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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 Sequence and MappSpeedET.dna
Map and Sequence File   
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) T7 promoter ATG TEV Site 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

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
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

The sticky ends produced by AflII are hard to ligate.
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
araC
4560 .. 5438  =  879 bp
292 amino acids  =  33.4 kDa
Product: L-arabinose regulatory protein
araC
4560 .. 5438  =  879 bp
292 amino acids  =  33.4 kDa
Product: L-arabinose regulatory protein
KanR
2396 .. 3211  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
2396 .. 3211  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
CmR
502 .. 1161  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
502 .. 1161  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
3382 .. 3970  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3382 .. 3970  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ccdB
1503 .. 1808  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
1503 .. 1808  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
araBAD promoter
120 .. 285  =  166 bp
promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction
araBAD promoter
120 .. 285  =  166 bp
promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction
AmpR promoter
2304 .. 2395  =  92 bp
AmpR promoter
2304 .. 2395  =  92 bp
rrnB T1 terminator
2079 .. 2165  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
2079 .. 2165  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
lac UV5 promoter
418 .. 448  =  31 bp
   Segment 1:  -35  
   418 .. 423  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
418 .. 448  =  31 bp
   Segment 2:  
   424 .. 441  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
418 .. 448  =  31 bp
   Segment 3:  -10  
   442 .. 448  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
418 .. 448  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
rrnB T2 terminator
2257 .. 2284  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
2257 .. 2284  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
TEV Site
382 .. 402  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV Site
382 .. 402  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
291 .. 309  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
291 .. 309  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
364 .. 381  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
364 .. 381  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
346 .. 348  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
346 .. 348  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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