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Plasmid Files

pSecTag/FRT/V5-His-TOPO® (linearized)

Linearized mammalian vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products, FLP recombination, and secretion of tagged proteins.

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pSecTag FRT V5-His-TOPO (linearized) Sequence and MappSecTag FRT V5-His-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SgrDI (4171) SspI (4054) XmnI (3849) FspI (3472) AhdI (3250) PciI (2357) BspQI - SapI (2241) BstZ17I (1978) BsmI (1926) PsiI (1865) PfoI (1781) SacII (1471) BglII (4185) MfeI (4334) Bpu10I (4353) NruI (4381) SpeI (4422) SnaBI (4763) NheI (5068) BmtI (5072) SfiI (5150) MauBI (5160) BsiWI (5168) End (5185) Start (1) Acc65I (13) KpnI (17) BamHI (25) BseRI (46) AgeI (79) PmeI (109) BclI * (118) BbsI (320) SphI (331) XbaI (666) MscI (691) PshAI (718) BsmBI (759) EcoRI (934) BfuAI - BspMI (1004) PstI (1028) AsiSI (1055) RsrII (1099) pSecTag/FRT/V5-His-TOPO® 5184 bp
SgrDI  (4171)
1 site
C G T C G A C G G C A G C T G C
SspI  (4054)
1 site
A A T A T T T T A T A A
XmnI  (3849)
1 site
G A A N N N N T T C C T T N N N N A A G
FspI  (3472)
1 site
T G C G C A A C G C G T
AhdI  (3250)
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.
PciI  (2357)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2241)
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  (2241)
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.
BstZ17I  (1978)
1 site
G T A T A C C A T A T G
BsmI  (1926)
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.
PsiI  (1865)
1 site
T T A T A A A A T A T T
PfoI  (1781)
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.
SacII  (1471)
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.
BglII  (4185)
1 site
A G A T C T T C T A G A
MfeI  (4334)
1 site
C A A T T G G T T A A C
Bpu10I  (4353)
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  (4381)
1 site
T C G C G A A G C G C T
SpeI  (4422)
1 site
A C T A G T T G A T C A
SnaBI  (4763)
1 site
T A C G T A A T G C A T
NheI  (5068)
1 site
G C T A G C C G A T C G
BmtI  (5072)
1 site
G C T A G C C G A T C G
SfiI  (5150)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
MauBI  (5160)
1 site
C G C G C G C G G C G C G C G C
BsiWI  (5168)
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.
End  (5185)
0 sites
Start  (1)
0 sites
Acc65I  (13)
1 site
G G T A C C C C A T G G
KpnI  (17)
1 site
G G T A C C C C A T G G
BamHI  (25)
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.
BseRI  (46)
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.
AgeI  (79)
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.
PmeI  (109)
1 site
G T T T A A A C C A A A T T T G
BclI  (118)
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  (320)
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  (331)
1 site
G C A T G C C G T A C G
XbaI  (666)
1 site
T C T A G A A G A T C T
MscI  (691)
1 site
T G G C C A A C C G G T
PshAI  (718)
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.
BsmBI  (759)
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.
EcoRI  (934)
1 site
G A A T T C C T T A A G
BfuAI  (1004)
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  (1004)
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.
PstI  (1028)
1 site
C T G C A G G A C G T C
AsiSI  (1055)
1 site
G C G A T C G C C G C T A G C G
RsrII  (1099)
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.
HygR
694 .. 1716  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
HygR
694 .. 1716  =  1023 bp
340 amino acids  =  37.9 kDa
Product: aminoglycoside phosphotransferase from E.
coli

confers resistance to hygromycin
AmpR
3177 .. 4037  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3177 .. 3968  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3177 .. 4037  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3969 .. 4037  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3177 .. 4037  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
2418 .. 3006  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2418 .. 3006  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
4408 .. 4787  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4408 .. 4787  =  380 bp
human cytomegalovirus immediate early enhancer
bGH poly(A) signal
131 .. 355  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
131 .. 355  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
4788 .. 4991  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
4788 .. 4991  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
1846 .. 1967  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1846 .. 1967  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4038 .. 4142  =  105 bp
AmpR promoter
4038 .. 4142  =  105 bp
Igκ leader
5078 .. 5140  =  63 bp
21 amino acids  =  2.4 kDa
Product: leader sequence from mouse
immunoglobulin κ light chain
Igκ leader
5078 .. 5140  =  63 bp
21 amino acids  =  2.4 kDa
Product: leader sequence from mouse
immunoglobulin κ light chain
FRT
639 .. 686  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
FRT
639 .. 686  =  48 bp
FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011).
V5 tag
34 .. 75  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
34 .. 75  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
T7 promoter
5036 .. 5054  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5036 .. 5054  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
85 .. 102  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
85 .. 102  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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