@possumtech/rummy 2.0.1 → 2.2.0

package/audit/sweep/dna-insert/turns/turn_004.md

@@ -0,0 +1,2214 @@
+# turn 004
+
+tokens: prompt=6433 cached=1749 completion=595 reasoning=0 total=7028 cost=0
+created: 2026-05-02 00:22:17
+
+update: status=155 body=distilling primer3 oligotm + discovering Q5 kit protocol/sequences.fasta
+
+---
+
+## reasoning (think)
+
+```
+* In Distillation Stage: resolve unknowns by discovering sources (search results, repo) and distilling into known:// entries.
+* Key unknowns: primer3_oligotm flags/usage (search has good hits like manpage 1352t, primer3 manual 101k t), Q5 kit details (no sources yet), sequences.fasta content (in repo? empty app/), primer design rules (can distill from prompt), output format.
+* Plan: 
+  1. Promote low-cost primer3 manpage (1352t < free) + 1-2 more for oligotm details to distill exact usage/flags.
+  2. Search for Q5 kit protocol (1 search/turn).
+  3. Manifest repo for sequences.fasta or biology files.
+  4. Distill 1-2 known:// from current unknowns + new sources (e.g. known://biology/tools/primer3_oligotm).
+  5. Demote nothing yet to save budget.
+* tokensFree 1.7M+ ample; continue 155.
+```
+
+## assistant emission
+
+```
+<think>
+* In Distillation Stage: resolve unknowns by discovering sources (search results, repo) and distilling into known:// entries.
+* Key unknowns: primer3_oligotm flags/usage (search has good hits like manpage 1352t, primer3 manual 101k t), Q5 kit details (no sources yet), sequences.fasta content (in repo? empty app/), primer design rules (can distill from prompt), output format.
+* Plan: 
+  1. Promote low-cost primer3 manpage (1352t < free) + 1-2 more for oligotm details to distill exact usage/flags.
+  2. Search for Q5 kit protocol (1 search/turn).
+  3. Manifest repo for sequences.fasta or biology files.
+  4. Distill 1-2 known:// from current unknowns + new sources (e.g. known://biology/tools/primer3_oligotm).
+  5. Demote nothing yet to save budget.
+* tokensFree 1.7M+ ample; continue 155.
+</think>
+
+<get path="https://manpages.debian.org/stretch/primer3/oligotm.1"/>
+<get path="https://primer3.org/manual.html" line="1" limit="2000"/>
+<get path="**" manifest>sequences.fasta</get>
+<get path="**" manifest>Q5</get>
+<search>neb q5 site-directed mutagenesis kit protocol circular plasmid primers minimal pairs</search>
+
+<set path="known://biology/tools/primer3_oligotm" visibility="visible" summary="biology,tools,primer3,oligotm,usage,flags,tm,ground_truth">
+# Related
+[prompt rules](prompt://1)
+[unknown resolved](unknown://biology/tools/primer3_oligotm)
+[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)
+
+# Primer3 oligotm exact usage and flags
+Standalone tool prints melting temperature (Tm) of deoxyribonucleotide sequence to stdout.
+
+**Flags from rules (ground truth):**
+- `-tp 1`: Temperature type? (pending manpage details)
+- `-sc 1`: Salt correction?
+- `-mv 50`: Monovalent cations 50 mM
+- `-dv 2`: Divalent cations 2 mM
+- `-n 0.8`: ? (pending)
+- `-d 500`: ? (pending, DNA conc?)
+
+Tm computed *only* on annealing part of primer (15-45nt to input).
+</set>
+
+<update status="155">distilling primer3 oligotm + discovering Q5 kit protocol/sequences.fasta</update>
+```
+
+## actions (chronological log entries)
+
+### `log://turn_4/get/https%3A/manpages.debian.org/stretch/primer3/oligotm.1`
+scheme=log action=get attrs={"path":"https://manpages.debian.org/stretch/primer3/oligotm.1","action":"get"}
+
+```
+https://manpages.debian.org/stretch/primer3/oligotm.1 promoted
+```
+
+### `log://turn_4/get/https%3A/primer3.org/manual.html`
+scheme=log action=get attrs={"path":"https://primer3.org/manual.html","lineStart":1,"lineEnd":2000,"totalLines":4371,"action":"get"}
+
+```
+https://primer3.org/manual.html
+[lines 1–2000 / 4371 total]
+# Primer3 - Manual
+
+## CONTENTS
+
+[1\. COPYRIGHT AND LICENSE](#copyrightLicense) [2\. INTRODUCTION](#introduction)
+[3\. CITING PRIMER3](#citationRequest)
+[4\. FAIR USE OF PRIMER3](#licenseExplain)
+[5\. DIFFERENCE BETWEEN PRIMER3 AND PRIMER3PLUS](#differencePrimer3vsPrimer3Plus)
+[6\. CHANGES FROM VERSION 2.2.3](#changesFrom2.2.3)
+[7\. INSTALLATION INSTRUCTIONS - UNIX/LINUX](#installLinux)
+[8\. INSTALLATION INSTRUCTIONS - Mac OSX](#installMac)
+[9\. INSTALLATION INSTRUCTIONS - WINDOWS](#installWindows)
+[10\. SYSTEM REQUIREMENTS](#systemRequirements)
+[11\. INVOKING primer3\_core](#invokingPrimer3)
+[12\. COMMAND LINE ARGUMENTS](#commandLineTags)
+[13\. INPUT AND OUTPUT CONVENTIONS](#inputOutputConventions)
+[14\. AN EXAMPLE](#example)
+[15\. HOW TO MIGRATE TAGS TO IO VERSION 4](#migrateTags)
+[16\. "SEQUENCE" INPUT TAGS](#sequenceTags)
+[17\. "GLOBAL" INPUT TAGS](#globalTags)
+[18\. "PROGRAM" INPUT TAGS](#programTags)
+[19\. HOW PRIMER3 CALCULATES THE PENALTY VALUE](#calculatePenalties)
+[20\. PRIMER3 SETTINGS FILE FORMAT](#fileFormat) [21\. OUTPUT TAGS](#outputTags)
+[22\. EXAMPLE OUTPUT](#exampleOutput)
+[23\. ADVICE FOR PICKING PRIMERS](#pickAdvice)
+[24\. GENERAL THOUGHTS ON PRIMER BINDING](#primerBinding)
+[25\. CAUTIONS](#cautions)
+[26\. WHAT TO DO IF PRIMER3 CANNOT FIND ANY PRIMERS?](#findNoPrimers)
+[27\. DIFFERENCES FROM EARLIER VERSIONS](#earlierVersions)
+[28\. EXIT STATUS CODES](#exitStatusCodes)
+[29\. PRIMER3 WWW INTERFACES](#webInterface)
+[30\. ACKNOWLEDGMENTS](#acknowledgments)
+
+## 1\. COPYRIGHT AND LICENSE
+
+Copyright (c) 1996 - 2022
+Whitehead Institute for Biomedical Research, Steve Rozen
+([http://purl.com/STEVEROZEN/](http://purl.com/STEVEROZEN/)), Helen Skaletsky, Triinu Koressaar,
+Maido Remm and Andreas Untergasser. All rights reserved.
+    This file is part of the Primer3 suite and libraries.
+    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.
+
+## 2\. INTRODUCTION
+
+Primer3 picks primers for PCR reactions, considering as criteria: o
+oligonucleotide melting temperature, size, GC content, and primer-dimer
+possibilities,
+
+o PCR product size,
+
+o positional constraints within the source (template) sequence, and
+
+o possibilities for ectopic priming (amplifying the wrong sequence)
+
+o many other constraints.
+
+All of these criteria are user-specifiable as constraints, and some are
+specifiable as terms in an objective function that characterizes an optimal
+primer pair.
+
+## 3\. CITING PRIMER3
+
+We request but do not require that use of this software be cited in publications
+as
+
+\* Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M and
+Rozen SG. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012 Aug
+1;40(15):e115. The paper is available at
+[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424584/](http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424584/)
+
+and
+
+\* Koressaar T and Remm M. Enhancements and modifications of primer design
+program Primer3. Bioinformatics 2007;23(10):1289-1291. The paper is available at
+[https://www.ncbi.nlm.nih.gov/pubmed/17379693](https://www.ncbi.nlm.nih.gov/pubmed/17379693)
+
+If you use masker function, please cite: \* Koressaar T, Lepamets M, Kaplinski
+L, Raime K, Andreson R and Remm M. Primer3\_masker: integrating masking of
+template sequence with primer design software. Bioinformatics
+2018;34(11):1937-1938. The paper is available at
+[https://www.ncbi.nlm.nih.gov/pubmed/29360956](https://www.ncbi.nlm.nih.gov/pubmed/29360956)
+
+Source code available at
+[https://github.com/primer3-org/primer3](https://github.com/primer3-org/primer3)
+.
+
+## 4\. FAIR USE OF PRIMER3
+
+The development of Primer3 is promoted by a small group of enthusiastic
+scientists mainly in their free time.They do not gain any financial profit with
+Primer3.
+
+There are two groups of Primer3 users: end users, who run Primer3 to pick their
+primers and programmers, who use Primer3 in their scripts or software packages.
+We encourage both to use Primer3.
+
+If you are an end user, we request but do not require that use of this software
+be cited in publications as listed above under CITING PRIMER3.
+
+If you are a programmer, you will see that Primer3 is now distributed under the
+GNU General Public License, version 2 or (at your option) any later version of
+the License (GPL2). As we understand it, if you include parts of the Primer3
+source code in your source code or link to Primer3 binary libraries in your
+executable, you have to release your software also under GPL2. If you only call
+Primer3 from your software and interpret its output, you can use any license you
+want for your software. If you modify Primer3 and then release your modified
+software, you have to release your modifications in source code under GPL2 as
+well.
+
+We chose GPL2 because we wanted Primer3 to evolve and for the improvements to
+find their way back into the main distribution. If you are programming a new web
+interface which runs Primer3, please include in the about page of the tool the
+sentence "<your software name> uses Primer3 version ...". Please consider
+releasing your software under GPL2 as well, especially if you do not want to
+maintain it in the future.
+
+There is no need to ask us for permission to include Primer3 in your tools.
+
+## 5\. DIFFERENCE BETWEEN PRIMER3 AND PRIMER3PLUS
+
+Primer3Plus is a web interface to Primer3, so if you pick primers with
+Primer3Plus, it will collect and reformat your input, run the command line tool
+Primer3, collet and reformat it's output and display it to you. In principle,
+both tools would give you the same output. In practice, the default settings ob
+both tools differ. While Primer3 default settings are usually kept for backward
+compatibility, the Primer3Plus default settings are adapted for regular wetlab
+use.
+
+#### Primer3 Values:
+
+  PRIMER\_PRODUCT\_SIZE\_RANGE=100-300
+  PRIMER\_SECONDARY\_STRUCTURE\_ALIGNMENT=0
+  PRIMER\_NUM\_RETURN=5
+  PRIMER\_MAX\_HAIRPIN\_TH=47.0
+  PRIMER\_INTERNAL\_MAX\_HAIRPIN\_TH=47.0
+  PRIMER\_MAX\_END\_STABILITY=100.0
+  PRIMER\_MIN\_LEFT\_THREE\_PRIME\_DISTANCE=-1
+  PRIMER\_MIN\_RIGHT\_THREE\_PRIME\_DISTANCE=-1
+  PRIMER\_EXPLAIN\_FLAG=0
+  PRIMER\_LIBERAL\_BASE=0
+  PRIMER\_FIRST\_BASE\_INDEX=0
+  PRIMER\_MAX\_TEMPLATE\_MISPRIMING=-1.00
+  PRIMER\_MAX\_TEMPLATE\_MISPRIMING\_TH=-1.00
+  PRIMER\_PAIR\_MAX\_TEMPLATE\_MISPRIMING=-1.00
+  PRIMER\_PAIR\_MAX\_TEMPLATE\_MISPRIMING\_TH=-1.00
+
+#### Primer3Plus Values:
+
+  PRIMER\_PRODUCT\_SIZE\_RANGE=501-600 601-700 401-500 701-850 851-1000 1001-1500
+                            1501-3000 3001-5000 401-500 301-400 201-300
+                            101-200 5001-7000 7001-10000 10001-20000
+  PRIMER\_SECONDARY\_STRUCTURE\_ALIGNMENT=1
+  PRIMER\_NUM\_RETURN=10
+  PRIMER\_MAX\_HAIRPIN\_TH=47.00
+  PRIMER\_INTERNAL\_MAX\_HAIRPIN\_TH=47.00
+  PRIMER\_MAX\_END\_STABILITY=9.0
+  PRIMER\_MIN\_LEFT\_THREE\_PRIME\_DISTANCE=3
+  PRIMER\_MIN\_RIGHT\_THREE\_PRIME\_DISTANCE=3
+  PRIMER\_EXPLAIN\_FLAG=1
+  PRIMER\_LIBERAL\_BASE=1
+  PRIMER\_FIRST\_BASE\_INDEX=1
+  PRIMER\_MAX\_TEMPLATE\_MISPRIMING=12.00
+  PRIMER\_MAX\_TEMPLATE\_MISPRIMING\_TH=47.00
+  PRIMER\_PAIR\_MAX\_TEMPLATE\_MISPRIMING=24.00
+  PRIMER\_PAIR\_MAX\_TEMPLATE\_MISPRIMING\_TH=47.00
+
+## 6\. CHANGES FROM VERSION 2.2.3
+
+1\. The tag PRIMER\_THERMODYNAMIC\_ALIGNMENT was replaced by two new tags:
+
+\-
+[PRIMER\_THERMODYNAMIC\_OLIGO\_ALIGNMENT](#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT)
+which governs hairpin and oligo-oligo interactions. Therefore, whenever it is
+set to 1 (the default) thermodynamic alignments will be used for oligo-oligo
+interactions and hairpins.
+
+\-
+[PRIMER\_THERMODYNAMIC\_TEMPLATE\_ALIGNMENT](#PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT)
+which governs the oligo-template interactions and, when set to 1 (default is 0),
+will cause Primer3 to use the thermodynamic alignment against templates.
+
+The reason for this change is to provide the option of using the old alignment
+for oligo-template interactions when the thermodynamic alignment is too slow or
+the template is too long (there is a hard limit THAL\_MAX\_SEQ=10k on the length
+of sequences involved in thermodynamic alignments).
+
+2\. We changed the following default values:
+
+2.1. Changing default oligo temperature calculations
+
+[PRIMER\_TM\_FORMULA](#PRIMER_TM_FORMULA) \=1 (was 0)
+[PRIMER\_SALT\_CORRECTIONS](#PRIMER_SALT_CORRECTIONS) \=1 (was 0)
+
+2.2. Making thermodynamic secondary structure calculations for oligos the
+default:
+
+[PRIMER\_THERMODYNAMIC\_OLIGO\_ALIGNMENT](#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT)
+\=1 (was 0)
+
+2.3. The following need reasonable values to support the changes in 2.1 and 2.2
+above:
+
+[PRIMER\_SALT\_DIVALENT](#PRIMER_SALT_DIVALENT) \=1.5 (was 0.0)
+[PRIMER\_DNTP\_CONC](#PRIMER_DNTP_CONC) \=0.6 (was 0.0)
+
+2.4. To make old defaults easily accessible, we added a command line argument,
+--default\_version=1, which directs primer3\_core to use the old defaults.
+--default\_version=2 directs primer3\_core to use the new defaults. The default
+is --default\_version=2.
+
+2.5. IMPORTANT: because
+[PRIMER\_THERMODYNAMIC\_OLIGO\_ALIGNMENT](#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT)
+\=1,
+[PRIMER\_THERMODYNAMIC\_PARAMETERS\_PATH](#PRIMER_THERMODYNAMIC_PARAMETERS_PATH)
+must point to the right location. This tag specifies the path to the directory
+that contains all the parameter files used by the thermodynamic approach. In
+Linux, there are two \*default\* locations that are tested if this tag is not
+defined: ./primer3\_config/ and /opt/primer3\_config/. For Windows, there is
+only one default location: .\\primer3\_config\\. If the the parameter files are
+not in one these locations, be sure to set
+[PRIMER\_THERMODYNAMIC\_PARAMETERS\_PATH](#PRIMER_THERMODYNAMIC_PARAMETERS_PATH)
+.
+
+2.6. Changed default for
+
+[PRIMER\_LIB\_AMBIGUITY\_CODES\_CONSENSUS](#PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS)
+\=0 (was 1)
+
+(0 is almost always the the behavior one wants.)
+
+2.7. To get the behavior of -default\_version=1 when -default\_version=2 set the
+following:
+
+[PRIMER\_TM\_FORMULA](#PRIMER_TM_FORMULA) \=0
+[PRIMER\_SALT\_CORRECTIONS](#PRIMER_SALT_CORRECTIONS) \=0
+[PRIMER\_THERMODYNAMIC\_OLIGO\_ALIGNMENT](#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT)
+\=0
+[PRIMER\_THERMODYNAMIC\_TEMPLATE\_ALIGNMENT](#PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT)
+\=0 [PRIMER\_SALT\_DIVALENT](#PRIMER_SALT_DIVALENT) \=0.0
+[PRIMER\_DNTP\_CONC](#PRIMER_DNTP_CONC) \=0.0
+[PRIMER\_LIB\_AMBIGUITY\_CODES\_CONSENSUS](#PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS)
+\=1
+
+2.8. To get the behavior of -default\_version=2 when -default\_version=1 set the
+following:
+
+[PRIMER\_TM\_FORMULA](#PRIMER_TM_FORMULA) \=1
+[PRIMER\_SALT\_CORRECTIONS](#PRIMER_SALT_CORRECTIONS) \=1
+[PRIMER\_THERMODYNAMIC\_OLIGO\_ALIGNMENT](#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT)
+\=1
+[PRIMER\_THERMODYNAMIC\_TEMPLATE\_ALIGNMENT](#PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT)
+\=0 [PRIMER\_SALT\_DIVALENT](#PRIMER_SALT_DIVALENT) \=1.5
+[PRIMER\_DNTP\_CONC](#PRIMER_DNTP_CONC) \=0.6
+[PRIMER\_LIB\_AMBIGUITY\_CODES\_CONSENSUS](#PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS)
+\=0
+
+3\. We changed the NULL value for SEQUENCE\_FORCE\_{LEFT,RIGHT}\_START\_END to
+-1000000, and made it an error to select
+[PRIMER\_FIRST\_BASE\_INDEX](#PRIMER_FIRST_BASE_INDEX) <= this value. This is to
+correct an error when SEQUENCE\_FORCE\_{LEFT,RIGHT}\_START\_END was -1 (value
+previously used to indicate a NULL) but
+[PRIMER\_FIRST\_BASE\_INDEX](#PRIMER_FIRST_BASE_INDEX) was < 0, which caused the
+intended NULL value (-1) to be treated as a constraint on primer location (a
+constraint that was not possible to satisfy).
+
+4\. We changed the [PRIMER\_TASK](#PRIMER_TASK) called
+'pick\_detection\_primers' to 'generic' while retaining
+'pick\_detection\_primers' as an alias for backward compatibility.
+
+5\. The code now uses 'end' alignments when assessing template mispriming using
+thermodynamic alignments. This is consistent with the approach taken with the
+previous alignment algorithm and with checking for mispriming against repeat
+libraries.
+
+6\. We removed PRIMER\_PAIR\_MAX\_HAIRPIN\_TH (which was ignored previously).
+
+7\. Primer3 now requires the user to set [SEQUENCE\_TARGET](#SEQUENCE_TARGET) ,
+not [SEQUENCE\_INCLUDED\_REGION](#SEQUENCE_INCLUDED_REGION) when
+[PRIMER\_TASK](#PRIMER_TASK) \=pick\_discriminative\_primers
+
+8\. When [PRIMER\_TASK](#PRIMER_TASK) \=pick\_discriminative\_primers or
+[PRIMER\_TASK](#PRIMER_TASK) \=pick\_cloning\_primers the value of
+[SEQUENCE\_INCLUDED\_REGION](#SEQUENCE_INCLUDED_REGION) is no longer changed to
+the entire input sequence.
+
+9\. The handling of divalent cations when
+[PRIMER\_SALT\_CORRECTIONS](#PRIMER_SALT_CORRECTIONS) \=2 (not the default and
+not the recommended value) has been updated. The rationale is that, when
+divalent cations are present, the formula by Owczarzy et al., 2004 (used
+previously by Primer3) can be improved upon as described in (Ahsen et al., 2010;
+Owczarzy et al., 2008). Therefore we have updated the melting temperature
+calculation to follow the scheme in Figure 9 of (Owczarzy et al., 2008). Please
+find references to these papers below in the manual.
+
+10\. We modified the error handling of non-memory related errors that can occur
+during the thermodynamic alignment. They are no longer fatal errors.
+
+11\. We removed io\_version 3, which was only kept for backward compatibility
+with very old versions of Primer3.
+
+12\. In addition, several error corrections:
+
+12.1. Corrected a short, fixed-size buffer for the file/path names specified by
+the command line arguments -output, -error and for the settings file.
+
+12.2. Two other corrected errors were in p3\_set\_gs\_primer\_self\_end and
+p3\_set\_gs\_primer\_internal\_oligo\_self\_end, which erroneously multiplied
+their 'val' arguments by 100.
+
+12.3. Primer3 now detects and handles the situation in which user-supplied
+primers ([SEQUENCE\_PRIMER](#SEQUENCE_PRIMER) and
+[SEQUENCE\_PRIMER\_REVCOMP](#SEQUENCE_PRIMER_REVCOMP) ) have the left primer to
+the right of the right primer. Also issue a warning if user-supplied primers
+occur in more than one location in the template.
+
+## 7\. INSTALLATION INSTRUCTIONS - UNIX/LINUX
+
+Unzip and untar the distribution. DO NOT do this on a PC if you are going to
+compile and/or test on a different operation system. Primer3\_core will not
+compile and various tests will fail if pc newlines get inserted into the code
+and test files. Instead, move the distribution (primer3-<release>.tar.gz) to
+Unix/Linux, and then
+
+$ unzip primer3-<release>.tar.gz $ tar xvf primer3-<release>.tar $ cd
+primer3-<release>/src
+
+If you do not use gcc, then you will probably have to modify the makefile to use
+your (ANSI) C compiler with the compile and link flags it understands.
+
+$ make all
+
+\# You should have created executables primer3\_core, ntdpal, \# olgotm, and
+long\_seq\_tm\_test
+
+$ make test
+
+\# You should not see 'FAILED' during the tests.
+
+If your perl command is not called perl (for example, if it is called perl5) you
+will have to modify the Makefile in the test/ directory.
+
+ntdpal (NucleoTide Dynamic Programming ALignment) is a stand-alone program that
+provides Primer3's alignment functionality (local, a.k.a. Smith-Waterman,
+global, a.k.a. Needleman-Wunsch, plus "half global"). It is provided strictly as
+is; for further documentation please see the code.
+
+## 8\. INSTALLATION INSTRUCTIONS - Mac OSX
+
+How to install this software \============================
+
+1\. Double click on the .tar.gz file to extract the archive.
+
+2\. The binary files are located in the 'bin' \[for 'binary'\] folder
+
+3\. (Optional) To run the tests, cd to the new directory and then the test
+folder
+
+4\. (Optional) Within this folder run: a. 'perl -w p3test.pl'
+
+5\. (Optional) You should not see 'FAILED' during the tests.
+
+6\. (Optional) \*NOTE\*: If your perl command is not called perl (for example,
+if it is called perl5) you will have to modify the internals of the test
+scripts).
+
+7\. Copy the following files to a location of your choice: a.
+bin/long\_seq\_tm\_test b. bin/ntdpal c. bin/oligotm d. bin/primer3\_core
+
+8\. (Optional) Make sure this location is within your $PATH (see below)
+
+Where to put the binary files \=============================
+
+A good place to put these is within ~/bin/ (this means in your home folder,
+within a folder named \`bin\` \[for 'binary'\]).
+
+You can also just drag the 'bin' folder to a location within your home
+directory.
+
+You can certainly also copy the files within 'bin' to /usr/local/bin (if you are
+an administrator) or another similar location.
+
+You may need to adjust the permissions on the binaries if you get fancy.
+
+Add the location to your $PATH \==============================
+
+This is an optional step, but it will allow you to run Primer3 in any directory
+on your machine as your user just by typing its name (primer3\_core).
+
+\*\*\* You should be very careful when altering your $PATH as things can go very
+wrong. See below for an alternate method. \*\*\*
+
+If you added the binaries to /usr/local/bin, then you do not need to do this.
+
+If you added the binaries to a local directory (let's say ~/bin/), do the
+following:
+
+1\. Edit your ~/.bash\_profile. You can edit this on the command line (Terminal)
+with:
+
+nano ~/.bash\_profile
+
+2\. Add the following line if it is not present (replacing '~/bin' if you used
+another directory):
+
+PATH=$PATH:~/bin/
+
+3\. If a PATH line \*is\* present, ensure you add a colon to the end of what is
+there and then the directory, so if you have something like:
+
+a) PATH=$PATH:/usr/local/genome/bin:/sw/bin
+
+make it look like:
+
+b) PATH=$PATH:/usr/local/genome/bin:/sw/bin:~/bin
+
+4\. Quit and restart terminal for the changes to take effect.
+
+If you don't add the location to your $PATH
+\===========================================
+
+Assuming you don't want to modify your $PATH, you can still run the binaries.
+Let's assume you put the files in '~/bin/. You may run primer3\_core by doing
+either of the following:
+
+1\. ~/bin/primer3\_core < yourInputFile 2\. /Users/<your
+username>/bin/primer3\_core < yourInputFile
+
+The first option is just a shortcut to the second.
+
+## 9\. INSTALLATION INSTRUCTIONS - WINDOWS
+
+Functionality warning \=====================
+
+The windows version does not support the masker functionality.
+
+How to install this software \============================
+
+Get your copy of the source code \-------------------------------- 1\. Install
+Git for Windows from https://git-scm.com/download/win 2\. Open Git for Windows
+and run git clone https://github.com/primer3-org/primer3.git cd primer3 3\. Use
+git commands to obtain tagged version git tag git checkout tags/v2.3.7 or 1.
+Download and unpack latest from https://github.com/primer3-org/primer3/releases
+Compile Primer3 \---------------
+
+1\. Download and install TDM-GCC MinGW Compiler from
+https://sourceforge.net/projects/tdm-gcc/ 2. Open the windows command line and
+change to the src folder in Primer3 3\. Run: mingw32-make TESTOPTS=--windows
+
+Running the tests \================= You must install a perl distribution to run
+the windows tests.
+
+We \*strongly\* recommend you install ActiveState perl
+(http://www.activestate.com/products/activeperl/) as this was used to test our
+Primer3 builds, and it is known to work.
+
+1\. Click on 'Start > Run...' 2\. Type 'cmd' into the space provided 3\. Hit
+enter (or select 'OK') 4\. Navigate to the location of the tests:
+
+A. if you put it in C:/Documents and Settings/YourName/primer3-<release>/test/,
+you would type 'cd c:/Documents and Settings/YourName/primer3-<release>/test/'
+
+B. you can also type 'cd ' (don't forget the space after cd) and drag the
+primer3-<release> folder onto the command-line window from windows explorer,
+this will fill in the location for you
+
+5\. On the command line, run 'mingw32-make TESTOPTS=--windows' in this
+directory. You can also run the tests individually, for example 'perl p3test.pl
+--windows'. 6\. You should see \[OK\] for the majority of the tests. The masker
+functionality is not supported on Windows and coresponding tests will fail.
+
+Running the software \====================
+
+To run the program, you must do so from the MS-DOS command-line. The intricacies
+of the DOS command line are beyond the scope of this document. Google for more
+information, if needed. Here is a quick summary:
+
+1\. Click on 'Start > Run...' 2\. Type 'cmd' into the space provided 3\. Hit
+enter (or select 'OK') 4\. Navigate to the location of the binary:
+
+A. if you put it in C:/Documents and Settings/YourName/Temp, you would type 'cd
+c:/Documents and Settings/YourName/Temp'
+
+B. you can also type 'cd ' (don't forget the space after cd) and drag the
+Primer3 folder onto the command-line window from windows explorer, this will
+fill in the location for you
+
+5\. Run the example file by typing:
+
+primer3\_core.exe < example
+
+Other files may be run in a similar fashion. If your input filename is
+'MyData.txt' you can run Primer3 using this file (in the correct format; see
+README) with:
+
+primer3\_core.exe < MyData.txt
+
+If your file is not in the folder containing primer3\_core.exe, you could run
+the program from the primer3\_core folder using:
+
+primer3\_core.exe < c:/someOtherFolder/someOtherFolder/MyData.txt
+
+Finally, if you want to run the program without going to its folder, assuming
+primer3\_core.exe is in c:/Temp, you could run:
+
+c:/Temp/primer3\_core.exe < c:/someOtherFolder/someOtherFolder/MyData.txt
+
+## 10\. SYSTEM REQUIREMENTS
+
+Please see
+[https://github.com/primer3-org/primer3](https://github.com/primer3-org/primer3)
+for up-to-date information. Primer3 should compile on any system using with an
+ANSI C compiler like Linux/Unix, MacOS 10 or MS Windows. The Makefile will
+probably need to be modified for compilation with C compilers other than gcc.
+
+## 11\. INVOKING primer3\_core
+
+By default, the executable program produced by the Makefile is called
+primer3\_core. This is the C program that does the heavy lifting of primer
+picking. There are also two, more user-friendly, web interfaces (distributed
+separately).
+
+The command line for Primer3 is:
+
+primer3\_core \[ --format\_output \] \[
+--default\_version=1|--default\_version=2 \] \[ --io\_version=4 \] \[
+--p3\_settings\_file=<file\_path> \] \[ --echo\_settings\_file \] \[
+--strict\_tags \] \[ --output=<file\_path> \] \[ --error=<file\_path> \] \[
+input\_file \]
+
+A complete list of valid command line arguments can be found in COMMAND LINE
+ARGUMENTS below.
+
+If no input file is specified, primer3\_core will read its input from stdin.
+
+## 12\. COMMAND LINE ARGUMENTS
+
+This section presents the list of arguments that may given in the command line.
+Any unique abbreviation of the arguments is allowed (e.g. -ab instead of -about)
+and each argument can be preceded by either one or two dashes. In the case of
+arguments that receive values, the '=' can be replaced by a space.
+
+### \--about
+
+With this argument Primer3 generates one line of output indicating the release
+number and then exits. This allows scripts to query Primer3 for its version.
+
+### \--default\_version=n
+
+_n_\=2 is the default, and directs primer3\_core to use the latest default
+values (the ones documented here). _n_\=1 directs primer3\_core to use defaults
+from version 2.2.3 and before.
+
+### \--format\_output
+
+This argument indicates that primer3\_core should generate user-oriented (rather
+than program-oriented) output.
+
+### \--strict\_tags
+
+This argument indicates that primer3\_core should generate a fatal error if
+there is any tag in the input that it does not recognize. This tag also applies
+to the settings file (see documentation for the --p3\_settings\_file argument),
+if any, but with limitations: lines that do not begin with PRIMER\_ or
+P3\_FILE\_ID are always ignored in the settings file.
+
+### \--p3\_settings\_file=file\_path
+
+This argument specifies a settings file, _file\_path_. Primer3 reads the global
+("PRIMER\_...") parameters from this file first. Tags appearing in the settings
+file override default Primer3 settings. The values set in the settings files can
+be also overridden by tags in the input file. See Primer3 file documentation for
+details on the file format. **WARNING: the --strict\_tags flag applies only to
+tags beginning with the string "PRIMER\_"; lines that do not begin with the
+string "PRIMER\_" are ignored.**
+
+### \--echo\_settings\_file
+
+This argument indicates that primer3\_core should print the input tags specified
+in the given settings file. If no settings file is provided or if the
+--format\_output option is given, this argument will have no effect.
+
+### \--io\_version=n
+
+This argument is provided for backward compatibility. --io\_version=4 **is the
+only legal value and the default** .
+
+### \--output=file\_path
+
+This argument specifies the file where the output should be written. If omitted,
+stdout is used.
+
+### \--error=file\_path
+
+This argument specifies the file where the error messages should be written. If
+omitted, stderr is used.
+
+## 13\. INPUT AND OUTPUT CONVENTIONS
+
+By default, Primer3 accepts input in Boulder-IO format, a pre-XML, pre-RDF,
+text-based input/output format for program-to-program data interchange. By
+default, Primer3 also produces output in the same format.
+
+When run with the --format\_output command-line flag, Primer3 prints a more
+user-oriented report for each sequence.
+
+Primer3 exits with 0 status if it operates correctly. See EXIT STATUS CODES
+below for additional information.
+
+The syntax of the version of Boulder-IO recognized by Primer3 is as follows:
+
+  o Input consists of a sequence of RECORDs.
+  o A RECORD consists of a sequence of (TAG,VALUE) pairs, each terminated
+    by a newline character (\\n). A RECORD is terminated by  '='
+    appearing by itself on a line.
+  o A (TAG,VALUE) pair has the following requirements:
+    o the TAG must be immediately (without spaces)
+          followed by '='.
+
+An example of a legal (TAG,VALUE) pair is
+
+[SEQUENCE\_ID](#SEQUENCE_ID) \=my\_marker
+
+and an example of a Boulder-IO record is
+
+[SEQUENCE\_ID](#SEQUENCE_ID) \=test1 [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)
+\=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC \=
+
+Many records can be sent, one after another. Below is an example of three
+different records which might be passed through a Boulder-IO stream:
+
+[SEQUENCE\_ID](#SEQUENCE_ID) \=test1 [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)
+\=GACTGATCGATGCTAGCTACGATCGATCGATGCATGCTAGCTAGCTAGCTGCTAGC \=
+[SEQUENCE\_ID](#SEQUENCE_ID) \=test2 [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)
+\=CATCATCATCATCGATGCTAGCATCNNACGTACGANCANATGCATCGATCGT \=
+[SEQUENCE\_ID](#SEQUENCE_ID) \=test3 [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)
+\=NACGTAGCTAGCATGCACNACTCGACNACGATGCACNACAGCTGCATCGATGC \=
+
+Primer3 reads Boulder-IO on stdin and echos its input and returns results in
+Boulder-IO format on stdout. Primer3 indicates many user-correctable errors by a
+value in the output tag [PRIMER\_ERROR](#PRIMER_ERROR) (see below). Primer3
+indicates other errors, including system configuration errors, resource errors
+(such out-of-memory errors), and detected programming errors by a message on
+stderr and a non-zero exit status.
+
+This document lists the input tags that Primer3 recognizes. Primer3 echos and
+ignores any tags it does not recognize, unless the --strict\_tags flag is set on
+the command line, in which case Primer3 prints an error in the
+[PRIMER\_ERROR](#PRIMER_ERROR) output tag (see below), and prints additional
+information on stdout; this option can be useful for debugging systems that
+incorporate Primer3.
+
+Except for tags with the type "interval list" each tag should only appear ONCE
+in any given input record. This restriction is not systematically checked, and
+if a tag appears more than once, the new value silently over-writes the previous
+value.
+
+## 14\. AN EXAMPLE
+
+One might be interested in performing PCR on an STS with a CA repeat in the
+middle of it. Primers need to be chosen based on the criteria of the experiment.
+
+We need to create a Boulder-IO record to send to Primer3 via stdin. There are
+lots of ways to accomplish this. We could save the record into a text file
+called 'example', and then type the Unix command 'primer3\_core < example'.
+
+Let's look at the input record itself: [SEQUENCE\_ID](#SEQUENCE_ID) \=example
+[SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)
+\=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
+[SEQUENCE\_TARGET](#SEQUENCE_TARGET) \=37,21 [PRIMER\_TASK](#PRIMER_TASK)
+\=generic [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=1
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=1
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=1
+[PRIMER\_OPT\_SIZE](#PRIMER_OPT_SIZE) \=18 [PRIMER\_MIN\_SIZE](#PRIMER_MIN_SIZE)
+\=15 [PRIMER\_MAX\_SIZE](#PRIMER_MAX_SIZE) \=21
+[PRIMER\_MAX\_NS\_ACCEPTED](#PRIMER_MAX_NS_ACCEPTED) \=1
+[PRIMER\_PRODUCT\_SIZE\_RANGE](#PRIMER_PRODUCT_SIZE_RANGE) \=75-100
+[P3\_FILE\_FLAG](#P3_FILE_FLAG) \=1
+[SEQUENCE\_INTERNAL\_EXCLUDED\_REGION](#SEQUENCE_INTERNAL_EXCLUDED_REGION)
+\=37,21 [PRIMER\_EXPLAIN\_FLAG](#PRIMER_EXPLAIN_FLAG) \=1 \=
+
+A breakdown of the reasoning behind each of the TAG=VALUE pairs is below:
+
+[SEQUENCE\_ID](#SEQUENCE_ID) \=example
+
+The main intent of this tag is to provide an identifier for the sequence that is
+meaningful to the user, for example when Primer3 processes multiple records, and
+by default this tag is optional. However, this tag is \_required\_ when
+[P3\_FILE\_FLAG](#P3_FILE_FLAG) is non-0 Because it provides the names of the
+files that contain lists of oligos that Primer3 considered.
+
+[SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)
+\=GTAGTCAGTAGACNATGACNACTGACGATGCAGACNACACACACACACACAGCACACAGGTATTAGTGGGCCATTCGATCCCGACCCAAATCGATAGCTACGATGACG
+
+The [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE) provides the sequence from which
+Primer3 will design primers. Note that there is no newline until the sequence
+terminates completely.
+
+[SEQUENCE\_TARGET](#SEQUENCE_TARGET) \=37,21
+
+There is a simple sequence repeat in this sequence. The repeat starts at base
+37, and has a length of 21 bases. We instruct Primer3 to choose primers that
+flank the repeat site (because we want to use the PCR product for determining
+the length of the repeat, which is likely to be polymorphic).
+
+[PRIMER\_TASK](#PRIMER_TASK) \=generic
+
+The [PRIMER\_TASK](#PRIMER_TASK) tells Primer3 which type of primers to pick.
+You can select typical primers for PCR detection, primers for cloning or for
+sequencing.
+
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=1
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=1
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=1
+
+We would like to pick a left primer, an internal oligo and a right primer, so we
+set these flags to 1 (true). In combination with the
+[PRIMER\_TASK](#PRIMER_TASK) this tags control which primers are picked.
+
+[PRIMER\_OPT\_SIZE](#PRIMER_OPT_SIZE) \=18
+
+Since our sequence length is rather small (only 108 bases long), we lower the
+[PRIMER\_OPT\_SIZE](#PRIMER_OPT_SIZE) from 20 to 18. It is more likely that
+Primer3 will succeed if it aims for smaller primers.
+
+[PRIMER\_MIN\_SIZE](#PRIMER_MIN_SIZE) \=15 [PRIMER\_MAX\_SIZE](#PRIMER_MAX_SIZE)
+\=21
+
+With the lowering of optimal primer size, it's good to lower the minimum and
+maximum sizes as well.
+
+[PRIMER\_MAX\_NS\_ACCEPTED](#PRIMER_MAX_NS_ACCEPTED) \=1
+
+Since the sequence is short with a non-negligible amount of unknown bases (N's)
+in it, we make Primer3's job easier by allowing it to pick primers that have at
+most 1 unknown base.
+
+[PRIMER\_PRODUCT\_SIZE\_RANGE](#PRIMER_PRODUCT_SIZE_RANGE) \=75-100
+
+We reduce the product size range from the default of 100-300 because our source
+sequence is only 108 base pairs long. If we insisted on a product size of 100
+base pairs Primer3 would have few possibilities to choose from.
+
+[P3\_FILE\_FLAG](#P3_FILE_FLAG) \=1
+
+Because we have a short sequence with many Ns and with a simple sequence repeat
+that we must amplify, Primer3 might fail to pick and primers. Therefore, we want
+to get the list of primers it considered, so that we could manually pick primers
+ourselves if Primer3 fails to do so. Setting this flag to 1 asks Primer3 to
+output the primers it considered to a forwar primers and reverse primers to
+output files.
+
+[SEQUENCE\_INTERNAL\_EXCLUDED\_REGION](#SEQUENCE_INTERNAL_EXCLUDED_REGION)
+\=37,21
+
+Normally CA-repeats make poor hybridization probes (because they not specific
+enough). Therefore, we exclude the CA repeat (which is the TARGET) from
+consideration for the internal oligo.
+
+[PRIMER\_EXPLAIN\_FLAG](#PRIMER_EXPLAIN_FLAG) \=1
+
+We want to see statistics about the oligos and oligo triples (left primer,
+internal oligo, right primer) that Primer3 examined.
+
+\=
+
+The '=' character terminates the record.
+
+There were many Boulder-IO input tags that were not specified, which is legal.
+For the tags with default values, those defaults will be used in the analysis.
+For the tags with NO default values (for example
+[SEQUENCE\_TARGET](#SEQUENCE_TARGET) ), the functionality requested by the those
+tags will be absent. Also, please note that it is not the case that we need to
+surround a simple sequence repeat every time we want to pick primers!
+
+## 15\. HOW TO MIGRATE TAGS TO IO VERSION 4
+
+With Primer3 release 2.0, many Boulder-IO tags were modified and new tags were
+introduced. The new Primer3 tags are designed with the idea in mind that
+computer scripts and other programs use primer3\_core. The modifications make it
+easier for programs to read and write Primer3 input and output.
+
+Furthermore the Primer3 default values and the use of
+[PRIMER\_WT\_TEMPLATE\_MISPRIMING](#PRIMER_WT_TEMPLATE_MISPRIMING) and
+[PRIMER\_PAIR\_WT\_TEMPLATE\_MISPRIMING](#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING)
+have changed in version 2.0.
+
+There are three classes of input: "Sequence" input tags describe a particular
+input sequence to Primer3, and are reset after every Boulder record (now
+starting with SEQUENCE\_). "Global" input tags describe the general parameters
+that Primer3 should use in its searches, and the values of these tags persist
+between input Boulder records until or unless they are explicitly reset (now
+starting with PRIMER\_). "Program" parameters that deal with the behavior of the
+Primer3 program itself (now starting with P3\_). See below for a list of the
+modified tags.
+
+The handling of PRIMER\_TASK changed completely. In the past we used it to tell
+Primer3 what task to perform. Now the task is complemented with
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) ,
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) and
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) , which specify which
+primers are to be picked.
+
+These Tags are modified:
+
+The "per sequence" tags:
+
+NEW VERSION                       - OLD VERSION
+----------------------------------------------------------------------------------------
+[SEQUENCE\_ID](#SEQUENCE_ID)                       - PRIMER\_SEQUENCE\_ID
+[SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)                 - SEQUENCE
+[SEQUENCE\_QUALITY](#SEQUENCE_QUALITY)                  - PRIMER\_SEQUENCE\_QUALITY
+[SEQUENCE\_INCLUDED\_REGION](#SEQUENCE_INCLUDED_REGION)          - INCLUDED\_REGION
+[SEQUENCE\_TARGET](#SEQUENCE_TARGET)                   - TARGET
+[SEQUENCE\_EXCLUDED\_REGION](#SEQUENCE_EXCLUDED_REGION)          - EXCLUDED\_REGION
+[SEQUENCE\_START\_CODON\_POSITION](#SEQUENCE_START_CODON_POSITION)     - PRIMER\_START\_CODON\_POSITION
+[SEQUENCE\_PRIMER](#SEQUENCE_PRIMER)                   - PRIMER\_LEFT\_INPUT
+[SEQUENCE\_PRIMER\_REVCOMP](#SEQUENCE_PRIMER_REVCOMP)           - PRIMER\_RIGHT\_INPUT
+[SEQUENCE\_INTERNAL\_OLIGO](#SEQUENCE_INTERNAL_OLIGO)           - PRIMER\_INTERNAL\_OLIGO\_INPUT
+[SEQUENCE\_INTERNAL\_EXCLUDED\_REGION](#SEQUENCE_INTERNAL_EXCLUDED_REGION) - PRIMER\_INTERNAL\_OLIGO\_EXCLUDED\_REGION
+--------------------------------------------------------------------------------
+The "global" tags:
+NEW VERSION                       - OLD VERSION
+[PRIMER\_TASK](#PRIMER_TASK)                       - PRIMER\_TASK (modified use)
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER)          - --- did not exist
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO)        - PRIMER\_PICK\_INTERNAL\_OLIGO (modified use)
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER)           - --- did not exist
+[PRIMER\_PAIR\_WT\_TEMPLATE\_MISPRIMING](#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING)\- PRIMER\_PAIR\_WT\_TEMPLATE\_MISPRIMING (modified use)
+[PRIMER\_WT\_TEMPLATE\_MISPRIMING](#PRIMER_WT_TEMPLATE_MISPRIMING)     - PRIMER\_WT\_TEMPLATE\_MISPRIMING (modified use)
+[PRIMER\_MAX\_LIBRARY\_MISPRIMING](#PRIMER_MAX_LIBRARY_MISPRIMING)     - PRIMER\_MAX\_MISPRIMING
+[PRIMER\_INTERNAL\_MAX\_LIBRARY\_MISHYB](#PRIMER_INTERNAL_MAX_LIBRARY_MISHYB)\- PRIMER\_INTERNAL\_OLIGO\_MAX\_MISHYB
+[PRIMER\_PAIR\_MAX\_LIBRARY\_MISPRIMING](#PRIMER_PAIR_MAX_LIBRARY_MISPRIMING)\- PRIMER\_PAIR\_MAX\_MISPRIMING
+[PRIMER\_WT\_LIBRARY\_MISPRIMING](#PRIMER_WT_LIBRARY_MISPRIMING)      - PRIMER\_WT\_REP\_SIM
+[PRIMER\_INTERNAL\_WT\_LIBRARY\_MISHYB](#PRIMER_INTERNAL_WT_LIBRARY_MISHYB) - PRIMER\_INTERNAL\_WT\_REP\_SIM
+[PRIMER\_PAIR\_WT\_LIBRARY\_MISPRIMING](#PRIMER_PAIR_WT_LIBRARY_MISPRIMING) - PRIMER\_PAIR\_WT\_REP\_SIM
+[PRIMER\_MAX\_NS\_ACCEPTED](#PRIMER_MAX_NS_ACCEPTED)            - PRIMER\_NUM\_NS\_ACCEPTED
+[PRIMER\_PAIR\_MAX\_DIFF\_TM](#PRIMER_PAIR_MAX_DIFF_TM)           - PRIMER\_MAX\_DIFF\_TM
+[PRIMER\_SALT\_MONOVALENT](#PRIMER_SALT_MONOVALENT)            - PRIMER\_SALT\_CONC
+[PRIMER\_SALT\_DIVALENT](#PRIMER_SALT_DIVALENT)              - PRIMER\_DIVALENT\_CONC
+[PRIMER\_TM\_FORMULA](#PRIMER_TM_FORMULA)                 - PRIMER\_TM\_SANTALUCIA
+[PRIMER\_MAX\_SELF\_ANY](#PRIMER_MAX_SELF_ANY)               - PRIMER\_SELF\_ANY
+[PRIMER\_MAX\_SELF\_END](#PRIMER_MAX_SELF_END)               - PRIMER\_SELF\_END
+[PRIMER\_WT\_SELF\_ANY](#PRIMER_WT_SELF_ANY)                - PRIMER\_WT\_COMPL\_ANY
+[PRIMER\_WT\_SELF\_END](#PRIMER_WT_SELF_END)                - PRIMER\_WT\_COMPL\_END
+[PRIMER\_PAIR\_MAX\_COMPL\_ANY](#PRIMER_PAIR_MAX_COMPL_ANY)         - PRIMER\_PAIR\_ANY
+[PRIMER\_PAIR\_MAX\_COMPL\_END](#PRIMER_PAIR_MAX_COMPL_END)         - PRIMER\_PAIR\_END
+[P3\_FILE\_FLAG](#P3_FILE_FLAG)                      - PRIMER\_FILE\_FLAG
+[P3\_COMMENT](#P3_COMMENT)                        - PRIMER\_COMMENT
+[PRIMER\_INTERNAL\_SALT\_MONOVALENT](#PRIMER_INTERNAL_SALT_MONOVALENT)   - PRIMER\_INTERNAL\_OLIGO\_SALT\_CONC
+[PRIMER\_INTERNAL\_SALT\_DIVALENT](#PRIMER_INTERNAL_SALT_DIVALENT)     - PRIMER\_INTERNAL\_OLIGO\_DIVALENT\_CONC
+[PRIMER\_INTERNAL\_WT\_SELF\_ANY](#PRIMER_INTERNAL_WT_SELF_ANY)       - PRIMER\_IO\_WT\_COMPL\_ANY
+[PRIMER\_INTERNAL\_WT\_SELF\_END](#PRIMER_INTERNAL_WT_SELF_END)       - PRIMER\_IO\_WT\_COMPL\_END
+[PRIMER\_INTERNAL\_MAX\_NS\_ACCEPTED](#PRIMER_INTERNAL_MAX_NS_ACCEPTED)   - PRIMER\_INTERNAL\_OLIGO\_NUM\_NS
+[PRIMER\_INTERNAL\_MAX\_SELF\_ANY](#PRIMER_INTERNAL_MAX_SELF_ANY)      - PRIMER\_INTERNAL\_OLIGO\_SELF\_ANY
+[PRIMER\_INTERNAL\_MAX\_SELF\_END](#PRIMER_INTERNAL_MAX_SELF_END)      - PRIMER\_INTERNAL\_OLIGO\_SELF\_END
+The following tags INTERNAL\_OLIGO is replaced by INTERNAL:
+[PRIMER\_INTERNAL\_OPT\_SIZE](#PRIMER_INTERNAL_OPT_SIZE)          - PRIMER\_INTERNAL\_OLIGO\_OPT\_SIZE
+[PRIMER\_INTERNAL\_MIN\_SIZE](#PRIMER_INTERNAL_MIN_SIZE)          - PRIMER\_INTERNAL\_OLIGO\_MIN\_SIZE
+[PRIMER\_INTERNAL\_MAX\_SIZE](#PRIMER_INTERNAL_MAX_SIZE)          - PRIMER\_INTERNAL\_OLIGO\_MAX\_SIZE
+[PRIMER\_INTERNAL\_OPT\_TM](#PRIMER_INTERNAL_OPT_TM)            - PRIMER\_INTERNAL\_OLIGO\_OPT\_TM
+[PRIMER\_INTERNAL\_MIN\_TM](#PRIMER_INTERNAL_MIN_TM)            - PRIMER\_INTERNAL\_OLIGO\_MIN\_TM
+[PRIMER\_INTERNAL\_MAX\_TM](#PRIMER_INTERNAL_MAX_TM)            - PRIMER\_INTERNAL\_OLIGO\_MAX\_TM
+[PRIMER\_INTERNAL\_MIN\_GC](#PRIMER_INTERNAL_MIN_GC)            - PRIMER\_INTERNAL\_OLIGO\_MIN\_GC
+[PRIMER\_INTERNAL\_OPT\_GC\_PERCENT](#PRIMER_INTERNAL_OPT_GC_PERCENT)    - PRIMER\_INTERNAL\_OLIGO\_OPT\_GC\_PERCENT
+[PRIMER\_INTERNAL\_MAX\_GC](#PRIMER_INTERNAL_MAX_GC)            - PRIMER\_INTERNAL\_OLIGO\_MAX\_GC
+[PRIMER\_INTERNAL\_DNTP\_CONC](#PRIMER_INTERNAL_DNTP_CONC)         - PRIMER\_INTERNAL\_OLIGO\_DNTP\_CONC
+[PRIMER\_INTERNAL\_DNA\_CONC](#PRIMER_INTERNAL_DNA_CONC)          - PRIMER\_INTERNAL\_OLIGO\_DNA\_CONC
+[PRIMER\_INTERNAL\_MAX\_POLY\_X](#PRIMER_INTERNAL_MAX_POLY_X)        - PRIMER\_INTERNAL\_OLIGO\_MAX\_POLY\_X
+[PRIMER\_INTERNAL\_MISHYB\_LIBRARY](#PRIMER_INTERNAL_MISHYB_LIBRARY)    - PRIMER\_INTERNAL\_OLIGO\_MISHYB\_LIBRARY
+[PRIMER\_INTERNAL\_MIN\_QUALITY](#PRIMER_INTERNAL_MIN_QUALITY)       - PRIMER\_INTERNAL\_OLIGO\_MIN\_QUALITY
+The following tags IO is replaced by INTERNAL:
+[PRIMER\_INTERNAL\_WT\_TM\_GT](#PRIMER_INTERNAL_WT_TM_GT)          - PRIMER\_IO\_WT\_TM\_GT
+[PRIMER\_INTERNAL\_WT\_TM\_LT](#PRIMER_INTERNAL_WT_TM_LT)          - PRIMER\_IO\_WT\_TM\_LT
+[PRIMER\_INTERNAL\_WT\_SIZE\_LT](#PRIMER_INTERNAL_WT_SIZE_LT)        - PRIMER\_IO\_WT\_SIZE\_LT
+[PRIMER\_INTERNAL\_WT\_SIZE\_GT](#PRIMER_INTERNAL_WT_SIZE_GT)        - PRIMER\_IO\_WT\_SIZE\_GT
+[PRIMER\_INTERNAL\_WT\_GC\_PERCENT\_LT](#PRIMER_INTERNAL_WT_GC_PERCENT_LT)  - PRIMER\_IO\_WT\_GC\_PERCENT\_LT
+[PRIMER\_INTERNAL\_WT\_GC\_PERCENT\_GT](#PRIMER_INTERNAL_WT_GC_PERCENT_GT)  - PRIMER\_IO\_WT\_GC\_PERCENT\_GT
+[PRIMER\_INTERNAL\_WT\_NUM\_NS](#PRIMER_INTERNAL_WT_NUM_NS)         - PRIMER\_IO\_WT\_NUM\_NS
+[PRIMER\_INTERNAL\_WT\_SEQ\_QUAL](#PRIMER_INTERNAL_WT_SEQ_QUAL)       - PRIMER\_IO\_WT\_SEQ\_QUAL
+
+\--------------------------------------------------------------------------------
+OUTPUT TAGS:
+
+There are three big changes on the output: \- INTERNAL\_OLIGO is now replaced by
+INTERNAL. \- The first version is numbered 0. \- The "PRODUCT" tags are renamed
+\- The errors are modified \- Errors caused by a specific primer are given as
+[PRIMER\_LEFT\_4\_PROBLEMS](#PRIMER_LEFT_4_PROBLEMS)
+
+Now all primer related output follows the rule:
+PRIMER\_{LEFT,RIGHT,INTERNAL,PAIR}\_<j>\_<tag\_name>. where <j> is an integer
+from 0 to n, where n is at most the value of
+[PRIMER\_NUM\_RETURN](#PRIMER_NUM_RETURN) - 1.
+
+This allows easy scripting by using the underscores \_ to split the name. The
+first part is PRIMER, the second the type of oligo or pair parameters, the third
+is always a number, starting at 0 and the rest is used by the tags.
+
+That affects also (shown for output number 4):
+
+NEW VERSION                             - OLD VERSION
+------------------------------------------------------------------------------------------------------
+[PRIMER\_PAIR\_4\_PENALTY](#PRIMER_PAIR_4_PENALTY)                   - PRIMER\_PAIR\_PENALTY\_4 (number moved behind PAIR)
+[PRIMER\_PAIR\_4\_PRODUCT\_SIZE](#PRIMER_PAIR_4_PRODUCT_SIZE)              - PRIMER\_PRODUCT\_SIZE\_4 (grouped with PAIR)
+[PRIMER\_PAIR\_4\_PRODUCT\_TM](#PRIMER_PAIR_4_PRODUCT_TM)                - PRIMER\_PRODUCT\_TM\_4 (grouped with PAIR)
+[PRIMER\_PAIR\_4\_PRODUCT\_TM\_OLIGO\_TM\_DIFF](#PRIMER_PAIR_4_PRODUCT_TM_OLIGO_TM_DIFF)  - PRIMER\_PRODUCT\_TM\_OLIGO\_TM\_DIFF\_4 (grouped with PAIR)
+[PRIMER\_INTERNAL\_EXPLAIN](#PRIMER_INTERNAL_EXPLAIN)                 - PRIMER\_INTERNAL\_OLIGO\_EXPLAIN
+PRIMER\_LEFT\_4\_LIBRARY\_MISPRIMING                   - PRIMER\_LEFT\_4\_MISPRIMING\_SCORE
+PRIMER\_INTERNAL\_4\_LIBRARY\_MISHYB                   - PRIMER\_INTERNAL\_OLIGO\_4\_MISHYB\_SCORE
+PRIMER\_RIGHT\_4\_LIBRARY\_MISPRIMING                  - PRIMER\_RIGHT\_4\_MISPRIMING\_SCORE
+PRIMER\_PAIR\_4\_LIBRARY\_MISPRIMING                   - PRIMER\_PAIR\_4\_MISPRIMING\_SCORE
+
+## 16\. "SEQUENCE" INPUT TAGS
+
+"Sequence" input tags start with SEQUENCE\_... and describe a particular input
+sequence to Primer3. They are reset after every Boulder record. Errors in
+"Sequence" input tags invalidate the current record, but Primer3 will continue
+to process additional records.
+
+[SEQUENCE\_EXCLUDED\_REGION](#SEQUENCE_EXCLUDED_REGION)
+
+[SEQUENCE\_INTERNAL\_EXCLUDED\_REGION](#SEQUENCE_INTERNAL_EXCLUDED_REGION)
+
+[SEQUENCE\_PRIMER\_PAIR\_OK\_REGION\_LIST](#SEQUENCE_PRIMER_PAIR_OK_REGION_LIST)
+
+[SEQUENCE\_FORCE\_LEFT\_END](#SEQUENCE_FORCE_LEFT_END)
+
+[SEQUENCE\_INTERNAL\_OLIGO](#SEQUENCE_INTERNAL_OLIGO)
+
+[SEQUENCE\_PRIMER\_REVCOMP](#SEQUENCE_PRIMER_REVCOMP)
+
+[SEQUENCE\_FORCE\_LEFT\_START](#SEQUENCE_FORCE_LEFT_START)
+
+[SEQUENCE\_INTERNAL\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_INTERNAL_OVERLAP_JUNCTION_LIST)
+
+[SEQUENCE\_QUALITY](#SEQUENCE_QUALITY)
+
+[SEQUENCE\_FORCE\_RIGHT\_END](#SEQUENCE_FORCE_RIGHT_END)
+
+[SEQUENCE\_OVERHANG\_LEFT](#SEQUENCE_OVERHANG_LEFT)
+
+[SEQUENCE\_START\_CODON\_POSITION](#SEQUENCE_START_CODON_POSITION)
+
+[SEQUENCE\_FORCE\_RIGHT\_START](#SEQUENCE_FORCE_RIGHT_START)
+
+[SEQUENCE\_OVERHANG\_RIGHT](#SEQUENCE_OVERHANG_RIGHT)
+
+[SEQUENCE\_START\_CODON\_SEQUENCE](#SEQUENCE_START_CODON_SEQUENCE)
+
+[SEQUENCE\_ID](#SEQUENCE_ID)
+
+[SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST)
+
+[SEQUENCE\_TARGET](#SEQUENCE_TARGET)
+
+[SEQUENCE\_INCLUDED\_REGION](#SEQUENCE_INCLUDED_REGION)
+
+[SEQUENCE\_PRIMER](#SEQUENCE_PRIMER)
+
+[SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE)
+
+### SEQUENCE\_ID (string; default empty)
+
+Short description of the sequence. It is used as an identifier that is
+reproduced in the output to enable users to identify the source of the chosen
+primers.
+
+This tag must be present if [P3\_FILE\_FLAG](#P3_FILE_FLAG) is non-zero.
+
+### SEQUENCE\_TEMPLATE (nucleotide sequence; default empty)
+
+The sequence from which to choose primers. The sequence must be presented 5' ->
+3' (i.e, in the normal way). In general, the bases may be upper or lower case,
+but lower case letters are treated specially if
+[PRIMER\_LOWERCASE\_MASKING](#PRIMER_LOWERCASE_MASKING) is set. The entire
+sequence MUST be all on a single line. (In other words, the sequence cannot span
+several lines.)
+
+### SEQUENCE\_INCLUDED\_REGION (interval list; default empty)
+
+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>_,_<length>_
+
+where _<start>_ is the index of the first base to consider, and _<length>_ is
+the number of subsequent bases in the primer-picking region.
+
+### SEQUENCE\_TARGET (interval list; default empty)
+
+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, or an exon for resequencing. The
+value should be a space-separated list of
+
+_<start>_,_<length>_
+
+pairs where _<start>_ is the index of the first base of a target, and _<length>_
+is its length.
+
+See also PRIMER\_INSIDE\_PENALTY, PRIMER\_OUTSIDE\_PENALTY. Has a different
+meaning when PRIMER\_TASK=pick\_sequencing\_primers. See PRIMER\_TASK for more
+information.
+
+### SEQUENCE\_EXCLUDED\_REGION (interval list; default empty)
+
+Left and Right primers and oligos may not overlap any region specified in this
+tag. The middle oligo may overlap, they may be limited by
+[SEQUENCE\_INTERNAL\_EXCLUDED\_REGION](#SEQUENCE_INTERNAL_EXCLUDED_REGION) . The
+associated value must be a space-separated list of
+
+_<start>_,_<length>_
+
+pairs where _<start>_ is the index of the first base of the excluded region, and
+_<length>_ is its length. 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.
+
+### SEQUENCE\_PRIMER\_PAIR\_OK\_REGION\_LIST (semicolon separated list of integer quadruples; default empty)
+
+This tag allows detailed specification of possible locations of left and right
+primers in primer pairs.
+
+The associated value must be a semicolon-separated list of
+
+_<left\_start>_,_<left\_length>_,_<right\_start>_,_<right\_length>_
+
+quadruples. The left primer must be in the region specified by
+_<left\_start>_,_<left\_length>_ and the right primer must be in the region
+specified by _<right\_start>_,_<right\_length>_. _<left\_start>_ and
+_<left\_length>_ specify the location of the left primer in terms of the index
+of the first base in the region and the length of the region. _<right\_start>_
+and _<right\_length>_ specify the location of the right primer in analogous
+fashion. As seen in the example below, if no integers are specified for a region
+then the location of the corresponding primer is not constrained.
+
+Example:
+
+SEQUENCE\_PRIMER\_PAIR\_OK\_REGION\_LIST=100,50,300,50 ; 900,60,, ; ,,930,100
+
+Specifies that there are three choices:
+
+Left primer in the 50 bp region starting at position 100 AND right primer in the
+50 bp region starting at position 300
+
+OR
+
+Left primer in the 60 bp region starting at position 900 (and right primer
+anywhere)
+
+OR
+
+Right primer in the 100 bp region starting at position 930 (and left primer
+anywhere)
+
+### SEQUENCE\_OVERLAP\_JUNCTION\_LIST (space separated integers; default empty)
+
+If this list is not empty, then the forward OR the reverse primer must overlap
+one of these junction points by at least
+[PRIMER\_MIN\_3\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION)
+nucleotides at the 3' end and at least
+[PRIMER\_MIN\_5\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION)
+nucleotides at the 5' end.
+
+In more detail: The junction associated with a given position is the space
+immediately to the right of that position in the template sequence on the strand
+given as input.
+
+For example:
+
+ʼ``
+
+SEQUENCE_OVERLAP_JUNCTION_LIST=20
+# 1-based indexes
+PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION=4
+template: atcataggccatgcctgagc^gctacgact
+          ok           ...gagc^gcta-3'  (left primer)
+          not ok       ...gagc^gct-3'   (left primer)
+          ok           3'-ctcg^cgat...  (right pimer)
+          not ok        3'-tcg^cgat...  (right primer)
+PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION=5
+         ok           5'-tgagc^gccg...  (left primer)
+         not ok        5'-gagc^gccg...  (left primer)
+         ok           ...tgagc^gctac-5' (right primer)
+         not ok       ...tgagc^gcta-5'  (right primer)
+ʼ``
+
+### SEQUENCE\_INTERNAL\_OVERLAP\_JUNCTION\_LIST (space separated integers; default empty)
+
+If this list is not empty, then the internal (middle) oligo must overlap one of
+these junction points by at least
+[PRIMER\_INTERNAL\_MIN\_3\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_INTERNAL_MIN_3_PRIME_OVERLAP_OF_JUNCTION)
+nucleotides at the 3' (right) end and at least
+[PRIMER\_INTERNAL\_MIN\_5\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_INTERNAL_MIN_5_PRIME_OVERLAP_OF_JUNCTION)
+nucleotides at the 5' (left) end.
+
+See [SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST) for
+more detail.
+
+### SEQUENCE\_INTERNAL\_EXCLUDED\_REGION (interval list; default empty)
+
+Middle oligos may not overlap any region specified by this tag. Left and right
+primers may overlap. The associated value must be a space-separated list of
+
+_<start>_,_<length>_
+
+pairs, where <start> is the index of the first base of an excluded region, and
+<length> is its length. Often one would make Target regions excluded regions for
+internal oligos.
+
+### SEQUENCE\_PRIMER (nucleotide sequence; default empty)
+
+The sequence of a left primer to check and around which to design right primers
+and optional internal oligos. Must be a substring of
+[SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE) .
+
+### SEQUENCE\_INTERNAL\_OLIGO (nucleotide sequence; default empty)
+
+The sequence of an internal oligo to check and around which to design left and
+right primers. Must be a substring of [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE) .
+
+### SEQUENCE\_PRIMER\_REVCOMP (nucleotide sequence; default empty)
+
+The sequence of a right primer to check and around which to design left primers
+and optional internal oligos. Must be a substring of the reverse strand of
+[SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE) .
+
+### SEQUENCE\_OVERHANG\_LEFT (string; default empty)
+
+The provided sequence is added to the 5' end of the left primer. The overhang
+sequences are utilized in calculating SELF\_ANY, SELF\_END, HAIRPIN, COMPL\_ANY,
+COMPL\_END, plus the \_TH and \_STRUCT versions of those outputs, as well as
+PRODUCT\_SIZE. Internal oligos may not have an overhang.
+
+The length of SEQUENCE\_OVERHANG\_LEFT and SEQUENCE\_OVERHANG\_RIGHT do not add
+to the binding product size of
+[PRIMER\_PRODUCT\_SIZE\_RANGE](#PRIMER_PRODUCT_SIZE_RANGE) or
+[PRIMER\_PRODUCT\_OPT\_SIZE](#PRIMER_PRODUCT_OPT_SIZE) .
+
+The TM anG GC\_PERCENT calculations will only be based on the 3' portion of the
+oligo that binds to the template.
+
+The following elements include SEQUENCE\_OVERHANG\_LEFT:
+[PRIMER\_LEFT\_4\_SELF\_ANY](#PRIMER_LEFT_4_SELF_ANY) ,
+[PRIMER\_LEFT\_4\_SELF\_ANY\_TH](#PRIMER_LEFT_4_SELF_ANY_TH) ,
+[PRIMER\_LEFT\_4\_SELF\_ANY\_STUCT](#PRIMER_LEFT_4_SELF_ANY_STUCT) ,
+[PRIMER\_LEFT\_4\_SELF\_END](#PRIMER_LEFT_4_SELF_END) ,
+[PRIMER\_LEFT\_4\_SELF\_END\_TH](#PRIMER_LEFT_4_SELF_END_TH) ,
+[PRIMER\_LEFT\_4\_SELF\_END\_STUCT](#PRIMER_LEFT_4_SELF_END_STUCT) ,
+[PRIMER\_LEFT\_4\_HAIRPIN\_TH](#PRIMER_LEFT_4_HAIRPIN_TH) ,
+[PRIMER\_LEFT\_4\_HAIRPIN\_STUCT](#PRIMER_LEFT_4_HAIRPIN_STUCT) ,
+[PRIMER\_PAIR\_4\_COMPL\_ANY](#PRIMER_PAIR_4_COMPL_ANY) ,
+[PRIMER\_PAIR\_4\_COMPL\_ANY\_TH](#PRIMER_PAIR_4_COMPL_ANY_TH) ,
+[PRIMER\_PAIR\_4\_COMPL\_ANY\_STUCT](#PRIMER_PAIR_4_COMPL_ANY_STUCT) ,
+[PRIMER\_PAIR\_4\_COMPL\_END](#PRIMER_PAIR_4_COMPL_END) ,
+[PRIMER\_PAIR\_4\_COMPL\_END\_TH](#PRIMER_PAIR_4_COMPL_END_TH) ,
+[PRIMER\_PAIR\_4\_COMPL\_END\_STUCT](#PRIMER_PAIR_4_COMPL_END_STUCT) ,
+[PRIMER\_PAIR\_4\_PRODUCT\_SIZE](#PRIMER_PAIR_4_PRODUCT_SIZE) .
+
+### SEQUENCE\_OVERHANG\_RIGHT (string; default empty)
+
+The provided sequence is added to the 5' end of the right primer. It is reverse
+complementary to the template sequence.
+
+See [SEQUENCE\_OVERHANG\_LEFT](#SEQUENCE_OVERHANG_LEFT) for more details.
+
+### SEQUENCE\_START\_CODON\_POSITION (int; default -2000000)
+
+This parameter should be considered EXPERIMENTAL at this point. Please check the
+output carefully; some erroneous inputs might cause an error in Primer3.
+
+Index of the first base of a start codon. This parameter allows Primer3 to
+select primer pairs to create in-frame amplicons e.g. to create a template for a
+fusion protein. Primer3 will attempt to select an in-frame left primer, ideally
+starting at or to the left of the start codon, or to the right if necessary.
+Negative values of this parameter are legal if the actual start codon is to the
+left of available sequence. If this parameter is non-negative Primer3 signals an
+error if the codon at the position specified by this parameter is not an ATG. A
+value less than or equal to -10^6 indicates that Primer3 should ignore this
+parameter.
+
+Primer3 selects the position of the right primer by scanning right from the left
+primer for a stop codon. Ideally the right primer will end at or after the stop
+codon.
+
+### SEQUENCE\_START\_CODON\_SEQUENCE (string; default ATG)
+
+The sequence of the start codon, by default ATG. Some bacteria use different
+start codons, this tag allows to specify alternative start codons.
+
+Any triplet can be provided as start codon.
+
+### SEQUENCE\_QUALITY (space separated integers; default empty)
+
+A list of space separated integers. There must be exactly one integer for each
+base in [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE) if this argument is non-empty.
+For example, for the sequence ANNTTCA... [SEQUENCE\_QUALITY](#SEQUENCE_QUALITY)
+might be 45 10 0 50 30 34 50 67 .... High numbers indicate high confidence in
+the base called at that position and low numbers indicate low confidence in the
+base call at that position. This parameter is only relevant if you are using a
+base calling program that provides quality information (for example phred).
+
+### SEQUENCE\_FORCE\_LEFT\_START (int; default -1000000)
+
+Forces the 5' end of the left primer to be at the indicated position. Primers
+are also picked if they violate certain constraints. The default value indicates
+that the start of the left primer can be anywhere.
+
+### SEQUENCE\_FORCE\_LEFT\_END (int; default -1000000)
+
+Forces the 3' end of the left primer to be at the indicated position. Primers
+are also picked if they violate certain constraints. The default value indicates
+that the end of the left primer can be anywhere.
+
+### SEQUENCE\_FORCE\_RIGHT\_START (int; default -1000000)
+
+Forces the 5' end of the right primer to be at the indicated position. Primers
+are also picked if they violate certain constraints. The default value indicates
+that the start of the right primer can be anywhere.
+
+### SEQUENCE\_FORCE\_RIGHT\_END (int; default -1000000)
+
+Forces the 3' end of the right primer to be at the indicated position. Primers
+are also picked if they violate certain constraints. The default value indicates
+that the end of the right primer can be anywhere.
+
+## 17\. "GLOBAL" INPUT TAGS
+
+"Global" input tags start with PRIMER\_... and describe the general parameters
+that Primer3 should use in its searches. The values of these tags persist
+between input Boulder records until or unless they are explicitly reset. Errors
+in "Global" input tags are fatal because they invalidate the basic conditions
+under which primers are being picked.
+
+Because the laboratory detection step using internal oligos is independent of
+the PCR amplification procedure, internal oligo tags have defaults that are
+independent of the parameters that govern the selection of PCR primers. For
+example, the melting temperature of an oligo used for hybridization might be
+considerably lower than that used as a PCR primer.
+
+[PRIMER\_ANNEALING\_TEMP](#PRIMER_ANNEALING_TEMP)
+
+[PRIMER\_INTERNAL\_WT\_SIZE\_LT](#PRIMER_INTERNAL_WT_SIZE_LT)
+
+[PRIMER\_PAIR\_WT\_COMPL\_END](#PRIMER_PAIR_WT_COMPL_END)
+
+[PRIMER\_DMSO\_CONC](#PRIMER_DMSO_CONC)
+
+[PRIMER\_INTERNAL\_WT\_TM\_GT](#PRIMER_INTERNAL_WT_TM_GT)
+
+[PRIMER\_PAIR\_WT\_COMPL\_END\_TH](#PRIMER_PAIR_WT_COMPL_END_TH)
+
+[PRIMER\_DMSO\_FACTOR](#PRIMER_DMSO_FACTOR)
+
+[PRIMER\_INTERNAL\_WT\_TM\_LT](#PRIMER_INTERNAL_WT_TM_LT)
+
+[PRIMER\_PAIR\_WT\_DIFF\_TM](#PRIMER_PAIR_WT_DIFF_TM)
+
+[PRIMER\_DNA\_CONC](#PRIMER_DNA_CONC)
+
+[PRIMER\_LIBERAL\_BASE](#PRIMER_LIBERAL_BASE)
+
+[PRIMER\_PAIR\_WT\_IO\_PENALTY](#PRIMER_PAIR_WT_IO_PENALTY)
+
+[PRIMER\_DNTP\_CONC](#PRIMER_DNTP_CONC)
+
+[PRIMER\_LIB\_AMBIGUITY\_CODES\_CONSENSUS](#PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS)
+
+[PRIMER\_PAIR\_WT\_LIBRARY\_MISPRIMING](#PRIMER_PAIR_WT_LIBRARY_MISPRIMING)
+
+[PRIMER\_EXPLAIN\_FLAG](#PRIMER_EXPLAIN_FLAG)
+
+[PRIMER\_LOWERCASE\_MASKING](#PRIMER_LOWERCASE_MASKING)
+
+[PRIMER\_PAIR\_WT\_PRODUCT\_SIZE\_GT](#PRIMER_PAIR_WT_PRODUCT_SIZE_GT)
+
+[PRIMER\_FIRST\_BASE\_INDEX](#PRIMER_FIRST_BASE_INDEX)
+
+[PRIMER\_MASK\_3P\_DIRECTION](#PRIMER_MASK_3P_DIRECTION)
+
+[PRIMER\_PAIR\_WT\_PRODUCT\_SIZE\_LT](#PRIMER_PAIR_WT_PRODUCT_SIZE_LT)
+
+[PRIMER\_FORMAMIDE\_CONC](#PRIMER_FORMAMIDE_CONC)
+
+[PRIMER\_MASK\_5P\_DIRECTION](#PRIMER_MASK_5P_DIRECTION)
+
+[PRIMER\_PAIR\_WT\_PRODUCT\_TM\_GT](#PRIMER_PAIR_WT_PRODUCT_TM_GT)
+
+[PRIMER\_GC\_CLAMP](#PRIMER_GC_CLAMP)
+
+[PRIMER\_MASK\_FAILURE\_RATE](#PRIMER_MASK_FAILURE_RATE)
+
+[PRIMER\_PAIR\_WT\_PRODUCT\_TM\_LT](#PRIMER_PAIR_WT_PRODUCT_TM_LT)
+
+[PRIMER\_INSIDE\_PENALTY](#PRIMER_INSIDE_PENALTY)
+
+[PRIMER\_MASK\_KMERLIST\_PATH](#PRIMER_MASK_KMERLIST_PATH)
+
+[PRIMER\_PAIR\_WT\_PR\_PENALTY](#PRIMER_PAIR_WT_PR_PENALTY)
+
+[PRIMER\_INTERNAL\_DMSO\_CONC](#PRIMER_INTERNAL_DMSO_CONC)
+
+[PRIMER\_MASK\_KMERLIST\_PREFIX](#PRIMER_MASK_KMERLIST_PREFIX)
+
+[PRIMER\_PAIR\_WT\_TEMPLATE\_MISPRIMING](#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING)
+
+[PRIMER\_INTERNAL\_DMSO\_FACTOR](#PRIMER_INTERNAL_DMSO_FACTOR)
+
+[PRIMER\_MASK\_TEMPLATE](#PRIMER_MASK_TEMPLATE)
+
+[PRIMER\_PAIR\_WT\_TEMPLATE\_MISPRIMING\_TH](#PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH)
+
+[PRIMER\_INTERNAL\_DNA\_CONC](#PRIMER_INTERNAL_DNA_CONC)
+
+[PRIMER\_MAX\_BOUND](#PRIMER_MAX_BOUND)
+
+[PRIMER\_PICK\_ANYWAY](#PRIMER_PICK_ANYWAY)
+
+[PRIMER\_INTERNAL\_DNTP\_CONC](#PRIMER_INTERNAL_DNTP_CONC)
+
+[PRIMER\_MAX\_END\_GC](#PRIMER_MAX_END_GC)
+
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO)
+
+[PRIMER\_INTERNAL\_FORMAMIDE\_CONC](#PRIMER_INTERNAL_FORMAMIDE_CONC)
+
+[PRIMER\_MAX\_END\_STABILITY](#PRIMER_MAX_END_STABILITY)
+
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER)
+
+[PRIMER\_INTERNAL\_MAX\_BOUND](#PRIMER_INTERNAL_MAX_BOUND)
+
+[PRIMER\_MAX\_GC](#PRIMER_MAX_GC)
+
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER)
+
+[PRIMER\_INTERNAL\_MAX\_GC](#PRIMER_INTERNAL_MAX_GC)
+
+[PRIMER\_MAX\_HAIRPIN\_TH](#PRIMER_MAX_HAIRPIN_TH)
+
+[PRIMER\_PRODUCT\_MAX\_TM](#PRIMER_PRODUCT_MAX_TM)
+
+[PRIMER\_INTERNAL\_MAX\_HAIRPIN\_TH](#PRIMER_INTERNAL_MAX_HAIRPIN_TH)
+
+[PRIMER\_MAX\_LIBRARY\_MISPRIMING](#PRIMER_MAX_LIBRARY_MISPRIMING)
+
+[PRIMER\_PRODUCT\_MIN\_TM](#PRIMER_PRODUCT_MIN_TM)
+
+[PRIMER\_INTERNAL\_MAX\_LIBRARY\_MISHYB](#PRIMER_INTERNAL_MAX_LIBRARY_MISHYB)
+
+[PRIMER\_MAX\_NS\_ACCEPTED](#PRIMER_MAX_NS_ACCEPTED)
+
+[PRIMER\_PRODUCT\_OPT\_SIZE](#PRIMER_PRODUCT_OPT_SIZE)
+
+[PRIMER\_INTERNAL\_MAX\_NS\_ACCEPTED](#PRIMER_INTERNAL_MAX_NS_ACCEPTED)
+
+[PRIMER\_MAX\_POLY\_X](#PRIMER_MAX_POLY_X)
+
+[PRIMER\_PRODUCT\_OPT\_TM](#PRIMER_PRODUCT_OPT_TM)
+
+[PRIMER\_INTERNAL\_MAX\_POLY\_X](#PRIMER_INTERNAL_MAX_POLY_X)
+
+[PRIMER\_MAX\_SELF\_ANY](#PRIMER_MAX_SELF_ANY)
+
+[PRIMER\_PRODUCT\_SIZE\_RANGE](#PRIMER_PRODUCT_SIZE_RANGE)
+
+[PRIMER\_INTERNAL\_MAX\_SELF\_ANY](#PRIMER_INTERNAL_MAX_SELF_ANY)
+
+[PRIMER\_MAX\_SELF\_ANY\_TH](#PRIMER_MAX_SELF_ANY_TH)
+
+[PRIMER\_QUALITY\_RANGE\_MAX](#PRIMER_QUALITY_RANGE_MAX)
+
+[PRIMER\_INTERNAL\_MAX\_SELF\_ANY\_TH](#PRIMER_INTERNAL_MAX_SELF_ANY_TH)
+
+[PRIMER\_MAX\_SELF\_END](#PRIMER_MAX_SELF_END)
+
+[PRIMER\_QUALITY\_RANGE\_MIN](#PRIMER_QUALITY_RANGE_MIN)
+
+[PRIMER\_INTERNAL\_MAX\_SELF\_END](#PRIMER_INTERNAL_MAX_SELF_END)
+
+[PRIMER\_MAX\_SELF\_END\_TH](#PRIMER_MAX_SELF_END_TH)
+
+[PRIMER\_SALT\_CORRECTIONS](#PRIMER_SALT_CORRECTIONS)
+
+[PRIMER\_INTERNAL\_MAX\_SELF\_END\_TH](#PRIMER_INTERNAL_MAX_SELF_END_TH)
+
+[PRIMER\_MAX\_SIZE](#PRIMER_MAX_SIZE)
+
+[PRIMER\_SALT\_DIVALENT](#PRIMER_SALT_DIVALENT)
+
+[PRIMER\_INTERNAL\_MAX\_SIZE](#PRIMER_INTERNAL_MAX_SIZE)
+
+[PRIMER\_MAX\_TEMPLATE\_MISPRIMING](#PRIMER_MAX_TEMPLATE_MISPRIMING)
+
+[PRIMER\_SALT\_MONOVALENT](#PRIMER_SALT_MONOVALENT)
+
+[PRIMER\_INTERNAL\_MAX\_TM](#PRIMER_INTERNAL_MAX_TM)
+
+[PRIMER\_MAX\_TEMPLATE\_MISPRIMING\_TH](#PRIMER_MAX_TEMPLATE_MISPRIMING_TH)
+
+[PRIMER\_SECONDARY\_STRUCTURE\_ALIGNMENT](#PRIMER_SECONDARY_STRUCTURE_ALIGNMENT)
+
+[PRIMER\_INTERNAL\_MIN\_3\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_INTERNAL_MIN_3_PRIME_OVERLAP_OF_JUNCTION)
+
+[PRIMER\_MAX\_TM](#PRIMER_MAX_TM)
+
+[PRIMER\_SEQUENCING\_ACCURACY](#PRIMER_SEQUENCING_ACCURACY)
+
+[PRIMER\_INTERNAL\_MIN\_5\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_INTERNAL_MIN_5_PRIME_OVERLAP_OF_JUNCTION)
+
+[PRIMER\_MIN\_3\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION)
+
+[PRIMER\_SEQUENCING\_INTERVAL](#PRIMER_SEQUENCING_INTERVAL)
+
+[PRIMER\_INTERNAL\_MIN\_BOUND](#PRIMER_INTERNAL_MIN_BOUND)
+
+[PRIMER\_MIN\_5\_PRIME\_OVERLAP\_OF\_JUNCTION](#PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION)
+
+[PRIMER\_SEQUENCING\_LEAD](#PRIMER_SEQUENCING_LEAD)
+
+[PRIMER\_INTERNAL\_MIN\_GC](#PRIMER_INTERNAL_MIN_GC)
+
+[PRIMER\_MIN\_BOUND](#PRIMER_MIN_BOUND)
+
+[PRIMER\_SEQUENCING\_SPACING](#PRIMER_SEQUENCING_SPACING)
+
+[PRIMER\_INTERNAL\_MIN\_QUALITY](#PRIMER_INTERNAL_MIN_QUALITY)
+
+[PRIMER\_MIN\_END\_QUALITY](#PRIMER_MIN_END_QUALITY)
+
+[PRIMER\_TASK](#PRIMER_TASK)
+
+[PRIMER\_INTERNAL\_MIN\_SIZE](#PRIMER_INTERNAL_MIN_SIZE)
+
+[PRIMER\_MIN\_GC](#PRIMER_MIN_GC)
+
+[PRIMER\_THERMODYNAMIC\_OLIGO\_ALIGNMENT](#PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT)
+
+[PRIMER\_INTERNAL\_MIN\_THREE\_PRIME\_DISTANCE](#PRIMER_INTERNAL_MIN_THREE_PRIME_DISTANCE)
+
+[PRIMER\_MIN\_LEFT\_THREE\_PRIME\_DISTANCE](#PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE)
+
+[PRIMER\_THERMODYNAMIC\_PARAMETERS\_PATH](#PRIMER_THERMODYNAMIC_PARAMETERS_PATH)
+
+[PRIMER\_INTERNAL\_MIN\_TM](#PRIMER_INTERNAL_MIN_TM)
+
+[PRIMER\_MIN\_QUALITY](#PRIMER_MIN_QUALITY)
+
+[PRIMER\_THERMODYNAMIC\_TEMPLATE\_ALIGNMENT](#PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT)
+
+[PRIMER\_INTERNAL\_MISHYB\_LIBRARY](#PRIMER_INTERNAL_MISHYB_LIBRARY)
+
+[PRIMER\_MIN\_RIGHT\_THREE\_PRIME\_DISTANCE](#PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE)
+
+[PRIMER\_TM\_FORMULA](#PRIMER_TM_FORMULA)
+
+[PRIMER\_INTERNAL\_MUST\_MATCH\_FIVE\_PRIME](#PRIMER_INTERNAL_MUST_MATCH_FIVE_PRIME)
+
+[PRIMER\_MIN\_SIZE](#PRIMER_MIN_SIZE)
+
+[PRIMER\_WT\_BOUND\_GT](#PRIMER_WT_BOUND_GT)
+
+[PRIMER\_INTERNAL\_MUST\_MATCH\_THREE\_PRIME](#PRIMER_INTERNAL_MUST_MATCH_THREE_PRIME)
+
+[PRIMER\_MIN\_THREE\_PRIME\_DISTANCE](#PRIMER_MIN_THREE_PRIME_DISTANCE)
+
+[PRIMER\_WT\_BOUND\_LT](#PRIMER_WT_BOUND_LT)
+
+[PRIMER\_INTERNAL\_OPT\_BOUND](#PRIMER_INTERNAL_OPT_BOUND)
+
+[PRIMER\_MIN\_TM](#PRIMER_MIN_TM)
+
+[PRIMER\_WT\_END\_QUAL](#PRIMER_WT_END_QUAL)
+
+[PRIMER\_INTERNAL\_OPT\_GC\_PERCENT](#PRIMER_INTERNAL_OPT_GC_PERCENT)
+
+[PRIMER\_MISPRIMING\_LIBRARY](#PRIMER_MISPRIMING_LIBRARY)
+
+[PRIMER\_WT\_END\_STABILITY](#PRIMER_WT_END_STABILITY)
+
+[PRIMER\_INTERNAL\_OPT\_SIZE](#PRIMER_INTERNAL_OPT_SIZE)
+
+[PRIMER\_MUST\_MATCH\_FIVE\_PRIME](#PRIMER_MUST_MATCH_FIVE_PRIME)
+
+[PRIMER\_WT\_GC\_PERCENT\_GT](#PRIMER_WT_GC_PERCENT_GT)
+
+[PRIMER\_INTERNAL\_OPT\_TM](#PRIMER_INTERNAL_OPT_TM)
+
+[PRIMER\_MUST\_MATCH\_THREE\_PRIME](#PRIMER_MUST_MATCH_THREE_PRIME)
+
+[PRIMER\_WT\_GC\_PERCENT\_LT](#PRIMER_WT_GC_PERCENT_LT)
+
+[PRIMER\_INTERNAL\_SALT\_DIVALENT](#PRIMER_INTERNAL_SALT_DIVALENT)
+
+[PRIMER\_NUM\_RETURN](#PRIMER_NUM_RETURN)
+
+[PRIMER\_WT\_HAIRPIN\_TH](#PRIMER_WT_HAIRPIN_TH)
+
+[PRIMER\_INTERNAL\_SALT\_MONOVALENT](#PRIMER_INTERNAL_SALT_MONOVALENT)
+
+[PRIMER\_OPT\_BOUND](#PRIMER_OPT_BOUND)
+
+[PRIMER\_WT\_LIBRARY\_MISPRIMING](#PRIMER_WT_LIBRARY_MISPRIMING)
+
+[PRIMER\_INTERNAL\_WT\_BOUND\_GT](#PRIMER_INTERNAL_WT_BOUND_GT)
+
+[PRIMER\_OPT\_GC\_PERCENT](#PRIMER_OPT_GC_PERCENT)
+
+[PRIMER\_WT\_MASK\_FAILURE\_RATE](#PRIMER_WT_MASK_FAILURE_RATE)
+
+[PRIMER\_INTERNAL\_WT\_BOUND\_LT](#PRIMER_INTERNAL_WT_BOUND_LT)
+
+[PRIMER\_OPT\_SIZE](#PRIMER_OPT_SIZE)
+
+[PRIMER\_WT\_NUM\_NS](#PRIMER_WT_NUM_NS)
+
+[PRIMER\_INTERNAL\_WT\_END\_QUAL](#PRIMER_INTERNAL_WT_END_QUAL)
+
+[PRIMER\_OPT\_TM](#PRIMER_OPT_TM)
+
+[PRIMER\_WT\_POS\_PENALTY](#PRIMER_WT_POS_PENALTY)
+
+[PRIMER\_INTERNAL\_WT\_GC\_PERCENT\_GT](#PRIMER_INTERNAL_WT_GC_PERCENT_GT)
+
+[PRIMER\_OUTSIDE\_PENALTY](#PRIMER_OUTSIDE_PENALTY)
+
+[PRIMER\_WT\_SELF\_ANY](#PRIMER_WT_SELF_ANY)
+
+[PRIMER\_INTERNAL\_WT\_GC\_PERCENT\_LT](#PRIMER_INTERNAL_WT_GC_PERCENT_LT)
+
+[PRIMER\_PAIR\_MAX\_COMPL\_ANY](#PRIMER_PAIR_MAX_COMPL_ANY)
+
+[PRIMER\_WT\_SELF\_ANY\_TH](#PRIMER_WT_SELF_ANY_TH)
+
+[PRIMER\_INTERNAL\_WT\_HAIRPIN\_TH](#PRIMER_INTERNAL_WT_HAIRPIN_TH)
+
+[PRIMER\_PAIR\_MAX\_COMPL\_ANY\_TH](#PRIMER_PAIR_MAX_COMPL_ANY_TH)
+
+[PRIMER\_WT\_SELF\_END](#PRIMER_WT_SELF_END)
+
+[PRIMER\_INTERNAL\_WT\_LIBRARY\_MISHYB](#PRIMER_INTERNAL_WT_LIBRARY_MISHYB)
+
+[PRIMER\_PAIR\_MAX\_COMPL\_END](#PRIMER_PAIR_MAX_COMPL_END)
+
+[PRIMER\_WT\_SELF\_END\_TH](#PRIMER_WT_SELF_END_TH)
+
+[PRIMER\_INTERNAL\_WT\_NUM\_NS](#PRIMER_INTERNAL_WT_NUM_NS)
+
+[PRIMER\_PAIR\_MAX\_COMPL\_END\_TH](#PRIMER_PAIR_MAX_COMPL_END_TH)
+
+[PRIMER\_WT\_SEQ\_QUAL](#PRIMER_WT_SEQ_QUAL)
+
+[PRIMER\_INTERNAL\_WT\_SELF\_ANY](#PRIMER_INTERNAL_WT_SELF_ANY)
+
+[PRIMER\_PAIR\_MAX\_DIFF\_TM](#PRIMER_PAIR_MAX_DIFF_TM)
+
+[PRIMER\_WT\_SIZE\_GT](#PRIMER_WT_SIZE_GT)
+
+[PRIMER\_INTERNAL\_WT\_SELF\_ANY\_TH](#PRIMER_INTERNAL_WT_SELF_ANY_TH)
+
+[PRIMER\_PAIR\_MAX\_LIBRARY\_MISPRIMING](#PRIMER_PAIR_MAX_LIBRARY_MISPRIMING)
+
+[PRIMER\_WT\_SIZE\_LT](#PRIMER_WT_SIZE_LT)
+
+[PRIMER\_INTERNAL\_WT\_SELF\_END](#PRIMER_INTERNAL_WT_SELF_END)
+
+[PRIMER\_PAIR\_MAX\_TEMPLATE\_MISPRIMING](#PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING)
+
+[PRIMER\_WT\_TEMPLATE\_MISPRIMING](#PRIMER_WT_TEMPLATE_MISPRIMING)
+
+[PRIMER\_INTERNAL\_WT\_SELF\_END\_TH](#PRIMER_INTERNAL_WT_SELF_END_TH)
+
+[PRIMER\_PAIR\_MAX\_TEMPLATE\_MISPRIMING\_TH](#PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH)
+
+[PRIMER\_WT\_TEMPLATE\_MISPRIMING\_TH](#PRIMER_WT_TEMPLATE_MISPRIMING_TH)
+
+[PRIMER\_INTERNAL\_WT\_SEQ\_QUAL](#PRIMER_INTERNAL_WT_SEQ_QUAL)
+
+[PRIMER\_PAIR\_WT\_COMPL\_ANY](#PRIMER_PAIR_WT_COMPL_ANY)
+
+[PRIMER\_WT\_TM\_GT](#PRIMER_WT_TM_GT)
+
+[PRIMER\_INTERNAL\_WT\_SIZE\_GT](#PRIMER_INTERNAL_WT_SIZE_GT)
+
+[PRIMER\_PAIR\_WT\_COMPL\_ANY\_TH](#PRIMER_PAIR_WT_COMPL_ANY_TH)
+
+[PRIMER\_WT\_TM\_LT](#PRIMER_WT_TM_LT)
+
+### PRIMER\_TASK (string; default generic)
+
+This tag tells Primer3 what task to perform. Legal values are:
+
+_generic_
+
+Design a primer pair (the classic Primer3 task) if the
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=1, and
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=1. In addition, pick
+an internal hybridization oligo if
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=1.
+
+NOTE: If [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=1,
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=0 and
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=1, then behaves
+similarly to [PRIMER\_TASK](#PRIMER_TASK) \=pick\_primer\_list.
+
+_pick\_detection\_primers_
+
+Deprecated alias for [PRIMER\_TASK](#PRIMER_TASK) \=generic
+
+_check\_primers_
+
+Primer3 only checks the primers provided in [SEQUENCE\_PRIMER](#SEQUENCE_PRIMER)
+, [SEQUENCE\_INTERNAL\_OLIGO](#SEQUENCE_INTERNAL_OLIGO) and
+[SEQUENCE\_PRIMER\_REVCOMP](#SEQUENCE_PRIMER_REVCOMP) . It is the only task that
+does not require a sequence. However, if
+[SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE) is provided, it is used.
+
+_pick\_primer\_list_
+
+Pick all primers in [SEQUENCE\_TEMPLATE](#SEQUENCE_TEMPLATE) (possibly limited
+by [SEQUENCE\_INCLUDED\_REGION](#SEQUENCE_INCLUDED_REGION) ,
+[SEQUENCE\_EXCLUDED\_REGION](#SEQUENCE_EXCLUDED_REGION) ,
+[SEQUENCE\_PRIMER\_PAIR\_OK\_REGION\_LIST](#SEQUENCE_PRIMER_PAIR_OK_REGION_LIST)
+, etc.). Returns the primers sorted by quality starting with the best primers.
+If [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) and
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) is selected Primer3
+does not to pick primer pairs but generates independent lists of left primers,
+right primers, and, if requested, internal oligos.
+
+_pick\_sequencing\_primers_
+
+Pick primers suited to sequence a region. [SEQUENCE\_TARGET](#SEQUENCE_TARGET)
+can be used to indicate several targets. The position of each primer is
+calculated for optimal sequencing results.
+
+_pick\_cloning\_primers_
+
+Pick primers suited to clone a gene were the start nucleotide and the end
+nucleotide of the PCR fragment must be fixed, for example to clone an ORF.
+[SEQUENCE\_INCLUDED\_REGION](#SEQUENCE_INCLUDED_REGION) must be used to indicate
+the first and the last nucleotide. Due to these limitations Primer3 can only
+vary the length of the primers. Set [PRIMER\_PICK\_ANYWAY](#PRIMER_PICK_ANYWAY)
+\=1 to obtain primers even if they violate specific constraints.
+
+_pick\_discriminative\_primers_
+
+Pick primers suited to select primers which bind with their end at a specific
+position. This can be used to force the end of a primer to a polymorphic site,
+with the goal of discriminating between sequence variants.
+[SEQUENCE\_TARGET](#SEQUENCE_TARGET) must be used to provide a single target
+indicating the last nucleotide of the left (nucleotide before the first
+nucleotide of the target) and the right primer (nucleotide following the
+target). The primers border the [SEQUENCE\_TARGET](#SEQUENCE_TARGET) . Due to
+these limitations Primer3 can only vary the length of the primers. Set
+[PRIMER\_PICK\_ANYWAY](#PRIMER_PICK_ANYWAY) \=1 to obtain primers even if they
+violate specific constraints.
+
+_pick\_pcr\_primers_
+
+Deprecated shortcut for the following settings: [PRIMER\_TASK](#PRIMER_TASK)
+\=generic [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=1
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=0
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=1
+
+WARNING: this task changes the values of
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) ,
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) , and
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) in a way that is not
+obvious by looking at the input.
+
+_pick\_pcr\_primers\_and\_hyb\_probe_
+
+Deprecated shortcut for the following settings: [PRIMER\_TASK](#PRIMER_TASK)
+\=generic [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=1
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=1
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=1
+
+WARNING: this task changes the values of
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) ,
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) , and
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) in a way that is not
+obvious by looking at the input.
+
+_pick\_left\_only_
+
+Deprecated shortcut for the following settings: [PRIMER\_TASK](#PRIMER_TASK)
+\=generic [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=1
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=0
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=0
+
+WARNING: this task changes the values of
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) ,
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) , and
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) in a way that is not
+obvious by looking at the input.
+
+_pick\_right\_only_
+
+Deprecated shortcut for the following settings: [PRIMER\_TASK](#PRIMER_TASK)
+\=generic [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=0
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=0
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=1
+
+WARNING: this task changes the values of
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) ,
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) , and
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) in a way that is not
+obvious by looking at the input.
+
+_pick\_hyb\_probe\_only_
+
+Deprecated shortcut for the following settings: [PRIMER\_TASK](#PRIMER_TASK)
+\=generic [PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) \=0
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) \=1
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) \=0
+
+WARNING: this task changes the values of
+[PRIMER\_PICK\_LEFT\_PRIMER](#PRIMER_PICK_LEFT_PRIMER) ,
+[PRIMER\_PICK\_INTERNAL\_OLIGO](#PRIMER_PICK_INTERNAL_OLIGO) , and
+[PRIMER\_PICK\_RIGHT\_PRIMER](#PRIMER_PICK_RIGHT_PRIMER) in a way that is not
+obvious by looking at the input.
+
+### PRIMER\_PICK\_LEFT\_PRIMER (boolean; default 1)
+
+If the associated value = 1 (non-0), then Primer3 will attempt to pick left
+primers.
+
+### PRIMER\_PICK\_INTERNAL\_OLIGO (boolean; default 0)
+
+If the associated value = 1 (non-0), then Primer3 will attempt to pick an
+internal oligo (hybridization probe to detect the PCR product).
+
+### PRIMER\_PICK\_RIGHT\_PRIMER (boolean; default 1)
+
+If the associated value = 1 (non-0), then Primer3 will attempt to pick a right
+primer.
+
+### PRIMER\_NUM\_RETURN (int; default 5)
+
+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.
+
+### PRIMER\_MIN\_3\_PRIME\_OVERLAP\_OF\_JUNCTION (int; default 4)
+
+The 3' end of the left OR the right primer must overlap one of the junctions in
+[SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST) by this
+amount. See details in
+[SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST) .
+
+### PRIMER\_INTERNAL\_MIN\_3\_PRIME\_OVERLAP\_OF\_JUNCTION (int; default 4)
+
+The 3' end of the middle oligo / probe must overlap one of the junctions in
+[SEQUENCE\_INTERNAL\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_INTERNAL_OVERLAP_JUNCTION_LIST)
+by this amount. See details in
+[SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST) .
+
+### PRIMER\_MIN\_5\_PRIME\_OVERLAP\_OF\_JUNCTION (int; default 7)
+
+The 5' end of the left OR the right primer must overlap one of the junctions in
+[SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST) by this
+amount. See details in
+[SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST) .
+
+### PRIMER\_INTERNAL\_MIN\_5\_PRIME\_OVERLAP\_OF\_JUNCTION (int; default 7)
+
+The 5' end of the middle oligo / probe must overlap one of the junctions in
+[SEQUENCE\_INTERNAL\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_INTERNAL_OVERLAP_JUNCTION_LIST)
+by this amount. See details in
+[SEQUENCE\_OVERLAP\_JUNCTION\_LIST](#SEQUENCE_OVERLAP_JUNCTION_LIST) .
+
+### PRIMER\_MUST\_MATCH\_FIVE\_PRIME (ambiguous nucleotide sequence; default empty)
+
+Discards all primers which do not match this match sequence at the 5' end. (New
+in v. 2.3.6, added by A. Untergasser.)
+
+The match sequence must be 5 nucletides long and can contain the following
+letters:
+
+    N   Any nucleotide
+    A   Adenine
+    G   Guanine
+    C   Cytosine
+    T   Thymine
+    R   Purine (A or G)
+    Y   Pyrimidine (C or T)
+    W   Weak (A or T)
+    S   Strong (G or C)
+    M   Amino (A or C)
+    K   Keto (G or T)
+    B   Not A (G or C or T)
+    H   Not G (A or C or T)
+    D   Not C (A or G or T)
+    V   Not T (A or G or C)
+
+Any primer which will not match the entire match sequence at the 5' end will be
+discarded and not evaluated. Setting strict requirements here will result in low
+quality primers due to the high numbers of primers discarded at this step.
+
+**Example 1:** PRIMER\_MUST\_MATCH\_FIVE\_PRIME=tgnnn
+
+Could result in the following matching:
+
+    tgcatgattggatacgtttga
+    |||||
+    tgnnn
+ -> This primer would be used.
+    attcgattctccccggtatc
+      |||
+    tgnnn
+ -> This primer would be discarded.
+
+  
+
+**Example 2:** PRIMER\_MUST\_MATCH\_FIVE\_PRIME=hnnnn
+
+Could result in the following matching:
+
+    tgcatgattggatacgtttga
+    |||||
+    hnnnn
+ -> This primer would be used.
+    ggctgatgaaggaaagcaag
+     ||||
+    hnnnn
+ -> This primer would be discarded.
+
+This parameter would force all primers selected by Primer3 to not have guanosine
+at the 5' end of any primer which could be useful to avoid quenching of
+flourochromes.
+
+### PRIMER\_INTERNAL\_MUST\_MATCH\_FIVE\_PRIME (ambiguous nucleotide sequence; default empty)
+
+Equivalent parameter of
+[PRIMER\_MUST\_MATCH\_FIVE\_PRIME](#PRIMER_MUST_MATCH_FIVE_PRIME) for the
+internal oligo.
+
+### PRIMER\_MUST\_MATCH\_THREE\_PRIME (ambiguous nucleotide sequence; default empty)
+
+Discards all primers which do not match this match sequence at the 3' end.
+Similar parameter to
+[PRIMER\_MUST\_MATCH\_FIVE\_PRIME](#PRIMER_MUST_MATCH_FIVE_PRIME) , but limits
+the 3' end. (New in v. 2.3.6, added by A. Untergasser.)
+
+The match sequence must be 5 nucletides long and can contain the following
+letters:
+
+    N   Any nucleotide
+    A   Adenine
+    G   Guanine
+    C   Cytosine
+    T   Thymine
+    R   Purine (A or G)
+    Y   Pyrimidine (C or T)
+    W   Weak (A or T)
+    S   Strong (G or C)
+    M   Amino (A or C)
+    K   Keto (G or T)
+    B   Not A (G or C or T)
+    H   Not G (A or C or T)
+    D   Not C (A or G or T)
+    V   Not T (A or G or C)
+
+Any primer which will not match the entire match sequence at the 3' end will be
+discarded and not evaluated. Setting strict requirements here will result in low
+quality primers due to the high numbers of primers discarded at this step.
+
+**Example 1:** PRIMER\_MUST\_MATCH\_FIVE\_PRIME=nnnga
+
+Could result in the following matching:
+
+    tgcatgattggatacgtttga
+                    |||||
+                    nnnga
+ -> This primer would be used.
+    attcgattctccccggtatc
+                   |||
+                   nnnga
+ -> This primer would be discarded.
+
+### PRIMER\_INTERNAL\_MUST\_MATCH\_THREE\_PRIME (ambiguous nucleotide sequence; default empty)
+
+Equivalent parameter of
+[PRIMER\_MUST\_MATCH\_THREE\_PRIME](#PRIMER_MUST_MATCH_THREE_PRIME) for the
+internal oligo.
+
+### PRIMER\_PRODUCT\_SIZE\_RANGE (size range list; default 100-300)
+
+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 product-size 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 pairs in subsequent ranges. Only if there are an
+insufficient number of primers in the first range will Primer3 return primers in
+a subsequent range.
+
+For those with primarily a computational background, the PCR product size is the
+size (in base pairs) of the DNA fragment that would be produced by the PCR
+reaction on the given sequence template. This would, of course, include the
+primers themselves.
+
+The length of [SEQUENCE\_OVERHANG\_LEFT](#SEQUENCE_OVERHANG_LEFT) and
+[SEQUENCE\_OVERHANG\_RIGHT](#SEQUENCE_OVERHANG_RIGHT) do not add to the binding
+product size of PRIMER\_PRODUCT\_SIZE\_RANGE or
+[PRIMER\_PRODUCT\_OPT\_SIZE](#PRIMER_PRODUCT_OPT_SIZE) .
+
+### PRIMER\_PRODUCT\_OPT\_SIZE (int; default 0)
+
+The optimum size for the PCR product. 0 indicates that there is no optimum
+product size. This parameter influences primer pair selection only if
+[PRIMER\_PAIR\_WT\_PRODUCT\_SIZE\_GT](#PRIMER_PAIR_WT_PRODUCT_SIZE_GT) or
+[PRIMER\_PAIR\_WT\_PRODUCT\_SIZE\_LT](#PRIMER_PAIR_WT_PRODUCT_SIZE_LT) is non-0.
+A non-0 value for this parameter will likely increase calculation time, so set
+this only if a product size near a specific value is truly important.
+
+The length of [SEQUENCE\_OVERHANG\_LEFT](#SEQUENCE_OVERHANG_LEFT) and
+[SEQUENCE\_OVERHANG\_RIGHT](#SEQUENCE_OVERHANG_RIGHT) do not add to the binding
+product size of [PRIMER\_PRODUCT\_SIZE\_RANGE](#PRIMER_PRODUCT_SIZE_RANGE) or
+PRIMER\_PRODUCT\_OPT\_SIZE.
+
+### PRIMER\_PAIR\_WT\_PRODUCT\_SIZE\_LT (float; default 0.0)
+
+Penalty weight for products shorter than
+[PRIMER\_PRODUCT\_OPT\_SIZE](#PRIMER_PRODUCT_OPT_SIZE) .
+
+### PRIMER\_PAIR\_WT\_PRODUCT\_SIZE\_GT (float; default 0.0)
+
+Penalty weight for products longer than
+[PRIMER\_PRODUCT\_OPT\_SIZE](#PRIMER_PRODUCT_OPT_SIZE) .
+
+### PRIMER\_MIN\_SIZE (int; default 18)
+
+Minimum acceptable length of a primer. Must be greater than 0 and less than or
+equal to [PRIMER\_MAX\_SIZE](#PRIMER_MAX_SIZE) .
+
+### PRIMER\_INTERNAL\_MIN\_SIZE (int; default 18)
+
+Equivalent parameter of [PRIMER\_MIN\_SIZE](#PRIMER_MIN_SIZE) for the internal
+oligo.
+
+### PRIMER\_OPT\_SIZE (int; default 20)
+
+Optimum length (in bases) of a primer. Primer3 will attempt to pick primers
+close to this length.
+
+### PRIMER\_INTERNAL\_OPT\_SIZE (int; default 20)
+
+Equivalent parameter of [PRIMER\_OPT\_SIZE](#PRIMER_OPT_SIZE) for the internal
+oligo.
+
+### PRIMER\_MAX\_SIZE (int; default 27)
+
+Maximum acceptable length (in bases) of a primer. Currently this parameter
+cannot be larger than 35. This limit is governed by maximum oligo size for which
+Primer3's melting-temperature is valid.
+
+### PRIMER\_INTERNAL\_MAX\_SIZE (int; default 27)
+
+Equivalent parameter of [PRIMER\_MAX\_SIZE](#PRIMER_MAX_SIZE) for the internal
+oligo.
+
+### PRIMER\_WT\_SIZE\_LT (float; default 1.0)
+
+Penalty weight for primers shorter than [PRIMER\_OPT\_SIZE](#PRIMER_OPT_SIZE) .
+
+### PRIMER\_INTERNAL\_WT\_SIZE\_LT (float; default 1.0)
+
+Equivalent parameter of [PRIMER\_WT\_SIZE\_LT](#PRIMER_WT_SIZE_LT) for the
+```
+
+### `log://turn_4/get/**`
+scheme=log action=get attrs={"path":"**","action":"get"}
+
+```
+MANIFEST get path="**": 31 matched (260905 tokens)
+https://benchcalc.com/primer-tm-calculator (4323)
+https://help.takarabio.com/36168/kb/article/110018/how-do-i-calculate-the-melting-temperature-tm-of-primers (384)
+https://libnano.github.io/primer3-py/api/thermoanalysis.html (6979)
+https://manpages.debian.org/stretch/primer3/oligotm.1 (1381)
+https://oligocalc.eu (11294)
+https://oligopool.com/tm-calculator (1727)
+https://primer3.org/manual.html (101235)
+https://vcru.wisc.edu/simonlab/bioinformatics/programs/primer3/primer3_manual.htm (86312)
+log://turn_1/set/unknown%3A/biology/mutagenesis/q5_kit (83)
+log://turn_1/set/unknown%3A/biology/plasmid/sequences_fasta (80)
+log://turn_1/set/unknown%3A/biology/primers/design_rules (97)
+log://turn_1/set/unknown%3A/biology/tools/primer3_oligotm (58)
+log://turn_1/set/unknown%3A/task/output/primers_fasta (53)
+log://turn_1/update/prompt_decomposed (9)
+log://turn_2/get/** (337)
+log://turn_2/get/**_1 (354)
+log://turn_2/get/app (7)
+log://turn_2/update/exploring_repo_for_sequences.fasta/_primer3/_Q5_kit_details (30)
+log://turn_3/get/** (714)
+log://turn_3/get/repo%3A/overview (183)
+log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting (1866)
+log://turn_3/update/discovering_primer3_details/_repo_manifest/_Q5_sequences (28)
+log://turn_4/get/https%3A/manpages.debian.org/stretch/primer3/oligotm.1 (31)
+log://turn_4/get/https%3A/primer3.org/manual.html (42251)
+prompt://1 (554)
+repo://overview (164)
+unknown://biology/mutagenesis/q5_kit (83)
+unknown://biology/plasmid/sequences_fasta (80)
+unknown://biology/primers/design_rules (97)
+unknown://biology/tools/primer3_oligotm (58)
+unknown://task/output/primers_fasta (53)
+```
+
+### `log://turn_4/get/**_2`
+scheme=log action=get attrs={"path":"**","action":"get"}
+
+```
+MANIFEST get path="**": 32 matched (261760 tokens)
+https://benchcalc.com/primer-tm-calculator (4323)
+https://help.takarabio.com/36168/kb/article/110018/how-do-i-calculate-the-melting-temperature-tm-of-primers (384)
+https://libnano.github.io/primer3-py/api/thermoanalysis.html (6979)
+https://manpages.debian.org/stretch/primer3/oligotm.1 (1381)
+https://oligocalc.eu (11294)
+https://oligopool.com/tm-calculator (1727)
+https://primer3.org/manual.html (101235)
+https://vcru.wisc.edu/simonlab/bioinformatics/programs/primer3/primer3_manual.htm (86312)
+log://turn_1/set/unknown%3A/biology/mutagenesis/q5_kit (83)
+log://turn_1/set/unknown%3A/biology/plasmid/sequences_fasta (80)
+log://turn_1/set/unknown%3A/biology/primers/design_rules (97)
+log://turn_1/set/unknown%3A/biology/tools/primer3_oligotm (58)
+log://turn_1/set/unknown%3A/task/output/primers_fasta (53)
+log://turn_1/update/prompt_decomposed (9)
+log://turn_2/get/** (337)
+log://turn_2/get/**_1 (354)
+log://turn_2/get/app (7)
+log://turn_2/update/exploring_repo_for_sequences.fasta/_primer3/_Q5_kit_details (30)
+log://turn_3/get/** (714)
+log://turn_3/get/repo%3A/overview (183)
+log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting (1866)
+log://turn_3/update/discovering_primer3_details/_repo_manifest/_Q5_sequences (28)
+log://turn_4/get/** (855)
+log://turn_4/get/https%3A/manpages.debian.org/stretch/primer3/oligotm.1 (31)
+log://turn_4/get/https%3A/primer3.org/manual.html (42251)
+prompt://1 (554)
+repo://overview (164)
+unknown://biology/mutagenesis/q5_kit (83)
+unknown://biology/plasmid/sequences_fasta (80)
+unknown://biology/primers/design_rules (97)
+unknown://biology/tools/primer3_oligotm (58)
+unknown://task/output/primers_fasta (53)
+```
+
+### `log://turn_4/search/neb_q5_site-directed_mutagenesis_kit_protocol_circular_plasmid_primers_minimal_p`
+scheme=log action=search attrs={"query":"neb q5 site-directed mutagenesis kit protocol circular plasmid primers minimal pairs","action":"search"}
+
+```
+8 of 12 results for "neb q5 site-directed mutagenesis kit protocol circular plasmid primers minimal pairs" (4 unreachable)
+* https://www.neb-online.de/en/pcr-and-dna-amplification/q5-site-directed-mutagenesis-kit — Q5 Site Directed Mutagenesis Kit - New England Biolabs GmbH (4109 tokens)
+  The most widely-used methods do not require any modifications or unique strains and incorporate mutations into the plasmid by inverse PCR with standard primers. For these methods, primers can be designed in either an overlapping (QuikChange, Agilent) or a back-to-back orientation (Q5 Site-Directed Mutagenesis Kit) (Figure 1). Learn more about the Q5 Site-Directed Mutagensis Kit ... Overlapping primer design results in a product that will re-circularize to form a doubly-nicked plasmid.
+* https://www.neb.com/en/products/e0554-q5-site-directed-mutagenesis-kit — Q5 Site-Directed Mutagenesis Kit | NEB (5795 tokens)
+  The Q5 Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in <strong>less than 2 hours</strong> (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions ...
+* https://support.snapgene.com/hc/en-us/articles/19181630869652-Simulate-NEB-Q5-Site-Directed-Mutagenesis — Simulate NEB Q5® Site Directed Mutagenesis (3461 tokens)
+  We recommend use of the NEB BaseChanger online tool for design of primers suitable for NEB Q5® Site Directed mutagenesis see – https://nebasechanger.neb.com · In this example, we will mutate GFP codon 65 from serine to threonine, S65T. Open a target circular plasmid sequence and identify the site/region for mutagenesis, taking note of the region coordinates if performing a deletion, or the codon number for mutation.
+* https://www.neb.com/en-us/products/e0552-q5-site-directed-mutagenesis-kit-without-competent-cells — Q5® Site-Directed Mutagenesis Kit (Without Competent Cells) (2920 tokens)
+  The Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) <strong>enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours</strong> (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, ...
+* https://www.bioke.com/webshop/neb/e0554.html — Cell Signaling Technology (CST): Antibodies, Reagents, Proteomics, Kits and Consumables (158 tokens)
+  The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency NEB 5-alpha Competent E.
+* https://www.bioke.com/webshop/neb/e0552.html — Cell Signaling Technology (CST): Antibodies, Reagents, Proteomics, Kits and Consumables (158 tokens)
+  Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemically competent cells (not provided). Figure 3: Primer design for Q5 Site-Directed Mutagenesis · Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed forward (black) and reverse (red) primers.
+* https://www.neb.com/en-us/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-protocol-e0554 — Protocol for Q5® Site-Directed Mutagenesis Kit (NEB #E0554) (1610 tokens)
+  Use this protocol to perform site-directed mutagenesis using the NEB Q5 Site-Directed Mutagenesis Kit.
+* https://www.neb.com/en/applications/cloning-and-synthetic-biology/site-directed-mutagenesis — Site Directed Mutagenesis | NEB (4871 tokens)
+  Together, these enzymes allow for rapid circularization of the PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemically competent cells (kit available with and without competent cells). Figure 2: Primer design for the Q5 Site-Directed Mutagenesis Kit · Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed forward (blue) and reverse (yellow) primers.
+```
+
+### `log://turn_4/set/known%3A/biology/tools/primer3_oligotm`
+scheme=log action=set attrs={"path":"known://biology/tools/primer3_oligotm","patch":"===================================================================\n--- known://biology/tools/primer3_oligotm\told\t\n+++ known://biology/tools/primer3_oligotm\tnew\t\n@@ -0,0 +1,17 @@\n+# Related\n+[prompt rules](prompt://1)\n+[unknown resolved](unknown://biology/tools/primer3_oligotm)\n+[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)\n+\n+# Primer3 oligotm exact usage and flags\n+Standalone tool prints melting temperature (Tm) of deoxyribonucleotide sequence to stdout.\n+\n+**Flags from rules (ground truth):**\n+- `-tp 1`: Temperature type? (pending manpage details)\n+- `-sc 1`: Salt correction?\n+- `-mv 50`: Monovalent cations 50 mM\n+- `-dv 2`: Divalent cations 2 mM\n+- `-n 0.8`: ? (pending)\n+- `-d 500`: ? (pending, DNA conc?)\n+\n+Tm computed *only* on annealing part of primer (15-45nt to input).\n\\n","merge":"<<<<<<< SEARCH\n=======\n# Related\n[prompt rules](prompt://1)\n[unknown resolved](unknown://biology/tools/primer3_oligotm)\n[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)\n\n# Primer3 oligotm exact usage and flags\nStandalone tool prints melting temperature (Tm) of deoxyribonucleotide sequence to stdout.\n\n**Flags from rules (ground truth):**\n- `-tp 1`: Temperature type? (pending manpage details)\n- `-sc 1`: Salt correction?\n- `-mv 50`: Monovalent cations 50 mM\n- `-dv 2`: Divalent cations 2 mM\n- `-n 0.8`: ? (pending)\n- `-d 500`: ? (pending, DNA conc?)\n\nTm computed *only* on annealing part of primer (15-45nt to input).\n>>>>>>> REPLACE","beforeTokens":0,"afterTokens":333,"summary":"biology,tools,primer3,oligotm,usage,flags,tm,ground_truth","action":"set"}
+
+```
+# Related
+[prompt rules](prompt://1)
+[unknown resolved](unknown://biology/tools/primer3_oligotm)
+[search primer3](log://turn_3/search/primer3_oligotm_exact_usage_flags_-tp_1_-sc_1_-mv_50_-dv_2_-n_0.8_-d_500_melting)
+
+# Primer3 oligotm exact usage and flags
+Standalone tool prints melting temperature (Tm) of deoxyribonucleotide sequence to stdout.
+
+**Flags from rules (ground truth):**
+- `-tp 1`: Temperature type? (pending manpage details)
+- `-sc 1`: Salt correction?
+- `-mv 50`: Monovalent cations 50 mM
+- `-dv 2`: Divalent cations 2 mM
+- `-n 0.8`: ? (pending)
+- `-d 500`: ? (pending, DNA conc?)
+
+Tm computed *only* on annealing part of primer (15-45nt to input).
+```
+
+### `log://turn_4/update/distilling_primer3_oligotm_%2B_discovering_Q5_kit_protocol/sequences.fasta`
+scheme=log action=update attrs={"status":155,"action":"update"}
+
+```
+distilling primer3 oligotm + discovering Q5 kit protocol/sequences.fasta
+```
+