ACGT Tm = -35.4345 (hand ~ -35.43)
AAAA Tm = -65.2419 (hand ~ -65.24)

junction at input index 215 ; insert = tagattagaagaagaattaagaagaagattaacagaaag len 39
upstream tail: ttaactttaagaaggagatatacatatgag
downstream head: caagggcgaggagctgttcaccggggtggt

coincidental 5' extension of fwd anneal into insert: 2 bp

Scan of candidate annealing lengths:
Lf  fwdSeq(anneal, intended)  Tm(intended)  Tm(effective=Lf+ext)   |  Lr  revSeq(anneal)  Tm(rev)
15  caagggcgaggagct  59.54  65.14   |
16  caagggcgaggagctg  61.24  66.27   |
17  caagggcgaggagctgt  63.28  67.95   |
18  caagggcgaggagctgtt  63.81  68.19   |
19  caagggcgaggagctgttc  64.67  68.71   |
20  caagggcgaggagctgttca  66.29  70.07   |
21  caagggcgaggagctgttcac  67.18  70.69   |
22  caagggcgaggagctgttcacc  69.15  72.39   |
23  caagggcgaggagctgttcaccg  71.14  74.08   |
24  caagggcgaggagctgttcaccgg  72.79  75.53   |
25  caagggcgaggagctgttcaccggg  74.34  76.88   |
26  caagggcgaggagctgttcaccgggg  75.77  78.15   |
---
15  ctcatatgtatatct  37.97  3'end=t
16  ctcatatgtatatctc  41.09  3'end=c
17  ctcatatgtatatctcc  45.08  3'end=c
18  ctcatatgtatatctcct  47.34  3'end=t
19  ctcatatgtatatctcctt  48.71  3'end=t
20  ctcatatgtatatctccttc  50.53  3'end=c
21  ctcatatgtatatctccttct  52.30  3'end=t
22  ctcatatgtatatctccttctt  53.25  3'end=t
23  ctcatatgtatatctccttctta  53.33  3'end=a
24  ctcatatgtatatctccttcttaa  54.16  3'end=a
25  ctcatatgtatatctccttcttaaa  54.92  3'end=a
26  ctcatatgtatatctccttcttaaag  56.01  3'end=g
