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Plasmid Files

pZIP-SFFV-RFP-Puro

Lentiviral vector with an SFFV promoter-driven cassette encoding TurboRFP plus a short hairpin RNA (shRNA) with an "UltramiR" microRNA scaffold.

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pZIP-SFFV-RFP-Puro Sequence and MappZIP-SFFV-RFP-Puro.dna
Map and Sequence File   
Sequence Author:  transOMIC
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 SgrDI (8194) SspI (8077) PvuI (7643) FspI (7495) AhdI (7273) DrdI (6491) PmeI (6015) PsiI (5965) MluI (4881) HpaI (4640) BamHI (4554) NotI (4544) BsmBI (4335) SpeI (41) NdeI (276) SnaBI (382) NruI * (867) BspQI - SapI (1136) FseI (1184) EcoNI (1204) MfeI (1223) AleI (1611) KflI (1968) BspDI - ClaI (2214) EcoRV (2338) AgeI (2644) BclI * (2666) BsrGI (2693) PasI (2874) SfiI (3119) PshAI (3283) EcoRI (3352) PspOMI (3478) ApaI (3482) PmlI (3681) AarI (3704) BmgBI (3908) BsiWI (3999) RsrII (4059) pZIP-SFFV-RFP-Puro 8196 bp
SgrDI  (8194)
1 site
C G T C G A C G G C A G C T G C
SspI  (8077)
1 site
A A T A T T T T A T A A
PvuI  (7643)
1 site
C G A T C G G C T A G C
FspI  (7495)
1 site
T G C G C A A C G C G T
AhdI  (7273)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
DrdI  (6491)
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.
PmeI  (6015)
1 site
G T T T A A A C C A A A T T T G
PsiI  (5965)
1 site
T T A T A A A A T A T T
MluI  (4881)
1 site
A C G C G T T G C G C A
HpaI  (4640)
1 site
G T T A A C C A A T T G
BamHI  (4554)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
NotI  (4544)
1 site
G C G G C C G C C G C C G G C G
BsmBI  (4335)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
SpeI  (41)
1 site
A C T A G T T G A T C A
NdeI  (276)
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.
SnaBI  (382)
1 site
T A C G T A A T G C A T
NruI  (867)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BspQI  (1136)
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  (1136)
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.
FseI  (1184)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
EcoNI  (1204)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
MfeI  (1223)
1 site
C A A T T G G T T A A C
AleI  (1611)
1 site
C A C N N N N G T G G T G N N N N C A C
KflI  (1968)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
BspDI  (2214)
1 site
A T C G A T T A G C T A
ClaI  (2214)
1 site
A T C G A T T A G C T A
EcoRV  (2338)
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.
AgeI  (2644)
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.
BclI  (2666)
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.
BsrGI  (2693)
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.
PasI  (2874)
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.
SfiI  (3119)
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.
PshAI  (3283)
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.
EcoRI  (3352)
1 site
G A A T T C C T T A A G
PspOMI  (3478)
1 site
G G G C C C C C C G G G
ApaI  (3482)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PmlI  (3681)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
AarI  (3704)
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.
BmgBI  (3908)
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.
BsiWI  (3999)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
RsrII  (4059)
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.
AmpR
7200 .. 8060  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7200 .. 7991  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7200 .. 8060  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7992 .. 8060  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7200 .. 8060  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TurboRFP
2656 .. 3351  =  696 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea
quadricolor

mammalian codon-optimized
TurboRFP
2656 .. 3351  =  696 bp
231 amino acids  =  26.1 kDa
Product: red fluorescent protein from Entacmaea
quadricolor

mammalian codon-optimized
PuroR
3940 .. 4542  =  603 bp
200 amino acids  =  21.6 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
3940 .. 4542  =  603 bp
200 amino acids  =  21.6 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
4901 .. 5489  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
4901 .. 5489  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
6444 .. 7029  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6444 .. 7029  =  586 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
IRES
3365 .. 3937  =  573 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
IRES
3365 .. 3937  =  573 bp
internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)
SFFV promoter
2236 .. 2643  =  408 bp
spleen focus-forming virus long terminal repeat
(LTR) promoter
SFFV promoter
2236 .. 2643  =  408 bp
spleen focus-forming virus long terminal repeat
(LTR) promoter
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
27 .. 404  =  378 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
6026 .. 6355  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
6026 .. 6355  =  330 bp
SV40 enhancer and early promoter
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
5' LTR (truncated)
411 .. 668  =  258 bp
truncated 5' long terminal repeat (LTR) from HIV-1
RRE
1337 .. 1570  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1337 .. 1570  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
3' LTR (ΔU3)
5697 .. 5930  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' LTR (ΔU3)
5697 .. 5930  =  234 bp
self-inactivating 3' long terminal repeat (LTR) from
HIV-1
3' miR-30a
4754 .. 4880  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
3' miR-30a
4754 .. 4880  =  127 bp
sequence downstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
715 .. 840  =  126 bp
packaging signal of human immunodeficiency virus
type 1
5' miR-30a
4563 .. 4684  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
5' miR-30a
4563 .. 4684  =  122 bp
sequence upstream of the 71-nt precursor of the
human miR-30a microRNA (Zeng et al., 2002)
cPPT/CTS
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
cPPT/CTS
2062 .. 2177  =  116 bp
central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)
AmpR promoter
8061 .. 8165  =  105 bp
AmpR promoter
8061 .. 8165  =  105 bp
shRNA
4685 .. 4753  =  69 bp
   Segment 1:  
   4685 .. 4711  =  27 bp
short hairpin RNA
shRNA
4685 .. 4753  =  69 bp
   Segment 2:  miR-30a loop  
   4712 .. 4726  =  15 bp
short hairpin RNA
shRNA
4685 .. 4753  =  69 bp
   Segment 3:  
   4727 .. 4753  =  27 bp
short hairpin RNA
shRNA
4685 .. 4753  =  69 bp
3 segments
short hairpin RNA
SV40 ori
6206 .. 6341  =  136 bp
SV40 origin of replication
SV40 ori
6206 .. 6341  =  136 bp
SV40 origin of replication
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