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Plasmid Files

pBluescript II SK(-)

Standard cloning vector (phagemid excised from lambda ZAPII). The f1 (–) orientation allows rescue of antisense strand ssDNA. pBluescript II KS(–) has a reversed MCS.

 
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 NgoMIV (129) XmnI (2645) BsaHI (2583) ScaI (2526) TatI (2524) NmeAIII (2194) BsaI (2107) AhdI (2046) AlwNI (1569) PspFI (1461) NaeI (131) BtgZI (229) BsaAI (234) DraIII (237) PsiI (362) M13 fwd Acc65I (653) KpnI (657) PspOMI (659) EcoO109I (660) ApaI (663) AbsI - PaeR7I - PspXI - XhoI (668) SalI (674) AccI (675) HincII (676) BspDI - ClaI (684) HindIII (689) EcoRV (697) EcoRI (701) PstI (711) TspMI - XmaI (713) SmaI (715) BamHI (719) SpeI (725) XbaI (731) EagI - NotI (738) BtgI (747) AleI (749) SacII (750) BstXI (751) Eco53kI (757) SacI (759) T3 promoter BspQI - SapI (1037) AflIII - PciI (1153) NspI (1157) BseYI (1457) pBluescript II SK(-) 2961 bp
NgoMIV  (129)
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.
XmnI  (2645)
1 site
G A A N N N N T T C C T T N N N N A A G
BsaHI  (2583)
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  (2526)
1 site
A G T A C T T C A T G A
TatI  (2524)
1 site
W G T A C W W C A T G W
NmeAIII  (2194)
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).
BsaI  (2107)
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  (2046)
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  (1569)
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.
PspFI  (1461)
1 site
C C C A G C G G G T C G
NaeI  (131)
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.
BtgZI  (229)
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.
BsaAI  (234)
1 site
Y A C G T R R T G C A Y
DraIII  (237)
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  (362)
1 site
T T A T A A A A T A T T
Acc65I  (653)
1 site
G G T A C C C C A T G G
KpnI  (657)
1 site
G G T A C C C C A T G G
PspOMI  (659)
1 site
G G G C C C C C C G G G
EcoO109I  (660)
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.
ApaI  (663)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AbsI  (668)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (668)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (668)
1 site
V C T C G A G B B G A G C T C V
XhoI  (668)
1 site
C T C G A G G A G C T C
SalI  (674)
1 site
G T C G A C C A G C T G
AccI  (675)
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  (676)
1 site
G T Y R A C C A R Y T G
BspDI  (684)
1 site
A T C G A T T A G C T A
ClaI  (684)
1 site
A T C G A T T A G C T A
HindIII  (689)
1 site
A A G C T T T T C G A A
EcoRV  (697)
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  (701)
1 site
G A A T T C C T T A A G
PstI  (711)
1 site
C T G C A G G A C G T C
TspMI  (713)
1 site
C C C G G G G G G C C C
XmaI  (713)
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  (715)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BamHI  (719)
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.
SpeI  (725)
1 site
A C T A G T T G A T C A
XbaI  (731)
1 site
T C T A G A A G A T C T
EagI  (738)
1 site
C G G C C G G C C G G C
NotI  (738)
1 site
G C G G C C G C C G C C G G C G
BtgI  (747)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
AleI  (749)
1 site
C A C N N N N G T G G T G N N N N C A C
SacII  (750)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BstXI  (751)
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.
Eco53kI  (757)
1 site
G A G C T C C T C G A G
SacI  (759)
1 site
G A G C T C C T C G A G
BspQI  (1037)
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  (1037)
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  (1153)
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  (1153)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (1157)
1 site
R C A T G Y Y G T A C R
BseYI  (1457)
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.
AmpR
1973 .. 2833  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1973 .. 2764  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1973 .. 2833  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2765 .. 2833  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1973 .. 2833  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1214 .. 1802  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1214 .. 1802  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4 .. 459  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4 .. 459  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
MCS
653 .. 760  =  108 bp
pBluescript multiple cloning site
MCS
653 .. 760  =  108 bp
pBluescript multiple cloning site
AmpR promoter
2834 .. 2938  =  105 bp
AmpR promoter
2834 .. 2938  =  105 bp
lac promoter
860 .. 890  =  31 bp
   Segment 3:  -10  
   860 .. 866  =  7 bp
promoter for the E. coli lac operon
lac promoter
860 .. 890  =  31 bp
   Segment 2:  
   867 .. 884  =  18 bp
promoter for the E. coli lac operon
lac promoter
860 .. 890  =  31 bp
   Segment 1:  -35  
   885 .. 890  =  6 bp
promoter for the E. coli lac operon
lac promoter
860 .. 890  =  31 bp
3 segments
promoter for the E. coli lac operon
T7 promoter
626 .. 644  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
626 .. 644  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T3 promoter
773 .. 791  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
773 .. 791  =  19 bp
promoter for bacteriophage T3 RNA polymerase
M13 fwd
600 .. 616  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
600 .. 616  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
812 .. 828  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
812 .. 828  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
836 .. 852  =  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
836 .. 852  =  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).
lacZα
451 .. 816  =  366 bp
121 amino acids  =  13.3 kDa
Product: LacZα fragment of β-galactosidase
lacZα
451 .. 816  =  366 bp
121 amino acids  =  13.3 kDa
Product: LacZα fragment of β-galactosidase
KS primer
670 .. 686  =  17 bp
common sequencing primer, one of multiple similar
variants
KS primer
670 .. 686  =  17 bp
common sequencing primer, one of multiple similar
variants
SK primer
720 .. 736  =  17 bp
common sequencing primer, one of multiple similar
variants
SK primer
720 .. 736  =  17 bp
common sequencing primer, one of multiple similar
variants
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