Difference between revisions of "Part:BBa K1680014"
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A ''Pst''I restriction site in the backbone was removed by mutagenesis PCR (see lane 1 & 2 in figure). Exchange of the wild type MCS for RFC10 compatible MCS coulb be shown by restriction with ''Sal''I restriction: The enzyme linearizes the vector with old MCS (lane 3) but does not cut the vector with the new MCS, as indicated by presence of supercoiled DNA (lane 4).
 
A ''Pst''I restriction site in the backbone was removed by mutagenesis PCR (see lane 1 & 2 in figure). Exchange of the wild type MCS for RFC10 compatible MCS coulb be shown by restriction with ''Sal''I restriction: The enzyme linearizes the vector with old MCS (lane 3) but does not cut the vector with the new MCS, as indicated by presence of supercoiled DNA (lane 4).
  
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[[PRS313_mutated.png|500x]]
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[[Image:PRS313_mutated.png|500x]]
  
 
Agarose Gel of different Versions of the pRS313 vector. Lane 1: wild type vector, cut with ''Pst''I; lane 2: mutagenised vector cut with ''Pst''I; lane 3: mutagenised vector cut with ''Sal''I; lane 4: vector with RFC10 MCS cut with ''Sal''I.
 
Agarose Gel of different Versions of the pRS313 vector. Lane 1: wild type vector, cut with ''Pst''I; lane 2: mutagenised vector cut with ''Pst''I; lane 3: mutagenised vector cut with ''Sal''I; lane 4: vector with RFC10 MCS cut with ''Sal''I.

Revision as of 00:44, 26 September 2015

pRS313 yeast shuttle vector with Biobrick MCS

This part is a biobricked shuttle vectors for Saccharomyces cerevisiae. The vector was made compatible with the RFC10 standard and also contains a RFC10 cloning site. It encode for an ampicillin resistance gene and replicates to high-copy numbers in E.Coli.

Furthermore, the CEN6/ARS4 (Chromosome VI centromere/Autonomously Replicating Sequence 4) cassette ensures that the plasmid stays at a low copy number in yeast, because they are treated as pseudo chromosomes. The vector also encodes for a HIS3 gene, which is used an auxotrophy marker in yeast.


This part is mutagenized version of the wild type pRS313 vector (Sikorski 1989).

A PstI restriction site in the backbone was removed by mutagenesis PCR (see lane 1 & 2 in figure). Exchange of the wild type MCS for RFC10 compatible MCS coulb be shown by restriction with SalI restriction: The enzyme linearizes the vector with old MCS (lane 3) but does not cut the vector with the new MCS, as indicated by presence of supercoiled DNA (lane 4).

500x

Agarose Gel of different Versions of the pRS313 vector. Lane 1: wild type vector, cut with PstI; lane 2: mutagenised vector cut with PstI; lane 3: mutagenised vector cut with SalI; lane 4: vector with RFC10 MCS cut with SalI.


Sequence and Features

This part is a RFC10 compatible shuttle vector for yeast.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2120
    Illegal XbaI site found at 2105
    Illegal SpeI site found at 2097
    Illegal PstI site found at 2083
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2120
    Illegal NheI site found at 1007
    Illegal SpeI site found at 2097
    Illegal PstI site found at 2083
    Illegal NotI site found at 2088
    Illegal NotI site found at 2112
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2120
    Illegal BglII site found at 898
    Illegal BglII site found at 958
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2120
    Illegal XbaI site found at 2105
    Illegal SpeI site found at 2097
    Illegal PstI site found at 2083
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 2120
    Illegal XbaI site found at 2105
    Illegal SpeI site found at 2097
    Illegal PstI site found at 2083
    Illegal NgoMIV site found at 1748
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 3494
    Illegal SapI site found at 2411
    Illegal SapI site found at 4515