pLIC-SGC1

Bacterial vector encoding an N-terminal 6xHis-TEV cassette and ampicillin resistance plus a SacB negative selection marker, for purification of recombinant proteins.

Sequence Author: Structural Genomics Consortium

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BlpI (7087) AvaI - BmeT110I - BsoBI - PaeR7I - PspXI - XhoI (7008) HindIII (6993) SalI (6987) SacI (6984) Eco53kI (6982) EcoRI (6974) BamHI (6968) BseRI (6952) SpeI (6946) SacII (6538) SnaBI (6208) BsrGI (6139) AanI - PsiI (6099) MscI (5722) StuI * (5700) BfuAI - BspMI (5029) BmtI (4944) NheI (4940) BseRI (4939) TEV site BglII (4902) 6xHis ATG NdeI (4868) RBS XbaI (4828) T7 promoter SgrAI (4717) SphI (4569) EcoNI (4504) PflMI (4461) BstAPI (4360) MluI (4036) BclI * (4022) BstEII (3854) ApaI (3833) PspOMI (3829) BssHII (3625) ScaI (566) PvuI (678) PstI (805) BsaI (981) AhdI (1047) AlwNI (1526) BspQI - SapI (2052) PflFI - Tth111I (2193) PpuMI (2930) FspAI (2958) PshAI (3195) pLIC-SGC1 7170 bp
BlpI  (7087)
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.
AvaI  (7008)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BmeT110I  (7008)
1 site
C Y C G R G G R G C Y C
BsoBI  (7008)
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  (7008)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (7008)
1 site
V C T C G A G B B G A G C T C V
XhoI  (7008)
1 site
C T C G A G G A G C T C
HindIII  (6993)
1 site
A A G C T T T T C G A A
SalI  (6987)
1 site
G T C G A C C A G C T G
SacI  (6984)
1 site
G A G C T C C T C G A G
Eco53kI  (6982)
1 site
G A G C T C C T C G A G
EcoRI  (6974)
1 site
G A A T T C C T T A A G
BamHI  (6968)
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.
BseRI  (6952)
2 sites
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.
SpeI  (6946)
1 site
A C T A G T T G A T C A
SacII  (6538)
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.
SnaBI  (6208)
1 site
T A C G T A A T G C A T
BsrGI  (6139)
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.
AanI  (6099)
1 site
T T A T A A A A T A T T
PsiI  (6099)
1 site
T T A T A A A A T A T T
MscI  (5722)
1 site
T G G C C A A C C G G T
StuI  (5700)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
BfuAI  (5029)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (5029)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
BmtI  (4944)
1 site
G C T A G C C G A T C G
NheI  (4940)
1 site
G C T A G C C G A T C G
BseRI  (4939)
2 sites
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.
BglII  (4902)
1 site
A G A T C T T C T A G A
NdeI  (4868)
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.
XbaI  (4828)
1 site
T C T A G A A G A T C T
SgrAI  (4717)
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  (4569)
1 site
G C A T G C C G T A C G
EcoNI  (4504)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
PflMI  (4461)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
BstAPI  (4360)
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  (4036)
1 site
A C G C G T T G C G C A
BclI  (4022)
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  (3854)
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  (3833)
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  (3829)
1 site
G G G C C C C C C G G G
BssHII  (3625)
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.
ScaI  (566)
1 site
A G T A C T T C A T G A
PvuI  (678)
1 site
C G A T C G G C T A G C
PstI  (805)
1 site
C T G C A G G A C G T C
BsaI  (981)
1 site
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
AhdI  (1047)
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.
AlwNI  (1526)
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.
BspQI  (2052)
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  (2052)
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.
PflFI  (2193)
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  (2193)
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  (2930)
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.
FspAI  (2958)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (3195)
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.
SacB
5459 .. 6880  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 1:  signal peptide  
   5459 .. 5545  =  87 bp
   29 amino acids  =  3.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
5459 .. 6880  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
   Segment 2:  
   5546 .. 6880  =  1335 bp
   444 amino acids  =  50.0 kDa
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
SacB
5459 .. 6880  =  1422 bp
473 amino acids  =  53.0 kDa
2 segments
Product: secreted levansucrase that renders bacterial growth sensitive to sucrose
negative selection marker
lacI
3309 .. 4391  =  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
3309 .. 4391  =  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).
AmpR
260 .. 1120  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   260 .. 331  =  72 bp
   24 amino acids  =  2.8 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
260 .. 1120  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   332 .. 1120  =  789 bp
   262 amino acids  =  28.8 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
260 .. 1120  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1291 .. 1879  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1291 .. 1879  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
sacB promoter
5013 .. 5458  =  446 bp
sacB promoter and control region
sacB promoter
5013 .. 5458  =  446 bp
sacB promoter and control region
rop
2309 .. 2500  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
2309 .. 2500  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
4392 .. 4469  =  78 bp
lacI promoter
4392 .. 4469  =  78 bp
ATG
4870 .. 4872  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
4870 .. 4872  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
4873 .. 4890  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4873 .. 4890  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
TEV site
4915 .. 4935  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
4915 .. 4935  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 terminator
7098 .. 7145  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
7098 .. 7145  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
4801 .. 4825  =  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
4801 .. 4825  =  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
4782 .. 4800  =  19 bp