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Plasmid Files

pcDNA™6.2/C-EmGFP-GW TOPO® (linearized)

Linearized vector with 3'-T overhangs and bound topoisomerase, for TOPO® TA cloning to add a C-terminal GFP tag and create a Gateway® expression clone.

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pcDNA6.2 C-EmGFP-GW TOPO (linearized) Sequence and MappcDNA6.2 C-EmGFP-GW TOPO (linearized).dna
Map and Sequence File   
Sequence Author:  Invitrogen (Life Technologies)
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 SgrDI (4859) SspI (4742) ScaI (4418) AhdI (3938) PciI (3045) BspQI - SapI (2929) BstZ17I (2666) BsmI (2614) BsaBI * (2276) PpuMI (2137) EagI (2094) BbsI (2063) MscI (1981) BclI * (1900) SmaI (1872) BmeT110I (1871) AvaI - BsoBI - TspMI - XmaI (1870) MfeI (5022) MluI (5089) SpeI (5110) NdeI (5345) SnaBI (5451) Eco53kI (5677) SacI (5679) End (5814) Start (1) HpaI (48) PmeI (781) SexAI * (1616) StuI (1848) AvrII (1849) pcDNA™6.2/C-EmGFP-GW TOPO® 5813 bp
SgrDI  (4859)
1 site
C G T C G A C G G C A G C T G C
SspI  (4742)
1 site
A A T A T T T T A T A A
ScaI  (4418)
1 site
A G T A C T T C A T G A
AhdI  (3938)
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.
PciI  (3045)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2929)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (2929)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be
mixed before removing an aliquot.
BstZ17I  (2666)
1 site
G T A T A C C A T A T G
BsmI  (2614)
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.
BsaBI  (2276)
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.
PpuMI  (2137)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
EagI  (2094)
1 site
C G G C C G G C C G G C
BbsI  (2063)
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.
MscI  (1981)
1 site
T G G C C A A C C G G T
BclI  (1900)
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.
SmaI  (1872)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BmeT110I  (1871)
1 site
C Y C G R G G R G C Y C
AvaI  (1870)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (1870)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
TspMI  (1870)
1 site
C C C G G G G G G C C C
XmaI  (1870)
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.
MfeI  (5022)
1 site
C A A T T G G T T A A C
MluI  (5089)
1 site
A C G C G T T G C G C A
SpeI  (5110)
1 site
A C T A G T T G A T C A
NdeI  (5345)
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.
SnaBI  (5451)
1 site
T A C G T A A T G C A T
Eco53kI  (5677)
1 site
G A G C T C C T C G A G
SacI  (5679)
1 site
G A G C T C C T C G A G
End  (5814)
0 sites
Start  (1)
0 sites
HpaI  (48)
1 site
G T T A A C C A A T T G
PmeI  (781)
1 site
G T T T A A A C C A A A T T T G
SexAI  (1616)
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.
StuI  (1848)
1 site
A G G C C T T C C G G A
AvrII  (1849)
1 site
C C T A G G G G A T C C
AmpR
3865 .. 4725  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   3865 .. 4656  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3865 .. 4725  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   4657 .. 4725  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
3865 .. 4725  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
EmGFP
52 .. 771  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 1:  
   52 .. 54  =  3 bp
   1 amino acid  =  149.2 Da
Product: Emerald GFP
mammalian codon-optimized
EmGFP
52 .. 771  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 2:  1a  
   55 .. 57  =  3 bp
   1 amino acid  =  117.2 Da
Product: Emerald GFP
mammalian codon-optimized
EmGFP
52 .. 771  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 3:  
   58 .. 771  =  714 bp
   237 amino acids  =  26.7 kDa
Product: Emerald GFP
mammalian codon-optimized
EmGFP
52 .. 771  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
Product: Emerald GFP
mammalian codon-optimized
ori
3106 .. 3694  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
3106 .. 3694  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1093 .. 1521  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1093 .. 1521  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
BSD
1978 .. 2376  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
1978 .. 2376  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
5096 .. 5475  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
5096 .. 5475  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
1535 .. 1864  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
1535 .. 1864  =  330 bp
SV40 enhancer and early promoter
CMV promoter
5476 .. 5679  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
5476 .. 5679  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
SV40 poly(A) signal
2534 .. 2655  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
2534 .. 2655  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4726 .. 4830  =  105 bp
AmpR promoter
4726 .. 4830  =  105 bp
HSV TK poly(A) signal
843 .. 891  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
843 .. 891  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
EM7 promoter
1912 .. 1959  =  48 bp
synthetic bacterial promoter
EM7 promoter
1912 .. 1959  =  48 bp
synthetic bacterial promoter
attB2
14 .. 38  =  25 bp
recombination site for the Gateway® BP reaction
attB2
14 .. 38  =  25 bp
recombination site for the Gateway® BP reaction
attB1
5776 .. 5800  =  25 bp
recombination site for the Gateway® BP reaction
attB1
5776 .. 5800  =  25 bp
recombination site for the Gateway® BP reaction
T7 promoter
5724 .. 5742  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5724 .. 5742  =  19 bp
promoter for bacteriophage T7 RNA polymerase
SV40 ori
1715 .. 1850  =  136 bp
SV40 origin of replication
SV40 ori
1715 .. 1850  =  136 bp
SV40 origin of replication
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