pKillerOrange-N

Vector for fusing KillerOrange to the C-terminus of a partner protein.

Sequence Author: Evrogen

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PfoI (3525) RsrII (3266) BsrDI (2983) PflFI - Tth111I (2868) FspI (2852) MscI (2832) PluTI (2753) SfoI (2751) NarI (2750) KasI (2749) BspDI * - ClaI * (2590) StuI (2571) BseRI (2568) SfiI (2525) AseI (7) NdeI (234) SnaBI (340) NheI (591) BmtI (595) AfeI (596) BglII (609) PaeR7I - XhoI (613) Eco53kI (618) SacI (620) HindIII (622) EcoRI (629) PstI (638) SalI (639) AccI (640) Acc65I (645) KpnI (649) SacII (652) PspOMI (653) TspMI - XmaI (656) ApaI (657) SmaI (658) BamHI (660) AgeI (666) Bpu10I (946) BlpI (1012) PaqCI (1030) PmlI (1317) NotI (1394) XbaI * (1404) MfeI (1500) HpaI (1513) BtsI - BtsαI (1589) AflII (1632) CsiI - SexAI * (2339) pKillerOrange-N 4726 bp
PfoI  (3525)
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.
RsrII  (3266)
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  (2983)
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  (2868)
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  (2868)
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  (2852)
1 site
T G C G C A A C G C G T
MscI  (2832)
1 site
T G G C C A A C C G G T
PluTI  (2753)
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  (2751)
1 site
G G C G C C C C G C G G
NarI  (2750)
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  (2749)
1 site
G G C G C C C C G C G G
BspDI  (2590)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (2590)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
StuI  (2571)
1 site
A G G C C T T C C G G A
BseRI  (2568)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
SfiI  (2525)
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
HindIII  (622)
1 site
A A G C T T T T C G A A
EcoRI  (629)
1 site
G A A T T C C T T A A G
PstI  (638)
1 site
C T G C A G G A C G T C
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
Bpu10I  (946)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BlpI  (1012)
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.
PaqCI  (1030)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
PmlI  (1317)
1 site
C A C G T G G T G C A C
NotI  (1394)
1 site
G C G G C C G C C G C C G G C G
XbaI  (1404)
1 site
T C T A G A A G A T C T
* Blocked by Dam methylation.
MfeI  (1500)
1 site
C A A T T G G T T A A C
HpaI  (1513)
1 site
G T T A A C C A A T T G
BtsI  (1589)
1 site
G C A G T G N N C G T C A C
BtsαI  (1589)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsαI sites may not be compatible.
AflII  (1632)
1 site
C T T A A G G A A T T C
CsiI  (2339)
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  (2339)
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.
NeoR/KanR
2622 .. 3416  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
NeoR/KanR
2622 .. 3416  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from Tn5
confers resistance to neomycin, kanamycin, and G418 (Geneticin®)
KillerOrange
679 .. 1389  =  711 bp
237 amino acids  =  26.4 kDa
Product: orange fluorescent protein KillerOrange, a genetically-encoded photosensitizer derived from KillerRed (Sarkisyan et al., 2015)
mammalian codon-optimized
KillerOrange
679 .. 1389  =  711 bp
237 amino acids  =  26.4 kDa
Product: orange fluorescent protein KillerOrange, a genetically-encoded photosensitizer derived from KillerRed (Sarkisyan et al., 2015)
mammalian codon-optimized
ori
4024 .. 4612  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4024 .. 4612  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
1642 .. 2097  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
1642 .. 2097  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 promoter
2230 .. 2587  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
2230 .. 2587  =  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
1514 .. 1635  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1514 .. 1635  =  122 bp
SV40 polyadenylation signal
AmpR promoter
2124 .. 2228  =  105 bp
AmpR promoter
2124 .. 2228  =  105 bp
MCS
591 .. 671  =  81 bp
multiple cloning site
MCS
591 .. 671  =  81 bp
multiple cloning site
HSV TK poly(A) signal
3648 .. 3695  =  48 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
3648 .. 3695  =  48 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
SV40 ori
2438 .. 2573  =  136 bp
SV40 origin of replication
SV40 ori
2438 .. 2573  =  136 bp
SV40 origin of replication
Kozak sequence
673 .. 682  =  10 bp
vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)
Kozak sequence
673 .. 682  =  10 bp
vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)
ORF:  679 .. 1392  =  714 bp
ORF:  237 amino acids  =  26.4 kDa
ORF:  2794 .. 3180  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  3437 .. 3886  =  450 bp
ORF:  149 amino acids  =  16.3 kDa
ORF:  2622 .. 3416  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  607 .. 918  =  312 bp
ORF:  103 amino acids  =  10.6 kDa
ORF:  447 .. 857  =  411 bp
ORF:  136 amino acids  =  14.7 kDa
ORF:  1005 .. 1373  =  369 bp
ORF:  122 amino acids  =  13.7 kDa
ORF:  2931 .. 3467