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Plasmid Files

pGlow-TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning of PCR products to measure promoter activity using GFP.

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pGlow-TOPO (linearized) Sequence and MappGlow-TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 AatII (5216) SgrDI (5215) ZraI (5214) SspI (5098) ScaI (4774) PvuI (4664) BglI (4414) AhdI (4294) AlwNI (3817) PciI (3401) BsmI (2970) PfoI (2825) RsrII (2566) BssHII (2447) BglII (5229) T7 promoter PmeI (5275) AflII (5279) Acc65I (5288) KpnI (5292) SpeI (5306) BstXI (5319) End (5333) Start (1) BglII (15) BspEI * (248) NdeI (256) BsrGI (302) MluI (348) MfeI (586) BstXI (759) NotI (764) EcoO109I - PspOMI (782) ApaI (786) PmeI (791) BbsI (1002) DraIII (1316) SexAI * (1607) BseRI (1836) StuI (1839) AvrII (1840) TspMI - XmaI (1861) SmaI (1863) BsaBI * (1909) KasI (2049) NarI (2050) SfoI (2051) PluTI (2053) PflFI - Tth111I (2168) pGlow-TOPO® 5332 bp
AatII  (5216)
1 site
G A C G T C C T G C A G
SgrDI  (5215)
1 site
C G T C G A C G G C A G C T G C
ZraI  (5214)
1 site
G A C G T C C T G C A G
SspI  (5098)
1 site
A A T A T T T T A T A A
ScaI  (4774)
1 site
A G T A C T T C A T G A
PvuI  (4664)
1 site
C G A T C G G C T A G C
BglI  (4414)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
AhdI  (4294)
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.
AlwNI  (3817)
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.
PciI  (3401)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BsmI  (2970)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
PfoI  (2825)
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.
RsrII  (2566)
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.
BssHII  (2447)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
BglII  (5229)
2 sites
A G A T C T T C T A G A
PmeI  (5275)
2 sites
G T T T A A A C C A A A T T T G
AflII  (5279)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
Acc65I  (5288)
1 site
G G T A C C C C A T G G
KpnI  (5292)
1 site
G G T A C C C C A T G G
SpeI  (5306)
1 site
A C T A G T T G A T C A
BstXI  (5319)
2 sites
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.
End  (5333)
0 sites
Start  (1)
0 sites
BglII  (15)
2 sites
A G A T C T T C T A G A
BspEI  (248)
1 site
T C C G G A A G G C C T
* Blocked by Dam methylation.
NdeI  (256)
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.
BsrGI  (302)
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.
MluI  (348)
1 site
A C G C G T T G C G C A
MfeI  (586)
1 site
C A A T T G G T T A A C
BstXI  (759)
2 sites
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.
NotI  (764)
1 site
G C G G C C G C C G C C G G C G
EcoO109I  (782)
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.
PspOMI  (782)
1 site
G G G C C C C C C G G G
ApaI  (786)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PmeI  (791)
2 sites
G T T T A A A C C A A A T T T G
BbsI  (1002)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
DraIII  (1316)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
SexAI  (1607)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BseRI  (1836)
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.
StuI  (1839)
1 site
A G G C C T T C C G G A
AvrII  (1840)
1 site
C C T A G G G G A T C C
TspMI  (1861)
1 site
C C C G G G G G G C C C
XmaI  (1861)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (1863)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BsaBI  (1909)
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.
KasI  (2049)
1 site
G G C G C C C C G C G G
NarI  (2050)
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  (2051)
1 site
G G C G C C C C G C G G
PluTI  (2053)
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.
PflFI  (2168)
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  (2168)
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.
AmpR
4221 .. 5081  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4221 .. 5012  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4221 .. 5081  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5013 .. 5081  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4221 .. 5081  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
1922 .. 2716  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
1922 .. 2716  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
Cycle 3 GFP
24 .. 743  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 1:  
   24 .. 26  =  3 bp
   1 amino acid  =  149.2 Da
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
Cycle 3 GFP
24 .. 743  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 2:  1a  
   27 .. 29  =  3 bp
   1 amino acid  =  89.1 Da
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
Cycle 3 GFP
24 .. 743  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 3:  
   30 .. 743  =  714 bp
   237 amino acids  =  26.7 kDa
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
Cycle 3 GFP
24 .. 743  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
Product: Cycle 3 GFP (Crameri et al., 1996)
mammalian codon-optimized
ori
3462 .. 4050  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3462 .. 4050  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1083 .. 1511  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1083 .. 1511  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 promoter
1525 .. 1855  =  331 bp
SV40 enhancer and early promoter
SV40 promoter
1525 .. 1855  =  331 bp
SV40 enhancer and early promoter
bGH poly(A) signal
813 .. 1037  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
813 .. 1037  =  225 bp
bovine growth hormone polyadenylation signal
SV40 poly(A) signal
2890 .. 3011  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2890 .. 3011  =  122 bp
SV40 polyadenylation signal
AmpR promoter
5082 .. 5186  =  105 bp
AmpR promoter
5082 .. 5186  =  105 bp
T7 promoter
5234 .. 5252  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5234 .. 5252  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
1706 .. 1841  =  136 bp
SV40 origin of replication
SV40 ori
1706 .. 1841  =  136 bp
SV40 origin of replication
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