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Plasmid Files

pCMV-3Tag-8

Mammalian expression vector with a hygromycin resistance marker  for tagging proteins with a C-terminal 3xFLAG epitope.

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pCMV-3Tag-8 Sequence and MappCMV-3Tag-8.dna
Map and Sequence File   
Sequence Author:  Agilent Technologies
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 PciI (5184) BseRI (4459) AfeI (4450) PluTI (4390) SfoI (4388) NarI (4387) KasI (4386) KflI - PpuMI (4181) XcmI (4162) HSV TK poly(A) signal AsiSI (3226) BfuAI - BspMI (3175) BsmBI (2930) PshAI (2889) BstBI (2713) NsiI (5) SnaBI (346) NheI (597) BmtI (601) T3 promoter Eco53kI (653) SacI (655) AleI (661) BstXI (663) NotI (668) SrfI (682) BamHI (687) EcoRV (713) HindIII (717) BspDI - ClaI (724) SalI (732) AccI (733) AbsI - PaeR7I - PspXI - XhoI (738) 3xFLAG PspOMI (818) ApaI (822) stop codons SexAI * (845) T7 promoter BclI * (909) bGH poly(A) signal BbsI (1111) SphI (1122) PsiI (1232) NgoMIV (1461) NaeI (1463) EarI (1711) XmnI (1910) DraI (1932) FspI (2287) AhdI (2510) pCMV-3Tag-8 5236 bp
PciI  (5184)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseRI  (4459)
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.
AfeI  (4450)
1 site
A G C G C T T C G C G A
PluTI  (4390)
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.
SfoI  (4388)
1 site
G G C G C C C C G C G G
NarI  (4387)
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.
KasI  (4386)
1 site
G G C G C C C C G C G G
KflI  (4181)
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  (4181)
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.
XcmI  (4162)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
AsiSI  (3226)
1 site
G C G A T C G C C G C T A G C G
BfuAI  (3175)
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  (3175)
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.
BsmBI  (2930)
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.
PshAI  (2889)
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.
BstBI  (2713)
1 site
T T C G A A A A G C T T
NsiI  (5)
1 site
A T G C A T T A C G T A
SnaBI  (346)
1 site
T A C G T A A T G C A T
NheI  (597)
1 site
G C T A G C C G A T C G
BmtI  (601)
1 site
G C T A G C C G A T C G
Eco53kI  (653)
1 site
G A G C T C C T C G A G
SacI  (655)
1 site
G A G C T C C T C G A G
AleI  (661)
1 site
C A C N N N N G T G G T G N N N N C A C
BstXI  (663)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
NotI  (668)
1 site
G C G G C C G C C G C C G G C G
SrfI  (682)
1 site
G C C C G G G C C G G G C C C G
BamHI  (687)
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.
EcoRV  (713)
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.
HindIII  (717)
1 site
A A G C T T T T C G A A
BspDI  (724)
1 site
A T C G A T T A G C T A
ClaI  (724)
1 site
A T C G A T T A G C T A
SalI  (732)
1 site
G T C G A C C A G C T G
AccI  (733)
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.
AbsI  (738)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (738)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (738)
1 site
V C T C G A G B B G A G C T C V
XhoI  (738)
1 site
C T C G A G G A G C T C
PspOMI  (818)
1 site
G G G C C C C C C G G G
ApaI  (822)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
SexAI  (845)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BclI  (909)
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.
BbsI  (1111)
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.
SphI  (1122)
1 site
G C A T G C C G T A C G
PsiI  (1232)
1 site
T T A T A A A A T A T T
NgoMIV  (1461)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NgoMIV
recognition sequence.
NaeI  (1463)
1 site
G C C G G C C G G C C G

Efficient cleavage requires at least two copies of the NaeI
recognition sequence.
EarI  (1711)
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.
XmnI  (1910)
1 site
G A A N N N N T T C C T T N N N N A A G
DraI  (1932)
1 site
T T T A A A A A A T T T
FspI  (2287)
1 site
T G C G C A A C G C G T
AhdI  (2510)
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.
HygR
2862 .. 3899  =  1038 bp
345 amino acids  =  38.5 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
2862 .. 3899  =  1038 bp
345 amino acids  =  38.5 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
1723 .. 2580  =  858 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1723 .. 1791  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1723 .. 2580  =  858 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1792 .. 2580  =  789 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1723 .. 2580  =  858 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
4540 .. 5128  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4540 .. 5128  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1136 .. 1591  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1136 .. 1591  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
66 .. 370  =  305 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
66 .. 370  =  305 bp
human cytomegalovirus immediate early enhancer
bGH poly(A) signal
922 .. 1129  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
922 .. 1129  =  208 bp
bovine growth hormone polyadenylation signal
CMV promoter
371 .. 574  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
371 .. 574  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
HSV TK promoter
2674 .. 2819  =  146 bp
herpes simplex virus thymidine kinase promoter
HSV TK promoter
2674 .. 2819  =  146 bp
herpes simplex virus thymidine kinase promoter
AmpR promoter
1618 .. 1722  =  105 bp
AmpR promoter
1618 .. 1722  =  105 bp
MCS
651 .. 743  =  93 bp
multiple cloning site
MCS
651 .. 743  =  93 bp
multiple cloning site
3xFLAG
744 .. 815  =  72 bp
24 amino acids  =  3.0 kDa
Product: three tandem FLAG epitope tags
3xFLAG
744 .. 815  =  72 bp
24 amino acids  =  3.0 kDa
Product: three tandem FLAG epitope tags
HSV TK poly(A) signal
3941 .. 3989  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
3941 .. 3989  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
T3 promoter
620 .. 638  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
620 .. 638  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T7 promoter
869 .. 887  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
869 .. 887  =  19 bp
promoter for bacteriophage T7 RNA polymerase
stop codons
831 .. 841  =  11 bp
stop codons in all three reading frames
stop codons
831 .. 841  =  11 bp
stop codons in all three reading frames
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