Mandatory qualifiers:  [-sequence]          seqall     The sequence from which to choose primers.                                  The sequence must be presented 5' to 3'  [-outfile]           outfile    Output file name   Optional qualifiers (* if not always prompted):   -task               list       Tell Primer3 what task to perform. Legal                                  values are 0: 'Pick PCR primers', 1: 'Pick                                  PCR primers and hybridization probe', 2:                                  'Pick forward primer only', 3: 'Pick reverse                                  primer only', 4: 'Pick hybridization probe                                  only'.                                  The tasks should be self explanatory.                                  Briefly, an 'internal oligo' is intended to                                  be used as a hybridization probe (hyb probe)                                  to detect the PCR product after                                  amplification.   -numreturn          integer    The maximum number of primer pairs to                                  return. Primer pairs returned are sorted by                                  their 'quality', in other words by the value                                  of the objective function (where a lower                                  number indicates a better primer pair).                                  Caution: setting this parameter to a large                                  value will increase running time.   -includedregion     range      A sub-region of the given sequence in which                                  to pick primers. For example, often the                                  first dozen or so bases of a sequence are                                  vector, and should be excluded from                                  consideration. The value for this parameter                                  has the form                                  (start),(end)                                  where (start) is the index of the first base                                  to consider, and (end) is the last in the                                  primer-picking region.   -target             range      If one or more Targets is specified then a                                  legal primer pair must flank at least one of                                  them. A Target might be a simple sequence                                  repeat site (for example a CA repeat) or a                                  single-base-pair polymorphism. The value                                  should be a space-separated list of                                  (start),(end)                                  pairs where (start) is the index of the                                  first base of a Target, and (end) is the                                  last                                  E.g. 50,51 requires primers to surround the                                  2 bases at positions 50 and 51.   -excludedregion     range      Primer oligos may not overlap any region                                  specified in this tag. The associated value                                  must be a space-separated list of                                  (start),(end)                                  pairs where (start) is the index of the                                  first base of the excluded region, and and                                  (end) is the last. This tag is useful for                                  tasks such as excluding regions of low                                  sequence quality or for excluding regions                                  containing repetitive elements such as ALUs                                  or LINEs.                                  E.g. 401,407 68,70 forbids selection of                                  primers in the 7 bases starting at 401 and                                  the 3 bases at 68.   -forwardinput       string     The sequence of a forward primer to check                                  and around which to design reverse primers                                  and optional internal oligos. Must be a                                  substring of SEQUENCE.   -reverseinput       string     The sequence of a reverse primer to check                                  and around which to design forward primers                                  and optional internal oligos. Must be a                                  substring of the reverse strand of SEQUENCE.*  -gcclamp            integer    Require the specified number of consecutive                                  Gs and Cs at the 3' end of both the forward                                  and reverse primer. (This parameter has no                                  effect on the internal oligo if one is                                  requested.)*  -osize              integer    Optimum length (in bases) of a primer oligo.                                  Primer3 will attempt to pick primers close                                  to this length.*  -minsize            integer    Minimum acceptable length of a primer. Must                                  be greater than 0 and less than or equal to                                  MAX-SIZE.*  -maxsize            integer    Maximum acceptable length (in bases) of a                                  primer. Currently this parameter cannot be                                  larger than 35. This limit is governed by                                  the maximum oligo size for which Primer3's                                  melting-temperature is valid.*  -otm                float      Optimum melting temperature(Celsius) for a                                  primer oligo. Primer3 will try to pick                                  primers with melting temperatures are close                                  to this temperature. The oligo melting                                  temperature formula in Primer3 is that given                                  in Rychlik, Spencer and Rhoads, Nucleic                                  Acids Research, vol 18, num 12, pp 6409-6412                                  and Breslauer, Frank, Bloeker and Marky,                                  Proc. Natl. Acad. Sci. USA, vol 83, pp                                  3746-3750. Please refer to the former paper                                  for background discussion.*  -mintm              float      Minimum acceptable melting                                  temperature(Celsius) for a primer oligo.*  -maxtm              float      Maximum acceptable melting                                  temperature(Celsius) for a primer oligo.*  -maxdifftm          float      Maximum acceptable (unsigned) difference                                  between the melting temperatures of the                                  forward and reverse primers.*  -ogcpercent         float      Primer optimum GC percent.*  -mingc              float      Minimum allowable percentage of Gs and Cs in                                  any primer.*  -maxgc              float      Maximum allowable percentage of Gs and Cs in                                  any primer generated by Primer.*  -saltconc           float      The millimolar concentration of salt                                  (usually KCl) in the PCR. Primer3 uses this                                  argument to calculate oligo melting                                  temperatures.*  -dnaconc            float      The nanomolar concentration of annealing                                  oligos in the PCR. Primer3 uses this                                  argument to calculate oligo melting                                  temperatures. The default (50nM) works well                                  with the standard protocol used at the                                  Whitehead/MIT Center for Genome                                  Research--0.5 microliters of 20 micromolar                                  concentration for each primer oligo in a 20                                  microliter reaction with 10 nanograms                                  template, 0.025 units/microliter Taq                                  polymerase in 0.1 mM each dNTP, 1.5mM MgCl2,                                  50mM KCl, 10mM Tris-HCL (pH 9.3) using 35                                  cycles with an annealing temperature of 56                                  degrees Celsius. This parameter corresponds                                  to 'c' in Rychlik, Spencer and Rhoads'                                  equation (ii) (Nucleic Acids Research, vol                                  18, num 12) where a suitable value (for a                                  lower initial concentration of template) is                                  'empirically determined'. The value of this                                  parameter is less than the actual                                  concentration of oligos in the reaction                                  because it is the concentration of annealing                                  oligos, which in turn depends on the amount                                  of template (including PCR product) in a                                  given cycle. This concentration increases a                                  great deal during a PCR; fortunately PCR                                  seems quite robust for a variety of oligo                                  melting temperatures.                                  See ADVICE FOR PICKING PRIMERS.*  -maxpolyx           integer    The maximum allowable length of a                                  mononucleotide repeat in a primer, for                                  example AAAAAA.*  -productosize       integer    The optimum size for the PCR product. 0                                  indicates that there is no optimum product                                  size.*  -productsizerange   range      The associated values specify the lengths of                                  the product that the user wants the primers                                  to create, and is a space separated list of                                  elements of the form                                  (x)-(y)                                  where an (x)-(y) pair is a legal range of                                  lengths for the product. For example, if one                                  wants PCR products to be between 100 to 150                                  bases (inclusive) then one would set this                                  parameter to 100-150. If one desires PCR                                  products in either the range from 100 to 150                                  bases or in the range from 200 to 250 bases                                  then one would set this parameter to                                  100-150 200-250.                                  Primer3 favors ranges to the left side of                                  the parameter string. Primer3 will return                                  legal primers pairs in the first range                                  regardless the value of the objective                                  function for these pairs. Only if there are                                  an insufficient number of primers in the                                  first range will Primer3 return primers in a                                  subsequent range.*  -productotm         float      The optimum melting temperature for the PCR                                  product. 0 indicates that there is no                                  optimum temperature.*  -productmintm       float      The minimum allowed melting temperature of