pAmCyan

Vector for expressing AmCyan in bacteria.

Sequence Author: Clontech (TaKaRa)

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BspQI - SapI (3317) AflIII - PciI (3200) PspFI (2900) BseYI (2896) AlwNI (2791) BmrI (2272) BsaI (2246) BpmI (2243) NmeAIII (2165) FspI (2089) PvuI (1943) TsoI (1914) ScaI (1831) XmnI (1712) 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) KpnI (277) BstXI (343) MscI (439) BsmI (457) BspDI - ClaI (501) BseRI (614) KflI - PpuMI (723) PflMI (876) NotI (981) ApoI - EcoRI (995) StuI (1055) SpeI (1059) BsiWI (1069) PfoI (1132) BstAPI (1271) AflII (1327) ZraI (1391) AatII (1393) pAmCyan 3325 bp
BspQI  (3317)
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  (3317)
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  (3200)
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  (3200)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PspFI  (2900)
1 site
C C C A G C G G G T C G
BseYI  (2896)
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  (2791)
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.
BmrI  (2272)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
BsaI  (2246)
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.
BpmI  (2243)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
NmeAIII  (2165)
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).
FspI  (2089)
1 site
T G C G C A A C G C G T
PvuI  (1943)
1 site
C G A T C G G C T A G C
TsoI  (1914)
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  (1831)
1 site
A G T A C T T C A T G A
XmnI  (1712)
1 site
G A A N N N N T T C C T T N N N N A A G
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
KpnI  (277)
1 site
G G T A C C C C A T G G
BstXI  (343)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
MscI  (439)
1 site
T G G C C A A C C G G T
BsmI  (457)
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.
BspDI  (501)
1 site
A T C G A T T A G C T A
ClaI  (501)
1 site
A T C G A T T A G C T A
BseRI  (614)
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.
KflI  (723)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (723)
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.
PflMI  (876)
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.
NotI  (981)
1 site
G C G G C C G C C G C C G G C G
ApoI  (995)
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  (995)
1 site
G A A T T C C T T A A G
StuI  (1055)
1 site
A G G C C T T C C G G A
SpeI  (1059)
1 site
A C T A G T T G A T C A
BsiWI  (1069)
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.
PfoI  (1132)
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  (1271)
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  (1327)
1 site
C T T A A G G A A T T C
ZraI  (1391)
1 site
G A C G T C C T G C A G
AatII  (1393)
1 site
G A C G T C C T G C A G
AmpR
1525 .. 2385  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   1525 .. 1593  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1525 .. 2385  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1594 .. 2385  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1525 .. 2385  =  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
AmCyan
289 .. 978  =  690 bp
229 amino acids  =  25.3 kDa
Product: enhanced variant of Anemonia majano cyan fluorescent protein
AmCyan
289 .. 978  =  690 bp
229 amino acids  =  25.3 kDa
Product: enhanced variant of Anemonia majano cyan fluorescent protein
ori
2556 .. 3144  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2556 .. 3144  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
1420 .. 1524  =  105 bp
AmpR promoter
1420 .. 1524  =  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 .. 978  =  762 bp
ORF:  253 amino acids  =  27.9 kDa
ORF:  1525 .. 2385  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  344 .. 628  =  285 bp
ORF:  94 amino acids  =  10.4 kDa
ORF:  568 .. 954  =  387 bp
ORF:  128 amino acids  =  14.8 kDa
ORF:  1989 .. 2255  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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