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Plasmid Files

pLIC-EGFP

Bacterial vector for expressing a protein with a C-terminal TEV-EGFP-8xHis cassette.

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pLIC-EGFP Sequence and MappLIC-EGFP.dna
Map and Sequence File   
Sequence Author:  Center for Structures of Membrane Proteins
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 PaeR7I - PspXI - XhoI (5915) EagI - NotI (5907) HindIII (5900) SalI (5894) EcoRI (5881) BamHI (5875) BsrGI (5840) BseRI (5161) PacI (5105) DraI - SwaI (5091) ATG NcoI (5069) XbaI (5030) T7 promoter BglII (4964) SgrAI (4923) SphI (4775) BstAPI (4566) MluI (4242) BclI * (4228) BstEII (4060) ApaI (4039) PspOMI (4035) BssHII (3831) EcoRV (3796) HpaI (3740) PshAI (3401) BglI (3185) FspI - FspAI (3164) BlpI (5994) DraIII (245) PsiI (370) SpeI (448) AsiSI - PvuI (945) TspMI - XmaI (1067) SmaI (1069) BspDI - ClaI (1250) NruI (1286) AlwNI (1732) PciI (2141) BspQI - SapI (2258) BstZ17I (2374) PflFI - Tth111I (2399) PpuMI (3136) pLIC-EGFP 6077 bp
PaeR7I  (5915)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5915)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5915)
1 site
C T C G A G G A G C T C
EagI  (5907)
1 site
C G G C C G G C C G G C
NotI  (5907)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5900)
1 site
A A G C T T T T C G A A
SalI  (5894)
1 site
G T C G A C C A G C T G
EcoRI  (5881)
1 site
G A A T T C C T T A A G
BamHI  (5875)
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.
BsrGI  (5840)
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.
BseRI  (5161)
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.
PacI  (5105)
1 site
T T A A T T A A A A T T A A T T
DraI  (5091)
1 site
T T T A A A A A A T T T
SwaI  (5091)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
NcoI  (5069)
1 site
C C A T G G G G T A C C
XbaI  (5030)
1 site
T C T A G A A G A T C T
BglII  (4964)
1 site
A G A T C T T C T A G A
SgrAI  (4923)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
SphI  (4775)
1 site
G C A T G C C G T A C G
BstAPI  (4566)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
MluI  (4242)
1 site
A C G C G T T G C G C A
BclI  (4228)
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.
BstEII  (4060)
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.
ApaI  (4039)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (4035)
1 site
G G G C C C C C C G G G
BssHII  (3831)
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.
EcoRV  (3796)
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.
HpaI  (3740)
1 site
G T T A A C C A A T T G
PshAI  (3401)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BglI  (3185)
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.
FspI  (3164)
1 site
T G C G C A A C G C G T
FspAI  (3164)
1 site
R T G C G C A Y Y A C G C G T R
BlpI  (5994)
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.
DraIII  (245)
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.
PsiI  (370)
1 site
T T A T A A A A T A T T
SpeI  (448)
1 site
A C T A G T T G A T C A
AsiSI  (945)
1 site
G C G A T C G C C G C T A G C G
PvuI  (945)
1 site
C G A T C G G C T A G C
TspMI  (1067)
1 site
C C C G G G G G G C C C
XmaI  (1067)
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  (1069)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
BspDI  (1250)
1 site
A T C G A T T A G C T A
ClaI  (1250)
1 site
A T C G A T T A G C T A
NruI  (1286)
1 site
T C G C G A A G C G C T
AlwNI  (1732)
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  (2141)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2258)
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  (2258)
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  (2374)
1 site
G T A T A C C A T A T G
PflFI  (2399)
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  (2399)
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.
PpuMI  (3136)
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.
lacI
3515 .. 4597  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
lacI
3515 .. 4597  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
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).
KanR
560 .. 1375  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
560 .. 1375  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
EGFP
5134 .. 5847  =  714 bp
238 amino acids  =  26.8 kDa
   Segment 1:  1a  
   5134 .. 5136  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
5134 .. 5847  =  714 bp
238 amino acids  =  26.8 kDa
   Segment 2:  
   5137 .. 5847  =  711 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
5134 .. 5847  =  714 bp
238 amino acids  =  26.8 kDa
2 segments
Product: enhanced GFP
mammalian codon-optimized
ori
1497 .. 2085  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1497 .. 2085  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
12 .. 467  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
12 .. 467  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
2515 .. 2706  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2515 .. 2706  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
4598 .. 4675  =  78 bp
lacI promoter
4598 .. 4675  =  78 bp
T7 terminator
6005 .. 6052  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
6005 .. 6052  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
5003 .. 5027  =  25 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
5003 .. 5027  =  25 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).
8xHis
5848 .. 5871  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
5848 .. 5871  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
TEV site
5113 .. 5133  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
5113 .. 5133  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
4984 .. 5002  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
4984 .. 5002  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
5921 .. 5938  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5921 .. 5938  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
5058 .. 5063  =  6 bp
ribosome binding site
RBS
5058 .. 5063  =  6 bp
ribosome binding site
ATG
5071 .. 5073  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5071 .. 5073  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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