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Plasmid Files

pNYCOMPS-LIC-FH10T+ (N term)

Bacterial vector for high-throughput expression and purification of membrane proteins with N-terminal FLAG® and 10xHis tags.

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pNYCOMPS-LIC-FH10T+ (N term).dna
Map and Sequence File:    Download    Open   
Sequence Author:  New York Structural Biology Center
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 SnaBI (6840) EagI - NotI (6798) EcoRI (6478) PasI (6247) ScaI (6065) BbvCI (5754) BsrGI (5634) SrfI (5611) BmgBI (5577) BsaI (5469) BfuAI - BspMI (5363) PstI (5360) SalI (5350) SnaBI (5313) TEV site FLAG AanI - AanI (5240) ATG RBS XbaI (5192) T7 promoter SgrAI (5085) SphI (4937) BstAPI (4728) MluI (4404) BclI * (4390) BstEII (4222) NmeAIII (4204) ApaI (4201) PspOMI (4197) EcoRV (3958) HpaI (3902) PshAI (3563) BglI (3347) PaeR7I - PspXI - XhoI (0) BlpI (79) DraIII (407) AanI - AanI (532) AsiSI - PvuI (1107) BspDI - ClaI (1412) NruI (1448) AcuI (1761) PciI (2303) BspQI - SapI (2420) PflFI - Tth111I (2561) PpuMI (3298) FspI - FspAI (3326) pNYCOMPS-LIC-FH10T+ (N term) 6858 bp
SnaBI  (6840)
2 sites
T A C G T A A T G C A T
EagI  (6798)
1 site
C G G C C G G C C G G C
NotI  (6798)
1 site
G C G G C C G C C G C C G G C G
EcoRI  (6478)
1 site
G A A T T C C T T A A G
PasI  (6247)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
ScaI  (6065)
1 site
A G T A C T T C A T G A
BbvCI  (5754)
1 site
C C T C A G C G G A G T C G
BsrGI  (5634)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
SrfI  (5611)
1 site
G C C C G G G C C G G G C C C G
BmgBI  (5577)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
BsaI  (5469)
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.
BfuAI  (5363)
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  (5363)
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.
PstI  (5360)
1 site
C T G C A G G A C G T C
SalI  (5350)
1 site
G T C G A C C A G C T G
SnaBI  (5313)
2 sites
T A C G T A A T G C A T
AanI  (5240)
2 sites
T T A T A A A A T A T T
AanI  (5240)
2 sites
T T A T A A A A T A T T
XbaI  (5192)
1 site
T C T A G A A G A T C T
SgrAI  (5085)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
SphI  (4937)
1 site
G C A T G C C G T A C G
BstAPI  (4728)
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  (4404)
1 site
A C G C G T T G C G C A
BclI  (4390)
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  (4222)
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.
NmeAIII  (4204)
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).
ApaI  (4201)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (4197)
1 site
G G G C C C C C C G G G
EcoRV  (3958)
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  (3902)
1 site
G T T A A C C A A T T G
PshAI  (3563)
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.
BglI  (3347)
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.
PaeR7I  (0)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (0)
1 site
V C T C G A G B B G A G C T C V
XhoI  (0)
1 site
C T C G A G G A G C T C
BlpI  (79)
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.
DraIII  (407)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
AanI  (532)
2 sites
T T A T A A A A T A T T
AanI  (532)
2 sites
T T A T A A A A T A T T
AsiSI  (1107)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1107)
1 site
C G A T C G G C T A G C
BspDI  (1412)
1 site
A T C G A T T A G C T A
ClaI  (1412)
1 site
A T C G A T T A G C T A
NruI  (1448)
1 site
T C G C G A A G C G C T
AcuI  (1761)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI
recognition sequence.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its
electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PciI  (2303)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2420)
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  (2420)
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.
PflFI  (2561)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (2561)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
PpuMI  (3298)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
FspI  (3326)
1 site
T G C G C A A C G C G T
FspAI  (3326)
1 site
R T G C G C A Y Y A C G C G T R
lacI
3677 .. 4759  =  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
3677 .. 4759  =  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).
KanR
722 .. 1537  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
722 .. 1537  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
CmR
6037 .. 6696  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
6037 .. 6696  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
1659 .. 2247  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1659 .. 2247  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
174 .. 629  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
174 .. 629  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
ccdB
5390 .. 5695  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
ccdB
5390 .. 5695  =  306 bp
101 amino acids  =  11.7 kDa
Product: CcdB, a bacterial toxin that poisons DNA
gyrase
Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains.
rop
2677 .. 2868  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2677 .. 2868  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
4760 .. 4837  =  78 bp
lacI promoter
4760 .. 4837  =  78 bp
T7 terminator
90 .. 137  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
90 .. 137  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac UV5 promoter
6750 .. 6780  =  31 bp
   Segment 3:  -10  
   6750 .. 6756  =  7 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
6750 .. 6780  =  31 bp
   Segment 2:  
   6757 .. 6774  =  18 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
6750 .. 6780  =  31 bp
   Segment 1:  -35  
   6775 .. 6780  =  6 bp
E. coli lac promoter with an "up" mutation
lac UV5 promoter
6750 .. 6780  =  31 bp
3 segments
E. coli lac promoter with an "up" mutation
10xHis
5260 .. 5289  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
5260 .. 5289  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
5165 .. 5189  =  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
5165 .. 5189  =  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).
FLAG
5236 .. 5259  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
5236 .. 5259  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
TEV site
5290 .. 5310  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
5290 .. 5310  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
5146 .. 5164  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5146 .. 5164  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
6 .. 23  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6 .. 23  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
5220 .. 5225  =  6 bp
ribosome binding site
RBS
5220 .. 5225  =  6 bp
ribosome binding site
ATG
5233 .. 5235  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5233 .. 5235  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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