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Plasmid Files

pCS2G

Gateway® library vector that allows high-level transient expression in vertebrate cells as well as in vitro transcription/translation.

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pCS2G Sequence and MappCS2G.dna
Map and Sequence File   
Sequence Author:  I.M.A.G.E. Consortium
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 HindIII (28) NdeI (3769) MscI * (3582) BseRI (3461) SalI (3216) NaeI (3017) NgoMIV (3015) DraIII (2914) XmnI (2371) SP6 promoter attB1 AvaI - BsoBI - PaeR7I - XhoI (118) BmeT110I (119) Eco53kI (123) SacI (125) BamHI (163) attB2 PstI (226) T7 promoter SnaBI (248) HpaI (386) MfeI (395) EagI - NotI (454) BtgI (457) SacII (460) BstXI - NsiI (472) PspOMI (475) ApaI (479) Acc65I (481) KpnI (485) BssHII (518) M13 rev lac operator PvuII (703) BspQI - SapI (763) AflIII - PciI (879) NspI (883) AlwNI (1295) AhdI (1772) NmeAIII (1920) pCS2G 4203 bp
HindIII  (28)
1 site
A A G C T T T T C G A A
NdeI  (3769)
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.
MscI  (3582)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
BseRI  (3461)
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.
SalI  (3216)
1 site
G T C G A C C A G C T G
NaeI  (3017)
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.
NgoMIV  (3015)
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.
DraIII  (2914)
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.
XmnI  (2371)
1 site
G A A N N N N T T C C T T N N N N A A G
AvaI  (118)
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  (118)
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.
PaeR7I  (118)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (118)
1 site
C T C G A G G A G C T C
BmeT110I  (119)
1 site
C Y C G R G G R G C Y C
Eco53kI  (123)
1 site
G A G C T C C T C G A G
SacI  (125)
1 site
G A G C T C C T C G A G
BamHI  (163)
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.
PstI  (226)
1 site
C T G C A G G A C G T C
SnaBI  (248)
1 site
T A C G T A A T G C A T
HpaI  (386)
1 site
G T T A A C C A A T T G
MfeI  (395)
1 site
C A A T T G G T T A A C
EagI  (454)
1 site
C G G C C G G C C G G C
NotI  (454)
1 site
G C G G C C G C C G C C G G C G
BtgI  (457)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
SacII  (460)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BstXI  (472)
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.
NsiI  (472)
1 site
A T G C A T T A C G T A
PspOMI  (475)
1 site
G G G C C C C C C G G G
ApaI  (479)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
Acc65I  (481)
1 site
G G T A C C C C A T G G
KpnI  (485)
1 site
G G T A C C C C A T G G
BssHII  (518)
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.
PvuII  (703)
1 site
C A G C T G G T C G A C
BspQI  (763)
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  (763)
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.
AflIII  (879)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (879)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
NspI  (883)
1 site
R C A T G Y Y G T A C R
AlwNI  (1295)
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.
AhdI  (1772)
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.
NmeAIII  (1920)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII
recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
CMV IE94 promoter
3219 .. 4203  =  985 bp
enhancer/promoter region of simian
cytomegalovirus major immediate early
transcription unit IE94
CMV IE94 promoter
3219 .. 4203  =  985 bp
enhancer/promoter region of simian
cytomegalovirus major immediate early
transcription unit IE94
AmpR
1699 .. 2559  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1699 .. 2490  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1699 .. 2559  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   2491 .. 2559  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1699 .. 2559  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
940 .. 1528  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
940 .. 1528  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
2690 .. 3145  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
2690 .. 3145  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
SV40 poly(A) signal
252 .. 386  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
252 .. 386  =  135 bp
SV40 polyadenylation signal
AmpR promoter
2560 .. 2664  =  105 bp
AmpR promoter
2560 .. 2664  =  105 bp
lac promoter
586 .. 616  =  31 bp
   Segment 3:  -10  
   586 .. 592  =  7 bp
promoter for the E. coli lac operon
lac promoter
586 .. 616  =  31 bp
   Segment 2:  
   593 .. 610  =  18 bp
promoter for the E. coli lac operon
lac promoter
586 .. 616  =  31 bp
   Segment 1:  -35  
   611 .. 616  =  6 bp
promoter for the E. coli lac operon
lac promoter
586 .. 616  =  31 bp
3 segments
promoter for the E. coli lac operon
attB1
93 .. 117  =  25 bp
recombination site for the Gateway® BP reaction
attB1
93 .. 117  =  25 bp
recombination site for the Gateway® BP reaction
attB2
169 .. 193  =  25 bp
recombination site for the Gateway® BP reaction
attB2
169 .. 193  =  25 bp
recombination site for the Gateway® BP reaction
SP6 promoter
35 .. 53  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
35 .. 53  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
T7 promoter
229 .. 247  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
229 .. 247  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T3 promoter
499 .. 517  =  19 bp
promoter for bacteriophage T3 RNA polymerase
T3 promoter
499 .. 517  =  19 bp
promoter for bacteriophage T3 RNA polymerase
M13 rev
538 .. 554  =  17 bp
common sequencing primer, one of multiple similar
variants
M13 rev
538 .. 554  =  17 bp
common sequencing primer, one of multiple similar
variants
lac operator
562 .. 578  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
562 .. 578  =  17 bp
The lac repressor binds to the lac operator to inhibit
transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-β-D-thiogalactopyranoside (IPTG).
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