pSpeedET

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

Sequence Author: Joint Center for Structural Genomics (JCSG)

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HomePlasmidsStructural Genomics VectorspSpeedET
AgeI (74) EcoRV (4850) AflII (4765) SphI (4471) MauBI (4449) PfoI (4381) PflFI - Tth111I (4282) BsaAI (4276) NdeI (4205) PciI (4026) BssSI - BssSαI (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) rrnB T2 terminator 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.
BssSI  (3853)
1 site		 C A C G A G G T G C T C
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 (50% GC)
Tm  =  52°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
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
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
TEV Site
382 .. 402  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV Site
382 .. 402  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
lac UV5 promoter
418 .. 448  =  31 bp
3 segments
   Segment 1:  -35  
   418 .. 423  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
418 .. 448  =  31 bp
3 segments
   Segment 2:  
   424 .. 441  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
418 .. 448  =  31 bp
3 segments
   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
T7 promoter
291 .. 309  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
291 .. 309  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  502 .. 1161  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  2396 .. 3211  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  1503 .. 1808  =  306 bp
ORF:  101 amino acids  =  11.7 kDa
ORF:  5124 .. 5372  =  249 bp
ORF:  82 amino acids  =  9.3 kDa
ORF:  4560 .. 25  =  930 bp
ORF:  309 amino acids  =  35.2 kDa
ORF:  1920 .. 2246  =  327 bp
ORF:  108 amino acids  =  12.1 kDa
ORF:  3006 .. 3233  =  228 bp
ORF:  75 amino acids  =  8.8 kDa
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Individual Sequences & Maps

p11
pMCSG19B (linearized)
pMCSG41
pMCSG68 (linearized)
p15Tv-L
pMCSG19C
pMCSG41 (linearized)
pMCSG69
p283
pMCSG19C (linearized)
pMCSG42
pMCSG69 (linearized)
pBEN1-SGC
pMCSG20
pMCSG42 (linearized)
pMCSG7
pBEN1-SGC (linearized)
pMCSG20 (linearized)
pMCSG43
pMCSG7 (linearized)
pDB-HisGST
pMCSG21
pMCSG43 (linearized)
pMCSG70
pDB.GST
pMCSG21 (linearized)
pMCSG46
pMCSG70 (linearized)
pDB.His.MBP
pMCSG22
pMCSG46 (linearized)
pMCSG71
pDB.His.NusA
pMCSG22 (linearized)
pMCSG48
pMCSG71 (linearized)
pDB.His.TRX
pMCSG23
pMCSG48 (linearized)
pMCSG73
pDB.TRX
pMCSG23 (linearized)
pMCSG49
pMCSG73 (linearized)
pEU-GST-FV
pMCSG24
pMCSG49 (linearized)
pMCSG75
pEU-His-FV
pMCSG24 (linearized)
pMCSG50
pMCSG75 (linearized)
pEU-HSBC
pMCSG25
pMCSG50 (linearized)
pMCSG76
pFB-LIC-Bse
pMCSG25 (linearized)
pMCSG51
pMCSG76 (linearized)
pFB-LIC-Bse (linearized)
pMCSG26
pMCSG51 (linearized)
pMCSG77
pH3c-LIC
pMCSG26 (linearized)
pMCSG52
pMCSG77 (linearized)
pH3c-LIC (linearized)
pMCSG27
pMCSG52 (linearized)
pMCSG79
pHM3C-LIC
pMCSG27 (linearized)
pMCSG53
pMCSG79 (linearized)
pHM3C-LIC (linearized)
pMCSG28
pMCSG53 (linearized)
pMCSG8
pLIC-CH
pMCSG28 (linearized)
pMCSG54
pMCSG8 (linearized)
pLIC-CH (linearized)
pMCSG29
pMCSG54 (linearized)
pMCSG81
pLIC-EGFP
pMCSG29 (linearized)
pMCSG55
pMCSG81 (linearized)
pLIC-EGFP (linearized)
pMCSG30
pMCSG55 (linearized)
pMCSG9
pLIC-SGC1
pMCSG30 (linearized)
pMCSG56
pMCSG9 (linearized)
pLIC-SGC1 (linearized)
pMCSG31
pMCSG56 (linearized)
pNH-TrxT
pMCentr1
pMCSG31 (linearized)
pMCSG57
pNH-TrxT (linearized)
pMCentr1 (linearized)
pMCSG32
pMCSG57 (linearized)
pNIC-CH
pMCentr2
pMCSG32 (linearized)
pMCSG58
pNIC-CH (linearized)
pMCentr2 (linearized)
pMCSG33
pMCSG58 (linearized)
pNIC-CTHF
pMCentr3
pMCSG33 (linearized)
pMCSG59
pNIC-CTHF (linearized)
pMCentr3 (linearized)
pMCSG34
pMCSG59 (linearized)
pNIC-Zb
pMCSG10
pMCSG34 (linearized)
pMCSG60
pNIC-Zb (linearized)
pMCSG10 (linearized)
pMCSG34B
pMCSG60 (linearized)
pNIC28-Bsa4
pMCSG11
pMCSG34B (linearized)
pMCSG61
pNIC28-Bsa4 (linearized)
pMCSG11 (linearized)
pMCSG35
pMCSG61 (linearized)
pNYCOMPS-LIC-FH10T+ (N term) - linearized
pMCSG12
pMCSG35 (linearized)
pMCSG62
pNYCOMPS-LIC-FH10T+ (N term)
pMCSG12 (linearized)
pMCSG35B
pMCSG62 (linearized)
pSpeedET
pMCSG13
pMCSG35B (linearized)
pMCSG63
pVP13
pMCSG13 (linearized)
pMCSG37
pMCSG63 (linearized)
pVP16
pMCSG14
pMCSG37 (linearized)
pMCSG64
pVP33A
pMCSG14 (linearized)
pMCSG38
pMCSG64 (linearized)
pVP33K
pMCSG17
pMCSG38 (linearized)
pMCSG65
pVP56A
pMCSG17 (linearized)
pMCSG38C
pMCSG65 (linearized)
pVP56K
pMCSG18
pMCSG38C (linearized)
pMCSG66
pVP65K
pMCSG18 (linearized)
pMCSG39
pMCSG66 (linearized)
pVP68K
pMCSG19
pMCSG39 (linearized)
pMCSG67
pVP81K
pMCSG19 (linearized)
pMCSG40
pMCSG67 (linearized)
pVP91A
pMCSG19B
pMCSG40 (linearized)
pMCSG68

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