pBAD-DEST49

Gateway® destination vector for the regulated expression of N-terminally tagged HP-thioredoxin fusion proteins in bacterial cells.

Sequence Author: Thermo Fisher (Invitrogen)

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HomePlasmidsGateway® Cloning VectorspBAD-DEST49
NruI (5868) EcoRV (5546) NsiI (5169) SphI (5167) MauBI (5145) PfoI (5077) PflFI - Tth111I (4978) BsaAI (4972) NdeI (4901) PciI (4722) AhdI (3834) BglI (3716) NmeAIII (3687) AseI (3659) FspI (3611) PvuI (3465) BstEII (77) RBS AleI (441) RsrII (448) HP-thioredoxin BspDI * - ClaI * (530) NgoMIV (673) NaeI (675) EagI - NotI (844) PvuII (1064) EcoRI (1164) PasI (1396) BbvCI (1889) SrfI (2035) BmgBI (2069) BstXI (2152) BfuAI - BspMI (2279) PstI (2290) SalI (2292) BclI * (2422) HindIII (2427) BseRI (2448) PmeI (2511) rrnB T2 terminator pBAD-DEST49 6160 bp
NruI  (5868)
1 site		 T C G C G A A G C G C T
EcoRV  (5546)
1 site		 G A T A T C C T A T A G
EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
NsiI  (5169)
1 site		 A T G C A T T A C G T A
SphI  (5167)
1 site		 G C A T G C C G T A C G
MauBI  (5145)
1 site		 C G C G C G C G G C G C G C G C
PfoI  (5077)
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.
PflFI  (4978)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (4978)
1 site		 G A C N N N G T C C T G N N N C A G
The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BsaAI  (4972)
1 site		 Y A C G T R R T G C A Y
NdeI  (4901)
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.
PciI  (4722)
1 site		 A C A T G T T G T A C A
PciI is inhibited by nonionic detergents.
AhdI  (3834)
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.
BglI  (3716)
1 site		 G C C N N N N N G G C C G G N N N N N C C G
Sticky ends from different BglI sites may not be compatible.
NmeAIII  (3687)
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).
AseI  (3659)
1 site		 A T T A A T T A A T T A
FspI  (3611)
1 site		 T G C G C A A C G C G T
PvuI  (3465)
1 site		 C G A T C G G C T A G C
BstEII  (77)
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.
AleI  (441)
1 site		 C A C N N N N G T G G T G N N N N C A C
RsrII  (448)
1 site		 C G G W C C G G C C W G G C
Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BspDI  (530)
1 site		 A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (530)
1 site		 A T C G A T T A G C T A
* Blocked by Dam methylation.
NgoMIV  (673)
1 site		 G C C G G C C G G C C G
Efficient cleavage requires at least two copies of the NgoMIV recognition sequence.
NaeI  (675)
1 site		 G C C G G C C G G C C G
Efficient cleavage requires at least two copies of the NaeI recognition sequence.
EagI  (844)
1 site		 C G G C C G G C C G G C
NotI  (844)
1 site		 G C G G C C G C C G C C G G C G
PvuII  (1064)
1 site		 C A G C T G G T C G A C
EcoRI  (1164)
1 site		 G A A T T C C T T A A G
PasI  (1396)
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.
BbvCI  (1889)
1 site		 C C T C A G C G G A G T C G
SrfI  (2035)
1 site		 G C C C G G G C C G G G C C C G
BmgBI  (2069)
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.
BstXI  (2152)
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.
BfuAI  (2279)
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  (2279)
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.
PstI  (2290)
1 site		 C T G C A G G A C G T C
SalI  (2292)
1 site		 G T C G A C C A G C T G
BclI  (2422)
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.
HindIII  (2427)
1 site		 A A G C T T T T C G A A
BseRI  (2448)
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.
PmeI  (2511)
1 site		 G T T T A A A C C A A A T T T G
araC
5256 .. 6134  =  879 bp
292 amino acids  =  33.4 kDa
Product: L-arabinose regulatory protein
araC
5256 .. 6134  =  879 bp
292 amino acids  =  33.4 kDa
Product: L-arabinose regulatory protein
AmpR
3047 .. 3907  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   3047 .. 3115  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3047 .. 3907  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3116 .. 3907  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3047 .. 3907  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
CmR
951 .. 1610  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
951 .. 1610  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
4078 .. 4666  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4078 .. 4666  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
HP-thioredoxin
346 .. 675  =  330 bp
110 amino acids  =  11.9 kDa
Product: His-Patch thioredoxin
The histidines at positions 8, 32, and 64 coordinate a divalent cation, allowing HP-thioredoxin to be purified on metal-chelating resins.
HP-thioredoxin
346 .. 675  =  330 bp
110 amino acids  =  11.9 kDa
Product: His-Patch thioredoxin
The histidines at positions 8, 32, and 64 coordinate a divalent cation, allowing HP-thioredoxin to be purified on metal-chelating resins.
enterokinase site
691 .. 705  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
691 .. 705  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
ccdB
1952 .. 2257  =  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
1952 .. 2257  =  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.
araBAD promoter
120 .. 285  =  166 bp
promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction
araBAD promoter
120 .. 285  =  166 bp
promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction
attR1
718 .. 842  =  125 bp
recombination site for the Gateway® LR reaction
attR1
718 .. 842  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2298 .. 2422  =  125 bp
recombination site for the Gateway® LR reaction
attR2
2298 .. 2422  =  125 bp
recombination site for the Gateway® LR reaction
AmpR promoter
2955 .. 3046  =  92 bp
AmpR promoter
2955 .. 3046  =  92 bp
rrnB T1 terminator
2730 .. 2816  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
rrnB T1 terminator
2730 .. 2816  =  87 bp
transcription terminator T1 from the E. coli rrnB gene
V5 tag
2436 .. 2477  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
2436 .. 2477  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
6xHis
2487 .. 2504  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2487 .. 2504  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
lac UV5 promoter
867 .. 897  =  31 bp
3 segments
   Segment 1:  -35  
   867 .. 872  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
867 .. 897  =  31 bp
3 segments
   Segment 2:  
   873 .. 890  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
867 .. 897  =  31 bp
3 segments
   Segment 3:  -10  
   891 .. 897  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
867 .. 897  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
rrnB T2 terminator
2908 .. 2935  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
rrnB T2 terminator
2908 .. 2935  =  28 bp
transcription terminator T2 from the E. coli rrnB gene
RBS
327 .. 333  =  7 bp
ribosome binding site
RBS
327 .. 333  =  7 bp
ribosome binding site
ORF:  346 .. 753  =  408 bp
ORF:  135 amino acids  =  14.5 kDa
ORF:  2926 .. 3150  =  225 bp
ORF:  74 amino acids  =  8.7 kDa
ORF:  1952 .. 2257  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  3047 .. 3907  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  951 .. 1610  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  5820 .. 6068  =  249 bp
ORF:  82 amino acids  =  9.3 kDa
ORF:  5256 .. 25  =  930 bp
ORF:  309 amino acids  =  35.2 kDa
ORF:  3511 .. 3777  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2571 .. 2897  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
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Individual Sequences & Maps

pAd BLOCK-iT-DEST
pDEST R4-R3 Vector II
pENTR11
pHELLSGATE 4
pAd CMV V5-DEST
pDEST10
pENTR1A
pHELLSGATE 8
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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