pECFP

Vector for expressing ECFP in bacteria.

Sequence Author: Clontech (TaKaRa)

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BspQI - SapI (3347) AflIII - PciI (3230) PspFI (2930) BseYI (2926) AlwNI (2821) AhdI (2342) BsaI (2276) FspI (2119) PvuI (1973) TsoI (1944) ScaI (1861) XmnI (1742) PvuII (55) lac operator HindIII (234) BfuAI - BspMI (239) SphI (244) PstI - SbfI (250) SalI (252) AccI (253) HincII (254) BamHI (264) AvaI - BsoBI - TspMI - XmaI (269) SmaI (271) Acc65I (273) AgeI (276) KpnI (277) BtgI - NcoI - StyI (287) BseRI (319) BtgZI (410) BsrGI (998) NotI (1011) ApoI - EcoRI (1025) StuI (1085) SpeI (1089) BsiWI (1099) PspOMI (1104) ApaI - BanII (1108) NdeI (1300) BstAPI (1301) AflII (1357) ZraI (1421) AatII (1423) SspI (1537) pECFP 3355 bp
BspQI  (3347)
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  (3347)
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  (3230)
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  (3230)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (2930)
1 site
C C C A G C G G G T C G
BseYI  (2926)
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.
AlwNI  (2821)
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.
AhdI  (2342)
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.
BsaI  (2276)
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.
FspI  (2119)
1 site
T G C G C A A C G C G T
PvuI  (1973)
1 site
C G A T C G G C T A G C
TsoI  (1944)
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).
ScaI  (1861)
1 site
A G T A C T T C A T G A
XmnI  (1742)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuII  (55)
1 site
C A G C T G G T C G A C
HindIII  (234)
1 site
A A G C T T T T C G A A
BfuAI  (239)
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  (239)
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.
SphI  (244)
1 site
G C A T G C C G T A C G
PstI  (250)
1 site
C T G C A G G A C G T C
SbfI  (250)
1 site
C C T G C A G G G G A C G T C C
SalI  (252)
1 site
G T C G A C C A G C T G
AccI  (253)
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.
HincII  (254)
1 site
G T Y R A C C A R Y T G
BamHI  (264)
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.
AvaI  (269)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (269)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
TspMI  (269)
1 site
C C C G G G G G G C C C
XmaI  (269)
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.
SmaI  (271)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Acc65I  (273)
1 site
G G T A C C C C A T G G
AgeI  (276)
1 site
A C C G G T T G G C C A
KpnI  (277)
1 site
G G T A C C C C A T G G
BtgI  (287)
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  (287)
1 site
C C A T G G G G T A C C
StyI  (287)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
BseRI  (319)
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.
BtgZI  (410)
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.
BsrGI  (998)
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.
NotI  (1011)
1 site
G C G G C C G C C G C C G G C G
ApoI  (1025)
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  (1025)
1 site
G A A T T C C T T A A G
StuI  (1085)
1 site
A G G C C T T C C G G A
SpeI  (1089)
1 site
A C T A G T T G A T C A
BsiWI  (1099)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PspOMI  (1104)
1 site
G G G C C C C C C G G G
ApaI  (1108)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BanII  (1108)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
NdeI  (1300)
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.
BstAPI  (1301)
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.
AflII  (1357)
1 site
C T T A A G G A A T T C
ZraI  (1421)
1 site
G A C G T C C T G C A G
AatII  (1423)
1 site
G A C G T C C T G C A G
SspI  (1537)
1 site
A A T A T T T T A T A A
AmpR
1555 .. 2415  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   1555 .. 1623  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1555 .. 2415  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1624 .. 2415  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1555 .. 2415  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ATG
217 .. 219  =  3 bp
1 amino acid  =  149.2 Da
Product: lacZ start codon
ATG
217 .. 219  =  3 bp
1 amino acid  =  149.2 Da
Product: lacZ start codon
ECFP
289 .. 1008  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 1:  
   289 .. 291  =  3 bp
   1 amino acid  =  149.2 Da
Product: enhanced CFP
mammalian codon-optimized
ECFP
289 .. 1008  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 2:  1a  
   292 .. 294  =  3 bp
   1 amino acid  =  117.1 Da
Product: enhanced CFP
mammalian codon-optimized
ECFP
289 .. 1008  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 3:  
   295 .. 1008  =  714 bp
   237 amino acids  =  26.7 kDa
Product: enhanced CFP
mammalian codon-optimized
ECFP
289 .. 1008  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
Product: enhanced CFP
mammalian codon-optimized
ori
2586 .. 3174  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2586 .. 3174  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
1450 .. 1554  =  105 bp
AmpR promoter
1450 .. 1554  =  105 bp
lac promoter
143 .. 173  =  31 bp
3 segments
   Segment 1:  -35  
   143 .. 148  =  6 bp
promoter for the E. coli lac operon
lac promoter
143 .. 173  =  31 bp
3 segments
   Segment 2:  
   149 .. 166  =  18 bp
promoter for the E. coli lac operon
lac promoter
143 .. 173  =  31 bp
3 segments
   Segment 3:  -10  
   167 .. 173  =  7 bp
promoter for the E. coli lac operon
lac promoter
143 .. 173  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
181 .. 197  =  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
181 .. 197  =  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:  217 .. 1008  =  792 bp
ORF:  263 amino acids  =  29.5 kDa
ORF:  1555 .. 2415  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2019 .. 2285  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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