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Plasmid Files

pT7CFE1-NHis-GST

Vector with N-terminal cleavable 9xHis and GST tags, for use in a human in vitro protein expression system.

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pT7CFE1-NHis-GST.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Thermo Fisher (Pierce)
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PvuII (4257) NaeI (4055) NgoMIV (4053) PsiI (3824) PfoI (3535) AatII (3424) ZraI (3422) NmeAIII (2650) BpmI (2572) BsaI (2563) AhdI (2502) AvrII (110) HindIII (183) PmlI (275) AarI - BfuAI - BspMI (298) Acc65I (400) KpnI (404) PflMI (412) BtgI (492) BmgBI (501) EcoNI (582) BspQI - SapI (651) BsgI (858) BstBI (969) SwaI (999) BclI * (1006) NdeI (1287) BamHI (1292) EcoRI (1298) EcoRV (1306) PacI (1314) PstI (1323) Eco53kI (1327) SacI (1329) SalI - SgrDI (1331) AccI (1332) HincII (1333) EagI - NotI (1338) AvaI - BsoBI - PaeR7I - PspXI - XhoI (1346) BmeT110I (1347) AleI (1348) BglII (1354) PmeI (1398) SpeI (1403) BlpI (1412) BseYI (1913) PspFI (1917) AlwNI (2025) pT7CFE1-NHis-GST 4350 bp
PvuII  (4257)
1 site
C A G C T G G T C G A C
NaeI  (4055)
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.
NgoMIV  (4053)
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.
PsiI  (3824)
1 site
T T A T A A A A T A T T
PfoI  (3535)
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.
AatII  (3424)
1 site
G A C G T C C T G C A G
ZraI  (3422)
1 site
G A C G T C C T G C A G
NmeAIII  (2650)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
BpmI  (2572)
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.
BsaI  (2563)
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.
AhdI  (2502)
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.
AvrII  (110)
1 site
C C T A G G G G A T C C
HindIII  (183)
1 site
A A G C T T T T C G A A
PmlI  (275)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (298)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
BfuAI  (298)
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  (298)
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.
Acc65I  (400)
1 site
G G T A C C C C A T G G
KpnI  (404)
1 site
G G T A C C C C A T G G
PflMI  (412)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
BtgI  (492)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
BmgBI  (501)
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.
EcoNI  (582)
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.
BspQI  (651)
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  (651)
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.
BsgI  (858)
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).
BstBI  (969)
1 site
T T C G A A A A G C T T
SwaI  (999)
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.
BclI  (1006)
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.
NdeI  (1287)
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.
BamHI  (1292)
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  (1298)
1 site
G A A T T C C T T A A G
EcoRV  (1306)
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.
PacI  (1314)
1 site
T T A A T T A A A A T T A A T T
PstI  (1323)
1 site
C T G C A G G A C G T C
Eco53kI  (1327)
1 site
G A G C T C C T C G A G
SacI  (1329)
1 site
G A G C T C C T C G A G
SalI  (1331)
1 site
G T C G A C C A G C T G
SgrDI  (1331)
1 site
C G T C G A C G G C A G C T G C
AccI  (1332)
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.
HincII  (1333)
1 site
G T Y R A C C A R Y T G
EagI  (1338)
1 site
C G G C C G G C C G G C
NotI  (1338)
1 site
G C G G C C G C C G C C G G C G
AvaI  (1346)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (1346)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (1346)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (1346)
1 site
V C T C G A G B B G A G C T C V
XhoI  (1346)
1 site
C T C G A G G A G C T C
BmeT110I  (1347)
1 site
C Y C G R G G R G C Y C
AleI  (1348)
1 site
C A C N N N N G T G G T G N N N N C A C
BglII  (1354)
1 site
A G A T C T T C T A G A
PmeI  (1398)
1 site
G T T T A A A C C A A A T T T G
SpeI  (1403)
1 site
A C T A G T T G A T C A
BlpI  (1412)
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.
BseYI  (1913)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
PspFI  (1917)
1 site
C C C A G C G G G T C G
AlwNI  (2025)
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.
AmpR
2429 .. 3289  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2429 .. 3220  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2429 .. 3289  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3221 .. 3289  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2429 .. 3289  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
GST
578 .. 1225  =  648 bp
216 amino acids  =  25.3 kDa
Product: glutathione S-transferase from Schistosoma japonicum
GST
578 .. 1225  =  648 bp
216 amino acids  =  25.3 kDa
Product: glutathione S-transferase from Schistosoma japonicum
ori
1670 .. 2258  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1670 .. 2258  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
IRES
70 .. 531  =  462 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
IRES
70 .. 531  =  462 bp
internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)
f1 ori
3728 .. 4183  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
3728 .. 4183  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
AmpR promoter
3290 .. 3394  =  105 bp
AmpR promoter
3290 .. 3394  =  105 bp
MCS
1286 .. 1353  =  68 bp
multiple cloning site
MCS
1286 .. 1353  =  68 bp
multiple cloning site
T7 terminator
1423 .. 1470  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1423 .. 1470  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
poly(A)
1365 .. 1394  =  30 bp
poly(A)
1365 .. 1394  =  30 bp
9xHis
551 .. 577  =  27 bp
9 amino acids  =  1.3 kDa
Product: 9xHis affinity tag
9xHis
551 .. 577  =  27 bp
9 amino acids  =  1.3 kDa
Product: 9xHis affinity tag
HRV 3C site
1256 .. 1279  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
HRV 3C site
1256 .. 1279  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ATG
533 .. 535  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
533 .. 535  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  533 .. 1354  =  822 bp
ORF:  273 amino acids  =  31.8 kDa
ORF:  2559 .. 2825  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2429 .. 3289  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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