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Plasmid Files

pME-HA (linearized)

Linearized mammalian cell expression vector for directional recombination-based cloning of a gene in the Expresso® system.

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pME-HA (linearized) Sequence and MappME-HA (linearized).dna
Map and Sequence File   
Sequence Author:  Lucigen
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 SacI (3326) Eco53kI (3324) CMV promoter SnaBI (3098) ApaI (2755) PspOMI (2751) ApaLI (2439) XcmI (2034) BsaI (1911) PfoI (1697) BstBI (1604) pME Forward (3344 .. 3365) Bts α I (3346) NheI (3382) BmtI (3386) End (3416) Start (0) BmgBI (12) AvaI - BsoBI - PaeR7I - XhoI (38) BmeT110I (39) pME Reverse (59 .. 80) PsiI (208) ApoI (263) AseI (277) XmnI (278) BspHI (348) SspI (383) Bsu36I (402) SexAI * (511) SfiI (697) BseRI (740) StuI (743) BsaBI * (781) EagI (828) KasI (921) NarI (922) SfoI (923) PluTI (925) DrdI (949) MscI (1004) FspI (1024) PflFI - Tth111I (1040) BsrDI (1155) BtgI (1354) RsrII (1438) pME-HA 3416 bp
SacI  (3326)
1 site
G A G C T C C T C G A G
Eco53kI  (3324)
1 site
G A G C T C C T C G A G
SnaBI  (3098)
1 site
T A C G T A A T G C A T
ApaI  (2755)
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  (2751)
1 site
G G G C C C C C C G G G
ApaLI  (2439)
1 site
G T G C A C C A C G T G
XcmI  (2034)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
BsaI  (1911)
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.
PfoI  (1697)
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.
BstBI  (1604)
1 site
T T C G A A A A G C T T
BtsαI  (3346)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsI sites may not be compatible.
NheI  (3382)
1 site
G C T A G C C G A T C G
BmtI  (3386)
1 site
G C T A G C C G A T C G
End  (3416)
0 sites
Start  (0)
0 sites
BmgBI  (12)
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.
AvaI  (38)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (38)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
PaeR7I  (38)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (38)
1 site
C T C G A G G A G C T C
BmeT110I  (39)
1 site
C Y C G R G G R G C Y C
PsiI  (208)
1 site
T T A T A A A A T A T T
ApoI  (263)
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.
AseI  (277)
1 site
A T T A A T T A A T T A
XmnI  (278)
1 site
G A A N N N N T T C C T T N N N N A A G
BspHI  (348)
1 site
T C A T G A A G T A C T
SspI  (383)
1 site
A A T A T T T T A T A A
Bsu36I  (402)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
SexAI  (511)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
SfiI  (697)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
BseRI  (740)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the
DNA.
StuI  (743)
1 site
A G G C C T T C C G G A
BsaBI  (781)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
EagI  (828)
1 site
C G G C C G G C C G G C
KasI  (921)
1 site
G G C G C C C C G C G G
NarI  (922)
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  (923)
1 site
G G C G C C C C G C G G
PluTI  (925)
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.
DrdI  (949)
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.
MscI  (1004)
1 site
T G G C C A A C C G G T
FspI  (1024)
1 site
T G C G C A A C G C G T
PflFI  (1040)
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  (1040)
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.
BsrDI  (1155)
1 site
G C A A T G N N C G T T A C

Sticky ends from different BsrDI sites may not be compatible.
BtgI  (1354)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
RsrII  (1438)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
pME Forward
22-mer  /  50% GC
1 binding site
3344 .. 3365  =  22 annealed bases
Tm  =  58°C
pME Reverse
22-mer  /  55% GC
1 binding site
59 .. 80  =  22 annealed bases
Tm  =  60°C
NeoR/KanR
794 .. 1588  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
NeoR/KanR
794 .. 1588  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin)
ori
2110 .. 2697  =  588 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
ori
2110 .. 2697  =  588 bp
high-copy-number colE1/pMB1/pBR322/pUC origin
of replication
SV40 promoter
402 .. 759  =  358 bp
SV40 enhancer and early promoter
SV40 promoter
402 .. 759  =  358 bp
SV40 enhancer and early promoter
CMV enhancer
2819 .. 3122  =  304 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
2819 .. 3122  =  304 bp
human cytomegalovirus immediate early enhancer
CMV promoter
3123 .. 3326  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
3123 .. 3326  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
bGH poly(A) signal
58 .. 169  =  112 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
58 .. 169  =  112 bp
bovine growth hormone polyadenylation signal
AmpR promoter
329 .. 400  =  72 bp
AmpR promoter
329 .. 400  =  72 bp
HSV TK poly(A) signal
1820 .. 1867  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
HSV TK poly(A) signal
1820 .. 1867  =  48 bp
herpesvirus thymidine kinase polyadenylation signal
soxR terminator
292 .. 328  =  37 bp
bidirectional E. coli soxR transcription terminator
soxR terminator
292 .. 328  =  37 bp
bidirectional E. coli soxR transcription terminator
T3Te terminator
2059 .. 2088  =  30 bp
phage T3 early transcription terminator
T3Te terminator
2059 .. 2088  =  30 bp
phage T3 early transcription terminator
T7Te terminator
2709 .. 2736  =  28 bp
phage T7 early transcription terminator
T7Te terminator
2709 .. 2736  =  28 bp
phage T7 early transcription terminator
HA
1 .. 27  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
1 .. 27  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
Forward Primer extension
3399 .. 3416  =  18 bp
Add this top strand sequence to the 5' end of the
forward primer for amplifying the gene of interest.
Forward Primer extension
3399 .. 3416  =  18 bp
Add this top strand sequence to the 5' end of the
forward primer for amplifying the gene of interest.
SV40 ori
610 .. 745  =  136 bp
SV40 origin of replication
SV40 ori
610 .. 745  =  136 bp
SV40 origin of replication
Reverse Primer extension
1 .. 18  =  18 bp
Add this bottom strand sequence to the 5' end of
the reverse primer for amplifying the gene of
interest.
Reverse Primer extension
1 .. 18  =  18 bp
Add this bottom strand sequence to the 5' end of
the reverse primer for amplifying the gene of
interest.
ATG
3414 .. 3416  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3414 .. 3416  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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