Part:BBa_K4574000

pcDNA3.4-TOPO vector

The pcDNA3.4-TOPO vector is a constitutive mammalian expression vector with high levels of transgene expression. Derived from the broader pcDNA series, this vector contains a CMV (cytomegalovirus) immediate-early promoter for expression in a variety of mammalian cells, an SV40 origin of replication, enabling replication of transiently transfected plasmid in mammalian hosts, a neomycin/kanamycin resistance gene, allowing for selection in mammalian cells using the antibiotic G418, a high-copy pUC bacterial origin of replication, and an ampicillin resistance (bla) gene for selection in bacterial cells among other features.

Source

The vector was developed by Invitrogen (a brand of Thermo Fisher Scientific; catalog number: A14697).

Design

Fig. 1. pcDNA3.4-TOPO plasmid map.

Usage and Biology

This vector and derived plasmids have been used in mammalian cell culture (HEK293T, Exi293, CHO, ExpiCHO, COS-7, RAMOS). The cloning has been performed in DH5α E. coli competent cells.

Sequence and Features

The vector contains a number of functional features in its sequence:

WPRE	woodchuck posttranscriptional regulatory element
HSV TK pA	herpesvirus thymidine kinase polyadenylation signal
f1 ori	f1 bacteriophage origin of replication
SV40 promoter	simian virus 40 enhancer and early promoter
SV40 ori	simian virus 40 origin of replication
NeoR/KanR	gene for aminoglycoside phosphotransferase from Tn5; confers resistance to neomycin, kanamycin, and G418 (Geneticin)
SV40 pA	simian virus 40 polyadenylation signal
pUC ori	high-copy-number ColE1-derived origin of replication
AmpR	gene for β-lactamase; confers resistance to ampicillin, carbenicillin, and related antibiotics
bla promoter	weak constitutive promoter for ampicillin resistance
CMV enhancer	human cytomegalovirus immediate early enhancer
CMV promoter	human cytomegalovirus immediate early promoter

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5993
  Illegal SpeI site found at 5334
  Illegal PstI site found at 1965
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal SpeI site found at 5334
  Illegal PstI site found at 1965
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BglII site found at 915
  Illegal BglII site found at 5097
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5993
  Illegal SpeI site found at 5334
  Illegal PstI site found at 1965
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 5993
  Illegal SpeI site found at 5334
  Illegal PstI site found at 1965
  Illegal NgoMIV site found at 558
  Illegal NgoMIV site found at 1075
  Illegal NgoMIV site found at 2416
  Illegal NgoMIV site found at 2701
  Illegal AgeI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 798
  Illegal BsaI.rc site found at 4229
  Illegal SapI site found at 3146
  Illegal SapI.rc site found at 2265
  Illegal SapI.rc site found at 2475