pd4EGFP-N1

Vector for fusing destabilized EGFP, with an in vivo half-life of ~4 hours, to the C-terminus of a partner protein.

Sequence Author: Clontech (TaKaRa)

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AflIII - PciI (4804) ApaLI (4490) EcoO109I (3984) BsaI (3875) RsrII (3402) BsrDI (3119) PflFI - Tth111I (3004) FspI (2988) MscI (2968) PluTI (2889) SfoI (2887) NarI (2886) KasI (2885) BspDI * - ClaI * (2726) StuI (2707) SfiI (2661) SV40 promoter AseI (7) CMV enhancer NdeI (234) SnaBI (340) NheI (591) BmtI (595) AfeI (596) BglII (609) PaeR7I - XhoI (613) Eco53kI (618) SacI (620) EcoRI (629) SalI (639) AccI (640) Acc65I (645) KpnI (649) SacII (652) PspOMI (653) TspMI - XmaI (656) ApaI (657) SmaI (658) BamHI (660) AgeI (666) BsrGI (1388) BlpI (1399) BssHII (1442) NotI (1530) XbaI * (1540) MfeI (1636) HpaI (1649) AflII (1768) DraIII (2002) CsiI - SexAI * (2475) pd4EGFP-N1 4862 bp
AflIII  (4804)
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  (4804)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
ApaLI  (4490)
1 site
G T G C A C C A C G T G
EcoO109I  (3984)
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.
BsaI  (3875)
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.
RsrII  (3402)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BsrDI  (3119)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
PflFI  (3004)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (3004)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
FspI  (2988)
1 site
T G C G C A A C G C G T
MscI  (2968)
1 site
T G G C C A A C C G G T
PluTI  (2889)
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  (2887)
1 site
G G C G C C C C G C G G
NarI  (2886)
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  (2885)
1 site
G G C G C C C C G C G G
BspDI  (2726)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (2726)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
StuI  (2707)
1 site
A G G C C T T C C G G A
SfiI  (2661)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
AseI  (7)
1 site
A T T A A T T A A T T A
NdeI  (234)
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.
SnaBI  (340)
1 site
T A C G T A A T G C A T
NheI  (591)
1 site
G C T A G C C G A T C G
BmtI  (595)
1 site
G C T A G C C G A T C G
AfeI  (596)
1 site
A G C G C T T C G C G A
BglII  (609)
1 site
A G A T C T T C T A G A
PaeR7I  (613)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (613)
1 site
C T C G A G G A G C T C
Eco53kI  (618)
1 site
G A G C T C C T C G A G
SacI  (620)
1 site
G A G C T C C T C G A G
EcoRI  (629)
1 site
G A A T T C C T T A A G
SalI  (639)
1 site
G T C G A C C A G C T G
AccI  (640)
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.
Acc65I  (645)
1 site
G G T A C C C C A T G G
KpnI  (649)
1 site
G G T A C C C C A T G G
SacII  (652)
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.
PspOMI  (653)
1 site
G G G C C C C C C G G G
TspMI  (656)
1 site
C C C G G G G G G C C C
XmaI  (656)
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.
ApaI  (657)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SmaI  (658)
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  (660)
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.
AgeI  (666)
1 site
A C C G G T T G G C C A
BsrGI  (1388)
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.
BlpI  (1399)
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.
BssHII  (1442)
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.
NotI  (1530)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1540)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
MfeI  (1636)
1 site
C A A T T G G T T A A C
HpaI  (1649)
1 site
G T T A A C C A A T T G
AflII  (1768)
1 site
C T T A A G G A A T T C
DraIII  (2002)
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.
CsiI  (2475)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (2475)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
d4EGFP
679 .. 1524  =  846 bp
281 amino acids  =  31.4 kDa
5 segments
   Segment 1:  
   679 .. 681  =  3 bp
   1 amino acid  =  149.2 Da
Product: EGFP destabilized by a mutated version of residues 422-461 of mouse ornithine decarboxylase
mammalian codon-optimized
d4EGFP
679 .. 1524  =  846 bp
281 amino acids  =  31.4 kDa
5 segments
   Segment 2:  1a  
   682 .. 684  =  3 bp
   1 amino acid  =  117.1 Da
Product: EGFP destabilized by a mutated version of residues 422-461 of mouse ornithine decarboxylase
mammalian codon-optimized
d4EGFP
679 .. 1524  =  846 bp
281 amino acids  =  31.4 kDa
5 segments
   Segment 3:  EGFP  
   685 .. 1395  =  711 bp
   237 amino acids  =  26.7 kDa
Product: EGFP destabilized by a mutated version of residues 422-461 of mouse ornithine decarboxylase
mammalian codon-optimized
d4EGFP
679 .. 1524  =  846 bp
281 amino acids  =  31.4 kDa
5 segments
   Segment 4:  
   1396 .. 1401  =  6 bp
   2 amino acids  =  259.3 Da
Product: EGFP destabilized by a mutated version of residues 422-461 of mouse ornithine decarboxylase
mammalian codon-optimized
d4EGFP
679 .. 1524  =  846 bp
281 amino acids  =  31.4 kDa
5 segments
   Segment 5:  destabilization domain  
   1402 .. 1524  =  123 bp
   40 amino acids  =  4.2 kDa
Product: EGFP destabilized by a mutated version of residues 422-461 of mouse ornithine decarboxylase
mammalian codon-optimized
d4EGFP
679 .. 1524  =  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
mammalian codon-optimized
NeoR/KanR
2758 .. 3552  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
NeoR/KanR
2758 .. 3552  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin)
ori
4160 .. 4748  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4160 .. 4748  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
1778 .. 2233  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1778 .. 2233  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 promoter
2366 .. 2723  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2366 .. 2723  =  358 bp
SV40 enhancer and early promoter
CMV enhancer
61 .. 364  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
61 .. 364  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
365 .. 568  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
365 .. 568  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
1650 .. 1771  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1650 .. 1771  =  122 bp
SV40 polyadenylation signal
AmpR promoter
2260 .. 2364  =  105 bp
AmpR promoter
2260 .. 2364  =  105 bp
MCS
591 .. 671  =  81 bp
multiple cloning site of fluorescent protein plasmids
MCS
591 .. 671  =  81 bp
multiple cloning site of fluorescent protein plasmids
HSV TK poly(A) signal
3784 .. 3831  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
3784 .. 3831  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
SV40 ori
2574 .. 2709  =  136 bp
SV40 origin of replication
SV40 ori
2574 .. 2709  =  136 bp
SV40 origin of replication
ORF:  679 .. 1524  =  846 bp
ORF:  281 amino acids  =  31.4 kDa
ORF:  2758 .. 3552  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  2930 .. 3316  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  3573 .. 4022  =  450 bp
ORF:  149 amino acids  =  16.3 kDa
ORF:  447 .. 722  =  276 bp
ORF:  91 amino acids  =  9.6 kDa
ORF:  607 .. 1407  =  801 bp
ORF:  266 amino acids  =  27.1 kDa
ORF:  3067 .. 3603  =  537 bp
ORF:  178 amino acids  =  19.8 kDa
ORF:  3778 .. 4011  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
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Download pd4EGFP-N1.dna file

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