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Plasmid Files

pET-16b

Bacterial vector for inducible expression of N-terminally 10xHis-tagged proteins with a Factor Xa site.

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pET-16b Sequence and MappET-16b.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 EcoRI (5709) AatII (5638) ZraI (5636) SspI (5520) ScaI (5196) PvuI (5086) PstI (4961) BsaI (4777) AhdI (4716) AlwNI (4239) PciI (3823) BspQI - SapI (3707) BstZ17I (3594) AccI (3593) BsaAI (3575) PflFI - Tth111I (3568) Bpu10I (2929) BspDI - ClaI (24) HindIII (29) BlpI (267) BamHI (319) PaeR7I - PspXI - XhoI (324) NdeI (331) Factor Xa site NcoI (392) ATG RBS XbaI (431) lac operator T7 promoter BglII (497) SgrAI (538) SphI (694) EcoNI (754) MluI (1219) BclI * (1233) BstEII (1400) PspOMI (1426) ApaI (1430) BssHII (1630) HpaI (1725) PshAI (2064) EagI (2287) NruI (2322) BfuAI - BspMI (2402) BsmI (2707) MscI * (2794) FspAI (2804) pET-16b 5711 bp
EcoRI  (5709)
1 site
G A A T T C C T T A A G
AatII  (5638)
1 site
G A C G T C C T G C A G
ZraI  (5636)
1 site
G A C G T C C T G C A G
SspI  (5520)
1 site
A A T A T T T T A T A A
ScaI  (5196)
1 site
A G T A C T T C A T G A
PvuI  (5086)
1 site
C G A T C G G C T A G C
PstI  (4961)
1 site
C T G C A G G A C G T C
BsaI  (4777)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (4716)
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  (4239)
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  (3823)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3707)
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  (3707)
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  (3594)
1 site
G T A T A C C A T A T G
AccI  (3593)
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.
BsaAI  (3575)
1 site
Y A C G T R R T G C A Y
PflFI  (3568)
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  (3568)
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.
Bpu10I  (2929)
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.
BspDI  (24)
1 site
A T C G A T T A G C T A
ClaI  (24)
1 site
A T C G A T T A G C T A
HindIII  (29)
1 site
A A G C T T T T C G A A
BlpI  (267)
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.
BamHI  (319)
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.
PaeR7I  (324)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (324)
1 site
V C T C G A G B B G A G C T C V
XhoI  (324)
1 site
C T C G A G G A G C T C
NdeI  (331)
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.
NcoI  (392)
1 site
C C A T G G G G T A C C
XbaI  (431)
1 site
T C T A G A A G A T C T
BglII  (497)
1 site
A G A T C T T C T A G A
SgrAI  (538)
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  (694)
1 site
G C A T G C C G T A C G
EcoNI  (754)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
MluI  (1219)
1 site
A C G C G T T G C G C A
BclI  (1233)
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  (1400)
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.
PspOMI  (1426)
1 site
G G G C C C C C C G G G
ApaI  (1430)
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  (1630)
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  (1725)
1 site
G T T A A C C A A T T G
PshAI  (2064)
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.
EagI  (2287)
1 site
C G G C C G G C C G G C
NruI  (2322)
1 site
T C G C G A A G C G C T
BfuAI  (2402)
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  (2402)
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.
BsmI  (2707)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
MscI  (2794)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
FspAI  (2804)
1 site
R T G C G C A Y Y A C G C G T R
lacI
869 .. 1951  =  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
869 .. 1951  =  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).
AmpR
4643 .. 5503  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   4643 .. 5434  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4643 .. 5503  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   5435 .. 5503  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4643 .. 5503  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
3884 .. 4472  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3884 .. 4472  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
rop
3263 .. 3454  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
rop
3263 .. 3454  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein
AmpR promoter
5504 .. 5608  =  105 bp
AmpR promoter
5504 .. 5608  =  105 bp
lacI promoter
791 .. 868  =  78 bp
lacI promoter
791 .. 868  =  78 bp
T7 terminator
213 .. 260  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
213 .. 260  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
10xHis
360 .. 389  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
360 .. 389  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
439 .. 463  =  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
439 .. 463  =  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
464 .. 482  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
464 .. 482  =  19 bp
promoter for bacteriophage T7 RNA polymerase
MCS
319 .. 335  =  17 bp
multiple cloning site
MCS
319 .. 335  =  17 bp
multiple cloning site
Factor Xa site
336 .. 347  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa recognition and cleavage site
Factor Xa site
336 .. 347  =  12 bp
4 amino acids  =  473.5 Da
Product: Factor Xa recognition and cleavage site
RBS
403 .. 407  =  5 bp
ribosome binding site
RBS
403 .. 407  =  5 bp
ribosome binding site
ATG
393 .. 395  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
393 .. 395  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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