pBiEx-3

Vector for high-level expression in both bacterial and insect cells of proteins with a C-terminal S-Tag-8xHis cassette.

Sequence Author: MilliporeSigma (Novagen)

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BspDI * - ClaI * (207) SmaI (5370) TspMI - XmaI (5368) BspQI - SapI (5124) PciI (5007) DrdI (4905) AlwNI (4598) AhdI (4119) BsaI (4053) ScaI (3638) XmnI (3519) EcoNI (3130) PfoI (3094) lacI promoter BstAPI (2986) BstEII (2480) PsiI (409) NheI (606) BmtI (610) SphI (614) BsmI (775) BseRI (781) BsiWI (784) SacII (1085) T7 promoter lac operator RBS NcoI (1177) ATG BmgBI (1194) Eco53kI (1202) SacI (1204) BamHI (1208) MfeI (1214) BglII (1222) BfuAI - BspMI (1229) AscI (1230) PstI - SbfI (1240) SalI (1242) AccI (1243) Acc65I (1248) AgeI (1251) KpnI (1252) BstBI (1257) HindIII (1260) NotI (1267) PaeR7I - PspXI - XhoI (1344) 8xHis DraIII (1376) BbvCI - Bpu10I (1386) BlpI (1423) EcoO109I (1450) Bsu36I (1500) PacI (1529) PflFI - Tth111I (1753) KasI (2023) NarI * (2024) SfoI (2025) PluTI (2027) BsmBI - Esp3I (2047) PspOMI (2455) ApaI (2459) pBiEx™-3 5373 bp
BspDI  (207)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (207)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SmaI  (5370)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (5368)
1 site
C C C G G G G G G C C C
XmaI  (5368)
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.
BspQI  (5124)
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  (5124)
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  (5007)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (4905)
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.
AlwNI  (4598)
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.
AhdI  (4119)
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.
BsaI  (4053)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
ScaI  (3638)
1 site
A G T A C T T C A T G A
XmnI  (3519)
1 site
G A A N N N N T T C C T T N N N N A A G
EcoNI  (3130)
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.
PfoI  (3094)
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.
BstAPI  (2986)
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.
BstEII  (2480)
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.
PsiI  (409)
1 site
T T A T A A A A T A T T
NheI  (606)
1 site
G C T A G C C G A T C G
BmtI  (610)
1 site
G C T A G C C G A T C G
SphI  (614)
1 site
G C A T G C C G T A C G
BsmI  (775)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
BseRI  (781)
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.
BsiWI  (784)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
SacII  (1085)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
NcoI  (1177)
1 site
C C A T G G G G T A C C
BmgBI  (1194)
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.
Eco53kI  (1202)
1 site
G A G C T C C T C G A G
SacI  (1204)
1 site
G A G C T C C T C G A G
BamHI  (1208)
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.
MfeI  (1214)
1 site
C A A T T G G T T A A C
BglII  (1222)
1 site
A G A T C T T C T A G A
BfuAI  (1229)
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  (1229)
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.
AscI  (1230)
1 site
G G C G C G C C C C G C G C G G
PstI  (1240)
1 site
C T G C A G G A C G T C
SbfI  (1240)
1 site
C C T G C A G G G G A C G T C C
SalI  (1242)
1 site
G T C G A C C A G C T G
AccI  (1243)
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.
Acc65I  (1248)
1 site
G G T A C C C C A T G G
AgeI  (1251)
1 site
A C C G G T T G G C C A
KpnI  (1252)
1 site
G G T A C C C C A T G G
BstBI  (1257)
1 site
T T C G A A A A G C T T
HindIII  (1260)
1 site
A A G C T T T T C G A A
NotI  (1267)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (1344)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1344)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1344)
1 site
C T C G A G G A G C T C
DraIII  (1376)
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.
BbvCI  (1386)
1 site
C C T C A G C G G A G T C G
Bpu10I  (1386)
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.
BlpI  (1423)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
EcoO109I  (1450)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
Bsu36I  (1500)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
PacI  (1529)
1 site
T T A A T T A A A A T T A A T T
PflFI  (1753)
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  (1753)
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.
KasI  (2023)
1 site
G G C G C C C C G C G G
NarI  (2024)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (2025)
1 site
G G C G C C C C G C G G
PluTI  (2027)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
BsmBI  (2047)
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  (2047)
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.
PspOMI  (2455)
1 site
G G G C C C C C C G G G
ApaI  (2459)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
lacI
1935 .. 3017  =  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
1935 .. 3017  =  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).
AmpR
3332 .. 4192  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3332 .. 3400  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3332 .. 4192  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3401 .. 4192  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3332 .. 4192  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
IE1 promoter
487 .. 1078  =  592 bp
promoter of the ie1 gene from the baculovirus Autographa californica
IE1 promoter
487 .. 1078  =  592 bp
promoter of the ie1 gene from the baculovirus Autographa californica
ori
4363 .. 4951  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
4363 .. 4951  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
hr5 enhancer
1 .. 483  =  483 bp
baculovirus early transcription enhancer
hr5 enhancer
1 .. 483  =  483 bp
baculovirus early transcription enhancer
ATG
1179 .. 1181  =  3 bp
1 amino acid  =  149.2 Da
ATG
1179 .. 1181  =  3 bp
1 amino acid  =  149.2 Da
S-Tag
1284 .. 1328  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
1284 .. 1328  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
8xHis
1350 .. 1373  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
1350 .. 1373  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
AmpR promoter
3227 .. 3331  =  105 bp
AmpR promoter
3227 .. 3331  =  105 bp
lacI promoter
3018 .. 3095  =  78 bp
lacI promoter
3018 .. 3095  =  78 bp
T7 terminator
1434 .. 1481  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1434 .. 1481  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
1111 .. 1135  =  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
1111 .. 1135  =  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
1092 .. 1110  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1092 .. 1110  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
1166 .. 1171  =  6 bp
ribosome binding site
RBS
1166 .. 1171  =  6 bp
ribosome binding site
ORF:  3193 .. 3435  =  243 bp
ORF:  80 amino acids  =  9.3 kDa
ORF:  1727 .. 2152  =  426 bp
ORF:  141 amino acids  =  15.5 kDa
ORF:  3332 .. 4192  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1015 .. 1275  =  261 bp
ORF:  86 amino acids  =  9.8 kDa
ORF:  1915 .. 2178  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  2767 .. 3132  =  366 bp
ORF:  121 amino acids  =  13.1 kDa
ORF:  3796 .. 4062  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1935 .. 2894  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  452 .. 1018  =  567 bp
ORF:  188 amino acids  =  21.4 kDa
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