pDB.His.NusA

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

Sequence Author: Berkeley Structural Genomics Center

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PaeR7I - PspXI - XhoI (6716) EagI - NotI (6708) HindIII (6701) 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

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
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.
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
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
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
TEV Site
6616 .. 6636  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV Site
6616 .. 6636  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage 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
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
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).
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
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
ORF:  3481 .. 3732  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4636 .. 4899  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  5071 .. 6756  =  1686 bp
ORF:  561 amino acids  =  62.1 kDa
ORF:  2738 .. 3106  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  4773 .. 5012  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  3495 .. 3758  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4347 .. 4847  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
ORF:  6258 .. 6494  =  237 bp
ORF:  78 amino acids  =  8.0 kDa
ORF:  560 .. 1375  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  3515 .. 4474  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  4844 .. 5182  =  339 bp
ORF:  112 amino acids  =  12.5 kDa
ORF:  2515 .. 2739  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  3103 .. 3459  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  5290 .. 6639  =  1350 bp
ORF:  449 amino acids  =  52.1 kDa
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