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Plasmid Files

pT7Blue-2

Multi-purpose cloning vector with T7 and SP6 promoters, and with a multiple cloning site that encodes a cleavable N-terminal S-tag.

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pT7Blue-2 Sequence and MappT7Blue-2.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 lac operator BspQI - SapI (2879) NspI (2766) PciI (2762) PspFI (2462) BseYI (2458) AlwNI (2353) AhdI (1874) BsaI (1808) BpmI (1805) NmeAIII (1727) MscI (94) BtgI - NcoI (98) BstBI (134) Acc65I (157) AgeI (160) KpnI (161) BseRI - TspMI - XmaI (189) SmaI - SrfI (191) PstI (212) MluI (214) SnaBI (222) BamHI (228) EcoNI (236) EcoRI (253) EcoRV (262) SalI (268) AccI (269) HincII (270) HindIII (274) PaeR7I - XhoI (280) AvrII (286) NheI (291) BmtI (295) XbaI (297) AleI - PmlI (308) BstXI (310) EcoO109I - PspOMI (315) ApaI (319) Eco53kI (323) SacI (325) EagI - NotI (328) PsiI (596) DraIII (724) NgoMIV (825) NaeI (827) XmnI (1274) BsaHI (1334) TatI (1391) ScaI (1393) pT7Blue-2 3103 bp
BspQI  (2879)
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  (2879)
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.
NspI  (2766)
1 site
R C A T G Y Y G T A C R
PciI  (2762)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (2462)
1 site
C C C A G C G G G T C G
BseYI  (2458)
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  (2353)
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.
AhdI  (1874)
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.
BsaI  (1808)
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.
BpmI  (1805)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI
recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its
electrophoretic mobility.
BpmI quickly loses activity at 37°C.
NmeAIII  (1727)
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).
MscI  (94)
1 site
T G G C C A A C C G G T
BtgI  (98)
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  (98)
1 site
C C A T G G G G T A C C
BstBI  (134)
1 site
T T C G A A A A G C T T
Acc65I  (157)
1 site
G G T A C C C C A T G G
AgeI  (160)
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.
KpnI  (161)
1 site
G G T A C C C C A T G G
BseRI  (189)
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.
TspMI  (189)
1 site
C C C G G G G G G C C C
XmaI  (189)
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  (191)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (191)
1 site
G C C C G G G C C G G G C C C G
PstI  (212)
1 site
C T G C A G G A C G T C
MluI  (214)
1 site
A C G C G T T G C G C A
SnaBI  (222)
1 site
T A C G T A A T G C A T
BamHI  (228)
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.
EcoNI  (236)
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.
EcoRI  (253)
1 site
G A A T T C C T T A A G
EcoRV  (262)
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.
SalI  (268)
1 site
G T C G A C C A G C T G
AccI  (269)
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.
HincII  (270)
1 site
G T Y R A C C A R Y T G
HindIII  (274)
1 site
A A G C T T T T C G A A
PaeR7I  (280)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (280)
1 site
C T C G A G G A G C T C
AvrII  (286)
1 site
C C T A G G G G A T C C
NheI  (291)
1 site
G C T A G C C G A T C G
BmtI  (295)
1 site
G C T A G C C G A T C G
XbaI  (297)
1 site
T C T A G A A G A T C T
AleI  (308)
1 site
C A C N N N N G T G G T G N N N N C A C
PmlI  (308)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BstXI  (310)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
EcoO109I  (315)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
PspOMI  (315)
1 site
G G G C C C C C C G G G
ApaI  (319)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
Eco53kI  (323)
1 site
G A G C T C C T C G A G
SacI  (325)
1 site
G A G C T C C T C G A G
EagI  (328)
1 site
C G G C C G G C C G G C
NotI  (328)
1 site
G C G G C C G C C G C C G G C G
PsiI  (596)
1 site
T T A T A A A A T A T T
DraIII  (724)
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.
NgoMIV  (825)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (827)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
XmnI  (1274)
1 site
G A A N N N N T T C C T T N N N N A A G
BsaHI  (1334)
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.
TatI  (1391)
1 site
W G T A C W W C A T G W
ScaI  (1393)
1 site
A G T A C T T C A T G A
AmpR
1087 .. 1947  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1087 .. 1155  =  69 bp
   23 amino acids  =  2.6 kDa
AmpR
1087 .. 1947  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1156 .. 1947  =  792 bp
   263 amino acids  =  28.9 kDa
AmpR
1087 .. 1947  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
ori
2118 .. 2706  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2118 .. 2706  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
LacZα
1 .. 549  =  549 bp
182 amino acids  =  20.4 kDa
Product: LacZα fragment of β-galactosidase
LacZα
1 .. 549  =  549 bp
182 amino acids  =  20.4 kDa
Product: LacZα fragment of β-galactosidase
AmpR promoter
982 .. 1086  =  105 bp
AmpR promoter
982 .. 1086  =  105 bp
lac promoter
3030 .. 3060  =  31 bp
   Segment 1:  -35  
   3030 .. 3035  =  6 bp
promoter for the E. coli lac operon
lac promoter
3030 .. 3060  =  31 bp
   Segment 2:  
   3036 .. 3053  =  18 bp
promoter for the E. coli lac operon
lac promoter
3030 .. 3060  =  31 bp
   Segment 3:  -10  
   3054 .. 3060  =  7 bp
promoter for the E. coli lac operon
lac promoter
3030 .. 3060  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
3068 .. 3084  =  17 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
3068 .. 3084  =  17 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).
f1 ori
500 .. 955  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
500 .. 955  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
MCS
92 .. 334  =  243 bp
multiple cloning site
MCS
92 .. 334  =  243 bp
multiple cloning site
T7 promoter
24 .. 42  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
24 .. 42  =  19 bp
promoter for bacteriophage T7 RNA polymerase
M13 fwd
342 .. 358  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
342 .. 358  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3092 .. 5  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3092 .. 5  =  17 bp
common sequencing primer, one of multiple similar
variants
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