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Plasmid Files

pET SUMO (linearized)

Linearized bacterial vector with 3'-T overhangs for TA cloning and inducible expression of an N-terminally 6xHis- and SUMO-tagged protein.

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pET SUMO (linearized) Sequence and MappET SUMO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 BstAPI (4781) MluI (4457) BstEII (4275) NmeAIII (4257) ApaI (4254) PspOMI (4250) BssHII (4046) EcoRV (4011) HincII - HpaI (3955) PshAI (3616) BglI (3400) FspI - FspAI (3379) PpuMI (3351) PflFI - Tth111I (2614) BstZ17I (2589) AccI (2588) TatI (2552) BspQI - SapI (2476) PciI (2359) BssS α I (2186) SphI (4990) SgrAI (5138) T7 promoter lac operator XbaI (5245) NdeI (5285) ATG 6xHis NheI (5344) BmtI (5348) DraI (5426) AflII (5535) SUMO Forward (5539 .. 5561) EcoRI (5561) End (5643) Start (1) HindIII (5) T7 Reverse (130 .. 149) StyI (158) DraIII (464) PsiI (589) AsiSI - PvuI (1163) AvaI - BsoBI - TspMI - XmaI (1285) BmeT110I (1286) SmaI (1287) BspDI - ClaI (1468) NruI (1504) AlwNI (1950) pET SUMO 5642 bp
BstAPI  (4781)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
MluI  (4457)
1 site
A C G C G T T G C G C A
BstEII  (4275)
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  (4257)
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  (4254)
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  (4250)
1 site
G G G C C C C C C G G G
BssHII  (4046)
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.
EcoRV  (4011)
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.
HincII  (3955)
1 site
G T Y R A C C A R Y T G
HpaI  (3955)
1 site
G T T A A C C A A T T G
PshAI  (3616)
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.
BglI  (3400)
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.
FspI  (3379)
1 site
T G C G C A A C G C G T
FspAI  (3379)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (3351)
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.
PflFI  (2614)
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  (2614)
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.
BstZ17I  (2589)
1 site
G T A T A C C A T A T G
AccI  (2588)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
TatI  (2552)
1 site
W G T A C W W C A T G W
BspQI  (2476)
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  (2476)
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.
PciI  (2359)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BssSαI  (2186)
1 site
C A C G A G G T G C T C
SphI  (4990)
1 site
G C A T G C C G T A C G
SgrAI  (5138)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
XbaI  (5245)
1 site
T C T A G A A G A T C T
NdeI  (5285)
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.
NheI  (5344)
1 site
G C T A G C C G A T C G
BmtI  (5348)
1 site
G C T A G C C G A T C G
DraI  (5426)
1 site
T T T A A A A A A T T T
AflII  (5535)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
EcoRI  (5561)
1 site
G A A T T C C T T A A G
End  (5643)
0 sites
Start  (1)
0 sites
HindIII  (5)
1 site
A A G C T T T T C G A A
StyI  (158)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
DraIII  (464)
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  (589)
1 site
T T A T A A A A T A T T
AsiSI  (1163)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1163)
1 site
C G A T C G G C T A G C
AvaI  (1285)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (1285)
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  (1285)
1 site
C C C G G G G G G C C C
XmaI  (1285)
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.
BmeT110I  (1286)
1 site
C Y C G R G G R G C Y C
SmaI  (1287)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BspDI  (1468)
1 site
A T C G A T T A G C T A
ClaI  (1468)
1 site
A T C G A T T A G C T A
NruI  (1504)
1 site
T C G C G A A G C G C T
AlwNI  (1950)
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.
SUMO Forward
23-mer  /  39% GC
1 binding site
5539 .. 5561  =  23 annealed bases
Tm  =  54°C
T7 Reverse
20-mer  /  50% GC
1 binding site
130 .. 149  =  20 annealed bases
Tm  =  56°C
lacI
3730 .. 4812  =  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
3730 .. 4812  =  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
778 .. 1593  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
778 .. 1593  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
1715 .. 2303  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1715 .. 2303  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
231 .. 686  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
231 .. 686  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SUMO
5350 .. 5643  =  294 bp
98 amino acids  =  11.3 kDa
Product: ubiquitin-like protein tag
SUMO
5350 .. 5643  =  294 bp
98 amino acids  =  11.3 kDa
Product: ubiquitin-like protein tag
rop
2730 .. 2921  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2730 .. 2921  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
4813 .. 4890  =  78 bp
lacI promoter
4813 .. 4890  =  78 bp
T7 terminator
147 .. 194  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
147 .. 194  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
5218 .. 5242  =  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
5218 .. 5242  =  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
5199 .. 5217  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5199 .. 5217  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
5299 .. 5316  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5299 .. 5316  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
thrombin site
5326 .. 5343  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
thrombin site
5326 .. 5343  =  18 bp
6 amino acids  =  627.8 Da
Product: thrombin recognition and cleavage site
RBS
5272 .. 5278  =  7 bp
ribosome binding site
RBS
5272 .. 5278  =  7 bp
ribosome binding site
ATG
5287 .. 5289  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5287 .. 5289  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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