phmKeima-Red-S1

Vector for expressing hmKeima-Red in bacteria.

Sequence Author: MBL International

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PfoI (3297) BstAPI (3166) NdeI (3162) HindIII (2945) SphI (2943) PstI (2937) BsaBI * (2903) BtgZI (2890) BmgBI (2854) EagI (2824) BsmI (2724) BbsI (2675) PasI (2674) Bpu10I (2435) BsrGI (2301) BtgI - 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) EcoO109I (8) ZraI (67) AatII (69) SspI (183) XmnI (388) ScaI (507) TsoI (590) NmeAIII (841) BsrFI (903) BsaI (922) AhdI (988) AlwNI (1467) phmKeima-Red-S1 3348 bp
PfoI  (3297)
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  (3166)
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  (3162)
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.
HindIII  (2945)
1 site
A A G C T T T T C G A A
SphI  (2943)
1 site
G C A T G C C G T A C G
PstI  (2937)
1 site
C T G C A G G A C G T C
BsaBI  (2903)
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.
BtgZI  (2890)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
BmgBI  (2854)
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.
EagI  (2824)
1 site
C G G C C G G C C G G C
BsmI  (2724)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BbsI  (2675)
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.
PasI  (2674)
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.
Bpu10I  (2435)
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.
BsrGI  (2301)
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.
BtgI  (2262)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
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.
EcoO109I  (8)
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.
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).
BsrFI  (903)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
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.
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.
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
hmKeima-Red
2264 .. 2932  =  669 bp
222 amino acids  =  25.1 kDa
3 segments
   Segment 1:  
   2264 .. 2266  =  3 bp
   1 amino acid  =  149.2 Da
Product: humanized monomeric Keima-Red fluorescent protein
mammalian codon-optimized
hmKeima-Red
2264 .. 2932  =  669 bp
222 amino acids  =  25.1 kDa
3 segments
   Segment 2:  1a  
   2267 .. 2269  =  3 bp
   1 amino acid  =  117.1 Da
Product: humanized monomeric Keima-Red fluorescent protein
mammalian codon-optimized
hmKeima-Red
2264 .. 2932  =  669 bp
222 amino acids  =  25.1 kDa
3 segments
   Segment 3:  
   2270 .. 2932  =  663 bp
   220 amino acids  =  24.8 kDa
Product: humanized monomeric Keima-Red fluorescent protein
mammalian codon-optimized
hmKeima-Red
2264 .. 2932  =  669 bp
222 amino acids  =  25.1 kDa
3 segments
Product: humanized monomeric Keima-Red 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 .. 3099  =  882 bp
ORF:  293 amino acids  =  33.1 kDa
ORF:  2264 .. 2932  =  669 bp
ORF:  222 amino acids  =  25.1 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 .. 3163  =  936 bp
ORF:  311 amino acids  =  31.6 kDa
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