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pMIB/V5-His B

Insect cell vector for expression of secreted, C-terminally V5-6xHis-tagged proteins. For other reading frames, use pMIB/V5‑His A or pMIB/V5‑His C.

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pMIB V5-His B Sequence and MappMIB V5-His B.dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AhdI (3432) BpmI (3363) BglI (3314) ScaI (2951) XmnI (2832) EarI (2633) BglII (2565) SmaI (2561) TspMI - XmaI (2559) BmgBI (2556) DraIII (2532) BsaBI * (2425) NruI * (2358) PvuII (2334) PpuMI (2286) BbsI (2212) MscI (2130) NcoI - StyI (2125) PmlI (2058) BspHI (0) BsiWI (317) RsrII (335) KasI (474) NarI (475) SfoI (476) PluTI (478) SphI (633) HindIII (636) Acc65I (642) KpnI (646) Eco53kI (650) SacI (652) BamHI (654) SpeI (660) EcoRI (677) EcoRV (689) NotI (704) PaeR7I - PspXI - XhoI (710) XbaI (716) SacII (729) AgeI (783) 6xHis PsiI (890) BspDI * - ClaI * (957) PciI (1020) BciVI (1223) pMIB/V5-His B 3600 bp
AhdI  (3432)
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.
BpmI  (3363)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI
recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its
electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BglI  (3314)
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.
ScaI  (2951)
1 site
A G T A C T T C A T G A
XmnI  (2832)
1 site
G A A N N N N T T C C T T N N N N A A G
EarI  (2633)
1 site
C T C T T C N G A G A A G N N N N

Efficient cleavage requires at least two copies of the EarI
recognition sequence.
Sticky ends from different EarI sites may not be compatible.
BglII  (2565)
1 site
A G A T C T T C T A G A
SmaI  (2561)
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  (2559)
1 site
C C C G G G G G G C C C
XmaI  (2559)
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.
BmgBI  (2556)
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.
DraIII  (2532)
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.
BsaBI  (2425)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
NruI  (2358)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
PvuII  (2334)
1 site
C A G C T G G T C G A C
PpuMI  (2286)
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.
BbsI  (2212)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
MscI  (2130)
1 site
T G G C C A A C C G G T
NcoI  (2125)
1 site
C C A T G G G G T A C C
StyI  (2125)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
PmlI  (2058)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BspHI  (0)
1 site
T C A T G A A G T A C T
BsiWI  (317)
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.
RsrII  (335)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
KasI  (474)
1 site
G G C G C C C C G C G G
NarI  (475)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
SfoI  (476)
1 site
G G C G C C C C G C G G
PluTI  (478)
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.
SphI  (633)
1 site
G C A T G C C G T A C G
HindIII  (636)
1 site
A A G C T T T T C G A A
Acc65I  (642)
1 site
G G T A C C C C A T G G
KpnI  (646)
1 site
G G T A C C C C A T G G
Eco53kI  (650)
1 site
G A G C T C C T C G A G
SacI  (652)
1 site
G A G C T C C T C G A G
BamHI  (654)
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.
SpeI  (660)
1 site
A C T A G T T G A T C A
EcoRI  (677)
1 site
G A A T T C C T T A A G
EcoRV  (689)
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.
NotI  (704)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (710)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (710)
1 site
V C T C G A G B B G A G C T C V
XhoI  (710)
1 site
C T C G A G G A G C T C
XbaI  (716)
1 site
T C T A G A A G A T C T
SacII  (729)
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.
AgeI  (783)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
PsiI  (890)
1 site
T T A T A A A A T A T T
BspDI  (957)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (957)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
PciI  (1020)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BciVI  (1223)
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.
AmpR
2645 .. 3505  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2645 .. 2713  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2645 .. 3505  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2714 .. 3505  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2645 .. 3505  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
1081 .. 1669  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1081 .. 1669  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
OpIE-2 promoter
1 .. 548  =  548 bp
strong constitutive baculovirus promoter for insect
cell expression
OpIE-2 promoter
1 .. 548  =  548 bp
strong constitutive baculovirus promoter for insect
cell expression
BSD
2127 .. 2525  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
2127 .. 2525  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
OpIE-1 promoter
1744 .. 2035  =  292 bp
moderate constitutive baculovirus promoter for
insect cell expression
OpIE-1 promoter
1744 .. 2035  =  292 bp
moderate constitutive baculovirus promoter for
insect cell expression
OpIE-2 poly(A) signal
824 .. 953  =  130 bp
baculovirus polyadenylation signal
OpIE-2 poly(A) signal
824 .. 953  =  130 bp
baculovirus polyadenylation signal
MCS
629 .. 731  =  103 bp
multiple cloning site
MCS
629 .. 731  =  103 bp
multiple cloning site
melittin signal sequence
565 .. 627  =  63 bp
21 amino acids  =  2.5 kDa
Product: signal sequence from honeybee melittin
melittin signal sequence
565 .. 627  =  63 bp
21 amino acids  =  2.5 kDa
Product: signal sequence from honeybee melittin
EM7 promoter
2061 .. 2108  =  48 bp
synthetic bacterial promoter
EM7 promoter
2061 .. 2108  =  48 bp
synthetic bacterial promoter
V5 tag
738 .. 779  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
738 .. 779  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
6xHis
789 .. 806  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
789 .. 806  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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