Asteraceae multi-omics information resource
——A multi-omics data platform for Asteraceae plants genetics and breeding research

Primer3 (Pick primers from a DNA sequence)

Select the Task for primer selection

Template masking before primer design
Primer failure rate cutoff<
Nucleotides to mask in 5' direction
Nucleotides to mask in 3' direction

Select the input mode:
Select the input genome:

You can enter a single or multiple LYG gene ID, Arabidopsis name or Arabidopsis gene ID (separated by ',').
Select the flanking region:

e.g. AlaNbV1_Chr01g0013571, AT1G03050, PICALM5A, Phosphatidylinositol binding clathrin assembly protein 5a

You can type a chromosome region, like this chr:start..end

e.g. Chr01:150000..151000

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)

e.g. example_1 or example_2

+ More config
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 IdA string to identify your output.
TargetsE.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 ListE.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 RegionsE.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 ListSee manual for help.
Included RegionE.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 StartForce Right Primer Start
Force Left Primer EndForce Right Primer End

Sequence Quality

Min Sequence QualityMin End Sequence QualitySequence Quality Range MinSequence Quality Range Max

General Primer Picking Conditions

Upload the settings from a file

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

Product Size Ranges
Number To ReturnMax 3' Stability
Max Library MisprimingPair Max Library Mispriming

Thermodynamic Secondary Structure Alignments
Use Thermodynamic Oligo Alignment
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 Self Complementarity
Max 3' Self Complementarity
Max Pair Complementarity
Max 3' Pair Complementarity

Thermodynamic Template Alignments
Use Thermodynamic Template Alignment
TH: Max Template Mispriming
TH: Pair Max Template Mispriming
Old Template Alignments
Max Template Mispriming
Pair Max Template Mispriming

Max #N's acceptedMax Poly-X
Inside Target PenaltyOutside Target PenaltyNote: you can set Inside Target Penalty to allow primers inside a target.
First Base IndexCG Clamp
Max GC in primer 3' end
3' End Distance Between Left Primers3' End Distance Between Right Primers
5 Prime Junction Overlap3 Prime Junction Overlap(Distance of the primer ends to one overlap position.)
Concentration of Monovalent CationsSalt Correction Formula
Concentration of Divalent CationsConcentration of dNTPs
Annealing Oligo Concentration(Not the concentration of oligos in the reaction mix but of those annealing to template.)
Sequencing SpacingSequencing Interval
Sequencing LeadSequencing 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

SizeLtGt
TmLtGt
GC%LtGt

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
Primer failure rate

Objective Function Penalty Weights for Primer Pairs

Product SizeLtGt
Product TmLtGt

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 SizeMin Opt Max
Internal Oligo TmMin Opt Max
Internal Oligo GC%Min Opt Max

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

Internal Oligo Self ComplementarityInternal Oligo Max 3' End Self Complementarity

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

Objective Function Penalty Weights for Internal Oligos (Hyb Oligos)

Internal Oligo SizeLtGt
Internal Oligo TmLtGt
Internal Oligo GC%LtGt

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