P3_SETTINGS_FILE_USED=../settings_files/primer3web_v4_0_0_default_settings.txt
P3_FILE_TYPE=settings
PRIMER_FIRST_BASE_INDEX=1
PRIMER_TASK=generic
P3_FILE_ID=Settings for PCR amplification followed by Sanger sequencing on both strands using PCR primers
PRIMER_MIN_THREE_PRIME_DISTANCE=3
PRIMER_EXPLAIN_FLAG=1
PRIMER_MISPRIMING_LIBRARY=
PRIMER_MAX_LIBRARY_MISPRIMING=12.00
PRIMER_PAIR_MAX_LIBRARY_MISPRIMING=20.00
PRIMER_PRODUCT_SIZE_RANGE=150-250 100-300 301-400 401-500 501-600 601-700 701-850 851-1000
PRIMER_NUM_RETURN=5
PRIMER_MAX_END_STABILITY=9.0
PRIMER_MAX_SELF_ANY_TH=45.00
PRIMER_MAX_SELF_END_TH=35.00
PRIMER_PAIR_MAX_COMPL_ANY_TH=45.00
PRIMER_PAIR_MAX_COMPL_END_TH=35.00
PRIMER_MAX_HAIRPIN_TH=24.00
PRIMER_MAX_TEMPLATE_MISPRIMING_TH=40.00
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=70.00
PRIMER_MIN_SIZE=18
PRIMER_OPT_SIZE=20
PRIMER_MAX_SIZE=23
PRIMER_MIN_TM=57.0
PRIMER_OPT_TM=59.0
PRIMER_MAX_TM=62.0
PRIMER_PAIR_MAX_DIFF_TM=5.0
PRIMER_TM_FORMULA=1
PRIMER_SALT_MONOVALENT=50.0
PRIMER_SALT_CORRECTIONS=1
PRIMER_SALT_DIVALENT=1.5
PRIMER_DNTP_CONC=0.6
PRIMER_DNA_CONC=50.0
PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT=1
PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT=0
PRIMER_LOWERCASE_MASKING=0
PRIMER_MIN_GC=30.0
PRIMER_MAX_GC=70.0
PRIMER_MAX_NS_ACCEPTED=0
PRIMER_MAX_POLY_X=4
PRIMER_OUTSIDE_PENALTY=0
PRIMER_GC_CLAMP=0
PRIMER_LIBERAL_BASE=1
PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=0
PRIMER_PICK_ANYWAY=1
PRIMER_WT_TM_LT=1.0
PRIMER_WT_TM_GT=1.0
PRIMER_WT_SIZE_LT=1.0
PRIMER_WT_SIZE_GT=1.0
PRIMER_WT_GC_PERCENT_LT=0.0
PRIMER_WT_GC_PERCENT_GT=0.0
PRIMER_WT_SELF_ANY_TH=0.0
PRIMER_WT_SELF_END_TH=0.0
PRIMER_WT_HAIRPIN_TH=0.0
PRIMER_WT_NUM_NS=0.0
PRIMER_WT_LIBRARY_MISPRIMING=0.0
PRIMER_WT_SEQ_QUAL=0.0
PRIMER_WT_END_QUAL=0.0
PRIMER_WT_POS_PENALTY=0.0
PRIMER_WT_END_STABILITY=0.0
PRIMER_WT_TEMPLATE_MISPRIMING_TH=0.0
PRIMER_PAIR_WT_PRODUCT_SIZE_LT=0.0
PRIMER_PAIR_WT_PRODUCT_SIZE_GT=0.0
PRIMER_PAIR_WT_PRODUCT_TM_LT=0.0
PRIMER_PAIR_WT_PRODUCT_TM_GT=0.0
PRIMER_PAIR_WT_DIFF_TM=0.0
PRIMER_PAIR_WT_COMPL_ANY_TH=0.0
PRIMER_PAIR_WT_COMPL_END_TH=0.0
PRIMER_PAIR_WT_LIBRARY_MISPRIMING=0.0
PRIMER_PAIR_WT_PR_PENALTY=1.0
PRIMER_PAIR_WT_IO_PENALTY=0.0
PRIMER_PAIR_WT_TEMPLATE_MISPRIMING=0.0
PRIMER_INTERNAL_WT_SIZE_LT=1.0
PRIMER_INTERNAL_WT_END_QUAL=0.0
PRIMER_INTERNAL_MAX_SELF_END=12.00
PRIMER_QUALITY_RANGE_MIN=0
PRIMER_PAIR_MAX_COMPL_END=3.00
PRIMER_PRODUCT_MAX_TM=1000000.0
PRIMER_INTERNAL_MAX_SIZE=27
PRIMER_INTERNAL_WT_SELF_ANY=0.0
PRIMER_INTERNAL_MAX_POLY_X=5
PRIMER_INTERNAL_WT_SIZE_GT=1.0
PRIMER_SEQUENCING_ACCURACY=20
PRIMER_INTERNAL_WT_TM_GT=1.0
PRIMER_INTERNAL_WT_LIBRARY_MISHYB=0.0
PRIMER_INTERNAL_MAX_GC=80.0
PRIMER_PAIR_WT_COMPL_ANY=0.0
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_MAX_SELF_END=3.00
PRIMER_QUALITY_RANGE_MAX=100
PRIMER_INTERNAL_DNTP_CONC=0.0
PRIMER_INTERNAL_MIN_SIZE=18
PRIMER_INTERNAL_MIN_QUALITY=0
PRIMER_SEQUENCING_INTERVAL=250
PRIMER_INTERNAL_SALT_DIVALENT=1.5
PRIMER_MAX_SELF_ANY=8.00
PRIMER_INTERNAL_WT_SEQ_QUAL=0.0
PRIMER_PAIR_WT_COMPL_END=0.0
PRIMER_INTERNAL_OPT_TM=60.0
PRIMER_SEQUENCING_SPACING=500
PRIMER_INTERNAL_MAX_SELF_ANY=12.00
PRIMER_MIN_END_QUALITY=0
PRIMER_INTERNAL_MIN_TM=57.0
PRIMER_PAIR_MAX_COMPL_ANY=8.00
PRIMER_SEQUENCING_LEAD=50
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_INTERNAL_OPT_SIZE=20
PRIMER_WT_TEMPLATE_MISPRIMING=0.0
PRIMER_MAX_END_GC=5
PRIMER_MIN_QUALITY=0
PRIMER_INTERNAL_MAX_LIBRARY_MISHYB=12.00
PRIMER_INTERNAL_WT_GC_PERCENT_GT=0.0
PRIMER_INTERNAL_MAX_NS_ACCEPTED=0
PRIMER_WT_SELF_ANY=0.0
PRIMER_MAX_TEMPLATE_MISPRIMING=12.00
PRIMER_INTERNAL_WT_NUM_NS=0.0
PRIMER_INTERNAL_WT_SELF_END=0.0
PRIMER_PRODUCT_OPT_SIZE=0
PRIMER_PRODUCT_OPT_TM=0.0
PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=24.00
PRIMER_INSIDE_PENALTY=-1.0
PRIMER_INTERNAL_MIN_GC=20.0
PRIMER_PRODUCT_MIN_TM=-1000000.0
PRIMER_INTERNAL_SALT_MONOVALENT=50.0
PRIMER_WT_SELF_END=0.0
PRIMER_INTERNAL_DNA_CONC=50.0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_INTERNAL_MAX_TM=63.0
PRIMER_INTERNAL_WT_GC_PERCENT_LT=0.0
PRIMER_INTERNAL_WT_TM_LT=1.0
P3_SETTINGS_FILE_END=
SEQUENCE_ID=test_settings
SEQUENCE_TEMPLATE=atggatttgatggacatggatgccatcaatgacctccacttctctggacacagctccctcaccaacacccctacttccgacgaggattatggttgcacctggaaccactggtctcccatcgtcaactgggacacctttacaggtgccccagatgactttcaccacctcatggacaccatcatcgaggacagaacaactgtcctcgaacagctcagtccatctatcaccacaaccaccaccaccaccacaaccacggatgaggaagaagaagaaatggaaacaacaaccacaacaacaacaacggcaataaaaacgcatgaagttggtgatgattcaaaagggctaaaactagtgcacctattgatggctggcgcggaagccttaaccggttcaaccaaaaaccgtgacttagctcgagtgatattgatccggctcaaggaattagtctcacaacatgctaacggctccaacatggagagacttgccgctcatttcaccgaagcccttcatggactactagaaggtgctgggggtgcgcataataaccaccatcaccacaacaacaacaaacattacctcaccacaaatggtccccacgacaaccaaaacgacacacttgctgctttccaactacttcaagacatgtcaccttacgtcaaattcggccacttcacagccaatcaagccatcatcgaagccgtggcccacgaacgccgcgtccatgtcatcgactacgatatcatggaaggggtccaatgggcctcattaatccaatcacttgcttccaacaacaacggtccacatcttcggataaccgcattatcgcggacaggaaccggccggaggtcaatcgccaccgtacaagaaaccggcaggcgattaacctcctttgccgcttccctcggacaaccattttcttttcatcactgcagattagactccgatgaaactttccgtccttctgcattaaagcttgtacgtggagaggctttggttttcaactgcatgctgaatttacctcaccttagttaccgtgcaccggaatcagttgcttcgtttttaaacggagcaaaaacgttgaatccaaagcttgtgacgttggttgaagaagaagttggttctgttattggtgggtttgtggaaaggttcatggactcacttcatcattattcagcggtttttgattcattggaggctggttttccgatgcagaaccgggcccggactttggtggagagggtattttttgggcctagaattgcaggctcgttgggccggatatatagaacgggtggtgaagaggagagaaggtcatggggggagtggttaggtgaggtagggtttaggggagttccggtgagctttgcaaatcattgtcaagcaaagctgttgttagggctttttaatgatgggtatagggttgaagaggtgggtgtgggtagtaataagttggtgttggattggaaatcaagacgtttgctttctgcttccctttggacttgttcttcttcagattctgatttatag
PRIMER_LEFT_EXPLAIN=considered 7867, GC content failed 203, low tm 3099, high tm 1432, high hairpin stability 7, long poly-x seq 168, high template mispriming score 1, ok 2957
PRIMER_RIGHT_EXPLAIN=considered 7867, GC content failed 226, low tm 3298, high tm 1334, high hairpin stability 4, long poly-x seq 168, ok 2837
PRIMER_PAIR_EXPLAIN=considered 495, unacceptable product size 488, primer in pair overlaps a primer in a better pair 67, ok 7
PRIMER_LEFT_NUM_RETURNED=5
PRIMER_RIGHT_NUM_RETURNED=5
PRIMER_INTERNAL_NUM_RETURNED=0
PRIMER_PAIR_NUM_RETURNED=5
PRIMER_PAIR_0_PENALTY=0.053176
PRIMER_LEFT_0_PENALTY=0.024271
PRIMER_RIGHT_0_PENALTY=0.028905
PRIMER_LEFT_0_SEQUENCE=gacacacttgctgctttcca
PRIMER_RIGHT_0_SEQUENCE=atgtggaccgttgttgttgg
PRIMER_LEFT_0=610,20
PRIMER_RIGHT_0=804,20
PRIMER_LEFT_0_TM=58.976
PRIMER_RIGHT_0_TM=58.971
PRIMER_LEFT_0_GC_PERCENT=50.000
PRIMER_RIGHT_0_GC_PERCENT=50.000
PRIMER_LEFT_0_SELF_ANY_TH=0.00
PRIMER_RIGHT_0_SELF_ANY_TH=0.00
PRIMER_LEFT_0_SELF_END_TH=0.00
PRIMER_RIGHT_0_SELF_END_TH=0.00
PRIMER_LEFT_0_HAIRPIN_TH=0.00
PRIMER_RIGHT_0_HAIRPIN_TH=0.00
PRIMER_LEFT_0_END_STABILITY=3.5300
PRIMER_RIGHT_0_END_STABILITY=3.7700
PRIMER_LEFT_0_TEMPLATE_MISPRIMING=8.0000
PRIMER_RIGHT_0_TEMPLATE_MISPRIMING=11.0000
PRIMER_PAIR_0_COMPL_ANY_TH=0.00
PRIMER_PAIR_0_COMPL_END_TH=0.00
PRIMER_PAIR_0_PRODUCT_SIZE=195
PRIMER_PAIR_0_PRODUCT_TM=86.0
PRIMER_PAIR_0_TEMPLATE_MISPRIMING=19.00
PRIMER_PAIR_1_PENALTY=0.082329
PRIMER_LEFT_1_PENALTY=0.028905
PRIMER_RIGHT_1_PENALTY=0.053424
PRIMER_LEFT_1_SEQUENCE=ccaacaacaacggtccacat
PRIMER_RIGHT_1_SEQUENCE=aaccaaagcctctccacgta
PRIMER_LEFT_1=785,20
PRIMER_RIGHT_1=996,20
PRIMER_LEFT_1_TM=58.971
PRIMER_RIGHT_1_TM=58.947
PRIMER_LEFT_1_GC_PERCENT=50.000
PRIMER_RIGHT_1_GC_PERCENT=50.000
PRIMER_LEFT_1_SELF_ANY_TH=0.00
PRIMER_RIGHT_1_SELF_ANY_TH=0.00
PRIMER_LEFT_1_SELF_END_TH=0.00
PRIMER_RIGHT_1_SELF_END_TH=0.00
PRIMER_LEFT_1_HAIRPIN_TH=0.00
PRIMER_RIGHT_1_HAIRPIN_TH=0.00
PRIMER_LEFT_1_END_STABILITY=3.2100
PRIMER_RIGHT_1_END_STABILITY=3.5700
PRIMER_LEFT_1_TEMPLATE_MISPRIMING=10.0000
PRIMER_RIGHT_1_TEMPLATE_MISPRIMING=9.0000
PRIMER_PAIR_1_COMPL_ANY_TH=0.00
PRIMER_PAIR_1_COMPL_END_TH=0.00
PRIMER_PAIR_1_PRODUCT_SIZE=212
PRIMER_PAIR_1_PRODUCT_TM=86.5
PRIMER_PAIR_1_TEMPLATE_MISPRIMING=18.00
PRIMER_PAIR_2_PENALTY=0.106163
PRIMER_LEFT_2_PENALTY=0.081892
PRIMER_RIGHT_2_PENALTY=0.024271
PRIMER_LEFT_2_SEQUENCE=caacatggagagacttgccg
PRIMER_RIGHT_2_SEQUENCE=tggaaagcagcaagtgtgtc
PRIMER_LEFT_2=468,20
PRIMER_RIGHT_2=629,20
PRIMER_LEFT_2_TM=58.918
PRIMER_RIGHT_2_TM=58.976
PRIMER_LEFT_2_GC_PERCENT=55.000
PRIMER_RIGHT_2_GC_PERCENT=50.000
PRIMER_LEFT_2_SELF_ANY_TH=0.00
PRIMER_RIGHT_2_SELF_ANY_TH=0.00
PRIMER_LEFT_2_SELF_END_TH=0.00
PRIMER_RIGHT_2_SELF_END_TH=0.00
PRIMER_LEFT_2_HAIRPIN_TH=0.00
PRIMER_RIGHT_2_HAIRPIN_TH=0.00
PRIMER_LEFT_2_END_STABILITY=5.6900
PRIMER_RIGHT_2_END_STABILITY=3.6700
PRIMER_LEFT_2_TEMPLATE_MISPRIMING=7.0000
PRIMER_RIGHT_2_TEMPLATE_MISPRIMING=9.0000
PRIMER_PAIR_2_COMPL_ANY_TH=13.09
PRIMER_PAIR_2_COMPL_END_TH=13.09
PRIMER_PAIR_2_PRODUCT_SIZE=162
PRIMER_PAIR_2_PRODUCT_TM=85.0
PRIMER_PAIR_2_TEMPLATE_MISPRIMING=15.00
PRIMER_PAIR_3_PENALTY=0.146938
PRIMER_LEFT_3_PENALTY=0.005753
PRIMER_RIGHT_3_PENALTY=0.141185
PRIMER_LEFT_3_SEQUENCE=tgccatcaatgacctccact
PRIMER_RIGHT_3_SEQUENCE=tggtggtggttgtggtgata
PRIMER_LEFT_3=21,20
PRIMER_RIGHT_3=241,20
PRIMER_LEFT_3_TM=59.006
PRIMER_RIGHT_3_TM=58.859
PRIMER_LEFT_3_GC_PERCENT=50.000
PRIMER_RIGHT_3_GC_PERCENT=50.000
PRIMER_LEFT_3_SELF_ANY_TH=0.00
PRIMER_RIGHT_3_SELF_ANY_TH=0.00
PRIMER_LEFT_3_SELF_END_TH=0.00
PRIMER_RIGHT_3_SELF_END_TH=0.00
PRIMER_LEFT_3_HAIRPIN_TH=0.00
PRIMER_RIGHT_3_HAIRPIN_TH=0.00
PRIMER_LEFT_3_END_STABILITY=4.0000
PRIMER_RIGHT_3_END_STABILITY=2.1500
PRIMER_LEFT_3_TEMPLATE_MISPRIMING=10.0000
PRIMER_RIGHT_3_TEMPLATE_MISPRIMING=12.0000
PRIMER_PAIR_3_COMPL_ANY_TH=18.23
PRIMER_PAIR_3_COMPL_END_TH=10.86
PRIMER_PAIR_3_PRODUCT_SIZE=221
PRIMER_PAIR_3_PRODUCT_TM=87.6
PRIMER_PAIR_3_TEMPLATE_MISPRIMING=22.00
PRIMER_PAIR_4_PENALTY=0.172306
PRIMER_LEFT_4_PENALTY=0.053424
PRIMER_RIGHT_4_PENALTY=0.118882
PRIMER_LEFT_4_SEQUENCE=tacgtggagaggctttggtt
PRIMER_RIGHT_4_SEQUENCE=ttctgcatcggaaaaccagc
PRIMER_LEFT_4=977,20
PRIMER_RIGHT_4=1214,20
PRIMER_LEFT_4_TM=58.947
PRIMER_RIGHT_4_TM=59.119
PRIMER_LEFT_4_GC_PERCENT=50.000
PRIMER_RIGHT_4_GC_PERCENT=50.000
PRIMER_LEFT_4_SELF_ANY_TH=0.00
PRIMER_RIGHT_4_SELF_ANY_TH=0.00
PRIMER_LEFT_4_SELF_END_TH=0.00
PRIMER_RIGHT_4_SELF_END_TH=0.00
PRIMER_LEFT_4_HAIRPIN_TH=0.00
PRIMER_RIGHT_4_HAIRPIN_TH=0.00
PRIMER_LEFT_4_END_STABILITY=3.6700
PRIMER_RIGHT_4_END_STABILITY=4.8500
PRIMER_LEFT_4_TEMPLATE_MISPRIMING=9.0000
PRIMER_RIGHT_4_TEMPLATE_MISPRIMING=8.0000
PRIMER_PAIR_4_COMPL_ANY_TH=0.00
PRIMER_PAIR_4_COMPL_END_TH=0.00
PRIMER_PAIR_4_PRODUCT_SIZE=238
PRIMER_PAIR_4_PRODUCT_TM=83.5
PRIMER_PAIR_4_TEMPLATE_MISPRIMING=17.00
=
