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Plasmid Files

pDB.His.NusA

Bacterial expression vector with a kanamycin resistance marker, for appending an N-terminal 6xHis-NusA tag followed by a TEV site.

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pDB.His.NusA Sequence and MappDB.His.NusA.dna
Map and Sequence File   
Sequence Author:  Berkeley Structural Genomics Center
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 PaeR7I - PspXI - XhoI (6716) EagI - NotI (6708) HindIII (6701) MCS SalI (6695) SacI (6692) Eco53kI (6690) EcoRI (6682) BamHI (6676) T7 tag (gene 10 leader) BmtI (6647) NheI (6643) NdeI (6638) TEV Site MscI (6346) StuI (6293) BfuAI - BspMI (6140) AatII (5237) ZraI (5235) BstBI (5174) 6xHis ATG RBS XbaI (5030) T7 promoter SphI (4775) MluI (4242) BstEII (4060) NmeAIII (4042) ApaI (4039) PspOMI (4035) HpaI (3740) 6xHis DraIII (245) PsiI (370) AsiSI (945) TspMI - XmaI (1067) SmaI (1069) AlwNI (1732) PciI (2141) BspQI - SapI (2258) TatI (2337) BstZ17I (2374) PflFI - Tth111I (2399) PpuMI (3136) FspI - FspAI (3164) BglI (3185) PshAI (3401) pDB.His.NusA 6878 bp
PaeR7I  (6716)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (6716)
1 site
V C T C G A G B B G A G C T C V
XhoI  (6716)
1 site
C T C G A G G A G C T C
EagI  (6708)
1 site
C G G C C G G C C G G C
NotI  (6708)
1 site
G C G G C C G C C G C C G G C G
HindIII  (6701)
1 site
A A G C T T T T C G A A
SalI  (6695)
1 site
G T C G A C C A G C T G
SacI  (6692)
1 site
G A G C T C C T C G A G
Eco53kI  (6690)
1 site
G A G C T C C T C G A G
EcoRI  (6682)
1 site
G A A T T C C T T A A G
BamHI  (6676)
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.
BmtI  (6647)
1 site
G C T A G C C G A T C G
NheI  (6643)
1 site
G C T A G C C G A T C G
NdeI  (6638)
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.
MscI  (6346)
1 site
T G G C C A A C C G G T
StuI  (6293)
1 site
A G G C C T T C C G G A
BfuAI  (6140)
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  (6140)
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.
AatII  (5237)
1 site
G A C G T C C T G C A G
ZraI  (5235)
1 site
G A C G T C C T G C A G
BstBI  (5174)
1 site
T T C G A A A A G C T T
XbaI  (5030)
1 site
T C T A G A A G A T C T
SphI  (4775)
1 site
G C A T G C C G T A C G
MluI  (4242)
1 site
A C G C G T T G C G C A
BstEII  (4060)
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.
NmeAIII  (4042)
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).
ApaI  (4039)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (4035)
1 site
G G G C C C C C C G G G
HpaI  (3740)
1 site
G T T A A C C A A T T G
DraIII  (245)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
PsiI  (370)
1 site
T T A T A A A A T A T T
AsiSI  (945)
1 site
G C G A T C G C C G C T A G C G
TspMI  (1067)
1 site
C C C G G G G G G C C C
XmaI  (1067)
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.
SmaI  (1069)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AlwNI  (1732)
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.
PciI  (2141)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2258)
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  (2258)
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.
TatI  (2337)
1 site
W G T A C W W C A T G W
BstZ17I  (2374)
1 site
G T A T A C C A T A T G
PflFI  (2399)
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  (2399)
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.
PpuMI  (3136)
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.
FspI  (3164)
1 site
T G C G C A A C G C G T
FspAI  (3164)
1 site
R T G C G C A Y Y A C G C G T R
BglI  (3185)
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.
PshAI  (3401)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
NusA
5107 .. 6588  =  1482 bp
494 amino acids  =  54.7 kDa
Product: transcription elongation protein (N
utilization substance protein A)
highly soluble in E. coli
NusA
5107 .. 6588  =  1482 bp
494 amino acids  =  54.7 kDa
Product: transcription elongation protein (N
utilization substance protein A)
highly soluble in E. coli
lacI
3515 .. 4597  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
lacI
3515 .. 4597  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
KanR
560 .. 1375  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
560 .. 1375  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
1497 .. 2085  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1497 .. 2085  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
12 .. 467  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
12 .. 467  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
2515 .. 2706  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2515 .. 2706  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
4598 .. 4675  =  78 bp
lacI promoter
4598 .. 4675  =  78 bp
T7 terminator
6806 .. 6853  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
6806 .. 6853  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
MCS
6676 .. 6721  =  46 bp
multiple cloning site
MCS
6676 .. 6721  =  46 bp
multiple cloning site
T7 tag (gene 10 leader)
6640 .. 6672  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene
10
promotes efficient translation in E. coli
T7 tag (gene 10 leader)
6640 .. 6672  =  33 bp
11 amino acids  =  1.1 kDa
Product: leader peptide from bacteriophage T7 gene
10
promotes efficient translation in E. coli
lac operator
5003 .. 5027  =  25 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
5003 .. 5027  =  25 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).
TEV Site
6616 .. 6636  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV Site
6616 .. 6636  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
4984 .. 5002  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
4984 .. 5002  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
5083 .. 5100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5083 .. 5100  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6722 .. 6739  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6722 .. 6739  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
5058 .. 5063  =  6 bp
ribosome binding site
RBS
5058 .. 5063  =  6 bp
ribosome binding site
ATG
5071 .. 5073  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5071 .. 5073  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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