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Plasmid Files

pF25K ICE T7

Flexi® vector with a kanamycin resistance marker, for cell-free translation in an insect cell extract.

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pF25K ICE T7 Sequence and MappF25K ICE T7.dna
Map and Sequence File   
Sequence Author:  Promega
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 AseI (20) AclI (3782) BsaI (3646) ScaI (3459) MluI (3375) BspEI (3162) BspHI (3118) ApaLI (2712) PciI (2398) AgeI (2330) BstBI (2303) RsrII (2137) NaeI (2123) NgoMIV (2121) T7 promoter AsiSI - PvuI - SgfI (90) BtgI - NcoI (93) PmeI (457) EcoRI (462) Acc65I (474) KpnI (478) BamHI (483) XbaI (489) SalI (495) AccI (496) HincII (497) SbfI (505) BlpI (570) AfeI (645) MfeI (676) SnaBI (687) AanI - PsiI (712) SwaI (777) EcoO109I (1034) NruI (1069) NotI (1074) StuI (1083) BstAPI (1319) XcmI (1437) BclI * (1462) MscI (1703) FspI (1723) PflFI - Tth111I (1739) BsrDI (1854) pF25K ICE T7 3942 bp
AseI  (20)
1 site
A T T A A T T A A T T A
AclI  (3782)
1 site
A A C G T T T T G C A A
BsaI  (3646)
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.
ScaI  (3459)
1 site
A G T A C T T C A T G A
MluI  (3375)
1 site
A C G C G T T G C G C A
BspEI  (3162)
1 site
T C C G G A A G G C C T
BspHI  (3118)
1 site
T C A T G A A G T A C T
ApaLI  (2712)
1 site
G T G C A C C A C G T G
PciI  (2398)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AgeI  (2330)
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.
BstBI  (2303)
1 site
T T C G A A A A G C T T
RsrII  (2137)
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.
NaeI  (2123)
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  (2121)
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.
AsiSI  (90)
1 site
G C G A T C G C C G C T A G C G
PvuI  (90)
1 site
C G A T C G G C T A G C
SgfI  (90)
1 site
G C G A T C G C C G C T A G C G
BtgI  (93)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (93)
1 site
C C A T G G G G T A C C
PmeI  (457)
1 site
G T T T A A A C C A A A T T T G
EcoRI  (462)
1 site
G A A T T C C T T A A G
Acc65I  (474)
1 site
G G T A C C C C A T G G
KpnI  (478)
1 site
G G T A C C C C A T G G
BamHI  (483)
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.
XbaI  (489)
1 site
T C T A G A A G A T C T
SalI  (495)
1 site
G T C G A C C A G C T G
AccI  (496)
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  (497)
1 site
G T Y R A C C A R Y T G
SbfI  (505)
1 site
C C T G C A G G G G A C G T C C
BlpI  (570)
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.
AfeI  (645)
1 site
A G C G C T T C G C G A
MfeI  (676)
1 site
C A A T T G G T T A A C
SnaBI  (687)
1 site
T A C G T A A T G C A T
AanI  (712)
1 site
T T A T A A A A T A T T
PsiI  (712)
1 site
T T A T A A A A T A T T
SwaI  (777)
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.
EcoO109I  (1034)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
NruI  (1069)
1 site
T C G C G A A G C G C T
NotI  (1074)
1 site
G C G G C C G C C G C C G G C G
StuI  (1083)
1 site
A G G C C T T C C G G A
BstAPI  (1319)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
XcmI  (1437)
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.
BclI  (1462)
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.
MscI  (1703)
1 site
T G G C C A A C C G G T
FspI  (1723)
1 site
T G C G C A A C G C G T
PflFI  (1739)
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  (1739)
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.
BsrDI  (1854)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
NeoR/KanR
1493 .. 2287  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1493 .. 2287  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
2459 .. 3047  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2459 .. 3047  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
polyhedrin 3' UTR
611 .. 983  =  373 bp
3' UTR from the Autographa californica
nucleopolyhedrovirus polyhedrin gene (Suzuki et al.,
2006)
polyhedrin 3' UTR
611 .. 983  =  373 bp
3' UTR from the Autographa californica
nucleopolyhedrovirus polyhedrin gene (Suzuki et al.,
2006)
barnase
117 .. 452  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
barnase
117 .. 452  =  336 bp
111 amino acids  =  12.5 kDa
Product: ribonuclease from Bacillus
amyloliquefaciens

The barnase gene is lethal in standard bacterial
transformation strains.
cer region
3163 .. 3446  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
cer region
3163 .. 3446  =  284 bp
ColE1-derived recombination site that helps to
maintain plasmids as monomers
rrnB T1 terminator
3676 .. 3762  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
3676 .. 3762  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
T7 terminator
1018 .. 1065  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
1018 .. 1065  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
polyhedrin 5' UTR
40 .. 85  =  46 bp
5' UTR from the Malacosoma neustria
nucleopolyhedrovirus polyhedrin gene (Ezure et al.,
2006)
polyhedrin 5' UTR
40 .. 85  =  46 bp
5' UTR from the Malacosoma neustria
nucleopolyhedrovirus polyhedrin gene (Ezure et al.,
2006)
poly(A)
988 .. 1017  =  30 bp
synthetic poly(A) tract
poly(A)
988 .. 1017  =  30 bp
synthetic poly(A) tract
rrnB T2 terminator
3854 .. 3881  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
3854 .. 3881  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
21 .. 39  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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