Skip to main content

Table 3 Oligonucleotides used in this study

From: The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

Primers

Sequence (5' to 3')a

Description/position

Plasmid construction

 

TM2064

CATGGTCTC AGATCTTTAAAACGCCATGCCTCG

BsaI; 5' end of P liaI

TM1980

CTTGTTGGATCCATCGAT GATCCTCCT TACGTTTTCCTTGTCTTC

Strong SD region; BamHI, ClaI; 3' end of P liaI

TM1991

ATCTGAATTC GGTTTTAAAACGCCATGCC

EcoRI; 5' end of P liaI

TM1992

ATTTTCTCTAGA ATCCTCCT TACGTTTTCCTTGTCTTC

Strong SD region; XbaI; 3' end of P liaI

TM1981

TCCTATCG ATG AGTAAAGGAGAAGAACTTTTCACTGG

ATG start codon; ClaI; 5' end of gfpmut1

TM1982

GGCCAAGCTT GAACTAGTTTCATTTATTTGTAGAGC

HindIII; 3' end of gfpmut1

TM1993

TTCCTCTAG ATG AGTAAAGGAGAAGAACTTTTC

ATG start codon; XbaI; 5' end of gfpmut1

TM1994

GGCCGTCGAC GAACTAGTTTCATTTATTTG

SalI; 3' end of gfpmut1

TM2535

CCATATCG ATG CAT CATCATCATCATCAC GAAACGAGATTTCTAATGGAAAAAG

ATG start codon; ClaI; His6-tag; 5’ end of ydfG

TM2536

CCATAAGCTT TCAATCTGCTGCGGGCATTTTC

HindIII; 3’ end of ydfG

TM2545

CCATTCTAG ATG CAT CATCATCATCATCAC GAAACGAGATTTCTAATGGAAAAAG

ATG start codon; XbaI; His6-tag; 5’ end of ydfG

Clean deletions

 

TM2130

GCGGGGATCC TCTTACATTTATTAGTCC

BamHI; upstream of P liaI

TM2131

CATTTGCCGCTTTTGTCTGG GCAGATCCTCCTTTCGTTTTC

3' end of P liaI ; 3' end of liaH

TM1055

CCAGACAAAAGCGGCAAATG

3' end of liaH

TM1058

CCATGAATTC GAATGCGGACGTCCGTCACGC

EcoRI; inside the liaG gene

TM2132

GCGAATTGATACGTGCGG GCAGATCCTCCTTTCGTTTTC

upstream of liaI gene; upstream of liaG

TM2133

CCGCACGTATCAATTCGC

upstream of liaG

TM2134

GCTAGAATTC TGCCGGCTGTTTTGGAG

EcoRI; center of liaG gene

  1. a Relevant restriction sites are shown in italics, complementary regions for joining PCR are underlined. The sequence for optimized SD sequences of the liaI promoter is indicated by bold type, the start codon is bold italic. The His-tags are in italics and underlined.