1 atgagactgt ttgttagcag aagagtcaaa agatggaaaa tttttcactt ttttgtcact
61 tgttttatat taagcttcat ggttttttgg agcccaatca ataattacat catgagccat
121 atgaagtcct actcctacag atacctcgta aatagctatg gctttgtaaa caattccctg
181 tctctcaagc acagctctgt gcagcctcac tacccatact tgatcaacca cagagagaag
241 tgtcaggctc aagatgtcct cctcttactg tttataaaga ctgcccctga aaactatggc
301 cgacgttctg caatcagaaa aacgtggggc aatgagaact atgttcagtc tcaactcaat
361 gccaacatca aaattctgtt tgcattagga actcctggtc cactgaaggg aaaagaactg
421 caaaaaagac taatcgggga agatcaagtg tacaaggata taattcagca agatttcatt
481 gattctttcc acaatcttac ttctaaattc cttcttcagt tcagctgggc aaataccttt
541 tgtccacatg ccaaattcct gatgactgct gatgatgata tatttatcca catgccaaat
601 ctcattgaat atcttcaagg gctagagcag attggagttc gagacttttg gattggtcac
661 gttcatcgag gtggccctcc tgttagggat aaaagcagca aatactatgt tccctatgaa
721 atgtacaagt ggccagccta ccctgactat acagctggtg ctgcctatgt tgtctccaga
781 gatgtagctg ccaaaatcta tgaggcatcg cagacactga attccagtat gtacatagat
841 gatgtattca tgggcctctg tgccaataaa gtgggaatct tgccacagga ccatgtattt
901 ttctctgggg aagggaaaat tccttatcac ccctgcatct atgaaaagat gatgacatct
961 cacggacact tacaagatct gcaggacctc tggatagagg ctacacatcc taaagtaaag
1021 aacatttcaa aagggttttt tggtcaaata tactgcaggt taattaagat agttcttctc
1081 tgcagactga cttacaggaa ttcataccct tgttgggctg catttgctta g

Primer3 Output

WARNING: Numbers in input sequence were deleted.

No mispriming library specified
Using 1-based sequence positions
OLIGO start len tm gc% any 3' seq
LEFT PRIMER 750 20 60.03 55.00 6.00 2.00 tacagctggtgctgcctatg
RIGHT PRIMER 824 20 59.40 50.00 6.00 2.00 gaattcagtgtctgcgatgc
SEQUENCE SIZE: 1131
INCLUDED REGION SIZE: 1131

PRODUCT SIZE: 75, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 1.00

1 atgagactgtttgttagcagaagagtcaaaagatggaaaatttttcacttttttgtcact

61 tgttttatattaagcttcatggttttttggagcccaatcaataattacatcatgagccat

121 atgaagtcctactcctacagatacctcgtaaatagctatggctttgtaaacaattccctg

181 tctctcaagcacagctctgtgcagcctcactacccatacttgatcaaccacagagagaag

241 tgtcaggctcaagatgtcctcctcttactgtttataaagactgcccctgaaaactatggc

301 cgacgttctgcaatcagaaaaacgtggggcaatgagaactatgttcagtctcaactcaat

361 gccaacatcaaaattctgtttgcattaggaactcctggtccactgaagggaaaagaactg

421 caaaaaagactaatcggggaagatcaagtgtacaaggatataattcagcaagatttcatt

481 gattctttccacaatcttacttctaaattccttcttcagttcagctgggcaaataccttt

541 tgtccacatgccaaattcctgatgactgctgatgatgatatatttatccacatgccaaat

601 ctcattgaatatcttcaagggctagagcagattggagttcgagacttttggattggtcac

661 gttcatcgaggtggccctcctgttagggataaaagcagcaaatactatgttccctatgaa

721 atgtacaagtggccagcctaccctgactatacagctggtgctgcctatgttgtctccaga
>>>>>>>>>>>>>>>>>>>>

781 gatgtagctgccaaaatctatgaggcatcgcagacactgaattccagtatgtacatagat
<<<<<<<<<<<<<<<<<<<<

841 gatgtattcatgggcctctgtgccaataaagtgggaatcttgccacaggaccatgtattt

901 ttctctggggaagggaaaattccttatcacccctgcatctatgaaaagatgatgacatct

961 cacggacacttacaagatctgcaggacctctggatagaggctacacatcctaaagtaaag

1021 aacatttcaaaagggttttttggtcaaatatactgcaggttaattaagatagttcttctc

1081 tgcagactgacttacaggaattcatacccttgttgggctgcatttgcttag

KEYS (in order of precedence):
>>>>>> left primer
<<<<<< right primer

ADDITIONAL OLIGOS
start len tm gc% any 3' seq

1 LEFT PRIMER 588 20 59.92 45.00 4.00 3.00 ccacatgccaaatctcattg
RIGHT PRIMER 667 20 59.35 45.00 4.00 0.00 gatgaacgtgaccaatccaa
PRODUCT SIZE: 80, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 3.00

2 LEFT PRIMER 750 20 60.03 55.00 6.00 2.00 tacagctggtgctgcctatg
RIGHT PRIMER 825 20 59.25 50.00 6.00 0.00 ggaattcagtgtctgcgatg
PRODUCT SIZE: 76, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00

3 LEFT PRIMER 588 20 59.92 45.00 4.00 3.00 ccacatgccaaatctcattg
RIGHT PRIMER 670 20 59.09 50.00 4.00 0.00 ctcgatgaacgtgaccaatc
PRODUCT SIZE: 83, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 3.00

4 LEFT PRIMER 588 20 59.92 45.00 4.00 3.00 ccacatgccaaatctcattg
RIGHT PRIMER 671 20 60.92 50.00 4.00 2.00 cctcgatgaacgtgaccaat
PRODUCT SIZE: 84, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 4.00

5 LEFT PRIMER 654 19 60.10 52.63 4.00 2.00 tggtcacgttcatcgaggt
RIGHT PRIMER 738 20 60.00 50.00 6.00 4.00 ggctggccacttgtacattt
PRODUCT SIZE: 85, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00

6 LEFT PRIMER 655 19 60.10 57.89 4.00 4.00 ggtcacgttcatcgaggtg
RIGHT PRIMER 738 20 60.00 50.00 6.00 4.00 ggctggccacttgtacattt
PRODUCT SIZE: 84, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

7 LEFT PRIMER 588 20 59.92 45.00 4.00 3.00 ccacatgccaaatctcattg
RIGHT PRIMER 668 19 59.91 52.63 4.00 0.00 cgatgaacgtgaccaatcc
PRODUCT SIZE: 81, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00

8 LEFT PRIMER 588 20 59.92 45.00 4.00 3.00 ccacatgccaaatctcattg
RIGHT PRIMER 672 19 60.10 52.63 4.00 2.00 acctcgatgaacgtgacca
PRODUCT SIZE: 85, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00

9 LEFT PRIMER 557 21 59.80 47.62 3.00 1.00 tcctgatgactgctgatgatg
RIGHT PRIMER 635 20 59.97 55.00 4.00 2.00 ccaatctgctctagcccttg
PRODUCT SIZE: 79, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00

Statistics
con too in in no tm tm high high high
sid many tar excl bad GC too too any 3' poly end
ered Ns get reg GC% clamp low high compl compl X stab ok
Left 2492 0 0 0 6 0 1713 246 5 3 346 28 145
Right 2576 0 0 0 2 0 1806 280 2 0 301 27 158
Pair Stats:
considered 5808, unacceptable product size 5785, tm diff too large 5, ok 18
primer3 release 1.0

Sequence to submit for M-fold: OK
tacagctggtgctgcctatgttgtctccaga
gatgtagctgccaaaatctatgaggcatcgcagacactgaattccagtat

Primers to test: worked

VALIDATED PRIMER:

LEFT PRIMER 750 20 60.03 55.00 6.00 2.00 tacagctggtgctgcctatg
RIGHT PRIMER 824 20 59.40 50.00 6.00 2.00 gaattcagtgtctgcgatgc