pMCSG81

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

Sequence Author: Midwest Center for Structural Genomics

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ScaI (3985) PvuI (3875) FspI (3727) BglI (3625) BsaI (3566) AhdI (3505) AlwNI (3028) DrdI (2720) PciI (2612) BspQI - SapI (2496) BstZ17I (2383) AccI (2382) BsaAI (2364) BspEI (2) BlpI (80) F primer (131 .. 157) R primer (158 .. 203) ATG NsiI (159) RBS XbaI (197) lac operator T7 promoter BspDI * - ClaI * (266) AgeI (308) AscI (525) SbfI (536) BstBI (541) BfuAI - BspMI (554) MreI (673) AfeI (759) NcoI (943) PsiI (958) EcoNI (1047) PfoI (1079) PflMI (1094) BstAPI (1195) MluI (1512) BclI * (1526) BstEII (1693) PspOMI (1719) ApaI (1723) ApoI (1787) EcoRV (1962) HpaI (2018) BsmBI - Esp3I (2127) PshAI (2357) pMCSG81 4680 bp
ScaI  (3985)
1 site
A G T A C T T C A T G A
PvuI  (3875)
1 site
C G A T C G G C T A G C
FspI  (3727)
1 site
T G C G C A A C G C G T
BglI  (3625)
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  (3566)
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  (3505)
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  (3028)
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  (2720)
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  (2612)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2496)
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  (2496)
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  (2383)
1 site
G T A T A C C A T A T G
AccI  (2382)
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  (2364)
1 site
Y A C G T R R T G C A Y
BspEI  (2)
1 site
T C C G G A A G G C C T
BlpI  (80)
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.
NsiI  (159)
1 site
A T G C A T T A C G T A
XbaI  (197)
1 site
T C T A G A A G A T C T
BspDI  (266)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (266)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
AgeI  (308)
1 site
A C C G G T T G G C C A
AscI  (525)
1 site
G G C G C G C C C C G C G C G G
SbfI  (536)
1 site
C C T G C A G G G G A C G T C C
BstBI  (541)
1 site
T T C G A A A A G C T T
BfuAI  (554)
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  (554)
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  (673)
1 site
C G C C G G C G G C G G C C G C
AfeI  (759)
1 site
A G C G C T T C G C G A
NcoI  (943)
1 site
C C A T G G G G T A C C
PsiI  (958)
1 site
T T A T A A A A T A T T
EcoNI  (1047)
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  (1079)
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  (1094)
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  (1195)
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  (1512)
1 site
A C G C G T T G C G C A
BclI  (1526)
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  (1693)
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  (1719)
1 site
G G G C C C C C C G G G
ApaI  (1723)
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  (1787)
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  (1962)
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  (2018)
1 site
G T T A A C C A A T T G
BsmBI  (2127)
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  (2127)
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.
PshAI  (2357)
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.
F primer
27-mer  /  48% GC
1 binding site
131 .. 157  =  27 annealed bases
Tm  =  61°C
R primer
46-mer  /  24% GC
1 binding site
158 .. 203  =  46 annealed bases
Tm  =  58°C
lacI
1162 .. 2244  =  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
1162 .. 2244  =  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
3432 .. 4292  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 2:  
   3432 .. 4223  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3432 .. 4292  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
   Segment 1:  signal sequence  
   4224 .. 4292  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
3432 .. 4292  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
2673 .. 3261  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
2673 .. 3261  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
AmpR promoter
4293 .. 4396  =  104 bp
AmpR promoter
4293 .. 4396  =  104 bp
lacI promoter
1084 .. 1161  =  78 bp
lacI promoter
1084 .. 1161  =  78 bp
argU
523 .. 599  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
argU
523 .. 599  =  77 bp
Product: E. coli tRNA-Arg recognizing AGA and AGG
ileX
869 .. 944  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
ileX
869 .. 944  =  76 bp
Product: E. coli tRNA-Ile recognizing AUA
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
26 .. 73  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
205 .. 229  =  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
205 .. 229  =  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).
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
140 .. 157  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
158 .. 160  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
158 .. 160  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
T7 promoter
230 .. 248  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
230 .. 248  =  19 bp
promoter for bacteriophage T7 RNA polymerase
RBS
169 .. 174  =  6 bp
ribosome binding site
RBS
169 .. 174  =  6 bp
ribosome binding site
ORF:  658 .. 912  =  255 bp
ORF:  84 amino acids  =  8.5 kDa
ORF:  1285 .. 2244  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  3562 .. 3828  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  140 .. 373  =  234 bp
ORF:  77 amino acids  =  8.6 kDa
ORF:  945 .. 1412  =  468 bp
ORF:  155 amino acids  =  16.8 kDa
ORF:  2001 .. 2264  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  3432 .. 4292  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  2027 .. 2278  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
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