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Plasmid Files

pBAD-DEST49

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

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pBAD-DEST49 Sequence and MappBAD-DEST49.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 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
   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
   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.
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
lac UV5 promoter
867 .. 897  =  31 bp
   Segment 1:  -35  
   867 .. 872  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
867 .. 897  =  31 bp
   Segment 2:  
   873 .. 890  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
867 .. 897  =  31 bp
   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
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
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
RBS
327 .. 333  =  7 bp
ribosome binding site
RBS
327 .. 333  =  7 bp
ribosome binding site
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