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Plasmid Files

d4EGFP

EGFP destabilized by a mutated version of residues 422-461 of mouse ornithine decarboxylase, giving an in vivo half-life of ~4 hours.

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d4EGFP Sequence and Mapd4EGFP.dna
Map and Sequence File   
Sequence Author:  Clontech
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 800 600 400 200 EGFP destabilizati... d4EGFP End (846) BstAPI (820) PstI (816) SfcI (812) BsrBI (794) BssHII (764) TsoI (739) BtgI - NcoI - StyI (726) BlpI (721) HindIII (718) BsrGI (710) TaqII (625) BsiHKAI (610) BmrI (606) BstYI (500) PfoI * (281) Bts α I (214) BssS α I (181) BsrFI (151) BtgZI (122) BanI (36) Start (0) d4EGFP 846 bp
End  (846)
0 sites
BstAPI  (820)
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.
PstI  (816)
1 site
C T G C A G G A C G T C
SfcI  (812)
1 site
C T R Y A G G A Y R T C

Sticky ends from different SfcI sites may not be compatible.
SfcI quickly loses activity at 37°C, but can be used at 25°C for long
incubations.
BsrBI  (794)
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.
BssHII  (764)
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.
TsoI  (739)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
BtgI  (726)
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  (726)
1 site
C C A T G G G G T A C C
StyI  (726)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BlpI  (721)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
HindIII  (718)
1 site
A A G C T T T T C G A A
BsrGI  (710)
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.
TaqII  (625)
1 site
G A C C G A ( N ) 9 N N C T G G C T ( N ) 9

Sticky ends from different TaqII sites may not be compatible.
BsiHKAI  (610)
1 site
G W G C W C C W C G W G

Sticky ends from different BsiHKAI sites may not be compatible.
BmrI  (606)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the
absence of magnesium.
BstYI  (500)
1 site
R G A T C Y Y C T A G R
PfoI  (281)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BtsαI  (214)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsI sites may not be compatible.
BssSαI  (181)
1 site
C A C G A G G T G C T C
BsrFI  (151)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI  (122)
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.
BanI  (36)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
Start  (0)
0 sites
d4EGFP
1 .. 846  =  846 bp
281 amino acids  =  31.4 kDa
   Segment 1:  
   1 .. 3  =  3 bp
   1 amino acid  =  149.2 Da
Product: EGFP destabilized by a mutated version of
residues 422-461 of mouse ornithine decarboxylase,
giving an in vivo half-life of ~4 hours
mammalian codon-optimized
d4EGFP
1 .. 846  =  846 bp
281 amino acids  =  31.4 kDa
   Segment 2:  1a  
   4 .. 6  =  3 bp
   1 amino acid  =  117.2 Da
Product: EGFP destabilized by a mutated version of
residues 422-461 of mouse ornithine decarboxylase,
giving an in vivo half-life of ~4 hours
mammalian codon-optimized
d4EGFP
1 .. 846  =  846 bp
281 amino acids  =  31.4 kDa
   Segment 3:  EGFP  
   7 .. 717  =  711 bp
   237 amino acids  =  26.7 kDa
Product: EGFP destabilized by a mutated version of
residues 422-461 of mouse ornithine decarboxylase,
giving an in vivo half-life of ~4 hours
mammalian codon-optimized
d4EGFP
1 .. 846  =  846 bp
281 amino acids  =  31.4 kDa
   Segment 4:  
   718 .. 723  =  6 bp
   2 amino acids  =  259.3 Da
Product: EGFP destabilized by a mutated version of
residues 422-461 of mouse ornithine decarboxylase,
giving an in vivo half-life of ~4 hours
mammalian codon-optimized
d4EGFP
1 .. 846  =  846 bp
281 amino acids  =  31.4 kDa
   Segment 5:  destabilization domain  
   724 .. 846  =  123 bp
   40 amino acids  =  4.2 kDa
Product: EGFP destabilized by a mutated version of
residues 422-461 of mouse ornithine decarboxylase,
giving an in vivo half-life of ~4 hours
mammalian codon-optimized
d4EGFP
1 .. 846  =  846 bp
281 amino acids  =  31.4 kDa
5 segments
Product: EGFP destabilized by a mutated version of
residues 422-461 of mouse ornithine decarboxylase,
giving an in vivo half-life of ~4 hours
mammalian codon-optimized
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