pETite N-His SUMO Kan

Vector for Expresso® cloning to add N-terminal 6xHis and SUMO tags, with inducible expression from the T7 promoter.

Sequence Author: Lucigen

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XbaI (2521) SwaI (2498) tonB terminator AvaI - BsoBI - PaeR7I - PspXI - XhoI (2314) NruI (2259) EarI (2100) TsoI (2077) PasI (2041) EcoNI (2003) SspI (1991) BsrFI (1958) AsiSI - PvuI (1918) Bpu10I - BsmBI - Esp3I (1896) TaqII (1664) PflMI (1656) XmnI (1413) PvuII (1380) ApaI (1336) PspOMI (1332) BanI (45) lac operator PmlI (92) NdeI (141) ATG 6xHis KflI - PpuMI (165) PstI - SbfI (174) DrdI (182) BglII (270) AclI (359) SUMO Forward (383 .. 406) BclI * (391) EaeI - EagI - NotI (472) StyI (506) pETite Reverse (513 .. 532) AlwNI (925) ApaLI (1020) BsiHKAI (1024) BseYI (1030) PspFI (1034) HaeII (1094) BciVI (1136) BssSI - BssSαI (1161) pETite N-His SUMO Kan 2535 bp
XbaI  (2521)
1 site
T C T A G A A G A T C T
SwaI  (2498)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
AvaI  (2314)
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  (2314)
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.
PaeR7I  (2314)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (2314)
1 site
V C T C G A G B B G A G C T C V
XhoI  (2314)
1 site
C T C G A G G A G C T C
NruI  (2259)
1 site
T C G C G A A G C G C T
EarI  (2100)
1 site
C T C T T C N G A G A A G N N N N

Cleavage may be enhanced when more than one copy of the EarI recognition sequence is present.
Sticky ends from different EarI sites may not be compatible.
TsoI  (2077)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PasI  (2041)
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.
EcoNI  (2003)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
SspI  (1991)
1 site
A A T A T T T T A T A A
BsrFI  (1958)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
AsiSI  (1918)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1918)
1 site
C G A T C G G C T A G C
Bpu10I  (1896)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BsmBI  (1896)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (1896)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
TaqII  (1664)
1 site
G A C C G A ( N ) 9 N N C T G G C T ( N ) 9

Sticky ends from different TaqII sites may not be compatible.
PflMI  (1656)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
XmnI  (1413)
1 site
G A A N N N N T T C C T T N N N N A A G
PvuII  (1380)
1 site
C A G C T G G T C G A C
ApaI  (1336)
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  (1332)
1 site
G G G C C C C C C G G G
BanI  (45)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
PmlI  (92)
1 site
C A C G T G G T G C A C
NdeI  (141)
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.
KflI  (165)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
PpuMI  (165)
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.
PstI  (174)
1 site
C T G C A G G A C G T C
SbfI  (174)
1 site
C C T G C A G G G G A C G T C C
DrdI  (182)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BglII  (270)
1 site
A G A T C T T C T A G A
AclI  (359)
1 site
A A C G T T T T G C A A
BclI  (391)
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.
EaeI  (472)
1 site
Y G G C C R R C C G G Y
EagI  (472)
1 site
C G G C C G G C C G G C
NotI  (472)
1 site
G C G G C C G C C G C C G G C G
StyI  (506)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
AlwNI  (925)
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.
ApaLI  (1020)
1 site
G T G C A C C A C G T G
BsiHKAI  (1024)
1 site
G W G C W C C W C G W G

Sticky ends from different BsiHKAI sites may not be compatible.
BseYI  (1030)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
PspFI  (1034)
1 site
C C C A G C G G G T C G
HaeII  (1094)
1 site
R G C G C Y Y C G C G R
BciVI  (1136)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
BssSI  (1161)
1 site
C A C G A G G T G C T C
BssSαI  (1161)
1 site
C A C G A G G T G C T C
SUMO Forward
24-mer  /  46% GC
1 binding site
383 .. 406  =  24 annealed bases
Tm  =  59°C
pETite Reverse
20-mer  /  55% GC
1 binding site
513 .. 532  =  20 annealed bases
Tm  =  56°C
KanR
1533 .. 2348  =  816 bp
271 amino acids  =  30.9 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
1533 .. 2348  =  816 bp
271 amino acids  =  30.9 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
691 .. 1278  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
691 .. 1278  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ATG
143 .. 145  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
143 .. 145  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
146 .. 163  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
146 .. 163  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
SUMO
176 .. 463  =  288 bp
96 amino acids  =  11.0 kDa
Product: ubiquitin-like protein tag
modified by Lucigen to prevent cleavage by eukaryotic SUMO proteases
SUMO
176 .. 463  =  288 bp
96 amino acids  =  11.0 kDa
Product: ubiquitin-like protein tag
modified by Lucigen to prevent cleavage by eukaryotic SUMO proteases
rop
1341 .. 1532  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
1341 .. 1532  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
cat promoter
2349 .. 2439  =  91 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
cat promoter
2349 .. 2439  =  91 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
T7 terminator
495 .. 542  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
495 .. 542  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
tonB terminator
2440 .. 2471  =  32 bp
bidirectional E. coli tonB-P14 transcription terminator
tonB terminator
2440 .. 2471  =  32 bp
bidirectional E. coli tonB-P14 transcription terminator
T3Te terminator
640 .. 669  =  30 bp
phage T3 early transcription terminator
T3Te terminator
640 .. 669  =  30 bp
phage T3 early transcription terminator
T7Te terminator
1290 .. 1317  =  28 bp
phage T7 early transcription terminator
T7Te terminator
1290 .. 1317  =  28 bp
phage T7 early transcription terminator
RBS
113 .. 135  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
RBS
113 .. 135  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
T7 promoter
51 .. 69  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
51 .. 69  =  19 bp
promoter for bacteriophage T7 RNA polymerase
lac operator
71 .. 86  =  16 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
71 .. 86  =  16 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).
ORF:  143 .. 466  =  324 bp
ORF:  107 amino acids  =  12.3 kDa
ORF:  1248 .. 1475  =  228 bp
ORF:  75 amino acids  =  8.6 kDa
ORF:  1533 .. 2348  =  816 bp
ORF:  271 amino acids  =  30.9 kDa
ORF:  953 .. 1438  =  486 bp
ORF:  161 amino acids  =  18.0 kDa
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