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pMCSG13 (linearized)

Linearized bacterial coexpression vector for ligation-independent cloning (LIC), with a 6xHis-MBP-TEV leader and a p15A origin.

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pMCSG13 (linearized).dna
Map and Sequence File:    Download    Open   
Sequence Author:  Midwest Center for Structural Genomics
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EcoNI (3684) ApaLI (3236) AflIII - MluI (3216) BstEII (3034) ApaI (3013) PspOMI (3009) HpaI (2714) PluTI (2581) SfoI (2579) NarI * (2578) KasI (2577) XbaI (2296) NspI (2033) BssS α I (1831) SacII (1707) SgrAI (1541) XmnI (1511) BstZ17I (1468) BmtI (1459) NheI (1455) Bpu10I (1101) AscI (3836) PstI - SbfI (3846) AflII (3874) BsrGI (3901) NdeI (4009) BsaI (4116) BsiWI (4343) PvuI (4378) PsiI (4405) BmgBI (4595) BsaBI (4728) EcoRV (5162) End (5183) Start (0) AvaI - BsoBI - PaeR7I - PspXI - XhoI (57) BmeT110I (58) PacI (132) AvrII (136) EcoO109I (181) Bsu36I (220) DrdI - PflFI - Tth111I (329) ScaI (460) MscI (610) pMCSG13 5183 bp
EcoNI  (3684)
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.
ApaLI  (3236)
1 site
G T G C A C C A C G T G
AflIII  (3216)
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  (3216)
1 site
A C G C G T T G C G C A
BstEII  (3034)
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  (3013)
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  (3009)
1 site
G G G C C C C C C G G G
HpaI  (2714)
1 site
G T T A A C C A A T T G
PluTI  (2581)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
SfoI  (2579)
1 site
G G C G C C C C G C G G
NarI  (2578)
1 site
G G C G C C C C G C G G
* Blocked by Dcm methylation.
Efficient cleavage requires at least two copies of the NarI recognition sequence.
KasI  (2577)
1 site
G G C G C C C C G C G G
XbaI  (2296)
1 site
T C T A G A A G A T C T
NspI  (2033)
1 site
R C A T G Y Y G T A C R
BssSαI  (1831)
1 site
C A C G A G G T G C T C
SacII  (1707)
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.
SgrAI  (1541)
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.
XmnI  (1511)
1 site
G A A N N N N T T C C T T N N N N A A G
BstZ17I  (1468)
1 site
G T A T A C C A T A T G
BmtI  (1459)
1 site
G C T A G C C G A T C G
NheI  (1455)
1 site
G C T A G C C G A T C G
Bpu10I  (1101)
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.
AscI  (3836)
1 site
G G C G C G C C C C G C G C G G
PstI  (3846)
1 site
C T G C A G G A C G T C
SbfI  (3846)
1 site
C C T G C A G G G G A C G T C C
AflII  (3874)
1 site
C T T A A G G A A T T C
BsrGI  (3901)
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.
NdeI  (4009)
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.
BsaI  (4116)
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.
BsiWI  (4343)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PvuI  (4378)
1 site
C G A T C G G C T A G C
PsiI  (4405)
1 site
T T A T A A A A T A T T
BmgBI  (4595)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
BsaBI  (4728)
1 site
G A T N N N N A T C C T A N N N N T A G
EcoRV  (5162)
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.
End  (5183)
0 sites
Start  (0)
0 sites
AvaI  (57)
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  (57)
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  (57)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (57)
1 site
V C T C G A G B B G A G C T C V
XhoI  (57)
1 site
C T C G A G G A G C T C
BmeT110I  (58)
1 site
C Y C G R G G R G C Y C
PacI  (132)
1 site
T T A A T T A A A A T T A A T T
AvrII  (136)
1 site
C C T A G G G G A T C C
EcoO109I  (181)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
Bsu36I  (220)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
DrdI  (329)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
PflFI  (329)
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  (329)
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.
ScaI  (460)
1 site
A G T A C T T C A T G A
MscI  (610)
1 site
T G G C C A A C C G G T
MBP
4056 .. 5153  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
MBP
4056 .. 5153  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
lacI
2489 .. 3571  =  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
2489 .. 3571  =  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).
CmR
432 .. 1091  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
432 .. 1091  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
p15A ori
1720 .. 2264  =  545 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
1720 .. 2264  =  545 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.
cat promoter
1092 .. 1194  =  103 bp
promoter of the E. coli cat gene
cat promoter
1092 .. 1194  =  103 bp
promoter of the E. coli cat gene
lacI promoter
3572 .. 3649  =  78 bp
lacI promoter
3572 .. 3649  =  78 bp
T7 terminator
165 .. 212  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
165 .. 212  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
S-Tag
69 .. 113  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
S-Tag
69 .. 113  =  45 bp
15 amino acids  =  1.7 kDa
Product: affinity and epitope tag derived from pancreatic ribonuclease A
lac operator
3714 .. 3738  =  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
3714 .. 3738  =  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
3944 .. 3968  =  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
3944 .. 3968  =  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).
TEV site
5175 .. 5195  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
5175 .. 5195  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 promoter
3695 .. 3713  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3695 .. 3713  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3925 .. 3943  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
3925 .. 3943  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
3794 .. 3811  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
3794 .. 3811  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4014 .. 4031  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4014 .. 4031  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
3769 .. 3774  =  6 bp
ribosome binding site
RBS
3769 .. 3774  =  6 bp
ribosome binding site
RBS
3997 .. 4002  =  6 bp
ribosome binding site
RBS
3997 .. 4002  =  6 bp
ribosome binding site
ATG
3782 .. 3784  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3782 .. 3784  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
4011 .. 4013  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
4011 .. 4013  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  2455 .. 2706  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  1635 .. 1913  =  279 bp
ORF:  92 amino acids  =  10.4 kDa
ORF:  4011 .. 5198  =  1188 bp
ORF:  396 amino acids  =  43.6 kDa
ORF:  432 .. 1091  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
ORF:  1602 .. 1874  =  273 bp
ORF:  90 amino acids  =  9.7 kDa
ORF:  2469 .. 2732  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3321 .. 3869  =  549 bp
ORF:  182 amino acids  =  19.6 kDa
ORF:  3924 .. 4172  =  249 bp
ORF:  82 amino acids  =  9.8 kDa
ORF:  4392 .. 5198  =  807 bp
ORF:  268 amino acids  =  31.0 kDa  (no start codon)
ORF:  2489 .. 3448  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
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