pMV158GFP |
Mobilizable, Gram-positive vector encoding gfp
|
Nieto and Espinosa (2003) |
pSMART-LCKan |
Low copy, transcription-free cloning vector |
Lucigen Corp., Middleton, WI, USA |
pGEM-T Easy |
PCR cloning vector |
Promega Corp., Madison, WI, USA |
pDL276 |
Shuttle vector for low %G + C Gram-positive bacteria |
Dunny et al. (1991) |
pCAMP17 |
Phage T12 derived integration vector with a cloning site under the control of the CAMP promoter |
Gase et al. (1999) |
pWM448 |
SpyCIM1 int clone into the CAMP promoter in pCAMP17 |
This work |
pWM481 |
SpyCIM5 int pseudogene and upstream (primers M5int5 and M5int4) fused to gfp in pSMART-LCKan |
This work |
pWM483 |
Insert from pWM481 cloned into pDL276 |
This work |
pWM485 |
SpyCIM5int pseudogene (primers M5int1 and M5int4) fused to gfp in pSMART-LCKan |
This work |
pWM486 |
SpyCIM5 int pseudogene subregion (primers M5int2 and M5int4) fused to gfp in pSMART-LCKan |
This work |
pWM487 |
SpyCIM5 int pseudogene subregion (primers M5int3 and M5int4) fused to gfp in pSMART-LCKan |
This work |
pWM488 |
Insert from pWM486 cloned into pDL276 |
This work |
pWM489 |
Insert from pWM485 cloned into pDL276 |
This work |
pWM490 |
Insert from pWM487 cloned into pDL276 |
This work |
|
Sequence |
Primer |
Gene target |
Product size (bp) |
|
PRIMERS FOR PCR |
5′AGCAGGGATTGAACGCTTTA |
attB-L |
int-mutL intergenic region (attB) |
655 (strain SF370) |
5′TCAATTTCCCCACCAGTAGC |
attB-R |
|
694 (strain Manfredo) |
5′CTTGCCTGCTGAACTCATTG |
attP-L |
SpyCI attP |
461 |
5′CACGCTTTTAGACACACTCA |
attP-R |
|
5′AAGCGTGAGGTCGTTCAAGT |
mutS-L |
mutS |
406 |
5′AGTGGCTGAGTTCTCGCATT |
mutS-R |
|
5′GAGGCTTTACCGTCTGTTGC |
mutL-L |
mutL |
413 |
5′CCGGAAACTTCAAAATCCAA |
mutL-R |
|
5′ATGAGTAAAGGAGAAGAACTTT |
gfp-L |
gfp ORF |
717 |
5′TTATTTGTATAGTTCATCCATGC |
gfp-R |
|
5′ACAAGCATCACAGGAAGAAC |
M5int1 |
SpyCIM5 int pseudogene and promoter |
Various lengths |
5′ATAAGATAACCGCTAGTGATA |
M5int2 |
|
5′TATTGATCCGTCTGATTTGA |
M5int3 |
|
5′AATTTTGTTTTTAACACTTTCTAAAC |
M5int4 |
|
5′CTTACTCCCATTCGGGAACA |
M5int5 |
|
PRIMERS FOR qRT-PCR |
5′ACTGCCAATCCAAGTCCAAT |
SpyCIM1 attP
|
96 |
5′TGAGAGGTGTAAACCTAATGAAACC |
5′TTGAGAAGTGTAAACCTAATGAAACC |
SpyCIM5attP
|
112 |
5′CAATACCCTGTAAAAACTGCCAAT |
5′ACACTCGCTGGCCTTTCTACAACTTCA |
attB |
65 |
5′CGAGGCCTTCGGCTATTTAT |
5′CTGCGACAGCAACCTTGTAA |
mutS |
108 |
5′GCAACAAGAATGCGGAAAAT |
5′CCGGAAACTTCAAAATCCAA |
mutL |
111 |
5′GACATCAGGAACTGGCGATT |
5′AGCGTTGTCCGGATTTATTG |
16S rRNA |
126 |
5′CACTCTCCCCTTCTGCACTC |