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 | Â | |
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 | Â | |
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 |