pMiniT (linearized)

Linearized compact bacterial vector that employs a toxic minigene for high-efficiency cloning of PCR products.

Sequence Author: New England Biolabs

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BspQI - SapI (1940) NspI (1827) AflIII - PciI (1823) DrdI (1721) PspFI (1523) BseYI (1519) AlwNI (1414) BanI (982) AhdI (935) BmrI (895) BsaI (869) BpmI (866) BglI (817) NmeAIII (788) FspI (712) BtgZI (2102) AfeI (2146) NdeI (2210) NruI (2228) Cloning Analysis Forward Primer (2394 .. 2415) BfuAI - BspMI (2403) PspXI (2489) BamHI (2495) Shine-Dalgarno sequence toxic minigene End (2525) Start (0) toxic minigene ZraI (14) AatII (16) Cloning Analysis Reverse Primer (92 .. 114) SspI (130) XmnI (335) TatI (452) ScaI (454) TsoI (537) PvuI (566) pMiniT 2525 bp
BspQI  (1940)
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  (1940)
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.
NspI  (1827)
1 site
R C A T G Y Y G T A C R
AflIII  (1823)
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  (1823)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (1721)
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  (1523)
1 site
C C C A G C G G G T C G
BseYI  (1519)
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  (1414)
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  (982)
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  (935)
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  (895)
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.
BsaI  (869)
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.
BpmI  (866)
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  (817)
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  (788)
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  (712)
1 site
T G C G C A A C G C G T
BtgZI  (2102)
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  (2146)
1 site
A G C G C T T C G C G A
NdeI  (2210)
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  (2228)
1 site
T C G C G A A G C G C T
BfuAI  (2403)
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  (2403)
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.
PspXI  (2489)
1 site
V C T C G A G B B G A G C T C V
BamHI  (2495)
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  (2525)
0 sites
Start  (0)
0 sites
ZraI  (14)
1 site
G A C G T C C T G C A G
AatII  (16)
1 site
G A C G T C C T G C A G
SspI  (130)
1 site
A A T A T T T T A T A A
XmnI  (335)
1 site
G A A N N N N T T C C T T N N N N A A G
TatI  (452)
1 site
W G T A C W W C A T G W
ScaI  (454)
1 site
A G T A C T T C A T G A
TsoI  (537)
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  (566)
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
2394 .. 2415  =  22 annealed bases
Tm  =  62°C
Cloning Analysis Reverse Primer
23-mer  /  48% GC
1 binding site
92 .. 114  =  23 annealed bases
Tm  =  59°C
AmpR
148 .. 1008  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   148 .. 216  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
148 .. 1008  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   217 .. 1008  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
148 .. 1008  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
1179 .. 1767  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1179 .. 1767  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
tnpA transposase
2255 .. 2485  =  231 bp
77 amino acids  =  9.0 kDa
Product: transposase encoded by the IS10 tnpA gene
tnpA transposase
2255 .. 2485  =  231 bp
77 amino acids  =  9.0 kDa
Product: transposase encoded by the IS10 tnpA gene
tnpA promoter
2148 .. 2254  =  107 bp
promoter of the IS10 transposase
tnpA promoter
2148 .. 2254  =  107 bp
promoter of the IS10 transposase
AmpR promoter
43 .. 147  =  105 bp
AmpR promoter
43 .. 147  =  105 bp
stop codons
2 .. 13  =  12 bp
stop codons
2 .. 13  =  12 bp
Shine-Dalgarno sequence
2507 .. 2513  =  7 bp
ribosome binding site
Shine-Dalgarno sequence
2507 .. 2513  =  7 bp
ribosome binding site
toxic minigene
2521 .. 2525  =  5 bp
1 amino acid  =  149.2 Da
Product: two-residue polypeptide that poisons the E. coli translation machinery
toxic minigene
2521 .. 2525  =  5 bp
1 amino acid  =  149.2 Da
Product: two-residue polypeptide that poisons the E. coli translation machinery
toxic minigene
1 .. 1  =  1 bp
0 codons
Product: two-residue polypeptide that poisons the E. coli translation machinery
toxic minigene
1 .. 1  =  1 bp
0 codons
Product: two-residue polypeptide that poisons the E. coli translation machinery
ORF:  148 .. 1008  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2255 .. 2515  =  261 bp
ORF:  86 amino acids  =  10.0 kDa
ORF:  3 .. 251  =  249 bp
ORF:  82 amino acids  =  9.4 kDa  (no start codon)
ORF:  612 .. 878  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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