pMCSG77 (linearized)

Linearized bacterial vector with a p15A origin for ligation-independent cloning (LIC), with a 6xHis-TEV leader, a second LIC site, and tRNA genes for rare Arg and Ile codons.

Sequence Author: Midwest Center for Structural Genomics

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BsrGI (5165) AlwNI (4823) p15A ori SacII (4472) PfoI * (4425) BstZ17I (4233) BmtI (4224) NheI (4220) DraIII (3992) NruI (3801) EcoNI (3545) AsiSI (3460) PaqCI (2487) MfeI (2237) BspEI (5172) BlpI (5250) EagI - NotI (5336) Eco53kI (5358) SacI (5360) EcoRI (5362) TspMI - XmaI (5402) SmaI (5404) RBS End (5430) Start (0) TEV site Acc65I (25) KpnI (29) BglII (32) ATG NdeI (68) RBS XbaI (106) T7 promoter AcuI (237) AscI (434) PstI - SbfI (445) MreI (582) NcoI (852) PsiI (867) AclI (1060) BstAPI (1081) ApaLI (1378) AflIII - MluI (1398) BclI * (1412) NmeAIII (1604) PspOMI (1605) ApaI (1609) EcoRV (1848) BciVI (1855) HpaI (1904) pMCSG77 5430 bp
BsrGI  (5165)
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.
AlwNI  (4823)
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.
SacII  (4472)
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.
PfoI  (4425)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
BstZ17I  (4233)
1 site
G T A T A C C A T A T G
BmtI  (4224)
1 site
G C T A G C C G A T C G
NheI  (4220)
1 site
G C T A G C C G A T C G
DraIII  (3992)
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.
NruI  (3801)
1 site
T C G C G A A G C G C T
EcoNI  (3545)
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.
AsiSI  (3460)
1 site
G C G A T C G C C G C T A G C G
PaqCI  (2487)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the PaqCI recognition sequence.
Sticky ends from different PaqCI sites may not be compatible.
Cleavage can be improved with PaqCI Activator.
MfeI  (2237)
1 site
C A A T T G G T T A A C
BspEI  (5172)
1 site
T C C G G A A G G C C T
BlpI  (5250)
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.
EagI  (5336)
1 site
C G G C C G G C C G G C
NotI  (5336)
1 site
G C G G C C G C C G C C G G C G
Eco53kI  (5358)
1 site
G A G C T C C T C G A G
SacI  (5360)
1 site
G A G C T C C T C G A G
EcoRI  (5362)
1 site
G A A T T C C T T A A G
TspMI  (5402)
1 site
C C C G G G G G G C C C
XmaI  (5402)
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  (5404)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
End  (5430)
0 sites
Start  (0)
0 sites
Acc65I  (25)
1 site
G G T A C C C C A T G G
KpnI  (29)
1 site
G G T A C C C C A T G G
BglII  (32)
1 site
A G A T C T T C T A G A
NdeI  (68)
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  (106)
1 site
T C T A G A A G A T C T
AcuI  (237)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Cleavage may be enhanced when more than one copy of the AcuI recognition sequence is present.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
AscI  (434)
1 site
G G C G C G C C C C G C G C G G
PstI  (445)
1 site
C T G C A G G A C G T C
SbfI  (445)
1 site
C C T G C A G G G G A C G T C C
MreI  (582)
1 site
C G C C G G C G G C G G C C G C
NcoI  (852)
1 site
C C A T G G G G T A C C
PsiI  (867)
1 site
T T A T A A A A T A T T
AclI  (1060)
1 site
A A C G T T T T G C A A
BstAPI  (1081)
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.
ApaLI  (1378)
1 site
G T G C A C C A C G T G
AflIII  (1398)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
MluI  (1398)
1 site
A C G C G T T G C G C A
BclI  (1412)
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.
NmeAIII  (1604)
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).
PspOMI  (1605)
1 site
G G G C C C C C C G G G
ApaI  (1609)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
EcoRV  (1848)
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.
BciVI  (1855)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
HpaI  (1904)
1 site
G T T A A C C A A T T G
lacI
1048 .. 2130  =  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
1048 .. 2130  =  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
3075 .. 3890  =  816 bp
271 amino acids  =  31.1 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
3075 .. 3890  =  816 bp
271 amino acids  =  31.1 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
p15A ori
4485 .. 5030  =  546 bp
Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin.
p15A ori
4485 .. 5030  =  546 bp
Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin.
lacIq promoter
970 .. 1047  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
lacIq promoter
970 .. 1047  =  78 bp
In the lacIq allele, a single base change in the promoter boosts expression of the lacI gene about 10-fold.
argU
432 .. 508  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
432 .. 508  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
778 .. 853  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
778 .. 853  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
4 .. 24  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
49 .. 66  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
67 .. 69  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
67 .. 69  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
T7 terminator
5196 .. 5243  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
5196 .. 5243  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
114 .. 138  =  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
114 .. 138  =  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
139 .. 157  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
139 .. 157  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
5310 .. 5327  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5310 .. 5327  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
78 .. 83  =  6 bp
ribosome binding site
RBS
78 .. 83  =  6 bp
ribosome binding site
RBS
5413 .. 5418  =  6 bp
ribosome binding site
RBS
5413 .. 5418  =  6 bp
ribosome binding site
ORF:  1 .. 282  =  282 bp
ORF:  93 amino acids  =  10.4 kDa  (no start codon)
ORF:  1171 .. 2130  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  2629 .. 2868  =  240 bp
ORF:  79 amino acids  =  8.7 kDa
ORF:  3532 .. 3771  =  240 bp
ORF:  79 amino acids  =  8.4 kDa
ORF:  4400 .. 4696  =  297 bp
ORF:  98 amino acids  =  10.9 kDa
ORF:  567 .. 821  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  894 .. 1298  =  405 bp
ORF:  134 amino acids  =  14.6 kDa
ORF:  1887 .. 2150  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  2274 .. 2699  =  426 bp
ORF:  141 amino acids  =  15.7 kDa
ORF:  3075 .. 3890  =  816 bp
ORF:  271 amino acids  =  31.1 kDa
ORF:  1913 .. 2164  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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