PHUSER v 1.1 (command line program behind the PHUSER webserver)
NAME
PHUSER - Primer helper for USER
SYNOPSIS
phuser [options...] file(s)
DESCRIPTION
PHUSER is a tool for designing primers for USER fusion
This tool ensures non-overlapping fusion tails, enabling
the user to fuse more than two DNA fragments together in
one reaction, as long as unique fusion tails exist.
PHUSER also ensures that the melting temperature (Tm) of the
primers are pairwise within 2 degrees celsius of each other,
optimizing the primers for PCR amplification.
The files containing the input DNA sequences are specified
seperately as commandline input or STDIN. The program can
handle almost any format of DNA (e.g. fasta, spaces, new
lines, etc.).
A cassette is chosen by the -c option (see below)
The needed primers will be printed to a user-defined output
file with the following three segments of information:
Overview of primers:
--------------------
Short print out of the needed primers.
Grafic overview of DNA fragments and primers:
---------------------------------------------
The first graphic illustrates the forward primer for
DNA fragment 1, i.e. binding region to biobrick 1,
and fusion tail to the selected USER cassette. The
following graphics (the number depends on how many
DNA sequences were submitted) illustrates the region
at which DNA fragment 1 is fused to DNA fragment 2
and so forth. The last 40 bases of DNA fragment 1 and
the first 40 bases of DNA fragment 2 are displayed,
along with the positioning of the fusion tails and
binding regions for both reverse and forward primers.
The last graphic of the overview illustrates the last
DNA fragment and its binding region to the selected
cassette, diplayed in a similar fashion as described
for the first block.
Primer details:
---------------
The primers are printed again with corresponding data
about DNA melting temperature (Tm) and GC ratio. Also
provided is the status of Tm or GC ratio in terms of
optimum The value of the two parameters is succeeded
by either '...YES' or '...NO', depending on the status.
OPTIONS
-c, --cassette
Select which cassete to use, by specifying one of the
predefined sets listed below, or LINEAR (0) for avoiding
the use of the restriction site primers at the borders
of the fused construct.
DEFAULT: "1" (PacI/Nt.BbvCI)
1 :
Name: PacI/Nt.BbvCI
Forward strand: GCTGAGGGTTTAATTAAGACCTCAGC
Reverse strand: CGACTCCCAAATTAATTCTGGAGTCG
Fusion tail forward: GGGTTTAAU
Fusion tail reverse: GGTCTTAAU
2 :
Name: XbaI2/Nt.BbvCI
Forward strand: GCTGAGGGAAAGTCTAGAGGATCCTCTAGATGTCTCCTCAGC
Reverse strand: CGACTCCCTTTCAGATCTCCTAGGAGATCTACAGAGGAGTCG
Fusion tail forward: GGGAAAGU
Fusion tail reverse: GGAGACAU
3 :
Name: PmeI/Nb.BbvCI
Forward strand: CCTCAGCCGTTTAAACAGCTGAGG
Reverse strand: GGAGTCGGCAAATTTGTCGACTCC
Fusion tail forward: GCCGTTU
Fusion tail reverse: GCTGTTU
4 :
Name: AsiSI/Nb.BsmI
Forward strand: GAATGCGTGCGATCGCGTGCATTC
Reverse strand: CTTACGCACGCTAGCGCACGTAAG
Fusion tail forward: CGTGCGAU
Fusion tail reverse: CACGCGAU
5 :
Name: AsiSI/Nb.BtsI
Forward strand: GCAGTGAGAGCGATCGCAGACACTGC
Reverse strand: CGTCACTCTCGCTAGCGTCTGTGACG
Fusion tail forward: AGAGCGAU
Fusion tail reverse: TCTGCGAU
-n, --nick
Specify nicking site - must the done together with
the -r option.
Nb.BbvCI : CCTCA.GC
Nb.BsmI : GAATG.CT
Nb.BsrDI : GCAATG.GG
Nb.BtsI : GCAGTG.CT
Nt.AlwI : C.TCTCGATCC
Nt.BbvCI : GCTGA.GG
Nt.BsmAI : C.AGAGAC
Nt.BspQI : .AGAAGAGC
Nt.BstNBI : T.TAGTCTGAT
-r, --restrict
Specify the respriction site - must be done together
with the -n option.
AsiSI : GCGAT.CGC
PacI : TTAAT.TAA
PmeI : GTTT.AAAC
SwaI : ATTT.AAAT
XbaI : T.CTAGA
MssI : GTTT.AAAC
RgaI : GCGAT.CGC
SgfI : GCGAT.CGC
SmiI : ATTT.AAAT
SrfI : GCCC.GGGC
--cassette_fwd
User defined cassette: FORWARD strand - must be used
together with the --cassette_rev option.
Example: GCTGAGGGTTTAAT.TAAGACC.TCAGC
--cassette_rev
User defined cassette: REVERSE strand - must be used
togehter with the --cassette_fwd option.
Example: CGACT.CCCAAAT.TAATTCTGGAGTCG
--fasta
Also export primer sequences in FASTA format to the
specified file.
--report
Also export the analysis report to the specified file.
-l, --maxlength
Fragment input sequences longer that the specied treshhold
into sub-sequences before running the main algorithm.
The aim is to automatically avoid overly long PCR products.
DEFAULT: 0 (disabled).
-h, --help
Print this help page and exit
KNOWN ISSUES
The program operates within the physical constraints of USER
fusion primers. Theoretically, the program can design primers
for fusion of as many DNA fragments in one reaction, as long
as unique fusion tails exists. However, experimental results
suggests that no more than 6-7 fragments can be fused in one
reaction.
REFERENCE
(Submitted to Nucleic Acids Research, Jan 2011).
WEB
http://www.cbs.dtu.dk/services/PHUSER
The webpage contains detailed instructions and examples.
The most recent version of this program is downloadable
from this web address.
AUTHORS
Lars Ronn Olsen, lronn@bio.dtu.dk (PERL part - main prog).
Rasmus Wernerson, raz@cbs.dtu.dk (Python & Web).
Dec 2009 - Dec 2010
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