Part:BBa_K4574032

pX459 vector

The pX459 vector is commonly used in the CRISPR-Cas9 system for genome editing. It contains both the Cas9 (Csn1) endonuclease from the Streptococcus pyogenes and a guide RNA (gRNA) scaffold in its expression cassette, along with a puromycin resistance gene (PuroR) for easy selection and tracking of transfected cells.

Source

The vector was developed by Zhang et al.. The provided sequence comprises strictly the plasmid's backbone, while the pX459 empty vector includes a double BsbI insertion site (GGGTCTTCGAGAAGACCT) at position 1 of the current sequence (see Fig. 1).

Design

Fig. 1. px458/px459 empty vector map.

Usage and Biology

This vector and derived plasmids have been used for CRISPR homology-directed repair and CRISPR knock-out in RAMOS B-cells.

Sequence and Features

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

gRNA scaffold	guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system
CMV enhancer	human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer
CBA promoter	promoter of chicken beta-actin gene
Hybrid intron	hybrid between chicken beta-actin and minute virus of mice introns
3xFLAG	Three tandem FLAG epitope tags, followed by an enterokinase cleavage site
SV40 NLS	nuclear localization signal of simian virus 40 large T antigen
Cas9	Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system which generates RNA-guided double-strand breaks in DNA
Nucleoplasmin NLS	bipartite nuclear localization signal from nucleoplasmin
T2A	2A-like peptide from Thosea asigna virus capsid protein functioning as ribosome skipping site
PuroR	gene from Streptomyces alboniger for puromycin N-acetyltransferase; confers resistance to puromycin
bGH pA	bovine growth hormone polyadenylation signal
AAV2 ITR	inverted terminal repeat of adeno-associated virus serotype 2
f1 ori	f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
bla promoter	weak constitutive promoter for ampicillin resistance
AmpR	gene for β-lactamase; confers resistance to ampicillin, carbenicillin, and related antibiotics
pUC ori	high-copy-number ColE1-derived origin of replication
U6 promoter	RNA polymerase III promoter for human U6 snRNA

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5252
  Illegal XbaI site found at 161
  Illegal PstI site found at 146
  Illegal PstI site found at 1889
  Illegal PstI site found at 3311
  Illegal PstI site found at 3515
  Illegal PstI site found at 3545
  Illegal PstI site found at 4757
  Illegal PstI site found at 6295
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5252
  Illegal PstI site found at 146
  Illegal PstI site found at 1889
  Illegal PstI site found at 3311
  Illegal PstI site found at 3515
  Illegal PstI site found at 3545
  Illegal PstI site found at 4757
  Illegal PstI site found at 6295
  Illegal NotI site found at 6153
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5252
  Illegal BglII site found at 1350
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5252
  Illegal XbaI site found at 161
  Illegal PstI site found at 146
  Illegal PstI site found at 1889
  Illegal PstI site found at 3311
  Illegal PstI site found at 3515
  Illegal PstI site found at 3545
  Illegal PstI site found at 4757
  Illegal PstI site found at 6295
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 5252
  Illegal XbaI site found at 161
  Illegal PstI site found at 146
  Illegal PstI site found at 1889
  Illegal PstI site found at 3311
  Illegal PstI site found at 3515
  Illegal PstI site found at 3545
  Illegal PstI site found at 4757
  Illegal PstI site found at 6295
  Illegal NgoMIV site found at 2177
  Illegal NgoMIV site found at 3281
  Illegal NgoMIV site found at 3354
  Illegal NgoMIV site found at 3839
  Illegal NgoMIV site found at 4748
  Illegal NgoMIV site found at 5209
  Illegal NgoMIV site found at 5228
  Illegal AgeI site found at 971
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 5801