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Plasmid Files

pET-17b

Bacterial vector for expression of N-terminally T7-tagged proteins.

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pET-17b Sequence and MappET-17b.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 AatII (3233) ZraI (3231) SspI (3115) HincII (2852) ScaI (2791) PvuI (2681) NmeAIII (2459) BglI (2431) BsrFI (2391) AhdI (2311) AlwNI (1834) PspFI (1726) StyI (59) BlpI (82) PaeR7I - PspXI - XhoI (141) NotI (147) EcoRV (166) ApoI - EcoRI (174) SpeI (199) BamHI (205) Eco53kI (213) BanII - SacI (215) Acc65I (217) KpnI (221) HindIII (223) NheI (261) BmtI (265) NdeI (268) RBS XbaI (306) BglII (364) MscI * (389) BtgI (390) FspAI (399) Bpu10I (524) BsgI * (578) BtgZI (602) AfeI (672) PvuII (1009) BsmBI (1059) PfoI (1060) PflFI - Tth111I (1163) BsaAI (1170) AccI (1188) BstZ17I (1189) BspQI - SapI (1302) AflIII - PciI (1418) BseYI (1722) pET-17b 3306 bp
AatII  (3233)
1 site
G A C G T C C T G C A G
ZraI  (3231)
1 site
G A C G T C C T G C A G
SspI  (3115)
1 site
A A T A T T T T A T A A
HincII  (2852)
1 site
G T Y R A C C A R Y T G
ScaI  (2791)
1 site
A G T A C T T C A T G A
PvuI  (2681)
1 site
C G A T C G G C T A G C
NmeAIII  (2459)
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).
BglI  (2431)
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.
BsrFI  (2391)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its
electrophoretic mobility.
AhdI  (2311)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
AlwNI  (1834)
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.
PspFI  (1726)
1 site
C C C A G C G G G T C G
StyI  (59)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BlpI  (82)
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.
PaeR7I  (141)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (141)
1 site
V C T C G A G B B G A G C T C V
XhoI  (141)
1 site
C T C G A G G A G C T C
NotI  (147)
1 site
G C G G C C G C C G C C G G C G
EcoRV  (166)
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.
ApoI  (174)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (174)
1 site
G A A T T C C T T A A G
SpeI  (199)
1 site
A C T A G T T G A T C A
BamHI  (205)
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.
Eco53kI  (213)
1 site
G A G C T C C T C G A G
BanII  (215)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (215)
1 site
G A G C T C C T C G A G
Acc65I  (217)
1 site
G G T A C C C C A T G G
KpnI  (221)
1 site
G G T A C C C C A T G G
HindIII  (223)
1 site
A A G C T T T T C G A A
NheI  (261)
1 site
G C T A G C C G A T C G
BmtI  (265)
1 site
G C T A G C C G A T C G
NdeI  (268)
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  (306)
1 site
T C T A G A A G A T C T
BglII  (364)
1 site
A G A T C T T C T A G A
MscI  (389)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
BtgI  (390)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
FspAI  (399)
1 site
R T G C G C A Y Y A C G C G T R
Bpu10I  (524)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BsgI  (578)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14
* Blocked by EcoKI methylation.
Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BtgZI  (602)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
AfeI  (672)
1 site
A G C G C T T C G C G A
PvuII  (1009)
1 site
C A G C T G G T C G A C
BsmBI  (1059)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
PfoI  (1060)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
PflFI  (1163)
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  (1163)
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.
BsaAI  (1170)
1 site
Y A C G T R R T G C A Y
AccI  (1188)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BstZ17I  (1189)
1 site
G T A T A C C A T A T G
BspQI  (1302)
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  (1302)
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.
AflIII  (1418)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (1418)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (1722)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
AmpR
2238 .. 3098  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   2238 .. 3029  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2238 .. 3098  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   3030 .. 3098  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2238 .. 3098  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1479 .. 2067  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1479 .. 2067  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
858 .. 1049  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
858 .. 1049  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
AmpR promoter
3099 .. 3203  =  105 bp
AmpR promoter
3099 .. 3203  =  105 bp
MCS
141 .. 228  =  88 bp
multiple cloning site
MCS
141 .. 228  =  88 bp
multiple cloning site
T7 terminator
28 .. 75  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
28 .. 75  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 tag
237 .. 269  =  33 bp
11 amino acids  =  1.1 kDa
Product: epitope tag from a T7 major capsid protein
T7 tag
237 .. 269  =  33 bp
11 amino acids  =  1.1 kDa
Product: epitope tag from a T7 major capsid protein
T7 promoter
331 .. 349  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
331 .. 349  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
278 .. 283  =  6 bp
ribosome binding site
RBS
278 .. 283  =  6 bp
ribosome binding site
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