Resources
Plasmid Files

Reading Frame Cassette A

DNA cassette for converting a custom vector into a Gateway® Destination Vector, if the vector blunt end terminates after a complete codon triplet.

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Reading Frame Cassette A Sequence and MapReading Frame Cassette A.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 1500 1000 500 attR1 lac UV5 promoter CmR attR2 End (1711) SalI (1578) PstI (1576) SfcI (1572) BfuAI - BspMI (1565) BsaI (1459) BsrFI (1450) Primer 2 (1444 .. 1466) BstXI (1438) BmgBI (1355) BaeGI - Bme1580I - BsiHKAI - Bsp1286I (1354) ApaLI (1350) BsaBI * (1334) EcoP15I (1330) SmaI - SrfI (1321) BmeT110I (1320) AvaI - BsoBI - TspMI - XmaI (1319) AlwNI (1229) BbvCI (1175) SmlI (1064) BpuEI (1049) BstZ17I (985) BssHII (944) AflIII - MluI (897) ScaI (867) SspI (762) BtgI - NcoI - StyI (751) PasI - PflMI * (682) BsmBI - TaqII (674) BpmI - Eco57MI (572) AclI (537) BsrDI (469) ApoI - EcoRI (450) BspEI (446) TsoI (351) PvuII (350) Primer 1 (163 .. 185) BanI (141) BsiEI (133) EagI - NotI (130) Start (0) Reading Frame Cassette A 1711 bp
End  (1711)
0 sites
SalI  (1578)
1 site
G T C G A C C A G C T G
PstI  (1576)
1 site
C T G C A G G A C G T C
SfcI  (1572)
1 site
C T R Y A G G A Y R T C

Sticky ends from different SfcI sites may not be compatible.
SfcI quickly loses activity at 37°C, but can be used at 25°C for long
incubations.
BfuAI  (1565)
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  (1565)
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.
BsaI  (1459)
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.
BsrFI  (1450)
1 site
R C C G G Y Y G G C C R

Efficient cleavage requires at least two copies of the BsrFI
recognition sequence.
After cleavage, BsrFI can remain bound to DNA and alter its
electrophoretic mobility.
BstXI  (1438)
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.
BmgBI  (1355)
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.
BaeGI  (1354)
1 site
G K G C M C C M C G K G

Sticky ends from different BaeGI sites may not be compatible.
Bme1580I  (1354)
1 site
G K G C M C C M C G K G

Sticky ends from different Bme1580I sites may not be compatible.
BsiHKAI  (1354)
1 site
G W G C W C C W C G W G

Sticky ends from different BsiHKAI sites may not be compatible.
Bsp1286I  (1354)
1 site
G D G C H C C H C G D G

Sticky ends from different Bsp1286I sites may not be compatible.
ApaLI  (1350)
1 site
G T G C A C C A C G T G
BsaBI  (1334)
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.
EcoP15I  (1330)
1 site
C A G C A G ( N ) 25 G T C G T C ( N ) 25 N N

Efficient cleavage requires two inversely oriented copies of the
EcoP15I recognition sequence.
Sticky ends from different EcoP15I sites may not be compatible.
EcoP15I requires ATP for activity.
SmaI  (1321)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (1321)
1 site
G C C C G G G C C G G G C C C G
BmeT110I  (1320)
1 site
C Y C G R G G R G C Y C
AvaI  (1319)
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  (1319)
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  (1319)
1 site
C C C G G G G G G C C C
XmaI  (1319)
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.
AlwNI  (1229)
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.
BbvCI  (1175)
1 site
C C T C A G C G G A G T C G
SmlI  (1064)
1 site
C T Y R A G G A R Y T C

Efficient cleavage requires at least two copies of the SmlI
recognition sequence.
Sticky ends from different SmlI sites may not be compatible.
BpuEI  (1049)
1 site
C T T G A G ( N ) 14 N N G A A C T C ( N ) 14

Sticky ends from different BpuEI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BstZ17I  (985)
1 site
G T A T A C C A T A T G
BssHII  (944)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
AflIII  (897)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (897)
1 site
A C G C G T T G C G C A
ScaI  (867)
1 site
A G T A C T T C A T G A
SspI  (762)
1 site
A A T A T T T T A T A A
BtgI  (751)
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  (751)
1 site
C C A T G G G G T A C C
StyI  (751)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PasI  (682)
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.
PflMI  (682)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
BsmBI  (674)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
TaqII  (674)
1 site
G A C C G A ( N ) 9 N N C T G G C T ( N ) 9

Sticky ends from different TaqII sites may not be compatible.
BpmI  (572)
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.
Eco57MI  (572)
1 site
C T G R A G ( N ) 14 N N G A C Y T C ( N ) 14

Sticky ends from different Eco57MI sites may not be compatible.
After cleavage, Eco57MI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
AclI  (537)
1 site
A A C G T T T T G C A A
BsrDI  (469)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
ApoI  (450)
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  (450)
1 site
G A A T T C C T T A A G
BspEI  (446)
1 site
T C C G G A A G G C C T
TsoI  (351)
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).
PvuII  (350)
1 site
C A G C T G G T C G A C
BanI  (141)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
BsiEI  (133)
1 site
C G R Y C G G C Y R G C

Sticky ends from different BsiEI sites may not be compatible.
EagI  (130)
1 site
C G G C C G G C C G G C
NotI  (130)
1 site
G C G G C C G C C G C C G G C G
Start  (0)
0 sites
Primer 2
23-mer  /  61% GC
1 binding site
1444 .. 1466  =  23 annealed bases
Tm  =  63°C
Primer 1
23-mer  /  48% GC
1 binding site
163 .. 185  =  23 annealed bases
Tm  =  59°C
attR1
4 .. 128  =  125 bp
recombination site for the Gateway® LR reaction
attR1
4 .. 128  =  125 bp
recombination site for the Gateway® LR reaction
lac UV5 promoter
153 .. 183  =  31 bp
   Segment 1:  -35  
   153 .. 158  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
153 .. 183  =  31 bp
   Segment 2:  
   159 .. 176  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
153 .. 183  =  31 bp
   Segment 3:  -10  
   177 .. 183  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
153 .. 183  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
CmR
237 .. 896  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
237 .. 896  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ccdB
1238 .. 1543  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
1238 .. 1543  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
attR2
1584 .. 1708  =  125 bp
recombination site for the Gateway® LR reaction
attR2
1584 .. 1708  =  125 bp
recombination site for the Gateway® LR reaction
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