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Plasmid Files

pETBlue™-2

Vector for blue/white screening and for inducible bacterial expression of tagged proteins.

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pETBlue-2 Sequence and MappETBlue-2.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 lac operator (symmetric) AgeI (3599) NspI (3267) PciI (3263) PspFI (2963) BseYI (2959) AlwNI (2854) BspHI (2543) XmnI (2338) ScaI (2219) NmeAIII (1887) T7 promoter XbaI (47) BglII (223) NheI (235) BmtI (239) BtgI - NcoI (276) EcoRV (285) TspMI - XmaI (288) SmaI (290) Eco53kI (295) SacI (297) BamHI (301) EcoRI (307) BsrGI (313) AscI - BssHII (320) PstI - SbfI (330) ZraI (334) SalI - SgrDI (335) AatII (336) HincII (337) Acc65I (341) KpnI (345) BspDI - ClaI (348) MluI (353) BstBI (359) HindIII (362) EagI - NotI (369) PvuII (379) BstZ17I (384) PmlI (389) PaeR7I - XhoI (431) PacI (464) tet promoter AvrII (597) BlpI (1001) BspEI (1078) BtgZI (1321) DraIII (1329) PsiI (1454) SspI (1534) AhdI (1739) pETBlue™-2 3653 bp
AgeI  (3599)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
NspI  (3267)
1 site
R C A T G Y Y G T A C R
PciI  (3263)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (2963)
1 site
C C C A G C G G G T C G
BseYI  (2959)
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.
AlwNI  (2854)
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.
BspHI  (2543)
1 site
T C A T G A A G T A C T
XmnI  (2338)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (2219)
1 site
A G T A C T T C A T G A
NmeAIII  (1887)
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).
XbaI  (47)
1 site
T C T A G A A G A T C T
BglII  (223)
1 site
A G A T C T T C T A G A
NheI  (235)
1 site
G C T A G C C G A T C G
BmtI  (239)
1 site
G C T A G C C G A T C G
BtgI  (276)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (276)
1 site
C C A T G G G G T A C C
EcoRV  (285)
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.
TspMI  (288)
1 site
C C C G G G G G G C C C
XmaI  (288)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (290)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Eco53kI  (295)
1 site
G A G C T C C T C G A G
SacI  (297)
1 site
G A G C T C C T C G A G
BamHI  (301)
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.
EcoRI  (307)
1 site
G A A T T C C T T A A G
BsrGI  (313)
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.
AscI  (320)
1 site
G G C G C G C C C C G C G C G G
BssHII  (320)
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.
PstI  (330)
1 site
C T G C A G G A C G T C
SbfI  (330)
1 site
C C T G C A G G G G A C G T C C
ZraI  (334)
1 site
G A C G T C C T G C A G
SalI  (335)
1 site
G T C G A C C A G C T G
SgrDI  (335)
1 site
C G T C G A C G G C A G C T G C
AatII  (336)
1 site
G A C G T C C T G C A G
HincII  (337)
1 site
G T Y R A C C A R Y T G
Acc65I  (341)
1 site
G G T A C C C C A T G G
KpnI  (345)
1 site
G G T A C C C C A T G G
BspDI  (348)
1 site
A T C G A T T A G C T A
ClaI  (348)
1 site
A T C G A T T A G C T A
MluI  (353)
1 site
A C G C G T T G C G C A
BstBI  (359)
1 site
T T C G A A A A G C T T
HindIII  (362)
1 site
A A G C T T T T C G A A
EagI  (369)
1 site
C G G C C G G C C G G C
NotI  (369)
1 site
G C G G C C G C C G C C G G C G
PvuII  (379)
1 site
C A G C T G G T C G A C
BstZ17I  (384)
1 site
G T A T A C C A T A T G
PmlI  (389)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
PaeR7I  (431)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (431)
1 site
C T C G A G G A G C T C
PacI  (464)
1 site
T T A A T T A A A A T T A A T T
AvrII  (597)
1 site
C C T A G G G G A T C C
BlpI  (1001)
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.
BspEI  (1078)
1 site
T C C G G A A G G C C T
BtgZI  (1321)
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.
DraIII  (1329)
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  (1454)
1 site
T T A T A A A A T A T T
SspI  (1534)
1 site
A A T A T T T T A T A A
AhdI  (1739)
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.
AmpR
1666 .. 2526  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1666 .. 2457  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1666 .. 2526  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2458 .. 2526  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1666 .. 2526  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2619 .. 3207  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2619 .. 3207  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1096 .. 1551  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1096 .. 1551  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
lacZα
54 .. 491  =  438 bp
145 amino acids  =  16.2 kDa
Product: LacZα fragment of β-galactosidase
lacZα
54 .. 491  =  438 bp
145 amino acids  =  16.2 kDa
Product: LacZα fragment of β-galactosidase
rrnB T1 terminator
739 .. 825  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
739 .. 825  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
T7 terminator
1012 .. 1059  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
1012 .. 1059  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
tet promoter
541 .. 569  =  29 bp
   Segment 3:  -10  
   541 .. 546  =  6 bp
E. coli promoter for tetracycline efflux protein gene
tet promoter
541 .. 569  =  29 bp
   Segment 2:  
   547 .. 563  =  17 bp
E. coli promoter for tetracycline efflux protein gene
tet promoter
541 .. 569  =  29 bp
   Segment 1:  -35  
   564 .. 569  =  6 bp
E. coli promoter for tetracycline efflux protein gene
tet promoter
541 .. 569  =  29 bp
3 segments
E. coli promoter for tetracycline efflux protein gene
rrnB T2 terminator
917 .. 944  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
917 .. 944  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
lac operator
20 .. 44  =  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
20 .. 44  =  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 (symmetric)
3606 .. 3625  =  20 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). The
symmetric lac operator was optimized for tight
binding of lac repressor.
lac operator (symmetric)
3606 .. 3625  =  20 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). The
symmetric lac operator was optimized for tight
binding of lac repressor.
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
HSV tag
398 .. 430  =  33 bp
11 amino acids  =  1.2 kDa
Product: HSV (herpes simplex virus) epitope tag
HSV tag
398 .. 430  =  33 bp
11 amino acids  =  1.2 kDa
Product: HSV (herpes simplex virus) epitope tag
6xHis
437 .. 454  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
437 .. 454  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
blunt sites
283 .. 298  =  16 bp
blunt cutter sites that allow ORF insertion in all
three reading frames
blunt sites
283 .. 298  =  16 bp
blunt cutter sites that allow ORF insertion in all
three reading frames
blunt sites
377 .. 392  =  16 bp
blunt cutter sites that allow ORF insertion in all
three reading frames
blunt sites
377 .. 392  =  16 bp
blunt cutter sites that allow ORF insertion in all
three reading frames
RBS
265 .. 270  =  6 bp
ribosome binding site
RBS
265 .. 270  =  6 bp
ribosome binding site
ATG
278 .. 280  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
278 .. 280  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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