pMCSG81 (linearized)

Linearized compact bacterial vector for ligation-independent cloning (LIC) and addition of a C-terminal 6xHis tag, with tRNA genes for rare Arg and Ile codons.

Sequence Author: Midwest Center for Structural Genomics

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BspEI (4525) ScaI (3828) PvuI (3718) FspI (3570) BglI (3468) BsaI (3409) AhdI (3348) AlwNI (2871) DrdI (2563) PciI (2455) BspQI - SapI (2339) BstZ17I (2226) AccI (2225) BsaAI (2207) PshAI (2200) BlpI (4603) End (4661) Start (0) ATG XbaI (40) T7 promoter BspDI * - ClaI * (109) AgeI (151) AscI (368) SbfI (379) BstBI (384) BfuAI - BspMI (397) MreI (516) AfeI (602) NcoI (786) PsiI (801) EcoNI (890) PfoI (922) PflMI (937) BstAPI (1038) MluI (1355) BclI * (1369) BstEII (1536) PspOMI (1562) ApaI (1566) ApoI (1630) EcoRV (1805) HpaI (1861) BsmBI - Esp3I (1970) pMCSG81 4661 bp
BspEI  (4525)
1 site
T C C G G A A G G C C T
ScaI  (3828)
1 site
A G T A C T T C A T G A
PvuI  (3718)
1 site
C G A T C G G C T A G C
FspI  (3570)
1 site
T G C G C A A C G C G T
BglI  (3468)
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  (3409)
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  (3348)
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  (2871)
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  (2563)
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  (2455)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2339)
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  (2339)
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  (2226)
1 site
G T A T A C C A T A T G
AccI  (2225)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the recognition sequence.
Sticky ends from different AccI sites may not be compatible.
BsaAI  (2207)
1 site
Y A C G T R R T G C A Y
PshAI  (2200)
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.
BlpI  (4603)
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.
End  (4661)
0 sites
Start  (0)
0 sites
XbaI  (40)
1 site
T C T A G A A G A T C T
BspDI  (109)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (109)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (151)
1 site
A C C G G T T G G C C A
AscI  (368)
1 site
G G C G C G C C C C G C G C G G
SbfI  (379)
1 site
C C T G C A G G G G A C G T C C
BstBI  (384)
1 site
T T C G A A A A G C T T
BfuAI  (397)
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  (397)
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  (516)
1 site
C G C C G G C G G C G G C C G C
AfeI  (602)
1 site
A G C G C T T C G C G A
NcoI  (786)
1 site
C C A T G G G G T A C C
PsiI  (801)
1 site
T T A T A A A A T A T T
EcoNI  (890)
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  (922)
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  (937)
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  (1038)
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  (1355)
1 site
A C G C G T T G C G C A
BclI  (1369)
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  (1536)
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  (1562)
1 site
G G G C C C C C C G G G
ApaI  (1566)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
ApoI  (1630)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
EcoRV  (1805)
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  (1861)
1 site
G T T A A C C A A T T G
BsmBI  (1970)
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  (1970)
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.
lacI
1005 .. 2087  =  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
1005 .. 2087  =  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
3275 .. 4135  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3275 .. 4066  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3275 .. 4135  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4067 .. 4135  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3275 .. 4135  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2516 .. 3104  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2516 .. 3104  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4136 .. 4239  =  104 bp
AmpR promoter
4136 .. 4239  =  104 bp
lacI promoter
927 .. 1004  =  78 bp
lacI promoter
927 .. 1004  =  78 bp
argU
366 .. 442  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
366 .. 442  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
712 .. 787  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
712 .. 787  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
T7 terminator
4549 .. 4596  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
4549 .. 4596  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
48 .. 72  =  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
48 .. 72  =  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
73 .. 91  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
73 .. 91  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
4663 .. 4680  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4663 .. 4680  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
12 .. 17  =  6 bp
ribosome binding site
RBS
12 .. 17  =  6 bp
ribosome binding site
ATG
1 .. 3  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1 .. 3  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  788 .. 1255  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  1844 .. 2107  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  501 .. 755  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1128 .. 2087  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3405 .. 3671  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  1870 .. 2121  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  3275 .. 4135  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
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