P5E-bactin2: Difference between revisions

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== Construction Details ==
== Construction Details ==
p5E-MCS was made by PCR of the pBSII SK+ multiple cloning site, from T7 to T3 primers, followed by a BP reaction with pDONR P4-P1R. Sequencing confirms that all bases of the insert are correct.
''bactin2'' genomic sequence was derived from a 5.3 kb clone (gift of Ken Poss), which was derived in turn from a 10 kb upstream construct from Shinichi Higashijima.


M13F, M13R, and T3 should all work as sequencing primers.
p5E-bactin2 was made by PCR of the bactin2 upstream genomic sequence (using primers to add att sites), followed by a BP reaction. Upstream sequence includes 3.8 kb upstream of transcriptional start, a small 5' UTR exon, a 1.4 kb intron, and then 38 bp of 5' UTR, terminating immediately before the native start codon. Sequencing confirms that the ends of the insert are correct.


Unfortunately, the backbone of P4-P1R contains recognition sites for several potentially useful restriction enzymes. The restriction sites from the Bluescript MCS that remain unique in p5E-MCS are:
== Crude Map ==
 
Screenshot from ApE showing locations of components:
''Asp718-KpnI-DraII-XhoI-SalI-Bsp106-HindIII-SmaI-BamHI-SpeI-SacII-BstXI''


== Crude Map ==
:[[File:299 p5E-bactin2.PNG|thumb|none|350px|299 p5E-bactin2]]
Screenshot from Sequencher showing locations of components:
P5E-MCS.png


==Download the Sequence ==
==Download the Sequence ==
* Annotated Sequence (Genbank format) and Full-Length Sequence with individual component sequences included (FASTA file)
* '''Annotated Sequence''' (Genbank format) and '''Full-Length Sequence with individual component sequences included''' (FASTA file)
: [[Media:233._pME-nlsmCherry.zip|Download p5E-MCS]]
: [[Media:299.p5E-bactin2.zip|Download p5E-''bactin2'']]
 




== Reference ==
== Reference ==
This template is used to display DNA sequence information in a standardized format.
Higashijima S, Okamoto H, Ueno N, Hotta Y, Eguchi G. (1997) Dev Biol.15;192:289-99. "High-frequency generation of transgenic zebrafish which
reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin."

Latest revision as of 04:01, 3 June 2025

Construction Details

bactin2 genomic sequence was derived from a 5.3 kb clone (gift of Ken Poss), which was derived in turn from a 10 kb upstream construct from Shinichi Higashijima.

p5E-bactin2 was made by PCR of the bactin2 upstream genomic sequence (using primers to add att sites), followed by a BP reaction. Upstream sequence includes 3.8 kb upstream of transcriptional start, a small 5' UTR exon, a 1.4 kb intron, and then 38 bp of 5' UTR, terminating immediately before the native start codon. Sequencing confirms that the ends of the insert are correct.

Crude Map

Screenshot from ApE showing locations of components:

299 p5E-bactin2

Download the Sequence

  • Annotated Sequence (Genbank format) and Full-Length Sequence with individual component sequences included (FASTA file)
Download p5E-bactin2


Reference

Higashijima S, Okamoto H, Ueno N, Hotta Y, Eguchi G. (1997) Dev Biol.15;192:289-99. "High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin."