pLIC-EGFP (linearized)

Linearized bacterial vector for ligation-independent cloning (LIC), with a C-terminal TEV-EGFP-8xHis cassette.

Sequence Author: Center for Structures of Membrane Proteins

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BlpI (903) PaeR7I - PspXI - XhoI (824) EagI - NotI (816) HindIII (809) SalI (803) EcoRI (790) BamHI (784) BsrGI (749) BseRI (70) Start (0) End (6061) ATG NcoI (6055) RBS XbaI (6016) T7 promoter BglII (5950) SgrAI (5909) SphI (5761) BstAPI (5552) MluI (5228) BclI * (5214) BstEII (5046) ApaI (5025) PspOMI (5021) BssHII (4817) EcoRV (4782) HpaI (4726) PshAI (4387) BglI (4171) DraIII (1231) PsiI (1356) SpeI (1434) AsiSI - PvuI (1931) TspMI - XmaI (2053) SmaI (2055) BspDI - ClaI (2236) NruI (2272) AlwNI (2718) PciI (3127) BspQI - SapI (3244) BstZ17I (3360) PflFI - Tth111I (3385) PpuMI (4122) FspI - FspAI (4150) pLIC-EGFP 6061 bp
BlpI  (903)
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.
PaeR7I  (824)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (824)
1 site
V C T C G A G B B G A G C T C V
XhoI  (824)
1 site
C T C G A G G A G C T C
EagI  (816)
1 site
C G G C C G G C C G G C
NotI  (816)
1 site
G C G G C C G C C G C C G G C G
HindIII  (809)
1 site
A A G C T T T T C G A A
SalI  (803)
1 site
G T C G A C C A G C T G
EcoRI  (790)
1 site
G A A T T C C T T A A G
BamHI  (784)
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  (749)
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  (70)
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.
Start  (0)
0 sites
End  (6061)
0 sites
NcoI  (6055)
1 site
C C A T G G G G T A C C
XbaI  (6016)
1 site
T C T A G A A G A T C T
BglII  (5950)
1 site
A G A T C T T C T A G A
SgrAI  (5909)
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  (5761)
1 site
G C A T G C C G T A C G
BstAPI  (5552)
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  (5228)
1 site
A C G C G T T G C G C A
BclI  (5214)
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  (5046)
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  (5025)
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  (5021)
1 site
G G G C C C C C C G G G
BssHII  (4817)
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  (4782)
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  (4726)
1 site
G T T A A C C A A T T G
PshAI  (4387)
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  (4171)
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.
DraIII  (1231)
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  (1356)
1 site
T T A T A A A A T A T T
SpeI  (1434)
1 site
A C T A G T T G A T C A
AsiSI  (1931)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1931)
1 site
C G A T C G G C T A G C
TspMI  (2053)
1 site
C C C G G G G G G C C C
XmaI  (2053)
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.
SmaI  (2055)
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  (2236)
1 site
A T C G A T T A G C T A
ClaI  (2236)
1 site
A T C G A T T A G C T A
NruI  (2272)
1 site
T C G C G A A G C G C T
AlwNI  (2718)
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  (3127)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3244)
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  (3244)
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  (3360)
1 site
G T A T A C C A T A T G
PflFI  (3385)
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  (3385)
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  (4122)
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.
FspI  (4150)
1 site
T G C G C A A C G C G T
FspAI  (4150)
1 site
R T G C G C A Y Y A C G C G T R
lacI
4501 .. 5583  =  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
4501 .. 5583  =  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
1546 .. 2361  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
1546 .. 2361  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
TEV site
22 .. 42  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
22 .. 42  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
EGFP
43 .. 756  =  714 bp
238 amino acids  =  26.8 kDa
2 segments
   Segment 1:  1a  
   43 .. 45  =  3 bp
   1 amino acid  =  117.1 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
43 .. 756  =  714 bp
238 amino acids  =  26.8 kDa
2 segments
   Segment 2:  
   46 .. 756  =  711 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
43 .. 756  =  714 bp
238 amino acids  =  26.8 kDa
2 segments
Product: enhanced GFP
mammalian codon-optimized
8xHis
757 .. 780  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
8xHis
757 .. 780  =  24 bp
8 amino acids  =  1.1 kDa
Product: 8xHis affinity tag
ori
2483 .. 3071  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2483 .. 3071  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
998 .. 1453  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
998 .. 1453  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
3501 .. 3692  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
3501 .. 3692  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
5584 .. 5661  =  78 bp
lacI promoter
5584 .. 5661  =  78 bp
T7 terminator
914 .. 961  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
914 .. 961  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
5989 .. 6013  =  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
5989 .. 6013  =  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).
T7 promoter
5970 .. 5988  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5970 .. 5988  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
830 .. 847  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
830 .. 847  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
6044 .. 6049  =  6 bp
ribosome binding site
RBS
6044 .. 6049  =  6 bp
ribosome binding site
ATG
6057 .. 6059  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
6057 .. 6059  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  1 .. 783  =  783 bp
ORF:  260 amino acids  =  29.5 kDa  (no start codon)
ORF:  3724 .. 4092  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  2003 .. 2242  =  240 bp
ORF:  79 amino acids  =  8.3 kDa
ORF:  5759 .. 5998  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  4467 .. 4718  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  5622 .. 5885  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  3501 .. 3725  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  4089 .. 4445  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  4481 .. 4744  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  5333 .. 5833  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
ORF:  10 .. 777  =  768 bp
ORF:  255 amino acids  =  25.8 kDa
ORF:  1546 .. 2361  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  4501 .. 5460  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  5830 .. 6075  =  246 bp
ORF:  81 amino acids  =  8.8 kDa
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