pKillerOrange-B

Vector for expressing KillerOrange in bacteria.

Sequence Author: Evrogen

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BbsI (4105) AatII (4049) ZraI (4047) XmnI (3726) PvuI (3497) FspI (3349) AseI (3299) NmeAIII (3275) BglI (3247) AhdI (3127) AlwNI (2650) BspQI - SapI (2118) BstAPI (2058) PaeR7I - XhoI (1) PsiI (49) MfeI (59) lac operator RBS BseRI (115) ATG BsaBI * (126) BamHI (127) BanII (335) BfuAI - BspMI - PaqCI (481) PmlI (768) HindIII (844) NheI (964) BmtI (968) BspEI (1212) PasI (1448) MscI (1483) NcoI - StyI (1517) XbaI (1821) PfoI (1878) PflFI - Tth111I (1981) AccI (2006) BstZ17I (2007) NdeI (2057) pKillerOrange-B 4119 bp
BbsI  (4105)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
AatII  (4049)
1 site
G A C G T C C T G C A G
ZraI  (4047)
1 site
G A C G T C C T G C A G
XmnI  (3726)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuI  (3497)
1 site
C G A T C G G C T A G C
FspI  (3349)
1 site
T G C G C A A C G C G T
AseI  (3299)
1 site
A T T A A T T A A T T A
NmeAIII  (3275)
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).
BglI  (3247)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
AhdI  (3127)
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.
AlwNI  (2650)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
BspQI  (2118)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (2118)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
BstAPI  (2058)
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.
PaeR7I  (1)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1)
1 site
C T C G A G G A G C T C
PsiI  (49)
1 site
T T A T A A A A T A T T
MfeI  (59)
1 site
C A A T T G G T T A A C
BseRI  (115)
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.
BsaBI  (126)
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.
BamHI  (127)
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.
BanII  (335)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
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.
PaqCI  (481)
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  (768)
1 site
C A C G T G G T G C A C
HindIII  (844)
1 site
A A G C T T T T C G A A
NheI  (964)
1 site
G C T A G C C G A T C G
BmtI  (968)
1 site
G C T A G C C G A T C G
BspEI  (1212)
1 site
T C C G G A A G G C C T
PasI  (1448)
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.
MscI  (1483)
1 site
T G G C C A A C C G G T
NcoI  (1517)
1 site
C C A T G G G G T A C C
StyI  (1517)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
XbaI  (1821)
1 site
T C T A G A A G A T C T
PfoI  (1878)
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.
PflFI  (1981)
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  (1981)
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.
AccI  (2006)
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.
BstZ17I  (2007)
1 site
G T A T A C C A T A T G
NdeI  (2057)
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.
AmpR
3054 .. 3914  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3054 .. 3845  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3054 .. 3914  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   3846 .. 3914  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3054 .. 3914  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ATG
115 .. 117  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
115 .. 117  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
KillerOrange
133 .. 843  =  711 bp
236 amino acids  =  26.2 kDa
Product: orange fluorescent protein KillerOrange, a genetically-encoded photosensitizer derived from KillerRed (Sarkisyan et al., 2015)
mammalian codon-optimized
KillerOrange
133 .. 843  =  711 bp
236 amino acids  =  26.2 kDa
Product: orange fluorescent protein KillerOrange, a genetically-encoded photosensitizer derived from KillerRed (Sarkisyan et al., 2015)
mammalian codon-optimized
CmR
1003 .. 1662  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
1003 .. 1662  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
2295 .. 2883  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2295 .. 2883  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
3915 .. 4019  =  105 bp
AmpR promoter
3915 .. 4019  =  105 bp
lambda t0 terminator
865 .. 959  =  95 bp
transcription terminator from phage lambda
lambda t0 terminator
865 .. 959  =  95 bp
transcription terminator from phage lambda
rrnB T1 terminator
1727 .. 1813  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
1727 .. 1813  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 1:  
   10 .. 24  =  15 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 2:  -35  
   25 .. 30  =  6 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 3:  
   31 .. 47  =  17 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
   Segment 4:  -10  
   48 .. 54  =  7 bp
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
T5 promoter
10 .. 54  =  45 bp
4 segments
bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator
lac operator
62 .. 78  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
62 .. 78  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
RBS
97 .. 108  =  12 bp
strong bacterial ribosome binding site (Elowitz and Leibler, 2000)
RBS
97 .. 108  =  12 bp
strong bacterial ribosome binding site (Elowitz and Leibler, 2000)
lac operator
30 .. 46  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
30 .. 46  =  17 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
ORF:  115 .. 843  =  729 bp
ORF:  242 amino acids  =  26.8 kDa
ORF:  1003 .. 1662  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  3184 .. 3450  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  109 .. 369  =  261 bp
ORF:  86 amino acids  =  8.4 kDa
ORF:  81 .. 308  =  228 bp
ORF:  75 amino acids  =  8.2 kDa
ORF:  456 .. 824  =  369 bp
ORF:  122 amino acids  =  13.7 kDa
ORF:  3054 .. 3914  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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