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

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

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

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