pDEST R4-R3 Vector II

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

Sequence Author: Thermo Fisher (Invitrogen)

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AflIII - PciI (4466) PspFI (4166) BseYI (4162) AhdI (3578) NmeAIII (3431) AseI (3403) BsrBI (2736) AatII (2659) ZraI (2657) EcoO109I (2598) PfoI (2539) BstAPI (2408) NdeI (2404) PluTI (2355) SfoI (2353) NarI (2352) KasI (2351) SalI (161) PstI (478) BfuAI - BspMI (481) BstXI (616) BmgBI (695) BsaBI * (716) AvaI - BsoBI - TspMI - XmaI (727) SmaI - SrfI (729) BsrGI (752) BbvCI (872) BstZ17I (1065) BssHII (1102) BamHI (1143) BtgI - NcoI - StyI (1297) ApoI - EcoRI (1598) BspEI (1602) BsaAI (1909) EagI (2058) M13 fwd pDEST™ R4-R3 Vector II 4555 bp
AflIII  (4466)
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  (4466)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (4166)
1 site
C C C A G C G G G T C G
BseYI  (4162)
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  (3578)
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  (3431)
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  (3403)
1 site
A T T A A T T A A T T A
BsrBI  (2736)
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  (2659)
1 site
G A C G T C C T G C A G
ZraI  (2657)
1 site
G A C G T C C T G C A G
EcoO109I  (2598)
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  (2539)
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.
BstAPI  (2408)
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.
NdeI  (2404)
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.
PluTI  (2355)
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.
SfoI  (2353)
1 site
G G C G C C C C G C G G
NarI  (2352)
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.
KasI  (2351)
1 site
G G C G C C C C G C G G
SalI  (161)
1 site
G T C G A C C A G C T G
PstI  (478)
1 site
C T G C A G G A C G T C
BfuAI  (481)
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  (481)
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  (616)
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  (695)
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  (716)
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  (727)
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  (727)
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  (727)
1 site
C C C G G G G G G C C C
XmaI  (727)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
SmaI  (729)
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  (729)
1 site
G C C C G G G C C G G G C C C G
BsrGI  (752)
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  (872)
1 site
C C T C A G C G G A G T C G
BstZ17I  (1065)
1 site
G T A T A C C A T A T G
BssHII  (1102)
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.
BamHI  (1143)
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.
BtgI  (1297)
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  (1297)
1 site
C C A T G G G G T A C C
StyI  (1297)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
ApoI  (1598)
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  (1598)
1 site
G A A T T C C T T A A G
BspEI  (1602)
1 site
T C C G G A A G G C C T
BsaAI  (1909)
1 site
Y A C G T R R T G C A Y
EagI  (2058)
1 site
C G G C C G G C C G G C
AmpR
2791 .. 3651  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   2791 .. 2859  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2791 .. 3651  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   2860 .. 3651  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2791 .. 3651  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
1163 .. 1816  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1163 .. 1816  =  654 bp
218 amino acids  =  25.6 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3822 .. 4410  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3822 .. 4410  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ccdB
508 .. 813  =  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
508 .. 813  =  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
2065 .. 2188  =  124 bp
recombination site for the Gateway® LR reaction
attR3
2065 .. 2188  =  124 bp
recombination site for the Gateway® LR reaction
AmpR promoter
2686 .. 2790  =  105 bp
AmpR promoter
2686 .. 2790  =  105 bp
cat promoter
1817 .. 1919  =  103 bp
promoter of the E. coli cat gene
cat promoter
1817 .. 1919  =  103 bp
promoter of the E. coli cat gene
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
2196 .. 2212  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
2196 .. 2212  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  2791 .. 3651  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  1133 .. 1816  =  684 bp
ORF:  227 amino acids  =  26.7 kDa
ORF:  508 .. 813  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  2154 .. 2405  =  252 bp
ORF:  83 amino acids  =  9.8 kDa
ORF:  3255 .. 3521  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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