pET-29c(+)

Bacterial vector for expression of N-terminally S-tagged proteins with a thrombin site. For other reading frames, use pET-29a(+) or pET-29b(+).

Sequence Author: MilliporeSigma (Novagen)

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EagI - NotI (166) PaeR7I - PspXI - XhoI (158) 6xHis BlpI (80) DraIII (5130) PsiI (5002) AsiSI - PvuI (4429) SmaI (4303) TspMI - XmaI (4301) NruI (4086) AcuI (3775) AlwNI (3643) BssSI - BssSαI (3400) PciI (3227) BspQI - SapI (3111) TatI (3031) BstZ17I (2998) PflFI - Tth111I (2972) HindIII (173) SalI (179) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) EcoRV (209) NcoI (213) thrombin site Acc65I (235) KpnI (239) BglII (242) BstBI (269) ATG NdeI (296) RBS XbaI (334) lac operator T7 promoter SgrAI (445) SphI (601) BstAPI (809) MluI (1126) BclI * (1140) BstEII (1307) NmeAIII (1332) PspOMI (1333) ApaI (1337) BssHII (1537) HpaI (1632) PshAI (1971) BglI (2190) FspI - FspAI (2208) PpuMI (2233) pET-29c(+) 5372 bp
EagI  (166)
1 site
C G G C C G G C C G G C
NotI  (166)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (158)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (158)
1 site
V C T C G A G B B G A G C T C V
XhoI  (158)
1 site
C T C G A G G A G C T C
BlpI  (80)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
DraIII  (5130)
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  (5002)
1 site
T T A T A A A A T A T T
AsiSI  (4429)
1 site
G C G A T C G C C G C T A G C G
PvuI  (4429)
1 site
C G A T C G G C T A G C
SmaI  (4303)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (4301)
1 site
C C C G G G G G G C C C
XmaI  (4301)
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.
NruI  (4086)
1 site
T C G C G A A G C G C T
AcuI  (3775)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
AlwNI  (3643)
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.
BssSI  (3400)
1 site
C A C G A G G T G C T C
BssSαI  (3400)
1 site
C A C G A G G T G C T C
PciI  (3227)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3111)
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  (3111)
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.
TatI  (3031)
1 site
W G T A C W W C A T G W
BstZ17I  (2998)
1 site
G T A T A C C A T A T G
PflFI  (2972)
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  (2972)
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.
HindIII  (173)
1 site
A A G C T T T T C G A A
SalI  (179)
1 site
G T C G A C C A G C T G
Eco53kI  (188)
1 site
G A G C T C C T C G A G
SacI  (190)
1 site
G A G C T C C T C G A G
EcoRI  (192)
1 site
G A A T T C C T T A A G
BamHI  (198)
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.
EcoRV  (209)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
NcoI  (213)
1 site
C C A T G G G G T A C C
Acc65I  (235)
1 site
G G T A C C C C A T G G
KpnI  (239)
1 site
G G T A C C C C A T G G
BglII  (242)
1 site
A G A T C T T C T A G A
BstBI  (269)
1 site
T T C G A A A A G C T T
NdeI  (296)
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  (334)
1 site
T C T A G A A G A T C T
SgrAI  (445)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
SphI  (601)
1 site
G C A T G C C G T A C G
BstAPI  (809)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
MluI  (1126)
1 site
A C G C G T T G C G C A
BclI  (1140)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
BstEII  (1307)
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  (1332)
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  (1333)
1 site
G G G C C C C C C G G G
ApaI  (1337)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BssHII  (1537)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
HpaI  (1632)
1 site
G T T A A C C A A T T G
PshAI  (1971)
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.
BglI  (2190)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
FspI  (2208)
1 site
T G C G C A A C G C G T
FspAI  (2208)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (2233)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
lacI
776 .. 1858  =  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
776 .. 1858  =  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
3998 .. 4813  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
3998 .. 4813  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
ori
3288 .. 3876  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3288 .. 3876  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
4906 .. 5361  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
4906 .. 5361  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
2667 .. 2858  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
2667 .. 2858  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
thrombin site
217 .. 234  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
thrombin site
217 .. 234  =  18 bp
6 amino acids  =  627.7 Da
Product: thrombin recognition and cleavage site
S-Tag
250 .. 294  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
250 .. 294  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
ATG
295 .. 297  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
295 .. 297  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
lacI promoter
698 .. 775  =  78 bp
lacI promoter
698 .. 775  =  78 bp
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
MCS
158 .. 203  =  46 bp
multiple cloning site
MCS
158 .. 203  =  46 bp
multiple cloning site
lac operator
342 .. 366  =  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
342 .. 366  =  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
367 .. 385  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
367 .. 385  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
306 .. 311  =  6 bp
ribosome binding site
RBS
306 .. 311  =  6 bp
ribosome binding site
ORF:  298 .. 1026  =  729 bp
ORF:  242 amino acids  =  25.5 kDa
ORF:  1615 .. 1878  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  899 .. 1858  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3998 .. 4813  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  1914 .. 2270  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  2634 .. 2858  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  474 .. 737  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  1641 .. 1892  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  2267 .. 2635  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  70 .. 297  =  228 bp
ORF:  75 amino acids  =  8.5 kDa
ORF:  349 .. 600  =  252 bp
ORF:  83 amino acids  =  8.6 kDa
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