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Duet Vectors

pETDuet™-1 is designed for the coexpression of two target genes. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter/lac operator and ribosome binding site (rbs). The vector also carries thepBR322-derived ColE1 replicon, lacI gene and ampicillin resistance gene. This vector can be used in tandem with pACYCDuet™-1 in the appropriate host strain for the coexpression of up to 4 target genes. Genes inserted into MCS-1 can be sequenced using the pETUpstream Primer and DuetDOWN1 Primer. Genes inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer.

pACYCDuet™-1 is designed for the coexpression of two target genes. The vector encodes two multiple cloning sites (MCS) each of which is receded by a T7 promoter/lac operator and ribosome binding site (rbs). The vector also carries the P15A replicon, lacI gene and chloramphenicol resistance gene. This vector can be used in tandem with pETDuet™-1 in the appropriate host strain for the coexpression of up to 4 target genes. Genes inserted into MCS-1 can be sequenced using the ACYCDuetUP1 Primer and DuetDOWN1 Primer. Genes inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer.

pCDFDuet™-1 is designed for the coexpression of two target genes. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). The vector also carries the pCloDF13 replicon, lacI gene and streptomycin/spectinomycin resistance marker. This vector can be transformed into the same cell with pETDuet™-1, pACYCDuet™-1, and/or pRSFDuet™-1 for the coexpression of up to eight target genes. ORFs inserted into MCS-1 can be sequenced using the pETUpstream Primer and DuetDOWN1 Primer. ORFs inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer.

pRSFDuet™-1 is designed for the coexpression of two target proteins. The vector encodes two multiple cloning sites (MCS) each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (rbs). The vector also carries the pRSF1030 replicon (also known as NTP1), lacI gene, and kanamycin resistance marker. This vector can be transformed into the same cell with pETDuet-1, pACYCDuet™-1, and/or pCDFDuet™-1 for the coexpression of up to eight target proteins. ORFs inserted into MCS-1 can be sequenced using the pETUpstream Primer and DuetDOWN1 Primer. ORFs inserted into MCS-2 can be sequenced using the DuetUP2 Primer and T7 Terminator Primer.

Powerful T7lac promoter driven expression
Coexpress 2 proteins per Duet vector
Cotransform up to 4 vectors for coexpression of 8 proteins
pACYCDuet, pCDF and pRSF are compatible with many existing pET constructs
Express protein subunits, interacting proteins, and pathway enzymes simultaneously
More information can be found in
TB340 "Duet Vectors"
TB336 "pAcYCDuet-1 Vector Map"
TB337 "pETDuet-1 Vector Map"
TB390 "pCDFDuet-1 Vector Map"
TB391 "pRSFDuet-1 Vector Map"

Table 1. Plasmid replicons in Novagen's E. coli expression systems

Plasmid(s)	Replicon (source)	Copy Number
pET (all) pETDuet™-1	ColE1 (pBR322)	~40
pACYCDuet™-1 pLysS pLysE pLacI pRARE	P15A (pACYC184)	10–12
pRSFDuet™-1	RSF1030	> 100
pCDFDuet™-1	CloDF13	20–40
pETBlue™ (all) pTriEx™ (all)	ColE1 (pUC)	> 500
pETcoco™ (all)	Mini-F/RK2 (1) (pBeloBAC11, RK2)	1, amplifiable to ~40
1. Wild, J., Hradecna, Z. and Szybalski, W. (2002) Genome Res. 12, 1434–1444.

Table 2. Vector and host strain compatibilities for coexpression from up to four plasmids

Compatible Vector Combinations				Target Proteins (number)	Compatible Host Strain Group
Vector 1	Vector 2	Vector 3	Vector 4
pETDuet™-1 (Aap^R)	pACYCDuet-1 (Cam^R)	pRSFDuet-1 (Kan^R)	pCDFDuet-1 (Sm^R)	8	A
pET (Amp^R)	pACYCDuet-1 (Cam^R)	pRSFDuet-1 (Kan^R)	pCDFDuet-1 (Sm^R)	7	A
pETDuet-1 (Amp^R)	pACYCDuet-1 (Cam^R)	pRSFDuet-1 (Kan^R)	none	6	A
pETDuet-1 (Amp^R)	pRSFDuet-1 (Kan^R)	pCDFDuet-1 (Sm^R)	none	6	B
pET (Amp^R)	pRSFDuet-1 (Kan^R)	pCDFDuet-1 (Sm^R)	none	5	B
pETDuet-1 (Amp^R)	pRSFDuet-1 (Kan^R)	none	none	4	B
pRSFDuet-1 (Kan^R)	pCDFDuet-1 (Sm^R)	none	none	4	B
pETDuet-1 (Amp^R)	pCDFDuet-1 (Sm^R)	none	none	4	C
Resistance markers: Amp^R: ampicillin/carbenicillin, Kan^R: kanamycin, Cam^R: chloramphenicol, Sm^R: streptomycin/spectinomycin

Host Strain Groups
Group A	Group B	Group C
B834(DE3) BL21(DE3) BLR(DE3) HMS174(DE3) Novablue(DE3) Tuner™(DE3)	B834(DE3) B834(DE3)pLysS BL21(DE3) BL21(DE3)pLysS BLR(DE3) BLR(DE3)pLysS HMS174(DE3) HMS174(DE3)pLysS Novablue(DE3) Novablue(DE3)pLysS Tuner(DE3) Tuner(DE3)pLysS Rosetta™(DE3) Rosetta(DE3)pLysS RosettaBlue™(DE3) RosettaBlue(DE3)pLys	B834(DE3) B834(DE3)pLysS BL21(DE3) BL21(DE3)pLysS BLR(DE3) BLR(DE3)pLysS HMS174(DE3) HMS174(DE3)pLysS Novablue(DE3) Novablue(DE3)pLysS Tuner(DE3) Tuner(DE3)pLysS Rosetta(DE3) Rosetta(DE3)pLysS RosettaBlue(DE3) RosettaBlue(DE3)pLysS	Origami™(DE3)* Origami(DE3)pLysS* Origami B(DE3) Origami B(DE3)pLysS Rosetta-gami(DE3)* Rosetta-gami(DE3)pLysS* Rosetta-gami B(DE3) Rosetta-gami B(DE3)pLysS
These strains carry the rpsL* mutation that confers resistance to streptomycin; therefore, spectinomycin must be used for selection of pCDFDuet recombinants.