pMiCy1-S1

Vector for expressing MiCy1 in bacteria.

Sequence Author: MBL International

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PfoI (3327) BstAPI (3196) PluTI (3143) SfoI (3141) NarI (3140) KasI (3139) HindIII (2975) SphI (2973) HpaI (2941) BbvCI - Bpu10I (2928) BstEII (2783) BstBI (2712) MfeI (2691) BseRI (2592) Bsu36I (2418) BclI * (2406) EcoNI (2379) BtgZI (2272) AleI (2264) BtgI - NcoI (2262) BamHI (2253) SmaI (2250) KpnI - TspMI - XmaI (2248) Acc65I (2244) BanII - SacI (2242) Eco53kI (2240) BspQI - SapI (1993) AflIII - PciI (1876) EcoO109I (8) ZraI (67) AatII (69) SspI (183) XmnI (388) ScaI (507) NmeAIII (841) BsaI (922) AlwNI (1467) pMiCy1-S1 3378 bp
PfoI  (3327)
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  (3196)
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.
PluTI  (3143)
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  (3141)
1 site
G G C G C C C C G C G G
NarI  (3140)
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  (3139)
1 site
G G C G C C C C G C G G
HindIII  (2975)
1 site
A A G C T T T T C G A A
SphI  (2973)
1 site
G C A T G C C G T A C G
HpaI  (2941)
1 site
G T T A A C C A A T T G
BbvCI  (2928)
1 site
C C T C A G C G G A G T C G
Bpu10I  (2928)
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.
BstEII  (2783)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
BstBI  (2712)
1 site
T T C G A A A A G C T T
MfeI  (2691)
1 site
C A A T T G G T T A A C
BseRI  (2592)
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.
Bsu36I  (2418)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BclI  (2406)
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.
EcoNI  (2379)
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.
BtgZI  (2272)
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.
AleI  (2264)
1 site
C A C N N N N G T G G T G N N N N C A 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
BanII  (2242)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
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
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
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.
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
MiCy
2264 .. 2962  =  699 bp
232 amino acids  =  26.2 kDa
Product: dimeric Midoriishi-Cyan fluorescent protein
MiCy
2264 .. 2962  =  699 bp
232 amino acids  =  26.2 kDa
Product: dimeric Midoriishi-Cyan fluorescent protein
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:  2264 .. 2962  =  699 bp
ORF:  232 amino acids  =  26.2 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:  2741 .. 3193  =  453 bp
ORF:  150 amino acids  =  17.3 kDa
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