Primer3web  version 4.0.0  -   Pick primers from a DNA sequence. disclaimer code
cautions

Select the Task for primer selection

Paste source sequence below (5'->3', string of ACGTNacgtn -- other letters treated as N -- numbers and blanks ignored). FASTA format ok. Please N-out undesirable sequence (vector, ALUs, LINEs, etc.) or use a Mispriming Library (repeat library)

Pick left primer, or use left primer below Pick hybridization probe (internal oligo), or use oligo below Pick right primer, or use right primer below (5' to 3' on opposite strand)

Sequence Id A string to identify your output.
Targets E.g. 50,2 requires primers to surround the 2 bases at positions 50 and 51. Or mark the source sequence with [ and ]: e.g. ...ATCT[CCCC]TCAT.. means that primers must flank the central CCCC.
Overlap Junction List E.g. 27 requires one primer to overlap the junction between positions 27 and 28. Or mark the source sequence with -: e.g. ...ATCTAC-TGTCAT.. means that primers must overlap the junction between the C and T.
Excluded Regions E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the source sequence with < and >: e.g. ...ATCT<CCCC>TCAT.. forbids primers in the central CCCC.
Pair OK Region List See manual for help.
Included Region E.g. 20,400: only pick primers in the 400 base region starting at position 20. Or use { and } in the source sequence to mark the beginning and end of the included region: e.g. in ATC{TTC...TCT}AT the included region is TTC...TCT.
Start Codon Position
Internal Oligo Excluded Region
Force Left Primer Start Force Right Primer Start
Force Left Primer End Force Right Primer End

Sequence Quality

Min Sequence Quality Min End Sequence Quality Sequence Quality Range Min Sequence Quality Range Max

General Primer Picking Conditions

Upload the settings from a file

Primer Size Min Opt Max
Primer Tm Min Opt Max Max Tm Difference Table of thermodynamic parameters
Product Tm Min Opt Max
Primer GC% Min Opt Max

Product Size Ranges
Number To Return Max 3' Stability
Max Library Mispriming Pair Max Library Mispriming

Thermodynamic Secondary Structure Alignments
Use Thermodynamic Oligo Alignment Use Thermodynamic Template Alignment
TH: Max Template Mispriming TH: Pair Max Template Mispriming
TH: Max Self Complementarity TH: Max 3' Self Complementarity
TH: Max Pair Complementarity TH: Max 3' Pair Complementarity
TH: Max Primer Hairpin

Old Secondary Structure Alignments
Max Template Mispriming Pair Max Template Mispriming
Max Self Complementarity Max 3' Self Complementarity
Max Pair Complementarity Max 3' Pair Complementarity

Max #N's accepted Max Poly-X
Inside Target Penalty Outside Target Penalty Note: you can set Inside Target Penalty to allow primers inside a target.
First Base Index CG Clamp
Max GC in primer 3' end
3' End Distance Between Left Primers 3' End Distance Between Right Primers
5 Prime Junction Overlap 3 Prime Junction Overlap (Distance of the primer ends to one overlap position.)
Concentration of Monovalent Cations Salt Correction Formula
Concentration of Divalent Cations Concentration of dNTPs
Annealing Oligo Concentration (Not the concentration of oligos in the reaction mix but of those annealing to template.)
Sequencing Spacing Sequencing Interval
Sequencing Lead Sequencing Accuracy
Liberal Base Show Debuging Info Treat ambiguity codes in libraries as consensus
Lowercase masking Pick anyway Print Statistics

Objective Function Penalty Weights for Primers

Size Lt Gt
Tm Lt Gt
GC% Lt Gt

TH: Self Complementarity
TH: 3' End Self Complementarity
TH: Hairpin
TH: Template Mispriming

Self Complementarity
3' End Self Complementarity
Template Mispriming

#N's
Library Mispriming
Sequence Quality
End Sequence Quality
Position Penalty
End Stability

Objective Function Penalty Weights for Primer Pairs

Product Size Lt Gt
Product Tm Lt Gt

TH: Any Complementarity
TH: 3' End Complementarity
TH: Template Mispriming Weight

Any Complementarity
3' End Complementarity
Template Mispriming Weight

Tm Difference
Library Mispriming
Primer Penalty Weight
Internal Oligo Penalty Weight

Internal Oligo (Hyb Oligo) General Conditions

Internal Oligo Size Min Opt Max
Internal Oligo Tm Min Opt Max
Internal Oligo GC% Min Opt Max

TH: Internal Oligo Self Complementarity TH: Internal Oligo Max 3' End Self Complementarity
TH: Internal Oligo Hairpin

Internal Oligo Self Complementarity Internal Oligo Max 3' End Self Complementarity

Internal Oligo Min Sequence Quality
Max #Ns Internal Oligo Max Poly-X
Internal Oligo Mishyb Library Internal Oligo Max Library Mishyb
Internal Oligo Conc of monovalent cations Internal Oligo DNA Concentration
Internal Oligo conc of divalent cations Internal Oligo [dNTP]

Objective Function Penalty Weights for Internal Oligos (Hyb Oligos)

Internal Oligo Size Lt Gt
Internal Oligo Tm Lt Gt
Internal Oligo GC% Lt Gt

TH: Internal Oligo Self Complementarity
TH: Internal Oligo 3' End Complementarity
TH: Internal Oligo Hairpin

Internal Oligo Self Complementarity
Internal Oligo 3' End Complementarity

Internal Oligo #N's
Internal Oligo Library Mishybing
Internal Oligo Sequence Quality
Internal Oligo Sequence End Quality

Copyright Notice and Disclaimer

Copyright (c) 1996,1997,1998,1999,2000,2001,2004,2006,2007,2008,2009,2010,2011,2012,2013
Whitehead Institute for Biomedical Research, Steve Rozen, Andreas Untergasser, Maido Remm, Triinu Koressaar and Helen Skaletsky.
All rights reserved.

    This file is part of the primer3web suite.

    The primer3 suite and libraries are free software;
    you can redistribute them and/or modify them under the terms
    of the GNU General Public License as published by the Free
    Software Foundation; either version 2 of the License, or (at
    your option) any later version.

    This software is distributed in the hope that it will be useful,
    but WITHOUT ANY WARRANTY; without even the implied warranty of
    MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
    GNU General Public License for more details.

    You should have received a copy of the GNU General Public License
    along with this software (file gpl-2.0.txt in the source
    distribution); if not, write to the Free Software
    Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA

THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS
"AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT
LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR
A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT
OWNERS OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL,
SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT
LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE,
DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY
THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT
(INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE
OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.

Citing Primer3

We request that use of this software be cited in publications as

Untergrasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3 - new capabilities and interfaces. Nucleic Acids Research 40(15):e115
Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3 Bioinformatics 23(10):1289-91

Source code available at http://sourceforge.net/projects/primer3/.

Acknowledgments

Initial development of Primer3 was funded by Howard Hughes Medical Institute and by the National Institutes of Health, National Human Genome Research Institute under grants R01-HG00257 (to David C. Page) and P50-HG00098 (to Eric S. Lander). Ongoing development and maintenance are partly funded by Centre of Excellence in Genomics at Estonian Biocentre (funding from EU European Regional Development Fund).

Primer3 was a complete re-implementation of an earlier program: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander).


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