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Plasmid Files

phmKO1-S1

Vector for expressing hmKO1 in bacteria.

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phmKO1-S1.dna
Map and Sequence File:    Download    Open   
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) BmgBI (2740) SgrAI (2633) PpuMI (2544) EcoNI (2539) StuI (2516) BsaAI - PmlI (2449) SexAI * (2436) MscI (2405) BclI * (2274) NcoI (2262) BamHI (2253) SmaI (2250) KpnI - TspMI - XmaI (2248) Acc65I (2244) SacI (2242) Eco53kI (2240) ApoI - EcoRI (2232) BspQI - SapI (1993) PciI (1876) PspFI (1576) ZraI (67) AatII (69) SspI (183) XmnI (388) ScaI (507) TsoI (590) NmeAIII (841) BsaI (922) AlwNI (1467) BseYI (1572) phmKO1-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.
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.
SgrAI  (2633)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI recognition sequence.
PpuMI  (2544)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
EcoNI  (2539)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
StuI  (2516)
1 site
A G G C C T T C C G G A
BsaAI  (2449)
1 site
Y A C G T R R T G C A Y
PmlI  (2449)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
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.
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

Efficient cleavage requires at least two copies of the XmaI recognition sequence.
Full cleavage with XmaI may require a long incubation.
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.
PciI  (1876)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (1576)
1 site
C C C A G C G G G T C G
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
XmnI  (388)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (507)
1 site
A G T A C T T C A T G 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.
AlwNI  (1467)
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.
BseYI  (1572)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
AmpR
201 .. 1061  =  861 bp
286 amino acids  =  31.6 kDa
   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
   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
hmKO
2264 .. 2920  =  657 bp
218 amino acids  =  24.5 kDa
Product: humanized monomeric Kusabira-Orange fluorescent protein
mammalian codon-optimized
hmKO
2264 .. 2920  =  657 bp
218 amino acids  =  24.5 kDa
Product: humanized monomeric 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
   Segment 1:  -35  
   2144 .. 2149  =  6 bp
promoter for the E. coli lac operon
lac promoter
2144 .. 2174  =  31 bp
   Segment 2:  
   2150 .. 2167  =  18 bp
promoter for the E. coli lac operon
lac promoter
2144 .. 2174  =  31 bp
   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:  2264 .. 2920  =  657 bp
ORF:  218 amino acids  =  24.5 kDa
ORF:  201 .. 1061  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  665 .. 931  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2228 .. 3151  =  924 bp
ORF:  307 amino acids  =  32.4 kDa
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