pMiniT 2.0

Compact bacterial vector that employs a toxic minigene for high-efficiency cloning and subsequent in vitro transcription of PCR products.

Sequence Author: New England Biolabs

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BspQI - SapI (2525) AflIII - PciI (2408) DrdI (2306) PspFI (2108) BseYI (2104) AlwNI (1999) BanI (1567) AhdI (1520) BmrI (1480) BpmI (1451) BglI (1402) NmeAIII (1373) FspI (1297) BtgZI (99) AfeI (143) NdeI (207) NruI (225) Cloning Analysis Forward Primer (391 .. 412) PstI - SbfI (463) AarI (466) PmeI (472) SP6 promoter PspXI - XhoI (509) BamHI (515) EcoRI (521) Shine-Dalgarno sequence toxic minigene PacI (560) ZraI (566) AatII (568) EcoRI (571) XhoI (576) EagI - NotI (583) BsmBI (601) Cloning Analysis Reverse Primer (677 .. 699) SspI (715) XmnI (920) TatI (1037) ScaI (1039) TsoI (1122) PvuI (1151) pMiniT 2.0 2588 bp
BspQI  (2525)
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  (2525)
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.
AflIII  (2408)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2408)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (2306)
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.
PspFI  (2108)
1 site
C C C A G C G G G T C G
BseYI  (2104)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
AlwNI  (1999)
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.
BanI  (1567)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
AhdI  (1520)
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.
BmrI  (1480)
1 site
A C T G G G ( N ) 4 N T G A C C C ( N ) 4

The 1-base overhangs produced by BmrI may be hard to ligate.
Sticky ends from different BmrI sites may not be compatible.
Unlike most restriction enzymes, BmrI can cleave DNA in the absence of magnesium.
BpmI  (1451)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
BglI  (1402)
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.
NmeAIII  (1373)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
FspI  (1297)
1 site
T G C G C A A C G C G T
BtgZI  (99)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
AfeI  (143)
1 site
A G C G C T T C G C G A
NdeI  (207)
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.
NruI  (225)
1 site
T C G C G A A G C G C T
PstI  (463)
1 site
C T G C A G G A C G T C
SbfI  (463)
1 site
C C T G C A G G G G A C G T C C
AarI  (466)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Cleavage may be enhanced when more than one copy of the AarI recognition sequence is present.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
PmeI  (472)
1 site
G T T T A A A C C A A A T T T G
PspXI  (509)
1 site
V C T C G A G B B G A G C T C V
XhoI  (509)
2 sites
C T C G A G G A G C T C
BamHI  (515)
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.
EcoRI  (521)
2 sites
G A A T T C C T T A A G
PacI  (560)
1 site
T T A A T T A A A A T T A A T T
ZraI  (566)
1 site
G A C G T C C T G C A G
AatII  (568)
1 site
G A C G T C C T G C A G
EcoRI  (571)
2 sites
G A A T T C C T T A A G
XhoI  (576)
2 sites
C T C G A G G A G C T C
EagI  (583)
1 site
C G G C C G G C C G G C
NotI  (583)
1 site
G C G G C C G C C G C C G G C G
BsmBI  (601)
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.
SspI  (715)
1 site
A A T A T T T T A T A A
XmnI  (920)
1 site
G A A N N N N T T C C T T N N N N A A G
TatI  (1037)
1 site
W G T A C W W C A T G W
ScaI  (1039)
1 site
A G T A C T T C A T G A
TsoI  (1122)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PvuI  (1151)
1 site
C G A T C G G C T A G C
Cloning Analysis Forward Primer
22-mer  /  55% GC
1 binding site
391 .. 412  =  22 annealed bases
Tm  =  62°C
Cloning Analysis Reverse Primer
23-mer  /  48% GC
1 binding site
677 .. 699  =  23 annealed bases
Tm  =  59°C
AmpR
733 .. 1593  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   733 .. 801  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
733 .. 1593  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   802 .. 1593  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
733 .. 1593  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1764 .. 2352  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1764 .. 2352  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
tnpA promoter
145 .. 251  =  107 bp
promoter of the IS10 transposase
tnpA promoter
145 .. 251  =  107 bp
promoter of the IS10 transposase
AmpR promoter
629 .. 732  =  104 bp
AmpR promoter
629 .. 732  =  104 bp
SP6 promoter
479 .. 497  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
479 .. 497  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
T7 promoter
601 .. 619  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
601 .. 619  =  19 bp
promoter for bacteriophage T7 RNA polymerase
stop codons
547 .. 555  =  9 bp
stop codons
547 .. 555  =  9 bp
Shine-Dalgarno sequence
527 .. 533  =  7 bp
ribosome binding site
Shine-Dalgarno sequence
527 .. 533  =  7 bp
ribosome binding site
toxic minigene
541 .. 546  =  6 bp
2 amino acids  =  262.4 Da
Product: two-residue polypeptide that poisons the E. coli translation machinery
toxic minigene
541 .. 546  =  6 bp
2 amino acids  =  262.4 Da
Product: two-residue polypeptide that poisons the E. coli translation machinery
ORF:  733 .. 1593  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  252 .. 488  =  237 bp
ORF:  78 amino acids  =  9.0 kDa
ORF:  1197 .. 1463  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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