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Plasmid Files

pET-20b(+)

Bacterial vector that encodes a signal sequence for expression of proteins in the periplasm.

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pET-20b(+).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Novagen (EMD Millipore)
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 DraIII (3474) PsiI (3346) BsaHI (2868) ScaI (2811) PvuI (2701) PstI (2576) NmeAIII (2479) AhdI (2331) BlpI (80) 6xHis PaeR7I - PspXI - XhoI (158) EagI - NotI (166) HindIII (173) SalI (179) HincII (181) Eco53kI (188) SacI (190) EcoRI (192) BamHI (198) EcoRV (217) NcoI (220) BseRI (260) BfuAI - BspMI (268) NdeI (288) XbaI (326) BglII (384) FspAI (419) Bpu10I (544) BbsI (556) BsgI * (598) AfeI (692) PvuII (1029) BsmBI (1079) PfoI (1080) PflFI - Tth111I (1183) BstZ17I (1209) BspQI - SapI (1322) AflIII - PciI (1438) AlwNI (1854) pET-20b(+) 3716 bp
DraIII  (3474)
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  (3346)
1 site
T T A T A A A A T A T T
BsaHI  (2868)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
ScaI  (2811)
1 site
A G T A C T T C A T G A
PvuI  (2701)
1 site
C G A T C G G C T A G C
PstI  (2576)
1 site
C T G C A G G A C G T C
NmeAIII  (2479)
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).
AhdI  (2331)
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.
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.
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
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
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
HincII  (181)
1 site
G T Y R A C C A R Y 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  (217)
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  (220)
1 site
C C A T G G G G T A C C
BseRI  (260)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
BfuAI  (268)
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  (268)
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.
NdeI  (288)
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  (326)
1 site
T C T A G A A G A T C T
BglII  (384)
1 site
A G A T C T T C T A G A
FspAI  (419)
1 site
R T G C G C A Y Y A C G C G T R
Bpu10I  (544)
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.
BbsI  (556)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
BsgI  (598)
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).
AfeI  (692)
1 site
A G C G C T T C G C G A
PvuII  (1029)
1 site
C A G C T G G T C G A C
BsmBI  (1079)
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  (1080)
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  (1183)
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  (1183)
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.
BstZ17I  (1209)
1 site
G T A T A C C A T A T G
BspQI  (1322)
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  (1322)
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  (1438)
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  (1438)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AlwNI  (1854)
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.
AmpR
2258 .. 3118  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   2258 .. 3049  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2258 .. 3118  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   3050 .. 3118  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2258 .. 3118  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1499 .. 2087  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1499 .. 2087  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
3250 .. 3705  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
3250 .. 3705  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
878 .. 1069  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
878 .. 1069  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
AmpR promoter
3119 .. 3223  =  105 bp
AmpR promoter
3119 .. 3223  =  105 bp
MCS
158 .. 225  =  68 bp
multiple cloning site
MCS
158 .. 225  =  68 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
T7 promoter
351 .. 369  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
351 .. 369  =  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
298 .. 302  =  5 bp
ribosome binding site
RBS
298 .. 302  =  5 bp
ribosome binding site
pelB signal sequence
224 .. 289  =  66 bp
22 amino acids  =  2.2 kDa
Product: leader peptide for secretion
pelB signal sequence
224 .. 289  =  66 bp
22 amino acids  =  2.2 kDa
Product: leader peptide for secretion
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