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Plasmid Files

pLMN-ZsGreen-Neomycin

Retroviral vector with a ZsGreen marker, for expressing a short hairpin RNA (shRNA) with an "UltramiR" microRNA scaffold.

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pLMN-ZsGreen-Neomycin Sequence and MappLMN-ZsGreen-Neomycin.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 NdeI (7515) PfoI (7377) PacI (4365) ApoI (873) BstEII (1088) SexAI * (1216) BglII (1410) HpaI (1496) MluI (1737) AgeI (1867) BlpI (2030) StuI (2051) BspEI (2176) RsrII (2923) PspOMI (3186) ApaI (3190) AvrII (3224) PmlI (3389) AarI (3412) DraIII (3436) PflMI (3526) BstXI (3658) BclI * (3741) pLMN-ZsGreen-Neomycin 8065 bp
NdeI  (7515)
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.
PfoI  (7377)
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.
PacI  (4365)
1 site
T T A A T T A A A A T T A A T T
ApoI  (873)
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.
BstEII  (1088)
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.
SexAI  (1216)
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.
BglII  (1410)
1 site
A G A T C T T C T A G A
HpaI  (1496)
1 site
G T T A A C C A A T T G
MluI  (1737)
1 site
A C G C G T T G C G C A
AgeI  (1867)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BlpI  (2030)
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.
StuI  (2051)
1 site
A G G C C T T C C G G A
BspEI  (2176)
1 site
T C C G G A A G G C C T
RsrII  (2923)
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.
PspOMI  (3186)
1 site
G G G C C C C C C G G G
ApaI  (3190)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
AvrII  (3224)
1 site
C C T A G G G G A T C C
PmlI  (3389)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (3412)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
DraIII  (3436)
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.
PflMI  (3526)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
BstXI  (3658)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BclI  (3741)
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.
AmpR
6271 .. 7131  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6271 .. 7062  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6271 .. 7131  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7063 .. 7131  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6271 .. 7131  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
2270 .. 3073  =  804 bp
267 amino acids  =  29.3 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
2270 .. 3073  =  804 bp
267 amino acids  =  29.3 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ZsGreen1
3659 .. 4354  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ZsGreen1
3659 .. 4354  =  696 bp
231 amino acids  =  26.1 kDa
Product: Zoanthus green fluorescent protein
mammalian codon-optimized
ori
5512 .. 6100  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5512 .. 6100  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
3096 .. 3647  =  552 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
3096 .. 3647  =  552 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
5' LTR
8064 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
5' LTR
8064 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
3' LTR
4458 .. 4972  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
3' LTR
4458 .. 4972  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
PGK promoter
1757 .. 2256  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
1757 .. 2256  =  500 bp
mouse phosphoglycerate kinase 1 promoter
gag (truncated)
987 .. 1403  =  417 bp
truncated Moloney murine leukemia virus (MMLV)
gag gene lacking the start codon
gag (truncated)
987 .. 1403  =  417 bp
truncated Moloney murine leukemia virus (MMLV)
gag gene lacking the start codon
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell
virus
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell
virus
3' miR-30a
1610 .. 1736  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
3' miR-30a
1610 .. 1736  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
5' miR-30a
1419 .. 1540  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
5' miR-30a
1419 .. 1540  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
AmpR promoter
7132 .. 7236  =  105 bp
AmpR promoter
7132 .. 7236  =  105 bp
shRNA
1541 .. 1609  =  69 bp
   Segment 1:  
   1541 .. 1567  =  27 bp
short hairpin RNA
shRNA
1541 .. 1609  =  69 bp
   Segment 2:  miR-30a loop  
   1568 .. 1582  =  15 bp
short hairpin RNA
shRNA
1541 .. 1609  =  69 bp
   Segment 3:  
   1583 .. 1609  =  27 bp
short hairpin RNA
shRNA
1541 .. 1609  =  69 bp
3 segments
short hairpin RNA
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