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Plasmid Files

pLMPd-Ametrine

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

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pLMPd-Ametrine Sequence and MappLMPd-Ametrine.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SgrAI (6562) NdeI (6190) PciI (4126) BspDI - ClaI (3057) ApoI (873) BstEII (1088) SexAI * (1216) BglII (1410) I-SceI (1430) HpaI (1520) AgeI (1884) BlpI (2047) StuI (2068) BspEI (2193) BsmI (2205) XcmI (2290) NcoI (2292) BtgZI (2415) NotI (3016) SalI (3032) AccI (3033) SbfI (3042) pLMPd-Ametrine 6740 bp
SgrAI  (6562)
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.
NdeI  (6190)
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.
PciI  (4126)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspDI  (3057)
1 site
A T C G A T T A G C T A
ClaI  (3057)
1 site
A T C G A T T A G C T A
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
I-SceI  (1430)
1 site
T A G G G A T A A C A G G G T A A T A T C C C T A T T G T C C C A T T A

I-SceI is a homing endonuclease that can recognize a variety of
similar recognition sequences.
After cleavage, I-SceI can remain bound to DNA and alter its
electrophoretic mobility.
HpaI  (1520)
1 site
G T T A A C C A A T T G
AgeI  (1884)
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  (2047)
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  (2068)
1 site
A G G C C T T C C G G A
BspEI  (2193)
1 site
T C C G G A A G G C C T
BsmI  (2205)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
XcmI  (2290)
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.
NcoI  (2292)
1 site
C C A T G G G G T A C C
BtgZI  (2415)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
NotI  (3016)
1 site
G C G G C C G C C G C C G G C G
SalI  (3032)
1 site
G T C G A C C A G C T G
AccI  (3033)
1 site
G T M K A C C A K M T G

Efficient cleavage with AccI requires ≥13 bp on each side of the
recognition sequence.
Sticky ends from different AccI sites may not be compatible.
SbfI  (3042)
1 site
C C T G C A G G G G A C G T C C
AmpR
4946 .. 5806  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4946 .. 5737  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4946 .. 5806  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5738 .. 5806  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4946 .. 5806  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
mAmetrine1.1
2294 .. 3013  =  720 bp
239 amino acids  =  26.8 kDa
   Segment 1:  
   2294 .. 2296  =  3 bp
   1 amino acid  =  149.2 Da
Product: photostable yellow fluorescent protein with
a large Stokes shift, excitable with violet light (Ai et
al., 2008)
mAmetrine1.1
2294 .. 3013  =  720 bp
239 amino acids  =  26.8 kDa
   Segment 2:  1a  
   2297 .. 2299  =  3 bp
   1 amino acid  =  117.2 Da
Product: photostable yellow fluorescent protein with
a large Stokes shift, excitable with violet light (Ai et
al., 2008)
mAmetrine1.1
2294 .. 3013  =  720 bp
239 amino acids  =  26.8 kDa
   Segment 3:  
   2300 .. 3013  =  714 bp
   237 amino acids  =  26.6 kDa
Product: photostable yellow fluorescent protein with
a large Stokes shift, excitable with violet light (Ai et
al., 2008)
mAmetrine1.1
2294 .. 3013  =  720 bp
239 amino acids  =  26.8 kDa
3 segments
Product: photostable yellow fluorescent protein with
a large Stokes shift, excitable with violet light (Ai et
al., 2008)
ori
4187 .. 4775  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
4187 .. 4775  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
5' LTR
6739 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
5' LTR
6739 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
3' LTR
3102 .. 3616  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
3' LTR
3102 .. 3616  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
PGK promoter
1774 .. 2273  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
1774 .. 2273  =  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
1634 .. 1760  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
3' miR-30a
1634 .. 1760  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
5' miR-30a
1443 .. 1564  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
5' miR-30a
1443 .. 1564  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
AmpR promoter
5807 .. 5911  =  105 bp
AmpR promoter
5807 .. 5911  =  105 bp
shRNA
1565 .. 1633  =  69 bp
   Segment 1:  
   1565 .. 1591  =  27 bp
short hairpin RNA
shRNA
1565 .. 1633  =  69 bp
   Segment 2:  miR-30a loop  
   1592 .. 1606  =  15 bp
short hairpin RNA
shRNA
1565 .. 1633  =  69 bp
   Segment 3:  
   1607 .. 1633  =  27 bp
short hairpin RNA
shRNA
1565 .. 1633  =  69 bp
3 segments
short hairpin RNA
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