Plasmid
APEV

Part:BBa_K596004

Designed by: Ebuzer Kalyoncu   Group: iGEM11_Bilkent_UNAM_Turkey   (2011-09-19)
Revision as of 14:20, 1 October 2011 by Aet (Talk | contribs)

Algae Protein Expression Vector

This Biobrick Plasmid is created from pRbcRL (which is also derived from pBluescriptKS-) by putting biobrick prefix and suffix between HSP70A/RbcS2 promoter and 3'UTR sequences from RbcS2 gene. We used pRbcRL(Hsp196)for the backbone plasmid. We remove luciferase protein coding sequence(which is represented in the figure as crluc)using XhoI/BamHI restriction. Then we ligated with excised plasmid with biobrick prefix and suffix. biobrick prefix and suffix contains four restriction enzymes namely XbaI, EcoRI, SpeI, PstI. This plasmid can be used for protein expression in C.Reinhardtii and for cloning purposes in E.coli. it contains ampicillin for selection in E.coli. F1 origin of replication is for double stranded replication process. HSP70A is a sequence for transcriptional activation that enhances expression of the gene.

References:

1. Schroda M, Blöcker D, Beck CF. The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. The Plant journal : for cell and molecular biology. 2000;21(2):121-31. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10743653.

2. Fuhrmann M, Hausherr A, Ferbitz L, et al. Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant molecular biology. 2004;55(6):869-81. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15604722.

Map: Algae protein expression vector pAPEV.png

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 3787
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3787
    Illegal NheI site found at 3376
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 3793
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3787
    Illegal BamHI site found at 23
    Illegal XhoI site found at 3779
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 3787
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 3787
    Illegal XbaI site found at 3802
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 2598
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1621
    Illegal SapI site found at 538


[edit]
Categories
Parameters
None