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Plasmid Files

pDEST™ R4-R3

Gateway® destination vector for generating an expression clone by three-fragment multisite recombination.

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pDEST R4-R3 Sequence and MappDEST R4-R3.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AflIII - PciI (4018) PspFI (3718) BseYI (3714) AhdI (3130) NmeAIII (2983) AseI (2955) XmnI (2530) BsrBI (2288) AatII (2211) ZraI (2209) EcoO109I (2150) PfoI (2091) SalI (161) PstI (171) BfuAI - BspMI (174) BstXI (309) BmgBI (388) BsaBI * (409) AvaI - BsoBI - TspMI - XmaI (420) BmeT110I (421) SmaI - SrfI (422) BsrGI (445) BbvCI (565) BstZ17I (758) BssHII (795) XbaI (842) BtgI - NcoI - StyI (988) PasI (1058) PflMI * (1064) ApoI - EcoRI (1289) BspEI (1293) EagI - NotI (1610) KasI (1903) NarI (1904) SfoI (1905) PluTI (1907) NdeI (1956) BstAPI (1960) pDEST™ R4-R3 4107 bp
AflIII  (4018)
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  (4018)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (3718)
1 site
C C C A G C G G G T C G
BseYI  (3714)
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.
AhdI  (3130)
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.
NmeAIII  (2983)
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).
AseI  (2955)
1 site
A T T A A T T A A T T A
XmnI  (2530)
1 site
G A A N N N N T T C C T T N N N N A A G
BsrBI  (2288)
1 site
C C G C T C G G C G A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BsrBI will not always regenerate a BsrBI site.
BsrBI is typically used at 37°C, but can be used at temperatures
up to 50°C.
AatII  (2211)
1 site
G A C G T C C T G C A G
ZraI  (2209)
1 site
G A C G T C C T G C A G
EcoO109I  (2150)
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.
PfoI  (2091)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI sites may not be compatible.
SalI  (161)
1 site
G T C G A C C A G C T G
PstI  (171)
1 site
C T G C A G G A C G T C
BfuAI  (174)
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  (174)
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.
BstXI  (309)
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.
BmgBI  (388)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
BsaBI  (409)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
AvaI  (420)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (420)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (420)
1 site
C C C G G G G G G C C C
XmaI  (420)
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.
BmeT110I  (421)
1 site
C Y C G R G G R G C Y C
SmaI  (422)
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  (422)
1 site
G C C C G G G C C G G G C C C G
BsrGI  (445)
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.
BbvCI  (565)
1 site
C C T C A G C G G A G T C G
BstZ17I  (758)
1 site
G T A T A C C A T A T G
BssHII  (795)
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.
XbaI  (842)
1 site
T C T A G A A G A T C T
BtgI  (988)
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  (988)
1 site
C C A T G G G G T A C C
StyI  (988)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PasI  (1058)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
PflMI  (1064)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
ApoI  (1289)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRI  (1289)
1 site
G A A T T C C T T A A G
BspEI  (1293)
1 site
T C C G G A A G G C C T
EagI  (1610)
1 site
C G G C C G G C C G G C
NotI  (1610)
1 site
G C G G C C G C C G C C G G C G
KasI  (1903)
1 site
G G C G C C C C G C G G
NarI  (1904)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (1905)
1 site
G G C G C C C C G C G G
PluTI  (1907)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
NdeI  (1956)
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.
BstAPI  (1960)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
AmpR
2343 .. 3203  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2343 .. 2411  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2343 .. 3203  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2412 .. 3203  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2343 .. 3203  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
CmR
848 .. 1507  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
848 .. 1507  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3374 .. 3962  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3374 .. 3962  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
201 .. 506  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
201 .. 506  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
attR4
36 .. 160  =  125 bp
recombination site for the Gateway® LR reaction
attR4
36 .. 160  =  125 bp
recombination site for the Gateway® LR reaction
attR3
1617 .. 1740  =  124 bp
recombination site for the Gateway® LR reaction
attR3
1617 .. 1740  =  124 bp
recombination site for the Gateway® LR reaction
AmpR promoter
2238 .. 2342  =  105 bp
AmpR promoter
2238 .. 2342  =  105 bp
lac UV5 promoter
1561 .. 1591  =  31 bp
   Segment 3:  -10  
   1561 .. 1567  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1561 .. 1591  =  31 bp
   Segment 2:  
   1568 .. 1585  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1561 .. 1591  =  31 bp
   Segment 1:  -35  
   1586 .. 1591  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
1561 .. 1591  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
M13 rev
1 .. 17  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
1 .. 17  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
1748 .. 1764  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 fwd
1748 .. 1764  =  17 bp
common sequencing primer, one of multiple similar
variants
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