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Plasmid Files

pSpeedET

Vector with a lethal ccdB gene, for polymerase incomplete primer extension (PIPE) cloning to express proteins with a cleavable 6xHis tag.

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pSpeedET.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Joint Center for Structural Genomics (JCSG)
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AgeI (74) EcoRV (4850) AflII (4765) SphI (4471) MauBI (4449) PfoI (4381) PflFI - Tth111I (4282) BsaAI (4276) NdeI (4205) PciI (4026) BssS α I (3853) AseI (3026) AsiSI - PvuI (2827) EcoNI (2739) BstEII (77) 5' Vector reverse primer (364 .. 399) KasI (400) NarI (401) SfoI (402) NgoMIV (403) PluTI (404) NaeI (405) PvuII (615) EcoRI (715) PasI (947) ScaI (1132) HincII (1307) BbvCI (1440) BsrGI (1559) SrfI (1586) BmgBI (1620) BstXI (1703) PacI (1814) XbaI (1819) 3' Vector forward primer (1857 .. 1874) PmeI (1860) XmnI (1901) BanII (2482) BspDI - ClaI (2518) pSpeedET 5464 bp
AgeI  (74)
1 site
A C C G G T T G G C C A
EcoRV  (4850)
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.
AflII  (4765)
1 site
C T T A A G G A A T T C
SphI  (4471)
1 site
G C A T G C C G T A C G
MauBI  (4449)
1 site
C G C G C G C G G C G C G C G C
PfoI  (4381)
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.
PflFI  (4282)
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  (4282)
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.
BsaAI  (4276)
1 site
Y A C G T R R T G C A Y
NdeI  (4205)
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.
PciI  (4026)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BssSαI  (3853)
1 site
C A C G A G G T G C T C
AseI  (3026)
1 site
A T T A A T T A A T T A
AsiSI  (2827)
1 site
G C G A T C G C C G C T A G C G
PvuI  (2827)
1 site
C G A T C G G C T A G C
EcoNI  (2739)
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.
BstEII  (77)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
KasI  (400)
1 site
G G C G C C C C G C G G
NarI  (401)
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  (402)
1 site
G G C G C C C C G C G G
NgoMIV  (403)
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.
PluTI  (404)
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.
NaeI  (405)
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.
PvuII  (615)
1 site
C A G C T G G T C G A C
EcoRI  (715)
1 site
G A A T T C C T T A A G
PasI  (947)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
ScaI  (1132)
1 site
A G T A C T T C A T G A
HincII  (1307)
1 site
G T Y R A C C A R Y T G
BbvCI  (1440)
1 site
C C T C A G C G G A G T C G
BsrGI  (1559)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
SrfI  (1586)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (1620)
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.
BstXI  (1703)
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.
PacI  (1814)
1 site
T T A A T T A A A A T T A A T T
XbaI  (1819)
1 site
T C T A G A A G A T C T
PmeI  (1860)
1 site
G T T T A A A C C A A A T T T G
XmnI  (1901)
1 site
G A A N N N N T T C C T T N N N N A A G
BanII  (2482)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
BspDI  (2518)
1 site
A T C G A T T A G C T A
ClaI  (2518)
1 site
A T C G A T T A G C T A
5' Vector reverse primer
36-mer  /  42% GC
1 binding site
364 .. 399  =  36 annealed bases
Tm  =  63°C
for PIPE cloning
3' Vector forward primer
35-mer  /  46% GC
1 binding site
1857 .. 1874  =  18 annealed bases
Tm  =  53°C
for PIPE cloning