pMCSG52 (linearized)

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

Sequence Author: Midwest Center for Structural Genomics

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6xHis BlpI (5670) T7 terminator ScaI (4895) PvuI (4785) BsaI (4476) AhdI (4415) AlwNI (3938) PciI (3522) BspQI - SapI (3406) BstZ17I (3293) BsaAI (3274) PflFI - Tth111I (3267) PpuMI (2528) FspAI (2503) PshAI (2266) AvaI - BsoBI - PaeR7I - PspXI - XhoI (5748) EagI - NotI (5756) HindIII (5763) SalI (5769) Eco53kI (5778) SacI (5780) EcoRI (5782) BamHI (5788) End (5794) Start (0) TEV site Acc65I (25) KpnI (29) BglII (32) 6xHis ATG NdeI (68) RBS XbaI (106) lac operator T7 promoter BspDI * - ClaI * (175) AgeI (217) AscI (434) SbfI (445) BstBI (450) BfuAI - BspMI (463) MreI (582) NcoI (852) PsiI (867) EcoNI (956) PflMI (1003) BstAPI (1104) MluI (1421) BclI * (1435) BstEII (1602) PspOMI (1628) ApaI (1632) EcoRV (1871) HpaI (1927) pMCSG52 5794 bp
BlpI  (5670)
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.
ScaI  (4895)
1 site
A G T A C T T C A T G A
PvuI  (4785)
1 site
C G A T C G G C T A G C
BsaI  (4476)
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  (4415)
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  (3938)
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  (3522)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (3406)
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  (3406)
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  (3293)
1 site
G T A T A C C A T A T G
BsaAI  (3274)
1 site
Y A C G T R R T G C A Y
PflFI  (3267)
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  (3267)
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  (2528)
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  (2503)
1 site
R T G C G C A Y Y A C G C G T R
PshAI  (2266)
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.
AvaI  (5748)
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  (5748)
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  (5748)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5748)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5748)
1 site
C T C G A G G A G C T C
EagI  (5756)
1 site
C G G C C G G C C G G C
NotI  (5756)
1 site
G C G G C C G C C G C C G G C G
HindIII  (5763)
1 site
A A G C T T T T C G A A
SalI  (5769)
1 site
G T C G A C C A G C T G
Eco53kI  (5778)
1 site
G A G C T C C T C G A G
SacI  (5780)
1 site
G A G C T C C T C G A G
EcoRI  (5782)
1 site
G A A T T C C T T A A G
BamHI  (5788)
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.
End  (5794)
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
BspDI  (175)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (175)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (217)
1 site
A C C G G T T G G C C A
AscI  (434)
1 site
G G C G C G C C C C G C G C G G
SbfI  (445)
1 site
C C T G C A G G G G A C G T C C
BstBI  (450)
1 site
T T C G A A A A G C T T
BfuAI  (463)
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  (463)
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.
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
EcoNI  (956)
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  (1003)
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  (1104)
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  (1421)
1 site
A C G C G T T G C G C A
BclI  (1435)
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  (1602)
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.
PspOMI  (1628)
1 site
G G G C C C C C C G G G
ApaI  (1632)
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  (1871)
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  (1927)
1 site
G T T A A C C A A T T G
lacI
1071 .. 2153  =  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
1071 .. 2153  =  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
4342 .. 5202  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   4342 .. 5133  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4342 .. 5202  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   5134 .. 5202  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
4342 .. 5202  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
3583 .. 4171  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
3583 .. 4171  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
rop
2962 .. 3153  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
2962 .. 3153  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
AmpR promoter
5203 .. 5306  =  104 bp
AmpR promoter
5203 .. 5306  =  104 bp
lacI promoter
993 .. 1070  =  78 bp
lacI promoter
993 .. 1070  =  78 bp
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
5616 .. 5663  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
5616 .. 5663  =  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
5730 .. 5747  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
5730 .. 5747  =  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
ORF:  1 .. 282  =  282 bp
ORF:  93 amino acids  =  10.4 kDa  (no start codon)
ORF:  2209 .. 2565  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  2929 .. 3153  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  854 .. 1321  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  1910 .. 2173  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  4472 .. 4738  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  567 .. 821  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1194 .. 2153  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  1936 .. 2187  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  4342 .. 5202  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2562 .. 2930  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
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