Project

Name

Purpose

Sequence 5'-3'

Size

Temperature

Notes

E.Coli RNAse deletion

PR_VV_001

Create linear fragment with HR2

5'- caggt ctgtt tcgtg tgctg aattg ttgac gcattt attta ttggt atcgc attcc gggga tccgt cgacc -3'

pkD13 - E. coli

74°C

E.Coli RNAse deletion

PR_VV_002

Create linear fragment with HR1

5'- ttttt tcaac gcctg ttcgg cggca gcctg ctcag cctta cgacg gcttg agtgt aggct ggagc tgctt c -3'

pkD13 - E. coli

79.1°C

E.Coli RNAse deletion

PR_VV_003

Check insertion of Kanamycin cassette into genome

5'- tgcgg gaagt gatgg agatt -3'

E. coli genome

55.4°C

E.Coli RNAse deletion

PR_VV_004

Check insertion of Kanamycin cassette into genome

5'- ccccg aagtt taagg ttggc -3'

E. coli genome

55.5°C

L. lactis RNAse gene deletion

PR_VV_005

50bp overhang with the region upstream rnc (forward)

5'- taagg gataa aacta aaaat taaag agaag tttta agtga aggga dattt tttgt gtggc tggag ctgct tc -3'

pKD13 / 1304 bp

57.2°C

L. lactis RNAse gene deletion

PR_VV_006

50bp overhang with the region at the end of rnc (reverse)

5'- tttta ttgca ttttc tgctg ctttt tgttc tgcaa ctttt ttagt ttcat tccgg ggatc cgacc -3'

pKD13 / 1304 bp

60.2°C

L. lactis RNAse gene deletion

PR_VV_007

Check rnc presence or removal on the genome

5'- tcatg gcggc tactt taggg -3'

1055 bp if rnc is in place, 1734 bp if rnc has been replaced by the KamR cassette, 512 bp if the KamR cassette has been removed

55.5°C

L. lactis RNAse gene deletion

PR_VV_008

Check rnc presence or removal on the genome

5'- gattg ctcgc ccaaa accc -3'

55.5°C

S. cerevisciae GFP reporter plasmid

PR_VV_009

PCR pRS314 backbone

5'- gggaa taagg gcgac acgga aatg -3'

pRS314 backbone / 3883 bp

60°C

Pair with PR_VV_011

S. cerevisciae GFP reporter plasmid

PR_VV_010

PCR pRS314 backbone

5'- cattt ccgtg tcgcc cttat tccc -3'

pRS314 backbone / 2091 bp

60°C

Pair with PR_VV_012

S. cerevisciae GFP reporter plasmid

PR_VV_011

PCR pRS314 backbone

5'- ctcga gTTAT TTGTA CAATT CATCC ATACC ATGGG -3'

pRS314 backbone / 3883 bp

60°C

Pair with PR_VV_009

S. cerevisciae GFP reporter plasmid

PR_VV_012

PCR pRS314 backbone

5'- gggac gctcg aaggc tttag atctg gtacc cagct ttttg ttccc tttag tgag -3'

pRS314 backbone / 2091 bp

60°C

Pair with PR_VV_010

S. cerevisciae GFP reporter plasmid

PR_VV_013

PCR tCYC1 + target site

5'- agatc taaag ccttc gargc gtccc -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR_VV_014 / 015 / 016

S. cerevisciae GFP reporter plasmid

PR_VV_014

PCR tCYC1 + target site 1

5'- ATGGA TGAAT TGTAC AAATA Actcg agCAC TAGAG TCTAG CTTGA GAtcg cccgt acatt catgt aatta gttat gtcac -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR_VV_013

S. cerevisciae GFP reporter plasmid

PR_VV_015

PCR tCYC1 + target site v3

5'- ATGGA TGAAT TGTAC AAATA Actcg agAGT CTGTA ACGTC CGCCA Ctcgc ccgta cattc atgta atta gtta tgtca c -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR_VV_013

S. cerevisciae GFP reporter plasmid

PR_VV_016

PCR tCYC1 + target site v10

5'- ATGGA TGAAT TGTAC AAATA Actcg agTCG CCAAT ATGCG TGTAC Ctcgc ccgta cattc atgta atta gtta tgtca c -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR_VV_013

S. cerevisciae GFP reporter plasmid

PR_VV_017

Diagnostic PCR for pRS314_pGAL1-GFP_target

5'- cagtc acgac gttgt aaaac gacgg -3'

Colony PCR pRS314 / 1642 bp

60°C

Pair with PR_VV_018

S. cerevisciae GFP reporter plasmid

PR_VV_018

Diagnostic PCR for pRS314_pGAL1-GFP_target

5'- gatta cgcca agctc ggaat taacc c -3'

Colony PCR pRS314 / 1642 bp

60°C

Pair with PR_VV_017

S. cerevisciae shRNA controls

PR_VV_019

PCR pRS426 backbone

5'- tatag ttttt tctcc ttgac gttaa agtat agagg tatat taaca atttt ttttg -3'

pRS426 backbone / 4168 bp

60°C

Pair with PR_VV_009

S. cerevisciae shRNA controls

PR_VV_023

PCR shRNA sequences

5'- ggata agaaa gcaaa cacct ggcaattcctta -3'

shRNA / ~120 bp

60°C

Pair with PR_VV_024

S. cerevisciae shRNA controls

PR_VV_024

PCR shRNA sequences

5'- acctc tatact ttaac gtcaa ggaga aaaa actata -3'

shRNA / ~120 bp

55°C

Pair with PR_VV_023

S. cerevisciae shRNA controls

PR_VV_025

shRNA1 with 9nt loop

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAACACTAG AGTCTAGCT TGAGATTCA AGAGATCTC AAGCTAGAC TCTAGTGTT taagg aattgc caggtt gcttt tcttatcc -3'

S. cerevisciae shRNA controls

PR_VV_026

shRNA1 mut-17

5'- cctct tatact ttaac gtcaa ggaga aaaa actat aAACACTA GAGTCTAG CTTGAGATG ATATGTGCA TCTCAAGC TAGACTC TAGTGTT taagg aattgc caggtt gcttt tcttatc -3'

S. cerevisciae shRNA controls

PR_VV_027

shRNA1 mut-25

5'- ctcta tatact ttaac gtcaa ggaga aaaa actat aAACACTA GAGTCTAG CTTGAGA CCTCTCA ACACTGGTC TCAAGC TAGACTCT AGTGTT taagg aattgc caggtt gcttt tcttatc -3'

S. cerevisciae shRNA controls

PR_VV_028

shRNAv3 with 9nt loop

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGTG GCGGACGTT ACAGAC TTTCAAGAGA AGTCTGT AACGTCC GCCACTT taagg aattgc caggtt gcttt tcttatcc -3'

S. cerevisciae shRNA controls

PR_VV_029

shRNAv3 mut-17

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGTGG CGGACGT TACAGACT TGATATGTGC AAGTCT GTACT CGCACC TTaagg aattgc caggtt gcttt tcttatcc -3'

S. cerevisciae shRNA controls

PR_VV_030

shRNAv3 mut-25

5'- ctcta tatact ttaac gtcaa ggaga aaaa actat aAAGTGG CGGACGTTAC AGACTCC TCTCAACAC TGGTCTC AAGACT TAGTG TTtaag gaa ttgccaggt tgctt tttctt atcc -3'

S. cerevisciae shRNA controls

PR_VV_031

shRNAv10 with 9nt loop

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGGTACAC GCATATT GGCGATTCAAGAGAT CGCCAATA TGCGTGTAC CTTtaagg aa ttgccaggt ttgct tttctt atcc -3'

S. cerevisciae shRNA controls

PR_VV_032

shRNAv10 mut-17

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGGT ACACGC ATATGG CGATG ATA TGTGCAC CAGTACT GTGTTTA TTAA-3'

S. cerevisciae shRNA controls

PR_VV_034

Diagnostic PCR

5' - CGAAA AACCG TCTAT CAGGG CGA TGG - 3'

S. cerevisciae shRNA controls

PR_VV_035

diagnostic PCR for pRS426_shRNA

5'- CGCAA TTAA TGTGA GTTAC CTCA CTCAT TAGGC -3'

colony PCR pRS426_shRNA/~1500 bp

60°C

Pair with PR_VV_034

Cloning shRNA in lactococcus

PR_VV_036

Open pMG36E backbone with homology arms for PTSIIC-celA promoter

5'- CCAAA ATTGT CATAT AAGTT GAATT CGATC GACCC ATATTT AAAAG C -3'

55.6°C

Paired with PR_VV_0037 : 3407 bp

Cloning shRNA in lactococcus

PR_VV_037

Open pMG36E backbone with homology arms for rrnBT1-T7TE double terminator

5'- TGGGC CTTTC TGCAG TTTAT AGTCG ACCTG CAGGC ATGC -3'

58.3°C

Cloning shRNA in lactococcus

PR_VV_038

Amplify PTSIIC-celA-shRNA-rrnBT1-T7TE

5'- AACTT ATATA GACAA TTTTG GTACA GGAGT C -3'

promoter-shRNA-terminator G-block

55.6°C

Cloning shRNA in lactococcus

PR_VV_039

5'- TATAA ACGCA GAAAG GCCCA CC -3'

57.1°C

Create promoter-shRNA-terminator part from long oligos

PR_VV_040

Long oligo corresponds to PTSIIC-celA promoter

5'- AACTT ATATA GACAA TTTTG GTACA GGAGT CTTC AAAAG TGGCA CAGAAC CAAAG TGATG GAAAA TAAAG AAAC TGC -3'

Create promoter-shRNA-terminator part from long oligos

PR_VV_041

Long oligo corresponds to (almost all) the reverse of rrnBT1-T7TE

5'- TATAA ACGCA GAAAG GCCCA CCCGA AGGTG AGCCG TGTGA TCTAG TAGAG AGCGT TCACC GACAA ACAAC AGATA A -3'

Create promoter-shRNA-terminator part from long oligos

PR_VV_042

Long oligo corresponds to shRNA_1 (control) with homology arms for promoter and terminator (to be used as template for amplification with VV_040 and 041)

5'- GCTT TACTT TGCCT ATTAG TGC TATAA TGA AAT AACAC TAGAG TCTAG CTGAG ATTCA AGAG ATCTC AAGCT AGACT CTAGT GTTCC AGG -3'

Create promoter-shRNA-terminator part from long oligos

PR_VV_043

Long oligo corresponds to shRNA_v3 with homology arms for promoter and terminator (to be used as template for amplification with VV_040 and 041)

5'- GCTT TACTT TGCCT ATTAG TGC TATAA TGA AAT AAGT GGCG GACGT TACAG ACTTT CAAGA GAAGT CTGT AACGT CCGC CACTT CCA -3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR_VV_044

Open pKD13 backbone, between KamR and one of the FRT sites

5'- ACTGG GCTAT CTGGA CAAG -3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR_VV_045

Open pKD13 backbone, between KamR and one of the FRT sites

5'- AGCTG ACATT CATCC GGGG -3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR_VV_046

Amplify GFP from pREXC3GH

5'- CCTTG TCCAG ATAGC CCAGT TTATT TGTAG AGCTC ATCCAT GCC -3'

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_047

PCR pPGK1 promoter

5'- ATAGG ATAGA TTCTG GAAAC TTTAC CGTCC CAGAC GCGA ATTTT TCGA AGAAG TACCT TC -3'

pVW222_ pRS305_ pPGK1_ UbiY.../ 1060 bp

60°C

Pair with PR_VV_048

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_048

PCR pPGK1 promoter

5'- GAGAG TTCTC TTCCG AGTTT GAGGA CATTT GTTTT ATATT TGTTG TAAAA AGTAG ATAAT TACTT CCTTG ATGAT CTG -3'

pVW222_ pRS305_ pPGK1_ UbiY.../ 1060 bp

60°C

Pair with PR_VV_047

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_051

PCR pN1-011 backbone

5'- CCATG GTTC AGCCA AAAAAC TTAAG ACC -3'

pN1-011 / 2603 bp

59°C

Pair with PR_VV_052

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_052

PCR pN1-011 backbone

5'- GGACG GTAAG TTTCC AGAAT CTATC CTAT -3'

pN1-011 / 2603 bp

57°C

Pair with PR_VV_051

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_053

diagnostic PCR1 for pN1-011_AGO1 & pN1-011_DCR1

5'- CACAT TTCCC CGAAA AGTGC CACCT -3'

colony PCR1 pN1-011_AGO1/2317 bp, colony PCR pN1-011_DCR1/3110 bp

60°C

AGO1: pair with PR_VV_055, DCR1: pair with PR_VV_054

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_054

diagnostic PCR1 for pN1-011_AGO1 & pN1-011_DCR1

5'- GCTAG TTATT GCTCA GCGGT GGCAG CA -3'

colony PCR2 pN1-011_AGO1/3451 bp, colony PCR pN1-011_DCR1/3110 bp

60°C

AGO1: pair with PR_VV_056, DCR1: pair with PR_VV_053

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_055

diagnostic PCR2 for pN1-011_AGO1

5'- ACTGT TTAG GCCCA CAACT TCCTG TC -3'

colony PCR1 pN1-011_AGO1/2317 bp

60°C

AGO1: pair with PR_VV_053

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_056

diagnostic PCR2 for pN1-011_AGO1

5'- AGAGC AAAG GAAAT TAATG ACCTT GCTGC -3'

colony PCR2 pN1-011_AGO1/3451 bp

60°C

AGO1: pair with PR_VV_054

S. cerevisciae intermediate plasmid pN1-011_DCR1

PR_VV_057

PCR pTEF2 promoter

5'- ATAGG ATAGA TTCTG GAAACT TTACC GTCCCA AAATG TTTC TACTC CTTTT TTACT CTTCC AGATT TTTC TC -3'

pSP329-SIV-TEFCherrySZ4 / 478 bp

60

pair with PR_VV_058

S. cerevisciae intermediate plasmid pN1-011_DCR1

PR_VV_058

PCR pTEF2 promoter

5'-ATGTT TTAG ATAGA GATCG GCGCT TTTTT TCCCT ATTC ATAGC TTTAG ATTAG ATTG CTAT GC-3'

pSP329-SIV-TEFCherrySZ4 / 478 bp

57.7

pair with PR_VV_057

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_061

PCR pSP599_pVB9 backbone

5'-CATGG TCATA GCTGT TTCC TGATA ACTTC G-3'

pSP599_pVB9 / 1982 bp

59.5

pair with PR_VV_062

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_062

PCR pSP599_pVB9 backbone

5'-AAAAA TTCGC GTCTG GAGCT CCAAT TCGCC CTAT AGTG AGTC-3'

pSP599_pVB9 / 1982 bp

60

pair with PR_VV_061

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_063

PCR AGO1

5'-CGAAT TGGAG CTCC AGAC GCGAA TTTTT TCGA AGAAG TAC-3'

pN1-011_AGO1 / 5418 bp

58.9

pair with PR_VV_064

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_066

PCR pSP599_pVB9 backbone

5'- ACTGG CCGTC GTTTT ACATG GAAAA AC-3'

pSP599_pVB9 / 2304 bp

60

pair with PR_VV_065

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_068

PCR DCR1 construct

5'- GAAAAT CTGGA AGAGT AAAAA AGGAG TAGAA ACATTT TGCAT GGTC ATAGC TGTTC CCTG ATAAC TTCG-3'

pN1-011_DCR1 / 2778 bp

58

pair with PR_VV_067

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR_VV_069

Amplify GFP from pREXC3GH

5'- GAAAAT AATAT TTAGG AGAAG GCAtg agcaa aggaga agaact ttttc ac-3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR_VV_070

Amplify PTSIIC-celA with homology arm for pKD13 backbone

5'- ACCCC GGATG AATGT CAGCT AACTT ATATA TGACA ATTTT GGTA CAGGA GTC-3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR_VV_071

Amplify PTSIIC-celA with homology arm for GFP

5'- GCTCA TGCCT TCTCC TAAAT ATTTA TTTTC ATTAT AGCATT AATAG GCAAG TAAGC-3'

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_49a

PCR tSIF2 terminator for pN1-011_AGO1

5'- GTTAC GGA CATC ATGT ATACT ACAT ATAAG CACTG AAGG AGTA CTTG AGGC ATTG C-3'

tSIF2 / 552 bp

60

pair with PR_VV_050a

S. cerevisciae intermediate plasmid pN1-011_AGO1

PR_VV_50a

PCR tSIF2 terminator for pN1-011_AGO1

5'- GTCTT AAGT TTTT TTGG CTGAA CCAT GGCG GCGGT CTTA AGTT TTTT TTGG CTGAA CC-3'

tSIF2 / 552 bp

60

pair with PR_VV_049a

S. cerevisciae shRNA controls

PR_VV_020a

PCR pRS426 backbone

5'-AGCTG TTTCC TGTG TGA AATT GTTAT CCGC-3'

pRS426 / 2005 bp

60

pair with PR_VV_10

S. cerevisciae shRNA controls

PR_VV_021a

PCR tSIF2 terminator for pRS426_shRNA

GCGG ATAA CAAT TTCAC ACAG GAAAC AGCT CGGCG GTCT TAAG TTTTT TTGG CTGAA CC-3'

tSIF2 / 559 bp

60

pair with PR_VV_022a

S. cerevisciae shRNA controls

PR_VV_022a

PCR tSIF2 terminator for pRS426_shRNA

5'- TAAGG AATG CCAG GTGT TGCT TTTCT TATCC GCACT GAA GGA GTACT TGAG GCATT GC-3'

tSIF2 / 559 bp

60

pair with PR_VV_021a

S. cerevisciae intermediate plasmid pN1-011_DCR1

PR_VV_059a

PCR tSTE2 terminator for pN1-011_DCR1

5'- GGAGT ACTTG AGGC ATTG CAACA ATCT GTAA AACAT TAAGG ATTT TAAAC CTCC ATAC ATTT ATTCT C-3'

tSTE2 / 629 bp

56.7

pair with PR_VV_060a

S. cerevisciae intermediate plasmid pN1-011_DCR1

PR_VV_060a

PCR tSTE2 terminator for pN1-011_DCR1

5'- GGTCT TAAGT TTTTT TGG CTGAA CCAT GGAA TCAGA AATT GGAG AAAGTC CTGG TTGA ACAAGA-3'

tSTE2 / 629 bp

60

pair with PR_VV_059a

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_064a

PCR AGO1 construct

5'- CTCA CTAAG GGAACA AAAG CTGG GTACC CGGCG GTCT TAAG TTTTT TTGG CTGAA CC-3'

pN1-011_AGO1 / 5442 bp

60

pair with PR_VV_063

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_067a

PCR DCR1 construct

5'- GTTTT TTCC ATGT AAAAC GACG GCCAG TAAT CAGA AATT GGAG AAAGTC CTGG TTGA ACAAGA AAAAG-3'

pN1-011_DCR1 / 2872 bp

60

pair with PR_VV_068

S. cerevisciae GFP reporter plasmid

PR_VV_009a

PCR pRS314 backbone (new primer instead of PR_VV_009)

5'- GCATC TTTTA CTTTC ACCAG CGTTT CTGG-3'

pRS314

60

pair with PR_VV_011

S. cerevisciae shRNA controls

PR_VV_075a

PCR pRS426 backbone

5'- CGGCG GCTTC TAATCC GTAC TTCA ATATAG GGTG ATGGT TCAC GTAG TGGG CCATC-3'

pRS426

60

pair with PR_VV_009a

S. cerevisciae shRNA controls

PR_VV_074a

PCR pGAL1 promoter

5'- CTAT ATTG AAGT ACGG ATTAG AAGCC GCCCG-3'

pRS314 (pGAL1)

60

pair with PR_VV_019

S. cerevisciae integrative plasmid pSP599_AGO1_DCR1

PR_VV_065a

PCR pSP599_pVB9 backbone

5'- AGCTT TTGTT CCCTT TAGTG AGGGTT AATTT CCTG TAAACT TTATA AATGA AATTC ATA TAG-3'

pSP599

60

pair with PR_VV_066

S. cerevisciae shRNA controls

PR_VV_022b

PCR tSIF2 gblock

5'- GCACT GAAGG AGTACT TGAGG CATTG-3'

tSIF2 gblock

59.7

pair with PR_VV_021a

lacto shRNA

PR_VV_081

Forward primer shRNA to check the insertion of fragment

5'- TTGGTT GATAAT GAAC TGTG CTG-3'

lacto shRNA

PR_VV_082

Reverse primer shRNA to check the insertion of fragment

5'- CTCCAA ATATC GTAG CGCCG-3'

AGO g-block amplification

PR_VV_083

Forward primer AGO1

5'- ATGTC CTC AACT CGGA AGAG AACT CTTC-3'

AGO g-block amplification

PR_VV_084

Reverse primer AGO1

5'- GTCAT TAATT TCCTT TGCT CTACT GTTT AGGCC-3'

AGO g-block amplification

PR_VV_085

Forward primer AGO2

5'- GACAG GAA GTTC TGTG GGC CTA AAC AG-3'

AGO g-block amplification

PR_VV_086

Reverse primer AGO2

5'- AGCTC GCCT TAA GTTC ATCC ATGACT-3'

AGO g-block amplification

PR_VV_087

Forward primer AGO3

5'- CAGTC ATGG ATGAA CTTAA GGC GAGC-3'

AGO g-block amplification

PR_VV_088a

Reverse primer AGO3

5'- TTATA TGTAG TACAT GATGT CCGT AACATT CTTAT GCAC-3'

pMG36E amplification

PR_VV_089

Amplify backbone of pMG36E (with promoter and terminator) Reverse

5'- ATTTTC ATTATA GCATTA ATAGG-3'

pMG36E amplification

PR_VV_090

Amplify backbone of pMG36E (with promoter and terminator) Forward

5'- CCAGGC ATCAA ATAAA ACGAA AGG-3'

Reverse of primer 42

PR_VV_093

42 reverse

Dilution

lacto shRNA - restriction enzymes tactic

PR_VV_094

Amplify promoter + backbone + terminator with overhangs for restriction enzymes

5'- AAACTT TACCG TCCCT CGAGC CAGGC ATCAA ATAAA ACGAA AGG-3'

lacto shRNA - restriction enzymes tactic

PR_VV_095

Amplify promoter + backbone + terminator

5'- CCAGAA TCTAT CCTAT GAGCT CATTTT CATTATA GCATTA ATAGG-3'

lacto shRNA - restriction enzymes tactic

PR_VV_096

SacI - shRNA - XhoI - v1 - control - Primer Reverse

5'- TCGAG AACAC TAGAG TCTAG CTGAG ATCTC TTGA ATCTC AAGCT AGACT CTAG TGTTG AGCT-3'

lacto shRNA - restriction enzymes tactic

PR_VV_097

SacI - shRNA - XhoI - v1 - control - Primer Forward

5'- CAACA CTAG AGTCT AGCT TGAG ATTCA AGAG ATCTC AAGCT AGACT CTAG TGTTC-3'

lacto shRNA - restriction enzymes tactic

PR_VV_098

SacI - shRNA - XhoI - v3 - Primer Forward

5'- CAAGT GGCGG ACGTT ACGAG ACTTT CAA GAGAA GTCTG TAACG TCCG CCACTTC-3'

lacto shRNA - restriction enzymes tactic

PR_VV_099

SacI - shRNA - XhoI - v3 - Primer Reverse

5'- TCGAG AAGT GGCGG ACGTT ACGAG ACTT CTCTT GAAAG TCTG TAACG TCCG CCACTTG AGCT-3'

Reverse of primer 43

PR_VV_100

43 reverse

Dilution

shRNA production in yeast

PR_VV_101

shRNA1, F

5'- TAACGT CAAGG AAAAA AACTA TAAAC ACTAG AGTCT AGCTTG AGATT CAAGAG ATCTC AAGCT AGACT CTAG TGTTG CACTG AAGG AGTACT TGAG-3'

97 bp

69.8

shRNA production in yeast

PR_VV_102

shRNA1, R

5'- CTCAAG TACTC CTTCA GTGCA ACAC TAACT AGAGT CTAGCT TGAGAT CTCTT GAATC TCAAG CTAGAC TCTAG TGTTT ATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

69.8

shRNA production in yeast

PR_VV_103

shRNA1, mut 17, F

5'- TAACGT CAAGG AAAAA AACTA TAAAC ACTAG AGTCT AGCTTG AGATT GATAT GTGCAT CTCAAG CTAGAC TCTAG TGTTG CACTG AAGG AGTACT TG-3'

97 bp

69.8

shRNA production in yeast

PR_VV_104

shRNA, mut 17, R

5'- CAAGT ACTC CTTCA GTGCA ACAC TAACT AGAGT CTAGCT TGAGAT GCACAT ATCAT CTCAAG CTAGAC TCTAG TGTTT ATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

69.8

shRNA production in yeast

PR_VV_105

shRNA, mut 25, F

5'- TAACGT CAAGG AAAAA AACTA TAAAC ACTAG AGTCT AGCTTG AGACCT CTCAAC ACTGG TCTCA AGCTAG ACTCT AGTGTTG CACTG AAGG AGTAC-3'

97 bp

71

shRNA production in yeast

PR_VV_106

shRNA, mut 25, R

5'- GTACTC CTTCA GTGCA ACAC TAACT AGAGT CTAGCT TGAGACC AGTGTT GAGAGG TCTCA AGCTAG ACTCT AGTGTTT ATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

71

shRNA production in yeast

PR_VV_107

shRNAv3, F

5'- CTATA CTTTA ACGTC AAGG AAAAA AACTA AAGTGG CGGAC GTTAC AGACT TTCAAG AGAAGT CTGTA ACGTCC GCCACT TGCACT GAAGG AGTA-3'

97 bp

71

shRNA production in yeast

PR_VV_108

shRNAv3, R

5'- TACTC CTTCA GTGCA AGTGG CGGAC GTTAC AGACT TCTCT TGAAG TCTGTA ACGTCC GCCACT TATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

71

shRNA production in yeast

PR_VV_109

shRNAv3, mut 17, F

5'- TAACGT CAAGG AAAAA AACTA AAGTGG CGGAC GTTAC AGACT TGAT ATGTGC AAGTCT GTAAC GTCCG CCACT TGCACT GAAGG AGTACT TG-3'

95 bp

71.7

shRNA production in yeast

PR_VV_110

shRNAv3, mut 17, R

5'- CAAGT ACTC CTTCA GTGCA AAGTGG CGGAC GTTAC AGACT TGCAC ATATCA AGTCT GTAAC GTCCG CCACT TATAGT TTTTT TCTC CTTGAC GTA-3'

95 bp

71.7

shRNA production in yeast

PR_VV_111

shRNAv3, mut 25, F

5'- ATACTT TTAAC GTCAAG GAAAA AACTA AAGTGG CGGAC GTTAC AGACT CCTCT CAAC ACTGG AGTCT GTAAC GTCCG CCACT TGCACT GAAGG AGT-3'

98 bp

72.6

shRNA production in yeast

PR_VV_112

shRNAv3, mut 25, R

5'- CTCCTT CAGTG CAAGT GGCGG ACCTG TTAAC ACTG TGTAG GAGGT CTGTA ACGTCC GACCT TATAGT TTTTT TCTC CTTGAC GTA-3'

98 bp

72.6

shRNA production in yeast

PR_VV_113

shRNAv10, F

5'- TCTATA CTTTA ACGTC AAGG AAAAA AACTA AAGGT ACACG CATATT GGCGAT TCAAG AGATC GCCAAT ATGCGT GTACCT TGCACT GAAGG AGTA-3'

98 bp

70.1

shRNA production in yeast

PR_VV_114

shRNAv10, R

5'- TACTC CTTCA GTGCA AGGTAC ACGCA TATTG GCGAT CTCTT GAATC GCCAAT ATGCGT GTACCT TATAGT TTTTT TCTC CTTGAC GTA-3'

98 bp

70.1

shRNA production in yeast

PR_VV_115

shRNAv10, mut 17, F

5'- TAACGT CAAGG AAAAA AACTA AAGGT ACACG CATATT GGCGAT GATAT GTGCAT CGCCA ATATG CGTGTACCT TGCACT GAAGG AGTACT TGA-3'

96 bp

70.7

shRNA production in yeast

PR_VV_116

shRNAv10, mut 17, R

5'- TCAAG TACTC CTTCA GTGCA AGGTAC ACACG CATATT GGCGAT GCACAT ATCAT CGCCA ATATG CGTGTACCT Ttatagttttttctccttgacgtta-3'

96 bp

70.7

shRNA production in yeast

PR_VV_117

shRNAv10, mut 25, F

5'- TAACGT CAAGG AAAAA AACTA AAGGT ACACG CATATT GGCGAC CCTCT CAAC ACTGG TCGCC AATAT GCGTGTACCT TGCACT GAAGG AGTACT-3'

96 bp

72

shRNA production in yeast

PR_VV_118

shRNAv10, mut 25, R

5'- AGTACT CCTTC AGTGCA AGGTAC ACACG CATATT GGCGAC CCTCT CAAC ACTGG TCGCCA ATATG CGTGTACCT Ttatagttttttctccttgacgtta-3'

96 bp

72

conjugation lacto

PR_VV_119a

PCR backbone pMG36E + overhang for oriT

5'- cctgc tctgc gaggc tggcc gtatc aggct tgatc cccag taagtc-3'

3467 bp

54.6°C

conjugation lacto

PR_VV_120

PCR backbone pMG36E + overhang for oriT

5'- gaagc agggt tatgc agcgg aaaag gaagta catcc gcaa cgtcc ata ctctg-3'

3467 bp

55.4°C

conjugation lacto

PR_VV_121

PCR to amplify oriT

5'- tacgg ccagc ctcgc agagc agg-3'

conjugation lacto

PR_VV_122

PCR to amplify oriT

5'- ctttt ccgct gcata accct gcttc-3'

Primers for target plasmid

PR_VV_011a

PCR pRS314 backbone

5'- ctcga gTTATT TGTAT AGTTC ATCCA TGCCA TGTG-3'

56

Primers for target plasmid

PR_VV_014a

PCR tCYC1 + target site 1

5'- ATGGA TGAACT ATACAA ATAAC tcgag CACTAG AGTCT AGCTT GAGAt cgccc ggtaca ttcat gtaat tagtt atgtc ac-3'

60

Primers for target plasmid

PR_VV_015a

PCR tCYC1 + target site v3

5'- ATGGA TGAACT ATACAA ATAAC tcgag AGTCT GTAAC GTCC GCCAC tcgcc cgtaca ttcat gtaat tagtt atgtc ac-3'

60

Primers for target plasmid

PR_VV_016a

PCR tCYC1 + target site v10

5'- ATGGA TGAACT ATACAA ATAAC tcgag TCGCC AATAT GCGT GTACC tcgcc ggtaca ttcat gtaat tagtt atgtc ac-3'

60

Primers for final AGO + DICR

PR_VV_061a

PCR pSP599_pVB9 backbone

5'- AAGAG ATTTCT ACACA CGATT GAGCA CTGT CTCA TGGTC ATAGC TGTTT CCCT GATAA CTTTC G-3'

Primers for final AGO + DICR

PR_VV_066a

PCR pSP599_pVB9 backbone

5'- AGCAA TCAATA GTGTT CGTAAG TGTAG TCCCT ggaa aaaaaca atagg tcctt aaata tttgt caag-3'

Primers for final AGO + DICR

PR_VV_067b

PCR DCR1 construct from pN1-011

5'- GGGAC TACA CTTA CGAA AACTA TTGA TTGCT aatc agaa atttg gaga aagtc ctgg ttgaac-3'

Primers for final AGO + DICR

PR_VV_068a

PCR DCR1 construct from pN1-011

5'- AGACA GTGCT CAATC GTGTAG AAATC TCTTC AAAAT GTTTC TACTC CTTTT TTACTC TTCCAG ATTTTC-3'

shRNA plasmid lacto, true targets

PR_VV_131

Target sequence hairpin 1, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT GATGC CTTGG GTGCA ATAGG TGTTC AAGAG ACACC TTATT GCACC CAAGG CATCA TTCc caggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR_VV_132

Target sequence hairpin 1, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AATGA TGCCT TGGGT GCAAT AGGTT GTCT CTTGA ACACC TTATT GCACC CAAGG CATCA TTat tttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

shRNA plasmid lacto, true targets

PR_VV_133

Target sequence hairpin 2, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT CGTAC AAATG ATGC TTTGG GTGTT CAAGA GACAC CCAAG GCATC ATTTG TACGA TTcc aggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR_VV_134

Target sequence hairpin 2, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AATCG TACAA ATGAT GCCTT GGGT GTCT CTCTT GAAC ACCCA AGGC ATCAT TTTG TACGA TTat tttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

shRNA plasmid lacto, true targets

PR_VV_135

Target sequence hairpin 3, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT GATGC CTTGG GTGCA ATAGG TTTCA AGAGA AACC TATTG CACCC AAGGC ATCAT TTTc caggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR_VV_136

Target sequence hairpin 3, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AAATG ATGC CTTGG GTGCA ATAGG TTCT CTCTG AAAC CCTAT TGCAC CCAAG GCATC ATTTT attttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

shRNA plasmid lacto, true targets

PR_VV_137

Target sequence hairpin 4, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT TTGG CGCATT GATA Cgtag tatTT CAAG AGAA TACT ACGT ATCA ATGC CCAA ATTcc aggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR_VV_138

Target sequence hairpin 4, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AATTT GGCG CATTG ATAC gtagt atTCT CTTG Aata ctac GTAT CAATG CGCC AAATT attttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_139

Binds on RBS in front of GFP on PN2-006, homology with PR_VV_089

5'- cctat taa tgc TATAA Tgaaa tTAAT TTTGT TTAAC TTTA AGAAG GAGA GATA TACA T-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_140

Binds on the C-term of GFP on PN2-006, homology with PR_VV_090

5'- ccttt cgttt tattt gatgc ctgg TTATT AGTC TTC AATCC AATCC AGG-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_141

Binds on RBS in front of GFP on pBTK503, homology with PR_VV_089

5'- cctat taa tgc TATAA Tgaaa atat acag aaac agagg agat attac atat g-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_142

Binds on the C-term of GFP on pBTK503, homology with PR_VV_090

5'- ccttt cgttt tattt gatgc ctgg ttat tTGT atag ttc atcc atgc cgtg-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_143

Binds just upstream PTSIICcelA promoter on pMG36E-shRNA, with overhang to include J23100 promoter

5'- gctag cac tgta ccta ggac tgag ctag ccgt caag aatt cgat cgac ccata tttaa aagc-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_144

Binds on RBS in front of GFP on PN2-006, homology with PR_VV_143 (J23100)

5'- ttgac ggc tagc tcagt ccta ggtac agtg ctag cTAAT TTTGT TTAAC TTTA AGAAG GAGA GATA TACA T-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_145

Binds on RBS in front of GFP on pBTK503, homology with PR_VV_143 (J23100)

5'- ttgac ggc tagc tcagt ccta ggtac agtg ctag catac agaa acag agg agat attac atat g-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR_VV_146

Binds on GFP without including MarA deg tag, adds the missing stop codon

5'- ccttt cgttt tattt gatgc ctgg TTATT ATTTA TACAG TTCGT CCA TGC CCG -3'

Plasmid

Characteristics

Source

pDA120

Plasmid containing pCYC + mCherry used as a comparaison point to pTEF1

Serge Pelet (DMF, University of Lausanne)

pDA122

Plasmid containing pADH1 + mCherry used as a comparaison point to pTEF1

Serge Pelet (DMF, University of Lausanne)

pDA95

Plasmid containing pTEF1 + mCherry used for the characterisation of this pTEF1

Serge Pelet (DMF, University of Lausanne)

pVW222

Plasmid containing pPGK1 taken for the regulation of the AGO protein

Serge Pelet (DMF, University of Lausanne)

pSP329

Plasmid containing pTEF1 taken for the regulation of the DICr protein

Serge Pelet (DMF, University of Lausanne)

pSP599

Plasmid used as the backbone for the final AGO + DICR assembly

Serge Pelet (DMF, University of Lausanne)

pMG36E

Used as the backbone for the shRNA producing plasmid in L. lactis

Ordered from Addgene

pREXC3GH

Backup plasmid for the shRNA production in L. lactis

Ordered from Addgene

pCP20

FLP helper plasmid /!\/!\ LOST AT 37°C (incubate at 30°C)

Given to us by the Schaerli Lab

pKD13

Plasmid containing the kanamycin cassette and the FRT sites

Given to us by the Schaerli Lab

pKD46

Red recombinase expression plasmid /!\/!\ LOST AT 37°C (incubate at 30°C), used for the recombination of the FRT sites and inducible by arabinose

Given to us by the Schaerli Lab

pRS314

Plasmid used as the backbone for the target sequence introduction in yeast

Given to us by the Schaerli Lab

pRS426

Plasmid used as the backbone for the shRNA production in yeast

Given to us by the Schaerli Lab

pN1-011 AGO

Plasmid coding for the Argonaute protein

Engineered by us

pN1-011 DICR

Plasmid coding for the Dicer protein

Engineered by us

pMG36 shRNA

Plasmid coding for the production of shRNA in L. lactis

Engineered by us