pET-50b(+)

Bacterial vector for expressing proteins with a cleavable N-terminal NusA tag and a cleavable C-terminal S-tag.

Sequence Author: MilliporeSigma (Novagen)

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HomePlasmidspET & Duet Vectors (Novagen)pET-50b(+)
S-Tag PaeR7I - XhoI (147) PacI (141) AvrII (131) DraIII (6491) PsiI (6363) AlwNI (5004) PciI (4588) BspQI - SapI (4472) BstZ17I (4359) PflFI - Tth111I (4333) thrombin site Eco53kI (236) SacI (238) EagI - NotI (242) HindIII (249) SalI (255) PshAI (262) AscI (270) BsrGI (283) EcoRI (289) BamHI (295) Acc65I (302) KpnI - TspMI - XmaI (306) SmaI (308) KflI (310) HRV 3C site SacII (338) 6xHis SpeI (369) BglII (484) MscI (616) BfuAI - BspMI (818) NcoI (944) 6xHis ATG NdeI (1894) RBS XbaI (1932) T7 promoter SphI (2199) MluI (2724) BstEII (2905) NmeAIII (2930) PspOMI (2931) ApaI (2935) HpaI (3230) pET-50b(+) 6733 bp
PaeR7I  (147)
1 site		 C T C G A G G A G C T C
PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (147)
1 site		 C T C G A G G A G C T C
PacI  (141)
1 site		 T T A A T T A A A A T T A A T T
AvrII  (131)
1 site		 C C T A G G G G A T C C
DraIII  (6491)
1 site		 C A C N N N G T G G T G N N N C A C
Sticky ends from different DraIII sites may not be compatible.
PsiI  (6363)
1 site		 T T A T A A A A T A T T
AlwNI  (5004)
1 site		 C A G N N N C T G G T C N N N G A C
Sticky ends from different AlwNI sites may not be compatible.
PciI  (4588)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
BspQI  (4472)
1 site		 G C T C T T C N C G A G A A G N N N N
Sticky ends from different BspQI sites may not be compatible.
SapI  (4472)
1 site		 G C T C T T C N C G A G A A G N N N N
Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
BstZ17I  (4359)
1 site		 G T A T A C C A T A T G
PflFI  (4333)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (4333)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
Eco53kI  (236)
1 site		 G A G C T C C T C G A G
SacI  (238)
1 site		 G A G C T C C T C G A G
EagI  (242)
1 site		 C G G C C G G C C G G C
NotI  (242)
1 site		 G C G G C C G C C G C C G G C G
HindIII  (249)
1 site		 A A G C T T T T C G A A
SalI  (255)
1 site		 G T C G A C C A G C T G
PshAI  (262)
1 site		 G A C N N N N G T C C T G N N N N C A G
PshAI quickly loses activity at 37°C, but can be used at 25°C for long incubations.
AscI  (270)
1 site		 G G C G C G C C C C G C G C G G
BsrGI  (283)
1 site		 T G T A C A A C A T G T
BsrGI is typically used at 37°C, but is even more active at 60°C.
EcoRI  (289)
1 site		 G A A T T C C T T A A G
BamHI  (295)
1 site		 G G A T C C C C T A G G
After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
Acc65I  (302)
1 site		 G G T A C C C C A T G G
KpnI  (306)
1 site		 G G T A C C C C A T G G
TspMI  (306)
1 site		 C C C G G G G G G C C C
XmaI  (306)
1 site		 C C C G G G G G G C C C
Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (308)
1 site		 C C C G G G G G G C C C
SmaI can be used at 37°C for brief incubations.
KflI  (310)
1 site		 G G G W C C C C C C W G G G
Sticky ends from different KflI sites may not be compatible.
SacII  (338)
1 site		 C C G C G G G G C G C C
Efficient cleavage requires at least two copies of the SacII recognition sequence.
SpeI  (369)
1 site		 A C T A G T T G A T C A
BglII  (484)
1 site		 A G A T C T T C T A G A
MscI  (616)
1 site		 T G G C C A A C C G G T
BfuAI  (818)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (818)
1 site		 A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4
Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
NcoI  (944)
1 site		 C C A T G G G G T A C C
NdeI  (1894)
1 site		 C A T A T G G T A T A C
Prolonged incubation with NdeI may lead to removal of additional nucleotides.
XbaI  (1932)
1 site		 T C T A G A A G A T C T
SphI  (2199)
1 site		 G C A T G C C G T A C G
MluI  (2724)
1 site		 A C G C G T T G C G C A
BstEII  (2905)
1 site		 G G T N A C C C C A N T G G
Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
NmeAIII  (2930)
1 site		 G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19
Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
PspOMI  (2931)
1 site		 G G G C C C C C C G G G
ApaI  (2935)
1 site		 G G G C C C C C C G G G
ApaI can be used between 25°C and 37°C.
HpaI  (3230)
1 site		 G T T A A C C A A T T G
S-Tag
168 .. 212  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
168 .. 212  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
thrombin site
213 .. 230  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
213 .. 230  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
HRV 3C site
309 .. 332  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C protease
HRV 3C site
309 .. 332  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C protease
6xHis
342 .. 359  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
342 .. 359  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
NusA
375 .. 1859  =  1485 bp
495 amino acids  =  54.9 kDa
Product: transcription elongation protein (N utilization substance protein A)
highly soluble in E. coli
NusA
375 .. 1859  =  1485 bp
495 amino acids  =  54.9 kDa
Product: transcription elongation protein (N utilization substance protein A)
highly soluble in E. coli
6xHis
1866 .. 1883  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1866 .. 1883  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
1893 .. 1895  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1893 .. 1895  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
lacI
2374 .. 3456  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
2374 .. 3456  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
KanR
5359 .. 6174  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
5359 .. 6174  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
ori
4649 .. 5237  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4649 .. 5237  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
6267 .. 6722  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
6267 .. 6722  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
4028 .. 4219  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
4028 .. 4219  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
lacI promoter
2296 .. 2373  =  78 bp
lacI promoter
2296 .. 2373  =  78 bp
MCS
234 .. 308  =  75 bp
multiple cloning site
MCS
234 .. 308  =  75 bp
multiple cloning site
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
1940 .. 1964  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
1940 .. 1964  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
T7 promoter
1965 .. 1983  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1965 .. 1983  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
1904 .. 1909  =  6 bp
ribosome binding site
RBS
1904 .. 1909  =  6 bp
ribosome binding site
ORF:  469 .. 705  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
ORF:  2497 .. 3456  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  5359 .. 6174  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  3995 .. 4219  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  345 .. 1673  =  1329 bp
ORF:  442 amino acids  =  51.3 kDa
ORF:  1866 .. 2624  =  759 bp
ORF:  252 amino acids  =  26.5 kDa
ORF:  3213 .. 3476  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  2072 .. 2335  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  3239 .. 3490  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  3628 .. 3996  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  141 .. 1895  =  1755 bp
ORF:  584 amino acids  =  64.3 kDa
ORF:  1947 .. 2198  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
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Individual Sequences & Maps

pACYCDuet-1
pET-24a(+)
pET-3a
pET-51b(+) Ek_LIC (linearized)
pCDF-1b
pET-24b(+)
pET-3b
pET-52b(+)
pCDF-2 Ek_LIC
pET-24c(+)
pET-3c
pET-52b(+) 3C_LIC
pCDF-2 Ek_LIC (linearized)
pET-24d(+)
pET-3d
pET-52b(+) 3C_LIC (linearized)
pCDFDuet-1
pET-25b(+)
pET-40b(+)
pET-53-DEST
pCOLA-2-DEST
pET-26b(+)
pET-41 Ek_LIC
pET-54-DEST
pCOLA-3-DEST
pET-27b(+)
pET-41 Ek_LIC (linearized)
pET-55-DEST
pCOLADuet-1
pET-28a(+)
pET-41a(+)
pET-56-DEST
pET-11a
pET-28b(+)
pET-41b(+)
pET-57-DEST
pET-11b
pET-28c(+)
pET-41c(+)
pET-58-DEST
pET-11c
pET-29a(+)
pET-42a(+)
pET-59-DEST
pET-11d
pET-29b(+)
pET-42b(+)
pET-60-DEST
pET-14b
pET-29c(+)
pET-42c(+)
pET-61-DEST
pET-15b
pET-30 Ek_LIC
pET-43.1 Ek_LIC
pET-62-DEST
pET-16b
pET-30 Ek_LIC (linearized)
pET-43.1 Ek_LIC (linearized)
pET-9a
pET-17b
pET-30 Xa_LIC
pET-43.1a(+)
pET-9b
pET-19b
pET-30 Xa_LIC (linearized)
pET-43.1b(+)
pET-9c
pET-20b(+)
pET-30a(+)
pET-43.1c(+)
pET-9d
pET-21(+)
pET-30b(+)
pET-44a(+)
pETBlue-1
pET-21a(+)
pET-30c(+)
pET-44b(+)
pETBlue-2
pET-21b(+)
pET-31b(+)
pET-44c(+)
pETcoco-1
pET-21c(+)
pET-32 Ek_LIC
pET-45b(+)
pETcoco-2
pET-21d(+)
pET-32 Ek_LIC (linearized)
pET-46 Ek_LIC
pETDuet-1
pET-22b(+)
pET-32 Xa_LIC
pET-46 Ek_LIC (linearized)
pLacI
pET-23(+)
pET-32 Xa_LIC (linearized)
pET-47b(+)
pLysE
pET-23a(+)
pET-32a(+)
pET-48b(+)
pLysS
pET-23b(+)
pET-32b(+)
pET-49b(+)
pRSF-1b
pET-23c(+)
pET-32c(+)
pET-50b(+)
pRSF-2 Ek_LIC
pET-23d(+)
pET-33b(+)
pET-51b(+)
pRSF-2 Ek_LIC (linearized)
pET-24(+)
pET-39b(+)
pET-51b(+) Ek_LIC
pRSFDuet-1

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