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.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsGateway® Cloning VectorsReading Frame Cassette A
1500 1000 500 End (1711) SalI (1578) PstI (1576) SfcI (1572) BfuAI - BspMI (1565) BsaI (1459) BsrFI (1450) Primer 2 (1444 .. 1466) BstXI (1438) BmgBI (1355) BaeGI - BsiHKAI - Bsp1286I - Bme1580I (1354) ApaLI (1350) BsaBI * (1334) EcoP15I (1330) SrfI - SmaI (1321) AvaI - XmaI - BsoBI - TspMI (1319) AlwNI (1229) BbvCI (1175) SmlI (1064) BpuEI (1049) BstZ17I (985) BssHII (944) MluI - AflIII (897) ScaI (867) SspI (762) StyI - BtgI - NcoI (751) PasI - PflMI * (682) Esp3I - BsmBI (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) NotI - EagI (130) Start (0) attR1 lac UV5 promoter CmR ccdB attR2 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
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.
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.
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.
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.
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.
SrfI  (1321)
1 site		 G C C C G G G C C G G G C C C G
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.
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.
XmaI  (1319)
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.
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
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
Cleavage may be enhanced when more than one copy of the SmlI recognition sequence is present.
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.
MluI  (897)
1 site		 A C G C G T T G C G C A
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.
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
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.
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
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.
Esp3I  (674)
1 site		 C G T C T C N G C A G A G N ( N ) 4
Sticky ends from different Esp3I 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.
BsmBI-v2 is an improved version of BsmBI.
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.
NotI  (130)
1 site		 G C G G C C G C C G C C G G C G
EagI  (130)
1 site		 C G G C C G G C C G G C
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
3 segments
   Segment 1:  -35  
   153 .. 158  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
153 .. 183  =  31 bp
3 segments
   Segment 2:  
   159 .. 176  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
153 .. 183  =  31 bp
3 segments
   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
ORF:  1238 .. 1543  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  237 .. 896  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
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Individual Sequences & Maps

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pHELLSGATE 4
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pDEST10
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pAd PL-DEST
pDEST14
pENTR2B
pIB V5-His-DEST
pBAD-DEST49
pDEST15
pENTR3C
pLenti4 BLOCK-iT-DEST
pcDNA-DEST40
pDEST17
pENTR4
pLenti4 V5-DEST
pcDNA-DEST47
pDEST20
pET-53-DEST
pLenti6.2 C-Lumio V5-DEST
pcDNA-DEST53
pDEST24
pET-54-DEST
pLenti6.2 N-Lumio V5-DEST
pcDNA3.1 nV5-DEST
pDEST26
pET-55-DEST
pLenti6.2 V5-DEST
pcDNA3.2 capTEV-CT V5-DEST
pDEST27
pET-56-DEST
pLenti6.3 TO V5-DEST
pcDNA3.2 capTEV-NT V5-DEST
pDEST8
pET-57-DEST
pLenti6.3 V5-DEST
pcDNA3.2 V5-DEST
pDONR221 P5-P2
pET-58-DEST
pLenti6.4 R4R2 V5-DEST
pcDNA6.2 C-EmGFP-DEST
pDONR P2r-P3
pET-59-DEST
pLenti6 BLOCK-iT-DEST
pcDNA6.2 C-YFP-DEST
pDONR P4-P1r
pET-60-DEST
pLenti6 capTEV-CT-DEST
pcDNA6.2 cGeneBLAzer-DEST
pDONR Zeo
pET-61-DEST
pLenti6 capTEV-NT-DEST1
pcDNA6.2 cLumio-DEST
pDONR201
pET-62-DEST
pLenti6 UbC V5-DEST
pcDNA6.2 EmGFP-Bsd V5-DEST
pDONR207
pET-DEST42
pLenti6 V5-DEST
pcDNA6.2 N-EmGFP-DEST
pDONR221
pET104-DEST
pLenti7.3 V5-DEST
pcDNA6.2 N-YFP-DEST
pDONR221 P1-P4
pET104.1-DEST
pMT-DEST48
pcDNA6.2 nGeneBLAzer-DEST
pDONR221 P1-P5r
pET160-DEST
pMT BioEase-DEST
pcDNA6.2 nLumio-DEST
pDONR221 P3-P2
pET161-DEST
pSCREEN-iT lacZ-DEST
pcDNA6.2 V5-DEST
pDONR221 P4r-P3r
pEXP1-DEST
pT-REx-DEST30
pcDNA6.2 V5-pL-DEST
pDONR221 P5-P4
pEXP2-DEST
pT-REx-DEST31
pCMV SPORT6
pDONR222
pEXP3-DEST
pYES-DEST52
pCMV SPORT6.1
pDONR223
pEXP4-DEST
Reading Frame Cassette A
pCOLA-2-DEST
pEF-DEST51
pHELLSGATE
Reading Frame Cassette B
pCOLA-3-DEST
pEF5 FRT V5-DEST
pHELLSGATE 12
Reading Frame Cassette C.1
pDEST R4-R3
pENTR GeneBLAzer

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