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Plasmid Files

pT7Blue-3

Cloning vector with a T7 promoter and dual ampicillin/kanamycin resistance, and with a multiple cloning site that includes an EcoRV site flanked by EcoRI sites.

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pT7Blue-3 Sequence and MappT7Blue-3.dna
Map and Sequence File   
Sequence Author:  Novagen (EMD Millipore)
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 Acc65I (51) M13 rev lac operator BspQI - SapI (3597) PciI (3480) PspFI (3180) BseYI (3176) AlwNI (3071) PflMI (2630) Bpu10I (2384) BsmBI (2383) AsiSI (2367) EcoNI (2279) SmaI (2241) TspMI - XmaI (2239) BspDI - ClaI (2058) NruI (2024) KpnI (55) SphI (63) PstI (68) MluI (70) SnaBI (78) BamHI (84) EcoRI (91) EcoRV (101) EcoRI (106) SalI (112) AccI (113) HincII (114) HindIII (118) PaeR7I - XhoI (124) AvrII - StyI (130) NheI (135) BmtI (139) XbaI (141) AleI - PmlI (152) BstXI (154) EcoO109I - PspOMI (159) ApaI (163) Eco53kI (167) SacI (169) EagI - NotI (172) M13 fwd AanI - PsiI (439) DraIII (567) BtgZI (568) NgoMIV (668) NaeI (670) BsaHI (1177) TatI (1234) ScaI (1236) NmeAIII (1570) BpmI (1648) BsaI (1651) AhdI (1717) pT7Blue-3 3821 bp
Acc65I  (51)
1 site
G G T A C C C C A T G G
BspQI  (3597)
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  (3597)
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.
PciI  (3480)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (3180)
1 site
C C C A G C G G G T C G
BseYI  (3176)
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  (3071)
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.
PflMI  (2630)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
Bpu10I  (2384)
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.
BsmBI  (2383)
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.
AsiSI  (2367)
1 site
G C G A T C G C C G C T A G C G
EcoNI  (2279)
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.
SmaI  (2241)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (2239)
1 site
C C C G G G G G G C C C
XmaI  (2239)
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.
BspDI  (2058)
1 site
A T C G A T T A G C T A
ClaI  (2058)
1 site
A T C G A T T A G C T A
NruI  (2024)
1 site
T C G C G A A G C G C T
KpnI  (55)
1 site
G G T A C C C C A T G G
SphI  (63)
1 site
G C A T G C C G T A C G
PstI  (68)
1 site
C T G C A G G A C G T C
MluI  (70)
1 site
A C G C G T T G C G C A
SnaBI  (78)
1 site
T A C G T A A T G C A T
BamHI  (84)
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  (91)
2 sites
G A A T T C C T T A A G
EcoRV  (101)
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.
EcoRI  (106)
2 sites
G A A T T C C T T A A G
SalI  (112)
1 site
G T C G A C C A G C T G
AccI  (113)
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  (114)
1 site
G T Y R A C C A R Y T G
HindIII  (118)
1 site
A A G C T T T T C G A A
PaeR7I  (124)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (124)
1 site
C T C G A G G A G C T C
AvrII  (130)
1 site
C C T A G G G G A T C C
StyI  (130)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
NheI  (135)
1 site
G C T A G C C G A T C G
BmtI  (139)
1 site
G C T A G C C G A T C G
XbaI  (141)
1 site
T C T A G A A G A T C T
AleI  (152)
1 site
C A C N N N N G T G G T G N N N N C A C
PmlI  (152)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BstXI  (154)
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  (159)
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  (159)
1 site
G G G C C C C C C G G G
ApaI  (163)
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  (167)
1 site
G A G C T C C T C G A G
SacI  (169)
1 site
G A G C T C C T C G A G
EagI  (172)
1 site
C G G C C G G C C G G C
NotI  (172)
1 site
G C G G C C G C C G C C G G C G
AanI  (439)
1 site
T T A T A A A A T A T T
PsiI  (439)
1 site
T T A T A A A A T A T T
DraIII  (567)
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.
BtgZI  (568)
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.
NgoMIV  (668)
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  (670)
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.
BsaHI  (1177)
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  (1234)
1 site
W G T A C W W C A T G W
ScaI  (1236)
1 site
A G T A C T T C A T G A
NmeAIII  (1570)
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).
BpmI  (1648)
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.
BsaI  (1651)
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  (1717)
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
930 .. 1790  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   930 .. 998  =  69 bp
   23 amino acids  =  2.6 kDa
AmpR
930 .. 1790  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   999 .. 1790  =  792 bp
   263 amino acids  =  28.9 kDa
AmpR
930 .. 1790  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
KanR
1936 .. 2751  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
1936 .. 2751  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
2836 .. 3424  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2836 .. 3424  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
343 .. 798  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
343 .. 798  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
MCS
51 .. 178  =  128 bp
multiple cloning site
MCS
51 .. 178  =  128 bp
multiple cloning site
AmpR promoter
825 .. 929  =  105 bp
AmpR promoter
825 .. 929  =  105 bp
lac promoter
3748 .. 3778  =  31 bp
   Segment 1:  -35  
   3748 .. 3753  =  6 bp
promoter for the E. coli lac operon
lac promoter
3748 .. 3778  =  31 bp
   Segment 2:  
   3754 .. 3771  =  18 bp
promoter for the E. coli lac operon
lac promoter
3748 .. 3778  =  31 bp
   Segment 3:  -10  
   3772 .. 3778  =  7 bp
promoter for the E. coli lac operon
lac promoter
3748 .. 3778  =  31 bp
3 segments
promoter for the E. coli lac operon
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
186 .. 202  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
186 .. 202  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
3786 .. 3802  =  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
3786 .. 3802  =  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).
M13 rev
3810 .. 5  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
3810 .. 5  =  17 bp
common sequencing primer, one of multiple similar
variants
LacZα
1 .. 351  =  351 bp
116 amino acids  =  13.3 kDa
Product: LacZα fragment of β-galactosidase
LacZα
1 .. 351  =  351 bp
116 amino acids  =  13.3 kDa
Product: LacZα fragment of β-galactosidase
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