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Plasmid Files

pT7CFE1-CGFP-HA-His

Vector with C-terminal cleavable GFP and HA and 6xHis tags, for use in a human in vitro protein expression system.

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pT7CFE1-CGFP-HA-His.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Thermo Fisher (Pierce)
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PvuII (4309) BtgZI (4005) PsiI (3876) PfoI (3587) AatII (3476) ZraI (3474) ScaI (3034) NmeAIII (2702) BsaI (2615) AhdI (2554) AvrII (110) HindIII (183) Acc65I (400) KpnI (404) MscI (536) XcmI (544) NdeI (546) BstXI (547) BamHI (551) EcoRI (557) EcoRV (565) PacI (573) PstI (582) Eco53kI (586) SacI (588) SalI - SgrDI (590) AccI (591) HincII (592) NotI (597) PaeR7I - PspXI - XhoI (605) BspQI - SapI (764) KasI (782) NarI (783) SfoI (784) PluTI (786) FseI (978) BsaBI * (1098) BseRI (1262) BglII (1406) PmeI (1450) SpeI (1455) AlwNI (2077) pT7CFE1-CGFP-HA-His 4402 bp
PvuII  (4309)
1 site
C A G C T G G T C G A C
BtgZI  (4005)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
PsiI  (3876)
1 site
T T A T A A A A T A T T
PfoI  (3587)
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  (3476)
1 site
G A C G T C C T G C A G
ZraI  (3474)
1 site
G A C G T C C T G C A G
ScaI  (3034)
1 site
A G T A C T T C A T G A
NmeAIII  (2702)
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).
BsaI  (2615)
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  (2554)
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
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
MscI  (536)
1 site
T G G C C A A C C G G T
XcmI  (544)
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.
NdeI  (546)
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.
BstXI  (547)
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.
BamHI  (551)
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  (557)
1 site
G A A T T C C T T A A G
EcoRV  (565)
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  (573)
1 site
T T A A T T A A A A T T A A T T
PstI  (582)
1 site
C T G C A G G A C G T C
Eco53kI  (586)
1 site
G A G C T C C T C G A G
SacI  (588)
1 site
G A G C T C C T C G A G
SalI  (590)
1 site
G T C G A C C A G C T G
SgrDI  (590)
1 site
C G T C G A C G G C A G C T G C
AccI  (591)
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  (592)
1 site
G T Y R A C C A R Y T G
NotI  (597)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (605)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (605)
1 site
V C T C G A G B B G A G C T C V
XhoI  (605)
1 site
C T C G A G G A G C T C
BspQI  (764)
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  (764)
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.
KasI  (782)
1 site
G G C G C C C C G C G G
NarI  (783)
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  (784)
1 site
G G C G C C C C G C G G
PluTI  (786)
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.
FseI  (978)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
BsaBI  (1098)
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.
BseRI  (1262)
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.
BglII  (1406)
1 site
A G A T C T T C T A G A
PmeI  (1450)
1 site
G T T T A A A C C A A A T T T G
SpeI  (1455)
1 site
A C T A G T T G A T C A
AlwNI  (2077)
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
2481 .. 3341  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   2481 .. 3272  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2481 .. 3341  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   3273 .. 3341  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
2481 .. 3341  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
TurboGFP
635 .. 1330  =  696 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
TurboGFP
635 .. 1330  =  696 bp
232 amino acids  =  25.7 kDa
Product: improved green fluorescent protein from Pontellina plumata (Evdokimov et al., 2006)
mammalian codon-optimized
ori
1722 .. 2310  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1722 .. 2310  =  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
3780 .. 4235  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
3780 .. 4235  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
AmpR promoter
3342 .. 3446  =  105 bp
AmpR promoter
3342 .. 3446  =  105 bp
MCS
534 .. 610  =  77 bp
multiple cloning site
MCS
534 .. 610  =  77 bp
multiple cloning site
T7 terminator
1475 .. 1522  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
1475 .. 1522  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
poly(A)
1417 .. 1446  =  30 bp
poly(A)
1417 .. 1446  =  30 bp
HA
1343 .. 1369  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin) epitope tag
HA
1343 .. 1369  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin) epitope tag
HRV 3C site
611 .. 634  =  24 bp
8 amino acids  =  902.1 Da
Product: recognition and cleavage site for human rhinovirus 3C and PreScission proteases
HRV 3C site
611 .. 634  =  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
6xHis
1382 .. 1399  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
1382 .. 1399  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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:  2611 .. 2877  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  533 .. 1402  =  870 bp
ORF:  289 amino acids  =  31.9 kDa
ORF:  1107 .. 1478  =  372 bp
ORF:  123 amino acids  =  12.8 kDa
ORF:  478 .. 756  =  279 bp
ORF:  92 amino acids  =  9.6 kDa
ORF:  2481 .. 3341  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
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