pcDNA6.2 N-EmGFP-GW TOPO (linearized)

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

Sequence Author: Thermo Fisher (Invitrogen)

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SgrDI (4187) SspI (4070) ScaI (3746) AhdI (3266) PciI (2373) BspQI - SapI (2257) BstZ17I (1994) BsmI (1942) BlpI (1701) BsaBI * (1604) PpuMI (1465) EagI (1422) MscI (1309) BclI * (1228) SmaI (1200) TspMI - XmaI (1198) MfeI (4350) SpeI (4438) NdeI (4673) SnaBI (4779) Eco53kI (5005) SacI (5007) BsmBI - Esp3I (5028) BspEI (5090) XbaI (5097) BamHI (5103) EcoRV (5119) PspXI (5851) End (5902) Start (1) attB2 PmeI (109) CsiI - SexAI * (944) StuI (1176) AvrII (1177) pcDNA™6.2/N-EmGFP-GW TOPO® 5901 bp
SgrDI  (4187)
1 site
C G T C G A C G G C A G C T G C
SspI  (4070)
1 site
A A T A T T T T A T A A
ScaI  (3746)
1 site
A G T A C T T C A T G A
AhdI  (3266)
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  (2373)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2257)
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  (2257)
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  (1994)
1 site
G T A T A C C A T A T G
BsmI  (1942)
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.
BlpI  (1701)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
BsaBI  (1604)
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  (1465)
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  (1422)
1 site
C G G C C G G C C G G C
MscI  (1309)
1 site
T G G C C A A C C G G T
BclI  (1228)
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  (1200)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (1198)
1 site
C C C G G G G G G C C C
XmaI  (1198)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
MfeI  (4350)
1 site
C A A T T G G T T A A C
SpeI  (4438)
1 site
A C T A G T T G A T C A
NdeI  (4673)
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  (4779)
1 site
T A C G T A A T G C A T
Eco53kI  (5005)
1 site
G A G C T C C T C G A G
SacI  (5007)
1 site
G A G C T C C T C G A G
BsmBI  (5028)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (5028)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
BspEI  (5090)
1 site
T C C G G A A G G C C T
XbaI  (5097)
1 site
T C T A G A A G A T C T
BamHI  (5103)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF® (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
EcoRV  (5119)
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.
PspXI  (5851)
1 site
V C T C G A G B B G A G C T C V
End  (5902)
0 sites
Start  (1)
0 sites
PmeI  (109)
1 site
G T T T A A A C C A A A T T T G
CsiI  (944)
1 site
A C C W G G T T G G W C C A

Sticky ends from different CsiI sites may not be compatible.
SexAI  (944)
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  (1176)
1 site
A G G C C T T C C G G A
AvrII  (1177)
1 site
C C T A G G G G A T C C
AmpR
3193 .. 4053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   3193 .. 3984  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3193 .. 4053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   3985 .. 4053  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3193 .. 4053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
EmGFP
5132 .. 5848  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 1:  
   5132 .. 5134  =  3 bp
   1 amino acid  =  149.2 Da
Product: Emerald GFP
mammalian codon-optimized
EmGFP
5132 .. 5848  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 2:  1a  
   5135 .. 5137  =  3 bp
   1 amino acid  =  117.1 Da
Product: Emerald GFP
mammalian codon-optimized
EmGFP
5132 .. 5848  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
   Segment 3:  
   5138 .. 5848  =  711 bp
   237 amino acids  =  26.7 kDa
Product: Emerald GFP
mammalian codon-optimized
EmGFP
5132 .. 5848  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
Product: Emerald GFP
mammalian codon-optimized
ori
2434 .. 3022  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
ori
2434 .. 3022  =  589 bp
high-copy-number colE1/pMB1/pBR322/pUC origin of replication
f1 ori
421 .. 849  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
421 .. 849  =  429 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
BSD
1306 .. 1704  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
BSD
1306 .. 1704  =  399 bp
132 amino acids  =  13.7 kDa
Product: blasticidin S deaminase
confers resistance to blasticidin
CMV enhancer
4424 .. 4803  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
4424 .. 4803  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
863 .. 1192  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
863 .. 1192  =  330 bp
SV40 enhancer and early promoter
CMV promoter
4804 .. 5007  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
CMV promoter
4804 .. 5007  =  204 bp
human cytomegalovirus (CMV) immediate early promoter
SV40 poly(A) signal
1862 .. 1983  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
1862 .. 1983  =  122 bp
SV40 polyadenylation signal
AmpR promoter
4054 .. 4158  =  105 bp
AmpR promoter
4054 .. 4158  =  105 bp
HSV TK poly(A) signal
171 .. 219  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
171 .. 219  =  49 bp
herpesvirus thymidine kinase polyadenylation signal
EM7 promoter
1240 .. 1287  =  48 bp
synthetic bacterial promoter
EM7 promoter
1240 .. 1287  =  48 bp
synthetic bacterial promoter
V5 tag
46 .. 87  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
V5 tag
46 .. 87  =  42 bp
14 amino acids  =  1.4 kDa
Product: epitope tag from simian virus 5
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
5864 .. 5888  =  25 bp
recombination site for the Gateway® BP reaction
attB1
5864 .. 5888  =&