Table 2. Plasmids and cosmids used in the current study.

Plasmid/cosmid	Antibiotic resistance marker(s)a	Description	Source/Reference
pGEMT-Easy	AmpR	General E. coli cloning vector	Promega
pET30b	KanR	E. coli protein expression vector	Novagen
pHM11a	HygR	Integrative Streptomyces expression vector containing the constitutive ermEp*	[36]
pSET152	AprR	Integrative Streptomyces cloning vector	[37]
pIJ773	AprR	Template plasmid for preparing the ReDirect apra disruption cassette	[34]
pIJ10700	HygR	Template plasmid for preparing the ReDirect hyg disruption cassette	[34]
pET30b-cpeSc	KanR	Plasmid vector used to express C-terminal 6×His-tagged Cpe in E. coli for purification	This study
12B8	AmpR, KanR	Cosmid clone containing the clavulanic acid biosynthetic gene cluster from S. clavuligerus	[19]
12B8-Δcpe::apra	AprR, AmpR, KanR	Mutant cosmid 12B8 in which cpe has been replaced by the disruption cassette from plasmid pIJ773 using the ReDirect system	This study
12B8-Δcpe-INF	AmpR, KanR	Mutant cosmid 12B8 in which cpe has been replaced by the 81-bp in-frame “scar” sequence using the ReDirect system	This study
12B8-Δcpe-INF-Δamp::hyg	HygR, KanR	Cosmid 12B8-Δcpe-INF in which ampicillin resistance gene replaced by the hyg cassette from plasmid pIJ10700 using the ReDirect system	This study
pHM:cpeSc	HygR	Expression plasmid pHM11a containing cpe from S. clavuligerus	This study
pHM:cpeSf	HygR	Expression plasmid pHM11a containing cpe from S. flavogriseus	This study
pHM:cpeSv	HygR	Expression plasmid pHM11a containing cpe from S. viridis	This study
pHM:cpeSj	HygR	Expression plasmid pHM11a containing cpe from S. jumonjinensis	This study
pHM:cpeSk	HygR	Expression plasmid pHM11a containing cpe from S. katsurahamanus	This study
pHM:blipFLAG	HygR	Expression plasmid pHM11a containing blip from S. clavuligerus with a C-terminal FLAG tag	This study
pHM:ccaRFLAG	HygR	Expression plasmid pHM11a containing ccaR from S. clavuligerus with a C-terminal FLAG tag	This study
pHM:cpeSc-FLAG	HygR	Expression plasmid pHM11a containing cpe from S. clavuligerus with a C-terminal FLAG tag	This study
pHM:cpeSc-6×his	HygR	Expression plasmid pHM11a containing cpe from S. clavuligerus with a C-terminal 6×His tag	This study
pHM:cpeCt	HygR	Expression plasmid pHM11a containing the C-terminal domain of cpe from S. clavuligerus	This study
pHM:cpeNt	HygR	Expression plasmid pHM11a containing the N-terminal domain of cpe from S. clavuligerus	This study
pHM-cpeCt+Nt	HygR	Expression plasmid pHM11a containing the N-terminal and C- terminal domains of cpe from S. clavuligerus, each expressed independently under the control of the ermEp*	This study
pSET:cpeSc	AprR	Plasmid pSET152 containing the S. clavuligerus cpe gene along with ermEp* from pHM11a was used as template to prepare all described cpeSc site directed mutants	This study

^aAmp^R, ampicillin resistance; Apr^R, apramycin resistance; Kan^R, kanamycin resistance, Hyg^R, hygromycin resistance.