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Plasmid Files

pREP4

Episomal mammalian vector with an RSV promoter and a hygromycin resistance marker, for high-level expression in primate and canine cells.

 
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pREP4.dna
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AsiSI (9114) PshAI (8777) BstBI (8601) PluTI (8205) SfoI (8203) NarI (8202) KasI (8201) AseI (6770) XmnI (6345) SspI (6140) BstEII (5854) EcoNI (5849) StuI * (5778) AvrII (5489) SphI (397) AleI (555) Acc65I (576) KpnI (580) PvuII (583) HindIII (592) NotI (603) PaeR7I - PspXI - XhoI (609) SfiI (621) BamHI (627) XbaI (1031) AfeI (1109) EcoRV (1649) SpeI (1971) NsiI (3199) SexAI * (3373) BbvCI (3814) SgrAI (4015) BsaAI (4053) Eco53kI (4073) SacI (4075) pREP4 10,338 bp
AsiSI  (9114)
1 site
G C G A T C G C C G C T A G C G
PshAI  (8777)
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.
BstBI  (8601)
1 site
T T C G A A A A G C T T
PluTI  (8205)
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.
SfoI  (8203)
1 site
G G C G C C C C G C G G
NarI  (8202)
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.
KasI  (8201)
1 site
G G C G C C C C G C G G
AseI  (6770)
1 site
A T T A A T T A A T T A
XmnI  (6345)
1 site
G A A N N N N T T C C T T N N N N A A G
SspI  (6140)
1 site
A A T A T T T T A T A A
BstEII  (5854)
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.
EcoNI  (5849)
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.
StuI  (5778)
1 site
A G G C C T T C C G G A
* Blocked by Dcm methylation.
AvrII  (5489)
1 site
C C T A G G G G A T C C
SphI  (397)
1 site
G C A T G C C G T A C G
AleI  (555)
1 site
C A C N N N N G T G G T G N N N N C A C
Acc65I  (576)
1 site
G G T A C C C C A T G G
KpnI  (580)
1 site
G G T A C C C C A T G G
PvuII  (583)
1 site
C A G C T G G T C G A C
HindIII  (592)
1 site
A A G C T T T T C G A A
NotI  (603)
1 site
G C G G C C G C C G C C G G C G
PaeR7I  (609)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (609)
1 site
V C T C G A G B B G A G C T C V
XhoI  (609)
1 site
C T C G A G G A G C T C
SfiI  (621)
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.
BamHI  (627)
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.
XbaI  (1031)
1 site
T C T A G A A G A T C T
AfeI  (1109)
1 site
A G C G C T T C G C G A
EcoRV  (1649)
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.
SpeI  (1971)
1 site
A C T A G T T G A T C A
NsiI  (3199)
1 site
A T G C A T T A C G T A
SexAI  (3373)
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.
BbvCI  (3814)
1 site
C C T C A G C G G A G T C G
SgrAI  (4015)
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.
BsaAI  (4053)
1 site
Y A C G T R R T G C A Y
Eco53kI  (4073)
1 site
G A G C T C C T C G A G
SacI  (4075)
1 site
G A G C T C C T C G A G
EBNA1
3607 .. 5532  =  1926 bp
641 amino acids  =  56.4 kDa
Product: Epstein-Barr nuclear antigen 1, also known as EBNA-1
crucial for latent viral infection, and for episomal amplification of vectors containing the oriP origin (Okita et al., 2013)
EBNA1
3607 .. 5532  =  1926 bp
641 amino acids  =  56.4 kDa
Product: Epstein-Barr nuclear antigen 1, also known as EBNA-1
crucial for latent viral infection, and for episomal amplification of vectors containing the oriP origin (Okita et al., 2013)
oriP
1517 .. 3305  =  1789 bp
Epstein-Barr virus oriP replication origin (Yates et al., 2000)
oriP
1517 .. 3305  =  1789 bp
Epstein-Barr virus oriP replication origin (Yates et al., 2000)
HygR
8750 .. 9787  =  1038 bp
345 amino acids  =  38.5 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
HygR
8750 .. 9787  =  1038 bp
345 amino acids  =  38.5 kDa
Product: hygromycin B phosphotransferase
confers resistance to hygromycin
AmpR
6158 .. 7018  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   6158 .. 6226  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6158 .. 7018  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   6227 .. 7018  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6158 .. 7018  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
7189 .. 7777  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7189 .. 7777  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
RSV promoter
305 .. 566  =  262 bp
Rous sarcoma virus enhancer/promoter
RSV promoter
305 .. 566  =  262 bp
Rous sarcoma virus enhancer/promoter
HSV TK promoter
8562 .. 8707  =  146 bp
herpes simplex virus thymidine kinase promoter
HSV TK promoter
8562 .. 8707  =  146 bp
herpes simplex virus thymidine kinase promoter
SV40 poly(A) signal
628 .. 762  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
628 .. 762  =  135 bp
SV40 polyadenylation signal
AmpR promoter
6053 .. 6157  =  105 bp
AmpR promoter
6053 .. 6157  =  105 bp
HSV TK poly(A) signal
9829 .. 9877  =  49 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
9829 .. 9877  =  49 bp
herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)
MCS
576 .. 632  =  57 bp
multiple cloning site
MCS
576 .. 632  =  57 bp
multiple cloning site
ORF:  4129 .. 4365  =  237 bp
ORF:  78 amino acids  =  8.9 kDa
ORF:  7747 .. 8079  =  333 bp
ORF:  110 amino acids  =  12.3 kDa
ORF:  10,084 .. 33  =  288 bp
ORF:  95 amino acids  =  10.7 kDa
ORF:  6158 .. 7018  =  861 bp
ORF:  286 amino acids  =  31.5 kDa
ORF:  8330 .. 8554  =  225 bp
ORF:  74 amino acids  =  8.0 kDa
ORF:  8750 .. 9787  =  1038 bp
ORF:  345 amino acids  =  38.5 kDa
ORF:  4320 .. 5534  =  1215 bp
ORF:  404 amino acids  =  45.2 kDa
ORF:  8475 .. 8753  =  279 bp
ORF:  92 amino acids  =  10.0 kDa
ORF:  10,077 .. 23  =  285 bp
ORF:  94 amino acids  =  10.8 kDa
ORF:  34 .. 546  =  513 bp
ORF:  170 amino acids  =  19.2 kDa
ORF:  934 .. 1242  =  309 bp
ORF:  102 amino acids  =  11.4 kDa
ORF:  3607 .. 5532  =  1926 bp
ORF:  641 amino acids  =  56.4 kDa
ORF:  6622 .. 6888  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  7696 .. 7932  =  237 bp
ORF:  78 amino acids  =  8.9 kDa
ORF:  507 .. 779  =  273 bp
ORF:  90 amino acids  =  9.6 kDa
ORF:  2229 .. 2468  =  240 bp
ORF:  79 amino acids  =  8.7 kDa
ORF:  3207 .. 3524  =  318 bp
ORF:  105 amino acids  =  11.7 kDa
ORF:  4305 .. 5417  =  1113 bp
ORF:  370 amino acids  =  40.7 kDa
ORF:  7452 .. 7850  =  399 bp
ORF:  132 amino acids  =  14.8 kDa
ORF:  7914 .. 8231  =  318 bp
ORF:  105 amino acids  =  11.9 kDa
ORF:  8135 .. 10,078  =  1944 bp
ORF:  647 amino acids  =  70.4 kDa
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