phKO1-S1

Vector for expressing humanized Kusabira-Orange in bacteria.

Sequence Author: MBL International

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PfoI (3285) BstAPI (3154) NdeI (3150) PluTI (3101) SfoI (3099) NarI (3098) KasI (3097) HindIII (2933) SphI (2931) PstI (2925) BbsI (2873) PflMI (2857) NaeI (2764) NgoMIV (2762) BmgBI (2740) BfuAI - BspMI - PaqCI (2723) Bpu10I (2531) StuI (2516) CsiI - SexAI * (2436) MscI (2405) BclI * (2274) AleI (2264) NcoI (2262) BamHI (2253) SmaI (2250) KpnI - TspMI - XmaI (2248) Acc65I (2244) SacI (2242) Eco53kI (2240) ApoI - EcoRI (2232) BspQI - SapI (1993) AflIII - PciI (1876) ZraI (67) AatII (69) SspI (183) TsoI (590) NmeAIII (841) BsaI (922) AhdI (988) phKO1-S1 3336 bp
PfoI  (3285)
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  (3154)
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  (3150)
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  (3101)
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  (3099)
1 site
G G C G C C C C G C G G
NarI  (3098)
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  (3097)
1 site
G G C G C C C C G C G G
HindIII  (2933)
1 site
A A G C T T T T C G A A
SphI  (2931)
1 site
G C A T G C C G T A C G
PstI  (2925)
1 site
C T G C A G G A C G T C
BbsI  (2873)
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.
PflMI  (2857)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
NaeI  (2764)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI recognition sequence.
NgoMIV  (2762)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
BmgBI  (2740)
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.
BfuAI  (2723)
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  (2723)
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  (2723)
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.
Bpu10I  (2531)
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.
StuI  (2516)
1 site
A G G C C T T C C G G A
CsiI  (2436)
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  (2436)
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.
MscI  (2405)
1 site
T G G C C A A C C G G T
BclI  (2274)
1 site
T G A T C A A C T A G T
* Blocked by Dam methylation.
BclI is typically used at 50-55°C, but is 50% active at 37°C.
AleI  (2264)
1 site
C A C N N N N G T G G T G N N N N C A C
NcoI  (2262)
1 site
C C A T G G G G T A C C
BamHI  (2253)
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.
SmaI  (2250)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
KpnI  (2248)
1 site
G G T A C C C C A T G G
TspMI  (2248)
1 site
C C C G G G G G G C C C
XmaI  (2248)
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.
Acc65I  (2244)
1 site
G G T A C C C C A T G G
SacI  (2242)
1 site
G A G C T C C T C G A G
Eco53kI  (2240)
1 site
G A G C T C C T C G A G
ApoI  (2232)
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  (2232)
1 site
G A A T T C C T T A A G
BspQI  (1993)
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  (1993)
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.
AflIII  (1876)
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  (1876)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
ZraI  (67)
1 site
G A C G T C C T G C A G
AatII  (69)
1 site
G A C G T C C T G C A G
SspI  (183)
1 site
A A T A T T T T A T A A
TsoI  (590)
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).
NmeAIII  (841)
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).
BsaI  (922)
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.
AhdI  (988)
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.
AmpR
201 .. 1061  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   201 .. 269  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
201 .. 1061  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   270 .. 1061  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
201 .. 1061  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
hKO
2264 .. 2920  =  657 bp
218 amino acids  =  24.4 kDa
Product: humanized Kusabira-Orange fluorescent protein
mammalian codon-optimized
hKO
2264 .. 2920  =  657 bp
218 amino acids  =  24.4 kDa
Product: humanized Kusabira-Orange fluorescent protein
mammalian codon-optimized
ori
1232 .. 1820  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1232 .. 1820  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
96 .. 200  =  105 bp
AmpR promoter
96 .. 200  =  105 bp
lac promoter
2144 .. 2174  =  31 bp
3 segments
   Segment 1:  -35  
   2144 .. 2149  =  6 bp
promoter for the E. coli lac operon
lac promoter
2144 .. 2174  =  31 bp
3 segments
   Segment 2:  
   2150 .. 2167  =  18 bp
promoter for the E. coli lac operon
lac promoter
2144 .. 2174  =  31 bp
3 segments
   Segment 3:  -10  
   2168 .. 2174  =  7 bp
promoter for the E. coli lac operon
lac promoter
2144 .. 2174  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
2182 .. 2198  =  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
2182 .. 2198  =  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:  2218 .. 3087  =  870 bp
ORF:  289 amino acids  =  32.5 kDa
ORF:  2264 .. 2920  =  657 bp
ORF:  218 amino acids  =  24.4 kDa
ORF:  201 .. 1061  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2503 .. 2748  =  246 bp
ORF:  81 amino acids  =  8.8 kDa
ORF:  665 .. 931  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2228 .. 3151  =  924 bp
ORF:  307 amino acids  =  31.5 kDa
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