Primer3 Input (version 0.4.0)

Primer3 (v. 0.4.0) Pick primers from a DNA sequence.	Checks for mispriming in template.	disclaimer	Primer3 Home
Primer3 (v. 0.4.0) Pick primers from a DNA sequence.	Primer3plus interface	cautions	FAQ/WIKI

There is a newer version of Primer3 available at http://primer3.ut.ee

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.
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.

Product Size Ranges

Number To Return		Max 3' Stability
Max Repeat Mispriming		Pair Max Repeat Mispriming
Max Template Mispriming		Pair Max Template Mispriming

General Primer Picking Conditions

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:

Max Self Complementarity:	Max 3' Self Complementarity:
Max #N's:	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:
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.)

Liberal Base

Show Debuging Info

Do not treat ambiguity codes in libraries as consensus

Lowercase masking

Other Per-Sequence Inputs

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:

Sequence Quality

Min Sequence Quality:

Min End Sequence Quality:

Sequence Quality Range Min:

Sequence Quality Range Max:

Objective Function Penalty Weights for Primers

Tm	Lt:	Gt:
Size	Lt:	Gt:
GC%	Lt:	Gt:

Self Complementarity
3' Self Complementarity
#N's
Mispriming
Sequence Quality
End Sequence Quality
Position Penalty
End Stability
Template Mispriming

Objective Function Penalty Weights for Primer Pairs

Product Size	Lt:		Gt:
Product Tm	Lt:		Gt:

Tm Difference
Any Complementarity
3' Complementarity
Pair Mispriming
Primer Penalty Weight
Hyb Oligo Penalty Weight
Primer Pair Template Mispriming Weight

Hyb Oligo (Internal Oligo) Per-Sequence Inputs

Hyb Oligo Excluded Region:

Hyb Oligo (Internal Oligo) General Conditions

Hyb Oligo Size:	Min	Opt	Max
Hyb Oligo Tm:	Min	Opt	Max
Hyb Oligo GC%	Min:	Opt:	Max:

Hyb Oligo Self Complementarity:				Hyb Oligo Max 3' Self Complementarity:
Max #Ns:				Hyb Oligo Max Poly-X:
Hyb Oligo Mishyb Library:				Hyb Oligo Max Mishyb:
Hyb Oligo Min Sequence Quality:
Hyb Oligo Conc of monovalent cations:				Hyb Oligo DNA Concentration:
Hyb Oligo conc of divalent cations:				Hyb Oligo [dNTP]

Objective Function Penalty Weights for Hyb Oligos (Internal Oligos)

Hyb Oligo Tm	Lt:	Gt:
Hyb Oligo Size	Lt:	Gt:
Hyb Oligo GC%	Lt:	Gt:

Hyb Oligo Self Complementarity
Hyb Oligo #N's
Hyb Oligo Mishybing
Hyb Oligo Sequence Quality

Copyright Notice and Disclaimer

Redistribution and use in source and binary forms, with or without
modification, are permitted provided that the following conditions are
met:

   * Redistributions of source code must retain the above copyright
notice, this list of conditions and the following disclaimer.
   * Redistributions in binary form must reproduce the above
copyright notice, this list of conditions and the following disclaimer
in the documentation and/or other materials provided with the
distribution.
   * Neither the names of the copyright holders nor contributors may
be used to endorse or promote products derived from this software
without specific prior written permission.

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

Untergasser 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 primer3.sourceforge.net/.

Acknowledgments

The development of Primer3 and the Primer3 web site 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).

We thank Centerline Software, Inc., for use of their TestCenter memory-error, -leak, and test-coverage checker.

Primer3 was a complete re-implementation of an earlier program: Primer 0.5 (Steve Lincoln, Mark Daly, and Eric S. Lander). Lincoln Stein championed the idea of making Primer3 a software component suitable for high-throughput primer design.
Web interface by Steve Rozen

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