a gtgggatcct
181 atgatcacta cattacctgg cctgtatctg gtgtcggtcg gtgtggtcaa acctgccagc
241 tggctccttg gatggtctga gcatgtgatc tgctccattg gggtgctcag atttgtcaat
301 cttctcttca gcgttggcaa cttctacttg ctgtatttgc tcttcaggaa ggtgcaaccc
361 agaaacaagg

Primer 3 Results

No mispriming library specified
Using 1-based sequence positions
OLIGO start len tm gc% any 3' seq
LEFT PRIMER 85 20 59.38 55.00 4.00 3.00 ggtctgagcatgtgatctgc
RIGHT PRIMER 149 20 59.58 50.00 4.00 2.00 gccaacgctgaagagaagat
SEQUENCE SIZE: 201
INCLUDED REGION SIZE: 201

PRODUCT SIZE: 65, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 4.00

1 agtgggatcctatgatcactacattacctggcctgtatctggtgtcggtcggtgtggtca

61 aacctgccagctggctccttggatggtctgagcatgtgatctgctccattggggtgctca
>>>>>>>>>>>>>>>>>>>>

121 gatttgtcaatcttctcttcagcgttggcaacttctacttgctgtatttgctcttcagga
<<<<<<<<<<<<<<<<<<<<

181 aggtgcaacccagaaacaagg

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

ADDITIONAL OLIGOS
start len tm gc% any 3' seq

1 LEFT PRIMER 48 19 59.99 57.89 6.00 4.00 gtcggtgtggtcaaacctg
RIGHT PRIMER 112 20 60.07 50.00 5.00 4.00 ccaatggagcagatcacatg
PRODUCT SIZE: 65, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00

2 LEFT PRIMER 128 21 60.00 47.62 4.00 2.00 caatcttctcttcagcgttgg
RIGHT PRIMER 200 20 60.15 50.00 4.00 2.00 cttgtttctgggttgcacct
PRODUCT SIZE: 73, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

3 LEFT PRIMER 128 21 60.00 47.62 4.00 2.00 caatcttctcttcagcgttgg
RIGHT PRIMER 199 20 60.53 45.00 4.00 0.00 ttgtttctgggttgcacctt
PRODUCT SIZE: 72, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 0.00

4 LEFT PRIMER 76 19 60.35 52.63 5.00 5.00 tccttggatggtctgagca
RIGHT PRIMER 149 20 59.58 50.00 4.00 2.00 gccaacgctgaagagaagat
PRODUCT SIZE: 74, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 2.00

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 362 0 0 0 0 0 162 145 6 0 18 9 22
Right 371 0 0 0 0 0 164 135 11 0 18 7 36
Pair Stats:
considered 254, unacceptable product size 243, tm diff too large 3, ok 8
primer3 release 1.0

Check primer in blast

LEFT PRIMER 85 20 59.38 55.00 4.00 3.00 ggtctgagcatgtgatctgc
RIGHT PRIMER 149 20 59.58 50.00 4.00 2.00 gccaacgctgaagagaagat

VALIDATED PRIMER SEQUENCES:

New Primers: Forward gtcggtgtggtcaaacctg

Reverse ccaatggagcagatcacatg