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Plasmid Files

pGS-21a

Bacterial vector for expression of 6xHis- and GST-tagged proteins. Two 6xHis sequences are encoded in the vector.

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pGS-21a Sequence and MappGS-21a.dna
Map and Sequence File   
Sequence Author:  GenScript
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 BspDI * - ClaI * (6149) SgrAI (6105) SphI (5957) PflMI (5849) BstAPI (5748) MluI (5424) BstEII (5242) ApaI (5221) PspOMI (5217) BssHII (5013) HpaI (4922) PshAI (4583) FspAI (4346) PpuMI (4318) Bpu10I (4218) PflFI - Tth111I (3581) BstZ17I (3556) T7 primer (1 .. 20) T7 promoter XbaI (47) NdeI (87) ATG 6xHis MscI (335) BstBI (525) SwaI (555) SfiI (798) BglII (806) Acc65I (812) KpnI (816) enterokinase site NcoI (834) EcoRV (844) BamHI (848) EcoRI (854) Eco53kI (862) SacI (864) SalI (866) HindIII (872) EagI - NotI (879) AvaI - BsoBI - PaeR7I - PspXI - XhoI (887) BmeT110I (888) 6xHis BlpI (966) T7 terminator primer (963 .. 981) T7 terminator DraIII (1294) PsiI (1419) PvuI (2066) PstI (2193) BsaI (2369) AhdI (2435) AlwNI (2914) pGS-21a 6169 bp
BspDI  (6149)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (6149)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (6105)
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  (5957)
1 site
G C A T G C C G T A C G
PflMI  (5849)
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  (5748)
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  (5424)
1 site
A C G C G T T G C G C A
BstEII  (5242)
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  (5221)
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  (5217)
1 site
G G G C C C C C C G G G
BssHII  (5013)
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.
HpaI  (4922)
1 site
G T T A A C C A A T T G
PshAI  (4583)
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.
FspAI  (4346)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (4318)
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.
Bpu10I  (4218)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I
recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends
generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
PflFI  (3581)
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  (3581)
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.
BstZ17I  (3556)
1 site
G T A T A C C A T A T G
XbaI  (47)
1 site
T C T A G A A G A T C T
NdeI  (87)
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  (335)
1 site
T G G C C A A C C G G T
BstBI  (525)
1 site
T T C G A A A A G C T T
SwaI  (555)
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.
SfiI  (798)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
BglII  (806)
1 site
A G A T C T T C T A G A
Acc65I  (812)
1 site
G G T A C C C C A T G G
KpnI  (816)
1 site
G G T A C C C C A T G G
NcoI  (834)
1 site
C C A T G G G G T A C C
EcoRV  (844)
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.
BamHI  (848)
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.
EcoRI  (854)
1 site
G A A T T C C T T A A G
Eco53kI  (862)
1 site
G A G C T C C T C G A G
SacI  (864)
1 site
G A G C T C C T C G A G
SalI  (866)
1 site
G T C G A C C A G C T G
HindIII  (872)
1 site
A A G C T T T T C G A A
EagI  (879)
1 site
C G G C C G G C C G G C
NotI  (879)
1 site
G C G G C C G C C G C C G G C G
AvaI  (887)
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  (887)
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  (887)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (887)
1 site
V C T C G A G B B G A G C T C V
XhoI  (887)
1 site
C T C G A G G A G C T C
BmeT110I  (888)
1 site
C Y C G R G G R G C Y C
BlpI  (966)
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  (1294)
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  (1419)
1 site
T T A T A A A A T A T T
PvuI  (2066)
1 site
C G A T C G G C T A G C
PstI  (2193)
1 site
C T G C A G G A C G T C
BsaI  (2369)
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  (2435)
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  (2914)
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.
T7 primer
20-mer  /  40% GC
1 binding site
1 .. 20  =  20 annealed bases
Tm  =  49°C
T7 terminator primer
19-mer  /  53% GC
1 binding site
963 .. 981  =  19 annealed bases
Tm  =  56°C
lacI
4697 .. 5779  =  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
4697 .. 5779  =  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
1648 .. 2508  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   1648 .. 1716  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1648 .. 2508  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   1717 .. 2508  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1648 .. 2508  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
GST
128 .. 781  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
GST
128 .. 781  =  654 bp
218 amino acids  =  25.5 kDa
Product: glutathione S-transferase from
Schistosoma japonicum
ori
2679 .. 3267  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2679 .. 3267  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1061 .. 1516  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1061 .. 1516  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
3697 .. 3888  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
3697 .. 3888  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
1543 .. 1647  =  105 bp
AmpR promoter
1543 .. 1647  =  105 bp
lacI promoter
5780 .. 5857  =  78 bp
lacI promoter
5780 .. 5857  =  78 bp
MCS
834 .. 892  =  59 bp
multiple cloning site
MCS
834 .. 892  =  59 bp
multiple cloning site
T7 terminator
977 .. 1024  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
977 .. 1024  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
20 .. 44  =  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
20 .. 44  =  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
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
1 .. 19  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
101 .. 118  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
101 .. 118  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
893 .. 910  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
893 .. 910  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
enterokinase site
818 .. 832  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
enterokinase site
818 .. 832  =  15 bp
5 amino acids  =  606.5 Da
Product: enterokinase recognition and cleavage site
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
89 .. 91  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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