pNEB206A (linearized)

Linearized bacterial vector with 8-base 3' overhangs, for cloning with the USER™ enzyme of PCR products amplified with dU-containing primers.

Sequence Author: New England Biolabs

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EcoO109I (2258) AatII (2204) ZraI (2202) SspI (2086) XmnI (1881) ScaI (1762) TsoI (1681) NmeAIII (1430) BsrFI (1362) BpmI (1352) BsaI (1343) AhdI (1282) AlwNI (805) PfoI (2315) NdeI (2453) BstAPI (2454) KasI (2504) NarI (2505) SfoI (2506) PluTI (2508) ApoI - EcoRI (2665) Eco53kI (2673) Bpu10I (2674) BanII - SacI (2675) AscI - BssHII (2679) PacI (2690) End (2706) Start (8) PmeI (17) PstI - SbfI (27) HindIII (30) BspQI - SapI (273) AflIII - PciI (389) BseYI (693) PspFI (697) pNEB206A 2698 bp
EcoO109I  (2258)
1 site
R G G N C C Y Y C C N G G R

Sticky ends from different EcoO109I sites may not be compatible.
AatII  (2204)
1 site
G A C G T C C T G C A G
ZraI  (2202)
1 site
G A C G T C C T G C A G
SspI  (2086)
1 site
A A T A T T T T A T A A
XmnI  (1881)
1 site
G A A N N N N T T C C T T N N N N A A G
ScaI  (1762)
1 site
A G T A C T T C A T G A
TsoI  (1681)
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).
NmeAIII  (1430)
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).
BsrFI  (1362)
1 site
R C C G G Y Y G G C C R

Cleavage may be enhanced when more than one copy of the BsrFI recognition sequence is present.
After cleavage, BsrFI can remain bound to DNA and alter its electrophoretic mobility.
BpmI  (1352)
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.
BsaI  (1343)
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  (1282)
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  (805)
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.
PfoI  (2315)
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.
NdeI  (2453)
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.
BstAPI  (2454)
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.
KasI  (2504)
1 site
G G C G C C C C G C G G
NarI  (2505)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (2506)
1 site
G G C G C C C C G C G G
PluTI  (2508)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
ApoI  (2665)
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.
EcoRI  (2665)
1 site
G A A T T C C T T A A G
Eco53kI  (2673)
1 site
G A G C T C C T C G A G
Bpu10I  (2674)
1 site
C C T N A G C G G A N T C G

Cleavage may be enhanced when more than one copy of the Bpu10I recognition sequence is present.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
BanII  (2675)
1 site
G R G C Y C C Y C G R G

Sticky ends from different BanII sites may not be compatible.
SacI  (2675)
1 site
G A G C T C C T C G A G
AscI  (2679)
1 site
G G C G C G C C C C G C G C G G
BssHII  (2679)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
PacI  (2690)
1 site
T T A A T T A A A A T T A A T T
End  (2706)
0 sites
Start  (8)
0 sites
PmeI  (17)
1 site
G T T T A A A C C A A A T T T G
PstI  (27)
1 site
C T G C A G G A C G T C
SbfI  (27)
1 site
C C T G C A G G G G A C G T C C
HindIII  (30)
1 site
A A G C T T T T C G A A
BspQI  (273)
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  (273)
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  (389)
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  (389)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BseYI  (693)
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.
PspFI  (697)
1 site
C C C A G C G G G T C G
AmpR
1209 .. 2069  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   1209 .. 2000  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1209 .. 2069  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   2001 .. 2069  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1209 .. 2069  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
450 .. 1038  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
450 .. 1038  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
lacZα
2415 .. 2698  =  284 bp
93 amino acids  =  10.7 kDa
Product: LacZα fragment of β-galactosidase
lacZα
2415 .. 2698  =  284 bp
93 amino acids  =  10.7 kDa
Product: LacZα fragment of β-galactosidase
AmpR promoter
2070 .. 2174  =  105 bp
AmpR promoter
2070 .. 2174  =  105 bp
lacZα
10 .. 52  =  43 bp
14 amino acids  =  1.5 kDa
Product: LacZα fragment of β-galactosidase
lacZα
10 .. 52  =  43 bp
14 amino acids  =  1.5 kDa
Product: LacZα fragment of β-galactosidase
lac promoter
96 .. 126  =  31 bp
3 segments
   Segment 3:  -10  
   96 .. 102  =  7 bp
promoter for the E. coli lac operon
lac promoter
96 .. 126  =  31 bp
3 segments
   Segment 2:  
   103 .. 120  =  18 bp
promoter for the E. coli lac operon
lac promoter
96 .. 126  =  31 bp
3 segments
   Segment 1:  -35  
   121 .. 126  =  6 bp
promoter for the E. coli lac operon
lac promoter
96 .. 126  =  31 bp
3 segments
promoter for the E. coli lac operon
lac operator
72 .. 88  =  17 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
72 .. 88  =  17 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).
M13 rev
48 .. 64  =  17 bp
common sequencing primer, one of multiple similar variants
M13 rev
48 .. 64  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
2648 .. 2664  =  17 bp
common sequencing primer, one of multiple similar variants
M13 fwd
2648 .. 2664  =  17 bp
common sequencing primer, one of multiple similar variants
ORF:  1339 .. 1605  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2455 .. 2706  =  252 bp
ORF:  84 amino acids  =  10.0 kDa
ORF:  1209 .. 2069  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  2415 .. 2705  =  291 bp
ORF:  96 amino acids  =  11.0 kDa  (no start codon)
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