pMCSG63 (linearized)

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

Sequence Author: Midwest Center for Structural Genomics

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T7 terminator BspEI (4591) ScaI (3894) PvuI (3784) FspI (3636) BglI (3534) BsaI (3475) AhdI (3414) AlwNI (2937) DrdI (2629) PciI (2521) BspQI - SapI (2405) BstZ17I (2292) BsaAI (2273) PshAI (2266) BsmBI - Esp3I (2036) HpaI (1927) EcoRV (1871) BlpI (4669) 6xHis PaeR7I - PspXI - XhoI (4747) EagI - NotI (4755) HindIII (4762) SalI (4768) Eco53kI (4777) SacI (4779) EcoRI (4781) BamHI (4787) TspMI - XmaI (4821) SmaI (4823) RBS End (4849) Start (0) TEV site Acc65I (25) KpnI (29) BglII (32) 6xHis ATG NdeI (68) RBS XbaI (106) T7 promoter BspDI * - ClaI * (175) AgeI (217) AscI (434) SbfI (445) BstBI (450) BfuAI - BspMI (463) MreI (582) AfeI (668) NcoI (852) PsiI (867) EcoNI (956) PfoI (988) PflMI (1003) BstAPI (1104) MluI (1421) BclI * (1435) BstEII (1602) PspOMI (1628) ApaI (1632) pMCSG63 4849 bp
BspEI  (4591)
1 site
T C C G G A A G G C C T
ScaI  (3894)
1 site
A G T A C T T C A T G A
PvuI  (3784)
1 site
C G A T C G G C T A G C
FspI  (3636)
1 site
T G C G C A A C G C G T
BglI  (3534)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
BsaI  (3475)
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  (3414)
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  (2937)
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.
DrdI  (2629)
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.
PciI  (2521)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2405)
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  (2405)
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  (2292)
1 site
G T A T A C C A T A T G
BsaAI  (2273)
1 site
Y A C G T R R T G C A Y
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.
BsmBI  (2036)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
BsmBI-v2 is an improved version of BsmBI.
Esp3I  (2036)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different Esp3I sites may not be compatible.
HpaI  (1927)
1 site
G T T A A C C A A T T G
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.
BlpI  (4669)
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.
PaeR7I  (4747)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (4747)
1 site
V C T C G A G B B G A G C T C V
XhoI  (4747)
1 site
C T C G A G G A G C T C
EagI  (4755)
1 site
C G G C C G G C C G G C
NotI  (4755)
1 site
G C G G C C G C C G C C G G C G
HindIII  (4762)
1 site
A A G C T T T T C G A A
SalI  (4768)
1 site
G T C G A C C A G C T G
Eco53kI  (4777)
1 site
G A G C T C C T C G A G
SacI  (4779)
1 site
G A G C T C C T C G A G
EcoRI  (4781)
1 site
G A A T T C C T T A A G
BamHI  (4787)
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.
TspMI  (4821)
1 site
C C C G G G G G G C C C
XmaI  (4821)
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  (4823)
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  (4849)
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
AfeI  (668)
1 site
A G C G C T T C G C G A
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.
PfoI  (988)
1 site
T C C N G G A A G G N C C T

Sticky ends from different PfoI 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.
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
3341 .. 4201  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3341 .. 4132  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3341 .. 4201  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4133 .. 4201  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3341 .. 4201  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2582 .. 3170  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2582 .. 3170  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4202 .. 4305  =  104 bp
AmpR promoter
4202 .. 4305  =  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
4615 .. 4662  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
4615 .. 4662  =  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
4729 .. 4746  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4729 .. 4746  =  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
4832 .. 4837  =  6 bp
ribosome binding site
RBS
4832 .. 4837  =  6 bp
ribosome binding site
ORF:  1 .. 282  =  282 bp
ORF:  93 amino acids  =  10.4 kDa  (no start codon)
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:  567 .. 821  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1194 .. 2153  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3471 .. 3737  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  3341 .. 4201  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  1936 .. 2187  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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