pSpark Done (linearized)

Linearized high copy vector for very efficient cloning of blunt-ended PCR products, with an MCS containing EcoRI and NotI sites on each side of the insertion site.

Sequence Author: Canvax Biotech

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<EcoRV> (3016) EcoRI (3008) SacII (3005) NotI (2999) NcoI - StyI (2993) SphI (2982) AatII (2976) ZraI (2974) ApaI (2970) PspOMI (2966) NaeI (2648) NgoMIV (2646) BtgZI (2546) BsaAI - DraIII (2545) PsiI (2417) XmnI (1950) ScaI (1831) TatI (1829) <EcoRV> (0) SpeI (5) EcoRI (11) BfuAI - BspMI - NotI (18) PstI - SbfI (29) SalI (31) AccI (32) HincII (33) NdeI (38) Eco53kI (48) SacI (50) MluI (55) BstXI (59) NsiI (68) BspQI - SapI (342) PciI (458) BseYI (762) PspFI (766) AlwNI (874) AhdI (1351) BsaI (1412) BpmI (1421) NmeAIII (1499) pSpark® Done 3016 bp
End  (3016)
0 sites
EcoRI  (3008)
2 sites
G A A T T C C T T A A G
SacII  (3005)
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.
NotI  (2999)
2 sites
G C G G C C G C C G C C G G C G
NcoI  (2993)
1 site
C C A T G G G G T A C C
StyI  (2993)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
SphI  (2982)
1 site
G C A T G C C G T A C G
AatII  (2976)
1 site
G A C G T C C T G C A G
ZraI  (2974)
1 site
G A C G T C C T G C A G
ApaI  (2970)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (2966)
1 site
G G G C C C C C C G G G
NaeI  (2648)
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.
NgoMIV  (2646)
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.
BtgZI  (2546)
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  (2545)
1 site
Y A C G T R R T G C A Y
DraIII  (2545)
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  (2417)
1 site
T T A T A A A A T A T T
XmnI  (1950)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (1831)
1 site
A G T A C T T C A T G A
TatI  (1829)
1 site
W G T A C W W C A T G W
Start  (0)
0 sites
SpeI  (5)
1 site
A C T A G T T G A T C A
EcoRI  (11)
2 sites
G A A T T C C T T A A G
BfuAI  (18)
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  (18)
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.
NotI  (18)
2 sites
G C G G C C G C C G C C G G C G
PstI  (29)
1 site
C T G C A G G A C G T C
SbfI  (29)
1 site
C C T G C A G G G G A C G T C C
SalI  (31)
1 site
G T C G A C C A G C T G
AccI  (32)
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  (33)
1 site
G T Y R A C C A R Y T G
NdeI  (38)
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.
Eco53kI  (48)
1 site
G A G C T C C T C G A G
SacI  (50)
1 site
G A G C T C C T C G A G
MluI  (55)
1 site
A C G C G T T G C G C A
BstXI  (59)
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.
NsiI  (68)
1 site
A T G C A T T A C G T A
BspQI  (342)
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  (342)
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  (458)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (762)
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.
PspFI  (766)
1 site
C C C A G C G G G T C G
AlwNI  (874)
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  (1351)
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  (1412)
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  (1421)
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  (1499)
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).
AmpR
1278 .. 2138  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1278 .. 2069  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1278 .. 2138  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   2070 .. 2138  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1278 .. 2138  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
519 .. 1107  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
519 .. 1107  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
2321 .. 2776  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
2321 .. 2776  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
lacZα
1 .. 121  =  121 bp
40 amino acids  =  4.3 kDa
Product: LacZα fragment of β-galactosidase
lacZα
1 .. 121  =  121 bp
40 amino acids  =  4.3 kDa
Product: LacZα fragment of β-galactosidase
AmpR promoter
2139 .. 2243  =  105 bp
AmpR promoter
2139 .. 2243  =  105 bp
MCS
2966 .. 3016  =  51 bp
multiple cloning site
MCS
2966 .. 3016  =  51 bp
multiple cloning site
lac promoter
165 .. 195  =  31 bp
3 segments
   Segment 3:  -10  
   165 .. 171  =  7 bp
promoter for the E. coli lac operon
lac promoter
165 .. 195  =  31 bp
3 segments
   Segment 2:  
   172 .. 189  =  18 bp
promoter for the E. coli lac operon
lac promoter
165 .. 195  =  31 bp
3 segments
   Segment 1:  -35  
   190 .. 195  =  6 bp
promoter for the E. coli lac operon
lac promoter
165 .. 195  =  31 bp
3 segments
promoter for the E. coli lac operon
T7 promoter
2940 .. 2958  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2940 .. 2958  =  19 bp
promoter for bacteriophage T7 RNA polymerase
lac operator
141 .. 157  =  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
141 .. 157  =  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 fwd
2917 .. 2933  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
2917 .. 2933  =  17 bp
common sequencing primer, one of multiple similar variants
lacZα
2753 .. 3015  =  263 bp
86 amino acids  =  9.8 kDa
Product: LacZα fragment of β-galactosidase
lacZα
2753 .. 3015  =  263 bp
86 amino acids  =  9.8 kDa
Product: LacZα fragment of β-galactosidase
MCS
1 .. 60  =  60 bp
multiple cloning site
MCS
1 .. 60  =  60 bp
multiple cloning site
SP6 promoter
81 .. 99  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
81 .. 99  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
M13 rev
117 .. 133  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
117 .. 133  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  1408 .. 1674  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2753 .. 3016