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Plasmid Files

pcDNA3.1+/N-GST(TEV)

Mammalian vector with a CMV promoter for expressing proteins with an N-terminal and TEV protease-cleavable GST tag.

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pcDNA3.1+ N-GST(TEV).dna
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Sequence Author:  GenScript
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 SgrDI (6090) SspI (5973) PvuI (5539) AhdI (5169) BstZ17I (3897) BsmI (3845) PfoI (3700) RsrII (3441) BssHII (3322) PflFI - Tth111I (3043) BglII (12) MfeI (161) Bpu10I (180) NruI (208) MluI (228) SpeI (249) CMV enhancer NdeI (484) SnaBI (590) Eco53kI (816) SacI (818) NheI (895) BmtI (899) AflII (908) HindIII (911) EcoNI (933) BsgI (1209) SwaI (1350) BamHI (1607) EcoRI (1613) EcoRV (1625) BstXI (1635) NotI (1640) PaeR7I - PspXI - XhoI (1646) XbaI (1652) PspOMI (1658) ApaI (1662) BbsI (1878) DraIII (2192) SexAI * (2482) BseRI (2711) StuI (2714) AvrII (2715) TspMI - XmaI (2736) SmaI (2738) BsaBI * (2784) KasI (2924) NarI (2925) SfoI (2926) PluTI (2928) pcDNA3.1+/N-GST(TEV) 6092 bp
SgrDI  (6090)
1 site
C G T C G A C G G C A G C T G C
SspI  (5973)
1 site
A A T A T T T T A T A A
PvuI  (5539)
1 site
C G A T C G G C T A G C
AhdI  (5169)
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.
BstZ17I  (3897)
1 site
G T A T A C C A T A T G
BsmI  (3845)
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.
PfoI  (3700)
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.
RsrII  (3441)
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.
BssHII  (3322)
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.
PflFI  (3043)
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  (3043)
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.
BglII  (12)
1 site
A G A T C T T C T A G A
MfeI  (161)
1 site
C A A T T G G T T A A C
Bpu10I  (180)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
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.
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
NdeI  (484)
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.
SnaBI  (590)
1 site
T A C G T A A T G C A T
Eco53kI  (816)
1 site
G A G C T C C T C G A G
SacI  (818)
1 site
G A G C T C C T C G A G
NheI  (895)
1 site
G C T A G C C G A T C G
BmtI  (899)
1 site
G C T A G C C G A T C G
AflII  (908)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
HindIII  (911)
1 site
A A G C T T T T C G A A
EcoNI  (933)
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.
BsgI  (1209)
1 site
G T G C A G ( N ) 14 N N C A C G T C ( N ) 14

Efficient cleavage requires at least two copies of the BsgI
recognition sequence.
Sticky ends from different BsgI sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
SwaI  (1350)
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.
BamHI  (1607)
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.
EcoRI  (1613)
1 site
G A A T T C C T T A A G
EcoRV  (1625)
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.
BstXI  (1635)
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  (1640)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (1646)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1646)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1646)
1 site
C T C G A G G A G C T C
XbaI  (1652)
1 site
T C T A G A A G A T C T
PspOMI  (1658)
1 site
G G G C C C C C C G G G
ApaI  (1662)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
BbsI  (1878)
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.
DraIII  (2192)
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.
SexAI  (2482)
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.
BseRI  (2711)
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.
StuI  (2714)
1 site
A G G C C T T C C G G A
AvrII  (2715)
1 site
C C T A G G G G A T C C
TspMI  (2736)
1 site
C C C G G G G G G C C C
XmaI  (2736)
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.
SmaI  (2738)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BsaBI  (2784)
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.
KasI  (2924)
1 site
G G C G C C C C G C G G
NarI  (2925)
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  (2926)
1 site
G G C G C C C C G C G G
PluTI  (2928)
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.
AmpR
5096 .. 5956  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5096 .. 5887  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5096 .. 5956  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5888 .. 5956  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
5096 .. 5956  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
2797 .. 3591  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
2797 .. 3591  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
GST
923 .. 1576  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
923 .. 1576  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
ori
4337 .. 4925  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4337 .. 4925  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1959 .. 2387  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1959 .. 2387  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
2401 .. 2730  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
2401 .. 2730  =  330 bp
SV40 enhancer and early promoter
bGH poly(A) signal
1689 .. 1913  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
1689 .. 1913  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
3765 .. 3886  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
3765 .. 3886  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5957 .. 6061  =  105 bp
AmpR promoter
5957 .. 6061  =  105 bp
MCS
1607 .. 1663  =  57 bp
multiple cloning site
MCS
1607 .. 1663  =  57 bp
multiple cloning site
TEV site
1586 .. 1606  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
1586 .. 1606  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
2581 .. 2716  =  136 bp
SV40 origin of replication
SV40 ori
2581 .. 2716  =  136 bp
SV40 origin of replication
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