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pNYCOMPS-LIC-FH10T+ (N term) - linearized

Linearized bacterial vector for ligation-independent cloning (LIC), with N-terminal FLAG® and 10xHis tags for expression and purification of membrane proteins.

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pNYCOMPS-LIC-FH10T+ (N term) - linearized.dna
Map and Sequence File:    Download    Open   
Sequence Author:  New York Structural Biology Center
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 Start (0) TEV site End (5316) FLAG AanI - AanI (5258) ATG RBS XbaI (5210) lac operator T7 promoter SgrAI (5103) SphI (4955) BstAPI (4746) MluI (4422) BclI * (4408) BstEII (4240) NmeAIII (4222) ApaI (4219) PspOMI (4215) EcoRV (3976) HpaI (3920) PshAI (3581) BglI (3365) FspI - FspAI (3344) PpuMI (3316) PaeR7I - XhoI (18) BlpI (97) DraIII (425) AanI - AanI (550) AsiSI - PvuI (1125) BspDI - ClaI (1430) NruI (1466) AcuI (1779) PciI (2321) BspQI - SapI (2438) PflFI - Tth111I (2579) pNYCOMPS-LIC-FH10T+ (N term) 5316 bp
Start  (0)
0 sites
End  (5316)
0 sites
AanI  (5258)
2 sites
T T A T A A A A T A T T
AanI  (5258)
2 sites
T T A T A A A A T A T T
XbaI  (5210)
1 site
T C T A G A A G A T C T
SgrAI  (5103)
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  (4955)
1 site
G C A T G C C G T A C G
BstAPI  (4746)
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  (4422)
1 site
A C G C G T T G C G C A
BclI  (4408)
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  (4240)
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  (4222)
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  (4219)
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  (4215)
1 site
G G G C C C C C C G G G
EcoRV  (3976)
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  (3920)
1 site
G T T A A C C A A T T G
PshAI  (3581)
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  (3365)
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.
FspI  (3344)
1 site
T G C G C A A C G C G T
FspAI  (3344)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (3316)
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.
PaeR7I  (18)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (18)
1 site
C T C G A G G A G C T C
BlpI  (97)
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  (425)
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  (550)
2 sites
T T A T A A A A T A T T
AanI  (550)
2 sites
T T A T A A A A T A T T
AsiSI  (1125)
1 site
G C G A T C G C C G C T A G C G
PvuI  (1125)
1 site
C G A T C G G C T A G C
BspDI  (1430)
1 site
A T C G A T T A G C T A
ClaI  (1430)
1 site
A T C G A T T A G C T A
NruI  (1466)
1 site
T C G C G A A G C G C T
AcuI  (1779)
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  (2321)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (2438)
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  (2438)
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  (2579)
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  (2579)
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.
lacI
3695 .. 4777  =  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
3695 .. 4777  =  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
740 .. 1555  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
740 .. 1555  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
1677 .. 2265  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
1677 .. 2265  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
192 .. 647  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
192 .. 647  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
2695 .. 2886  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
2695 .. 2886  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
4778 .. 4855  =  78 bp
lacI promoter
4778 .. 4855  =  78 bp
T7 terminator
108 .. 155  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
108 .. 155  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
10xHis
5278 .. 5307  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
10xHis
5278 .. 5307  =  30 bp
10 amino acids  =  1.4 kDa
Product: 10xHis affinity tag
lac operator
5183 .. 5207  =  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
5183 .. 5207  =  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
5254 .. 5277  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
FLAG
5254 .. 5277  =  24 bp
8 amino acids  =  1.0 kDa
Product: FLAG® epitope tag, followed by an
enterokinase cleavage site
TEV site
5308 .. 5328  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
5308 .. 5328  =  21 bp
7 amino acids  =  900.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
5164 .. 5182  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
5164 .. 5182  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
24 .. 41  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
24 .. 41  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
RBS
5238 .. 5243  =  6 bp
ribosome binding site
RBS
5238 .. 5243  =  6 bp
ribosome binding site
ATG
5251 .. 5253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5251 .. 5253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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