Difference between revisions of "Part:BBa K165063"

Latest revision as of 19:38, 31 October 2008

pRS306 yeast shuttle vector, URA3 selection

Ampicillin resistance when grown in bacteria. This plasmid is not currently Biobrick compatible, but is useful for inserting a final construct into a yeast cell.

Useful for chromosomal integration of a device into yeast strains. This will insert the vector at the specific locus of the URA3 gene. To be used in conjunction with uracil drop-out media for positive selection of transformed cells. Transformation using this vector requires linearization of the plasmid by cutting with PstI.

To incorporate a single Biobrick part into this vector for subsequent transformation into yeast, one should cut both vector and part with EcoRI and SpeI.

Alternatively, one can do the final ligation step between two Biobrick parts into this vector by cutting with the following:

Vector: EcoRI, Not I

Prefix: EcoRI, SpeI

Suffix: XbaI, NotI

Original Sikorski vector was described in R. S. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=2659436 Sikorski and P. Hieter, Genetics, 122: 19-27 (1989)].

Vector NTI annotated sequence: File:PRS306.gb

Yeast transformation protocol: R Daniel Gietz & Robert H Schiestl. [http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method]. Nature protocols (2007)

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 2047
Illegal XbaI site found at 2017
Illegal SpeI site found at 2023
Illegal PstI site found at 389
Illegal PstI site found at 2041
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 2047
Illegal SpeI site found at 2023
Illegal PstI site found at 389
Illegal PstI site found at 2041
Illegal NotI site found at 2009
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 2047
Illegal BamHI site found at 2029
Illegal XhoI site found at 2080
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 2047
Illegal XbaI site found at 2017
Illegal SpeI site found at 2023
Illegal PstI site found at 389
Illegal PstI site found at 2041
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 2047
Illegal XbaI site found at 2017
Illegal SpeI site found at 2023
Illegal PstI site found at 389
Illegal PstI site found at 2041
Illegal NgoMIV site found at 1668
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1079
Illegal BsaI.rc site found at 3453
Illegal SapI site found at 2370
Illegal SapI.rc site found at 926

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K165063 short</partinfo>
-Useful for chromosomal integration of a part into yeast strains.  Insertion of a single Biobrick (or biofusion) part requires cutting with EcoRI and SpeI.  Another option for this vector is to insert two Biobrick/biofusion parts by cutting the first part with NotI and SpeI and the second part with XbaI and PstI, while cutting the vector with NotI and PstI.  Note that this part does not conform to Standard Assembly requirements and will maintain idempotency.  Once a construct of interest has been inserted into the vector, it should be linearized with a digest using PstI.  This vector will supplement a URA3 auxotrophic yeast strain.  The part is then transformed into yeast according to the protocol outlined in this paper:
+Ampicillin resistance when grown in bacteria.  This plasmid is not currently Biobrick compatible, but is useful for inserting a final construct into a yeast cell.
+Useful for chromosomal integration of a device into yeast strains.  This will insert the vector at the specific locus of the URA3 gene.  To be used in conjunction with uracil drop-out media for positive selection of transformed cells.  Transformation using this vector requires linearization of the plasmid by cutting with PstI.
+To incorporate a single Biobrick part into this vector for subsequent transformation into yeast, one should cut both vector and part with EcoRI and SpeI.
+Alternatively, one can do the final ligation step between two Biobrick parts into this vector by cutting with the following:
+Vector: EcoRI, Not I
+Prefix: EcoRI, SpeI
+Suffix: XbaI, NotI
+Original Sikorski vector was described in R. S. [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=2659436 Sikorski and P. Hieter, Genetics, 122: 19-27 (1989)].
+Vector NTI annotated sequence: [[Image:PRS306.gb]]
-R Daniel Gietz & Robert H Schiestl. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method . Nature protocols
+Yeast transformation protocol:
+R Daniel Gietz & Robert H Schiestl. [http://www.natureprotocols.com/2007/01/31/highefficiency_yeast_transform.php High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method]. Nature protocols (2007)