
TAP purification of Raa7 and mass spectrometry analysis of trans-splicing factors copurified with Raa7 768
Three independent TAP experiments were performed using Raa7 as bait (P1-P3). Non-specific interactors were identified by control 769
purifications using three RST-1 cultures and one non-tagged wild type culture for TAP experiments (C1-C4). Proteins were analyzed with the 770
tools PredAlgo (PA), TargetP (TP), and ChloroP (CP) for the prediction of subcellular localization. Proteins which were found with at least two 771
peptides in all three Raa7-TAP experiments and exhibit a putative mitochondrial/chloroplast transit peptide were considered as specific (32 772
proteins). Amongst 32 proteins (Table S3), the trans-splicing factors Raa1, Raa2, and Raa7 were detected (Columns “Accession” and 773
“Protein”). The number of unique peptides is given in column “Σ ≠ peptides”. Column “kDa” represents the predicted molecular mass of 774
corresponding proteins in kDa.
775
Raa7-TAP Σ ≠ peptides
Accession Protein kDa PA TP CP P1 P2 P3
Trans-splicing factors
Cre03.g201103 Raa7 (bait) 130 M M C 8 12 14
Cre16.g665750 Raa2, pseudouridine synthase 45 M M - 4 4 4
Cre09.g394150 Raa1 130 M C C 17 19 22
Plastid ribosomal proteins
Cre12.g494750 Plastid ribosomal protein S20 18 C C M 5 3 3
Cre02.g118950 Plastid ribosomal protein S17 12 C C M 3 5 3
Cre12.g494450 Plastid ribosomal protein S16 14 C C M 3 3 2
Not annotated
Cre17.g728850 no functional annotations 58 C M C 8 7 7
Cre17.g698750 no functional annotations 92 C M M 7 10 11
Cre10.g426450 no functional annotations 67 C C C 6 6 5
Cre03.g179000 no functional annotations 52 C M M 4 5 5
Cre02.g086100 no functional annotations 16 C C M 2 3 4
Photosynthesis
DAA00964.1 PsbD 39
plastid
3 2 2
DAA00966.1 PsbC 51 5 4 4
DAA00933.1 PsbB 56 4 3 2
DAA00911.1 PetB 24 2 3 3
DAA00955.1 AtpF 20 2 2 2
Cre11.g481450 CF0 ATP synthase subunit II precursor 22 C C M 2 2 3
Cre01.g045902 ParA/MinD ATPase like, putative PSI stabilizing protein 58 C C M 3 3 4 Others
Cre11.g467723 3-ketoacyl-CoA-synthase 49 - C M 3 3 2
Cre08.g382800 Ca2+/calmodulin-dependent protein kinase 76 C C M 6 2 2
Cre08.g373100 Cytochrome P450 63 C C C 8 7 3
Cre07.g343700 Dihydrolipoamide succinyltransferase 48 C M M 9 6 6
Cre06.g271200 NADH oxidase-related 63 - C M 2 6 6
Cre06.g257601 2-cys peroxiredoxin 26 C C C 3 2 3
Cre04.g217942 Acyl-CoA desaturase 64 C O C 2 2 2
Cre03.g199800 Iron hydrogenase, HyDA1 53 C C C 4 4 3
Cre03.g182150 TPM domain (TLP18.3, Psb32 and MOLO-1) 28 C C C 4 5 6
Cre03.g161400 Tryptophan synthase beta subunit 49 C C C 2 2 5
Cre02.g079700 Aspartate carbamoyltransferase 41 C C M 4 2 2
Cre02.g073200 Threonine dehydratase 67 C C M 14 8 12
Cre12.g546050 1-deoxy-D-xylulose 5-phosphate reductoisomerase 50 C C C 7 8 7
Cre12.g497300 Rhodanese-like Ca-sensing receptor 39 C C M 8 2 2
FIGURE LEGENDS 776
Figure 1. RNA and protein analysis of raa7 777
A. RNA-blot analysis of psaA transcripts in raa7 778
Total RNA extracts were subjected to denaturing agarose gel electrophoresis, blotted to 779
Nylon membranes and hybridized with radiolabelled probes for exon 3 of psaA, exon 1 of 780
psaA or psbD as a control.
781
B. Immunoblot analysis of photosynthetic complexes in raa7 782
Total protein extracts were subjected to SDS-PAGE and immunoblotting with anti-sera 783
against the PsaA, PsaD, PsaF subunits of photosystem I, the RbcL subunit of Rubisco, the 784
Cytf subunit of the b6f complex, the D1 subunit of photosystem II, the cF1 fraction of 785
ATPsynthase, subunit p17.2 of the LHCI antenna and CP29 of the LHCII antenna. The algae 786
were grown in medium containing acetate (TAP) in the dark.
787 788
Figure 2. Chloroplast transformation with intron-less psaA rescues raa7 789
A. Growth tests 790
Chlamydomonas cultures were spotted on minimal medium (HSM) or on medium containing 791
acetate (TAP) under normal light (60 μE m-2 s-1, NL) or dim light (6 μE m-2 s-1, DL).
792
B. Immunoblot analysis of PsaA 793
Total protein extracts were subjected to SDS-PAGE and immunoblotting with anti-sera 794
against PsaA, or the D1 protein of photosystem II as a loading control. The algae were grown 795
in medium containing acetate (TAP) in the dark.
796 797
Figure 3. Identification of the RAA7 gene 798
A. Mapping of the RAA7 gene 799
The upper line represents a segment of chromosome III with the different markers that were 800
used for genetic mapping. The numbers below each marker indicate the percentage of co-801
segregation with the raa7 mutation in the progeny of the cross to C. grossii. The second line 802
is a magnification of the region of chromosome III comprised between the markers CAPS1 803
and CAPS2. The different BAC clones (24N15, 30D16, 39C20, 27B18, 20E5, 15I14, 32B4, 804
3K11, and 2F17) covering the region are represented as segments below. In the lower part 805
of the panel, a magnification of BAC 3K11 is represented. Each subfragment of 3K11 served 806
was used to transform the raa7 mutant. Complementation is reported on the right of each 807
subfragment as the number of colonies obtained in duplicate transformation (colonies / 808
plate).
809
B. Map of the RAA7 gene 810
The first line is a schematic representation of the predicted structure of the RAA7 gene as 811
annotated on the Phytozome server (Cr03.g201103), with black bars representing the coding 812
sequence of the exons and white bars the 5’ and 3’ untranslated regions. The second line 813
represents the structure of the RAA7 gene as determined by RT-PCR. The third line (raa7) 814
shows the location of the frameshift mutation in raa7, with a hatched bar denoting the 815
aberrant polypeptide sequence beyond the position of the mutation. The fourth line (raa7-rev) 816
shows the position of the intragenic suppressor mutation that restores the reading frame. The 817
fifth line (RAA7-HA) represents the structure of the midigene construct. The 5’ part of the 818
genomic DNA is fused to the 3’part of the cDNA as shown with lines. The flag represents the 819
position of the triple HA epitope tag.
820 821
Figure 4. Raa7 localizes to the chloroplast 822
A. Chloroplasts 823
Chloroplasts were prepared from the strain raa7;cw15;Raa7-HA. The lanes contain total cell 824
extract (total) and the chloroplast fraction (chloroplast). Equal amounts of protein were 825
loaded. Immunoblots were performed with antibodies against the HA epitope, PRK (a stromal 826
chloroplast protein), PsaA (an integral membrane protein of Photosystem I) and the cytosolic 827
ribosomal protein Rpl37.
828
B. Mitochondria 829
Mitochondria were prepared from the strain raa7;cw15;Raa7-HA. Equal amounts of protein 830
were loaded. For the immunoblotting, antibodies against the HA epitope, PsaA and the 831
mitochondrial alternative oxidase (mAOX1) were used.
832
C. Membrane association 833
Whole-cell extracts (total) of raa7;cw15;Raa7-HA were separated into a membrane fraction 834
(Mb) and a soluble fraction (S). Equal amounts of protein were loaded. Immunoblots were 835
performed with the same antibodies as in panel A.
836 837
Figure 5. Raa7 interacts with Raa1 and Raa2 838
A. Raa7 is part of a large complex 839
Total soluble proteins from the raa7;cw15;Raa7-HA strain were fractionated by 840
sedimentation in sucrose density gradients. A total of 11 fractions were collected through the 841
bottom of each tube. Fractions 2 to 10 were analyzed by SDS-PAGE and immunoblotting 842
with anti-HA and anti-Raa2 sera. The position of Raa2 is indicated with an asterisk, as 843
validated in panel B. Total proteins from raa1/RAA1-HA were analyzed similarly with anti-HA 844
monoclonal antibody, the position of Raa1-HA is indicated with a triangle, as identified in 845
panel B. For calibration, a parallel gradient was loaded with protein markers. The migration of 846
the markers is indicated with the corresponding molecular masses at the bottom of the panel.
847
B. Identification of Raa2 and Raa1.
848
Because the polyclonal Raa2 anti-serum is not mono-specific, fraction 4 from panel A was 849
run alongside the wild type and the raa2 mutant to identify the Raa2 band (asterisk).
850
Likewise the Raa1-HA band in fraction 4 (triangle) was identified by comparison with the 851
raa1/RAA1-HA and the raa1 strains.
852
C Yeast two-hybrid interactions 853
Diploid strains carrying combinations of Raa1, Raa2 or Raa7 fused to the GAL4 activation 854
(AD) and DNA-binding domain (BD) were obtained by mating. Serial dilutions of the diploids 855
were spotted on minimal medium (CSM) lacking leucine and tryptophan to allow growth of all 856
diploids (left panels) and onto medium lacking leucine, tryptophan, adenine and histidine 857
where growth reveals an interaction between the two fusion proteins (right panels). All yeast 858
strains were mated against control strains carrying the empty plasmids (-) as controls for the 859
lack of activation in the absence of an interacting partner.
860
Figure 6. The psaA trans-splicing complexes in Chlamydomonas 861
Maturation of the psaA-mRNA in the chloroplast of C. reinhardtii comprises splicing of two 862
discontinuous group II introns, psaA-i1 and psaA-i2. This process is dependent on nucleus-863
encoded factors, which are categorized according to splicing deficiencies in corresponding 864
mutants into three classes. Class B factors (Raa1, yellow) are required for both splicing 865
reactions, class A factors (Raa2, Raa7, orange) for psaA-i2 splicing and class C factors 866
(Rat2, Raa3, Raa4, Raa8) for splicing of psaA-i1. TAP-MS analysis with trans-splicing factor 867
Raa7 as bait confirmed the formation of a protein complex necessary for psaA-i2 splicing and 868
consisting of at least Raa1, Raa2, and Raa7. Previous TAP-MS analyses with Raa4 as bait 869
uncovered the composition of a further protein ribonucleoprotein complex comprising the 870
known splicing factors Raa1, Raa3, Raa4, Raa8, Rat2, as well as 18 uncharacterized 871
proteins (Jacobs, et al. 2013). Uncharacterized proteins identified in TAP-MS analysis are 872
indicated in grey.
873
exons 1-2-3 pre-exon 3 pre-exon 1
psaA exon3 psaA exon1 psbD
Figure 1
A
B
WT raa7
100%
PsaA
PsaF
PsaD
RbcL
Cytf
cF1
D1
LHCI (p17.2)
LHCII (CP29)
10%
25%
50%
raa7 WT
75%
100%
A
B Figure 2
TAP DL TAP NL HSM NL
WT
PsaA D1
WT raa7 raa7/
psaA-Δi raa7
raa7/psaA-Δi
24N15
A B C Figure 4
Raa7-HA PRK
PsaA RPL37
total chloroplast total mitochondria total Sn Mb
Raa7-HA PsaA mAOX1
Raa7-HA PsaA PRK
A
2B
C
CSM -LEU -TRPBD-Raa7/- AD-Raa7/-BD-Raa7/AD-Raa7 BD-Raa7/- AD-Raa1/-BD-Raa7/AD-Raa1 BD-Raa1/- AD-Raa7/-BD-Raa1/AD-Raa7 BD-Raa2/- AD-Raa7/-BD-Raa2/AD-Raa7
CSM -LEU -TRP -ADE -HIS
Raa2
✱
▲
▲
✱
Raa7-HA
Figure 5
3 4 5 6 7 8 9 10
bottom top
Raa1-HA kDa
Raa2 fraction 4WT raa2
Raa1-HA fraction 4raa1/RAA1-HAraa1
670 550 158 75 44
Figure 6
Figure S1
WT MEAACGTHLRGLGPLPRVQGGGLSGPSGLFLTFWLPSAARSRSSPSPAVVRAPNESWDCRAGGVPPAHRPGASGPAYGTFKPLRATAGAAPATTAMWPASPLEGASGRHRASICRGSDSG 120 raa7-rev MEAACGTHLRGLGPLPRVQGGGLSGPSGLFLTFWLPSAARSRSSPSPAVVRAPNESWDCRAGGVPPAHRPGASGPAYGTFKPLRATAGAAPATTAMWPASPLEGASGRHRASICRGSDSG 120 raa7 MEAACGTHLRGLGPLPRVQGGGLSGPSGLFLTFWLPSAARSRSSPSPAVVRAPNESWDCRAGGVPPAHRPGASGPAYGTFKPLRATAGAAPATTAMWPASPLEGASGRHRASICRGSDSG 120 WT VVWAAAAAPPTASARAVAAAAASSRGATGGGGGSSTSSSSSSRARHLGSPPPSPGADKESTNTAQGGGGAVAQGAPLQRVAAAAPASRAALLRPGWSPALRAATGRGRGRGRGPGRGRGR 240 raa7-rev VVWAAAAAPPTASARAVAAAAASSRGATGGGGGSSTSSSSSSRARHLGSPPPSPGADKESTNTAQGGGGAVAQGAPLQRVAAAAPASRAALLRPGWSPALRAATGRGRGRGRGPGRGRGR 240 raa7 VVWAAAAAPPTASARAVAAAAASSRGATGGGGGSSTSSSSSSRARHLGSPPPSPGADKESTNTAQGGGGAVAQGAPLQRVAAAAPASRAALLRPGWSPALRAATGRGRGRGRGPGRGRGR 240 WT GEGSGVVEAQWGARGQPQGSRGAAAGLPLPLALLPTDPWRSQPQPQPPVRPSPAADGPAQEEDWDWAGDGEGADEQAGGQEWGSNWEEHVDAGQQAAHGSSAATSSSSGGGTAESPWAVA 360 raa7-rev GEGSGVVEAQWGARGQPQGSRGAAAGLPLPLALLPTDPWRSQPQPQPPVRPSPAADGPAQEEDWDWAGDGEGADEQAGGQEWGSNWEEHVDAGQQAAHGSSAATSSSSGGGTAESPWAVA 360 raa7 GEGSGVVEAQWGARGQPQGSRGAAAGLPLPLALLPTDPWRSQPQPQPPVRPSPAADGPAQEEDWDWAGDGEGADEQAGGQEWGSNWEEHVDAGQQAAHGSSAATSSSSGGGTAESPWAVA 360 WT DSGWLAQQRPLRPLRALPQRPPTAPGLEQAALQRLQQQWARGAGGGGSSRGLQAGGGAEAEREEQQLLQQVVAAASPEEVVACLSPALPPLPAASAPPSRLCSARLLLVAAERLAALQQS 480 raa7-rev DSGWLAQQRPLRPLRALPQRPPTAPGLEQAALQRLQQQWARGAGGGGSSRGLQAGGGAEAEREEQQLLQQVVAAASPEEVVACLSPALPPLPAASAPPSRLCSARLLLVAAERLAALQQS 480 raa7 DSGWLAQQRPLRPLRALPQRPPTAPGLEQAALQRLQQQWARGAGGGGSSRGLQAGGGAEAEREEQQLLQQVVAAASPEEVVACLSPALPPLPAASAPPSRLCSARLLLVAAERLAALQQS 480 WT GQQAQVARPAWSEPPPALAALCDAILRSAAAGGFSWPQLEWQQLEWQQQQQWEWPAREGGSSLGPSAPHAGDGGNGGVGAGQAYGRLAALLWRGAELQLQLGPGAGSSGGAAAGAGVVAA 600 raa7-rev GQQAQVARPAWSEPPPALAALCDAILRSAAAGGFSWPQLEWQQLEWQQQQQWEWPAREGGSSLGPSAPHAGDGGNGGVGAGQAYGRLAALLWRGAELQLQLGPGAGSSGGAAAGAGVVAA 600 raa7 GQQAQVARPAWSEPPPALAALCDAILRSAAAGGFSWPQLEWQQLEWQQQQQWEWPAREGGSSLGPSAPHAGDGGNGGVGAGQAYGRLAALLWRGAELQLQLGPGAGSSGGAAAGAGVVAA 600 WT APPPGVPRTAAAIAAAAAAPGDPGWRQQRRCGGLLAPWRRSSTAAAFWPQLLTCAVRQAGASLQPAELADLLEATAAAAAACHEQDEEDEDEREEDVGSRQRDGGGGGGGSSGLNPRGGS 720 raa7-rev APPPGVPRTAAAIAAAAAAPGDPGWRQQRRCGGLLAPWRRSSTAAAFWPQLLTCAVRQAGASLQPAELADLLEATAAAAAACHEQDEEDEDEREEDVGSRQRDGGGGGGGSSGLNPRGGS 720 raa7 APPPGVPRTAAAIAAAAAAPGDPGWRQQRRCGGLLAPWRRSSTAAAFWPQLLTCAVRQAGASLQPAELADLLEATAAAAAACHEQDEEDEDEREEDVGSRQRDGGGGGGGSSGLNPRGGS 720 WT SPLPLPLPPGALVAAEERLLAVGVHGIAPSDAVRLLAALAALRHPPRAATTRLLTAAAGAGFRGWRWRPPAELQQRLVLTRPQGAAVGATGSDAAVAAAAAEAEQAAAEPVAEAMQLGER 840 raa7-rev SPLPLPLPPGALVAAEERLLAVGVHGIAPSDAVRLLAALAALRHPPRAATTRLLTAAAGAGFRGWRWRPPAELQQRLVLTRPQGAAVGATGSDAAVAAAAAEAEQAAAEPVAEAMQLGER 840 raa7 SPLPLPLPPGALVAAEERLLAVGVHGIAPSDAVRLLAALAALRHPPRAATTRLLTAAAGAGFRGWRWRPPAELQQRLVLTRPQGAAVGATGSDAAVAAAAAEAEQAAAEPVAEAMQLGER 840 WT ELRSLLRSVCRLDGAYAAAGAEAWLADWAAAYWAHLHPRPLHCEAHTQHTGPHAHTHTEAEAEAEVGELTAGAGAGAAAEVLYLLATLQFVPPWEAWEAAALAAVQPRLMPWRRLLAAAR 960 raa7-rev ELRSLLRSVCRLDGAYAAAGAEAWLADWAAAYWAHLHPRPLHCEAHTQHTGPHAHTHTEAEAEAEVGELTAGAGAGAAAEVLYLLATLQFVPPWEAWEAAALAAVQPRLMPWRR----PG 957 raa7 ELRSLLRSVCRLDGAYAAAGAEAWLADWAAAYWAHLHPRPLHCEAHTQHTGPHAHTHTEAEAEAEVGELTAGAGAGAAAEVLYLLATLQFVPPWEAWEAAALAAVQPRLMPWRRPGPVGG 957 WT PGRRQQQWRQQRGRQDRAPATPAIPATAVEAQATLDCWLSHSPSPVPPQPRSEQHTHQPPHPQSSQPPPQSPPPLPKPLPPLALVQLAWAVGAHAAACPVPHPPILPPAAPAIESSDGGS 1080 raa7-rev PVGGSSSGGSSGGGKTGRLPPLPSLLLLWRRRRHWTAGCRTRPARCRPSHGASSTTHQPPHPQSSQPPPQSPPPLPKPLPPLALVQLAWAVGAHAAACPVPHPPILPPAAPAIESSDGGS 1076 raa7 SSSGGSSGGGKTGRLPPLPSLLLLWRRRRHWTAGCRTRPARCRPSHGASSTHTSRHIHSRRSRRRSHPRHCPSRCPPWPWCSWPGQWAHTPPPAPCRTRQSCRLPHLRLSQAMAAAVAAA 1080 WT SGGGGVSPVHAAGLAPGDGHGPAAAGRLTPRQQLRLSAHRHRLRVFGAALAALAAWAEAGSAEGAVAAAAATAAAAGSGGAPGAAGTAARQGAPGMARGRSGSGGGGHTIGMPPVSSAGG 1200 raa7-rev SGGGGVSPVHAAGLAPGDGHGPAAAGRLTPRQQLRLSAHRHRLRVFGAALAALAAWAEAGSAEGAVAAAAATAAAAGSGGAPGAAGTAARQGAPGMARGRSGSGGGGHTIGMPPVSSAGG 1196 raa7 ASPRSMRRAWRPETATAPPPPAA 1103
WT VAGAGRGPVDAQAAAWAACVRDVAWGAAAAGLDAGWVWPAAAGARLQLQAHLGPGGGGDGGSGGGEDRHGAGGLVECMLETAAARGWLL 1289 raa7-rev VAGAGRGPVDAQAAAWAACVRDVAWGAAAAGLDAGWVWPAAAGARLQLQAHLGPGGGGDGGSGGGEDRHGAGGLVECMLETAAARGWLL 1285
A
B Figure S2
TAP DL HSM NL cw15
PsaA cF1 Raa7-HA
cw15 raa7-cw15raa7-cw15RAA7-HA raa7-cw15
raa7-cw15/
RAA7-HA
Figure S3
RAA7_Ex1 (595 bp)
junction RAA7-TAP-tag
(590 bp)
TAP-tag (525 bp)
Cal-BP PsaA RbcL
pL52 T7.2 +RT T7.2 -RT pL52 T7.2 +RT T7.2 -RT
WT raa7 T7.2
pL52 T7.2 +RT T7.2 -RT
A
B
Supplementary Table S1.
Polymorphic markers used for mapping or RAA7
For mapping of RAA7, segregation of polymorphic markers was monitored in the progeny of a cross between the raa7 mutant in the C. reinhardtii background with the C. grossii strain S1D2.
The table lists the oligonucleotides used for PCR amplification with their respective sequence, and the size (bp: base pairs) of the amplicons in the two parental strains (C.r.: C. reinhardtii;
S1D2: C. grossii srain S1D2). For Cleaved Amplified Polymorphic Sequence markers (CAPS), the restriction enzyme used for cleavage is indicated.
Supplementary Table S2.
Oligonucleotides used in this work
The table lists the names and respective sequences of the oligonucleotides used for PCR amplification (other than for mapping of RAA7, see Table S1) as described in Experimental Procedures.
Supplementary Table S1
Polymorphic markers used for mapping or RAA7.
Name Sequence C.r product size /
S1D2 product size (bp)
Restriction enzyme for CAPS
CAPS1.1 AACTAAATGCTTAGGGTTGGTGTGAAG 301 EcoRV
CAPS1.2 CCTCGTTCCGAGCAACGTGAAA
STS14.A CATCTATCCACCACTATATGCCCC 115 / 150
STS14.B AAAATCTCAGAGCAAACCAGCAGA
STS14.C TACATATACCCGAACCAGGGGTGA
CAPS2 For CATCACCACTGCAAAACCACCAC 301 XhoI
CAPS2 Rev GCCTGGATCGCATACCTCACTGT
STS20.A TTCCTCATTGAGCTCCTTGCTCTT 199 / 148
STS20.B CTACATCGTTCGTGCTTTCTCGGG
STS20.C ATTGCTAATTGCGTTGGGCAGATA
STS15.A ATAGCCGCAGCCGTGTGAACCGTA 171 / 131
STS15.B CCACACACCAGTATAGCAACACAC
STS15.C GCGTGGGGTCATGTACTGTTGAC
STS10.1 TTGCATACACCGCCATGGGCCACG 298 / 195
STS10.2 AAGGCCAAGTAGTAAGAAGCCCAA
STS10.3 GTGGCAACCATTTTGATTGTGTGT
STS8.1 TACTGATAACCATGATCCTGTCCG 170 / 105
STS8.2 GGATTTACGTCCCAACAATGTGCA
STS8.3 ACAGCGGTGTCTGCATCTGATGTT
STS6.1 GGGTTGCAGCTAGTTTGCAACACT 376 / 194
STS6.2 TAGCAACGAGCTGGTGTTCGGACG
STS6.3 GTCCTGCTTGTTGCTATTACGGCT
KINESINA.1 CACGCGGCAAGACTACGGCGAC 417 / 187
KINESINA.2 TCTGCTATCGCCTCCGCGGATAGAG KINESINA.3 TGCAGGGCATAGCAGCAAGAGGGAC
Supplementary Table S2
Oligonucleotides used in this work.
Name Sequence
13For2 GCACTTTCAAGCCGCTGCG
13Rev2 GACCCCAAATGACGTGCTCG
13For3 GTCGCCAGCTGCTGACGG
13Rev3 CCACCACTTGCTGCAACAGC
ACC Rev8 GTGCCGTCTCCCTACGGCAT
ACC For4 CGGAAGTGCTTTACCTGCTGGC
ACC Rev9 AGGCGGGGCTGCACTG
ACC For6 TGAGGAGGATGGAGATGAGCGTGAG
ACC Rev5 GCCAGCAGGTAAAGCACTTCCG
ACC For8 CGGGAGGAGCAGCAGCTGT
ACC 13ATG bis For ATGGAAGCAGCTTGTGGCACG
ACC BAC3 Rev CGTCACACAGCGCTGCCA
TAP tag For CTCGAGAGCAGATCTAAGCGCCG
TAP tag Rev CTCGAGTACAAACCCGGGAGATCTCTTG
ACC DH1 For GAATTCAGATCCAGCCCCAGCCCTG
ACC DH1 Rev GGATCCCCACCACTTGCTGCAACAG