>test_sequence aatacTCCCTCCGTCCCTAAATATTTGACGCCGTTgactttttaaaacatatttgaccgttcgtcttattcaaaaaaattaagtaattattaattcctttcctatcatttgattcattgttaaatatacttttatgtatacatatagttttacatatttcacaaaagtttttgaataagacgaacggtcaaacatgtttaaaaaagtcAACGGCGTCAAATATTTAGGGAAGGAGGGAgtagcttacctcgcttgtcctcaagcgaaaaccaagtgcttacctcgcttgtcctcaagcgaaaaccaatccacaaacatgttcacatgtttggggacgaaggtgtcgataaaacataatacggacatgagggcatcatgatcacacatagaacagcaatatatcataaatattcttatgggaaagtaacaattccttcagaaagcaaagcatgaagcaaaaaccttaccgagaagtaaccaacaacagtccatagtcattgaagcaattgtaatttatcacaacatcagaaagagtcaaataagagcttgtaaggcaaacccacatactcaatcatctcttttgttttccacaattcttacaactcacgtggtactcatggtgtcaaagtttcagctcagaggaagataggggcttatagttttgcctcccaacggtttacctcaagggtaaagtcaacaacaataaaacatgagttactccctccgttccaaaatagatgactcaactttgtactaactttagtacaaagttagtataaggttgggtcatctattttggaacggatgtcgcgaccttatgagccgacgaaaccatcatcttagggtggcgcttgagctttctaattcgcccatgtggatagatagatagatagatagatagatagatagatagatagatatcattcctgtcacgaccttatgagccgacgaaaccatcatctatggtggcgcttgagctttctgactcgccccatcctgtcgcgaccttatgagccgacgaatccgtcagcttatggtggcgcttgagctttctgactcgccccatcctgtcgcgaccttatgagaacgctgcgacgccgttgactttttaaagcagcgaccgacgaaaccatcatctt _______________________________ In silico Primer(s) search for: 65 5'-tccctccgtccctaaatatttgacgccgtt Position: 6->35 30bp 100% Tm = 63.7°C 5-tccctccgtccctaaatatttgacgccgtt-> |||||||||||||||||||||||||||||| tgagggaggcagggatttataaactgcggcaactga Position: 209<-238 30bp 97% Tm = 61.9°C <-ttgccgcagtttataaatccctgcctccct-5 |||||||||||||||||||||| ||||||| tcaacggcgtcaaatatttagggaaggagggagtag 65 5'-tccctccgtccctaaatatttgacgccgtt 65 5'-tccctccgtccctaaatatttgacgccgtt PCR product size: 233bp Here Primo Pro 3.4: PCR Primer Design Output: Direction 5'-Pos Tm Primer Sequence (5'->3') Product Length Forward 5 65 TCCCTCCGTCCCTAAATATTTGACGCCGTT 1135 ******************** Reverse 1139 65 AAGATGATGGTTTCGTCGGTCGCTGCT Forward 5 65 TCCCTCCGTCCCTAAATATTTGACGCCGTT 1135 Reverse 1139 65 AAGATGATGGTTTCGTCGGTCGCTGCTTT Forward 5 65 TCCCTCCGTCCCTAAATATTTGACGCCGTT 1134 Reverse 1138 65 AGATGATGGTTTCGTCGGTCGCTGCT Forward 5 65 TCCCTCCGTCCCTAAATATTTGACGCCGTT 976 Reverse 980 64 AAGCTCAAGCGCCACCATAGATGATGGT Forward 5 65 TCCCTCCGTCCCTAAATATTTGACGCCGTT 330 Reverse 334 64 CCCCAAACATGTGAACATGTTTGTGGATTGGT Forward 245 64 ACCTCGCTTGTCCTCAAGCGAAAACC 856 Reverse 1100 66 CGGCGTCGCAGCGTTCTCATAAGGTC Forward 245 64 ACCTCGCTTGTCCTCAAGCGAAAACC 810 Reverse 1054 65 AGAAAGCTCAAGCGCCACCATAAGCTGAC Forward 245 64 ACCTCGCTTGTCCTCAAGCGAAAACC 706 Reverse 950 65 CGTCGGCTCATAAGGTCGTGACAGGAATGA Forward 245 64 ACCTCGCTTGTCCTCAAGCGAAAACC 95 Reverse 339 65 TTCGTCCCCAAACATGTGAACATGTTTGTGGA Forward 272 66 AGTGCTTACCTCGCTTGTCCTCAAGCGAAAAC 829 Reverse 1100 66 CGGCGTCGCAGCGTTCTCATAAGGTC Forward 272 66 AGTGCTTACCTCGCTTGTCCTCAAGCGAAAAC 783 Reverse 1054 65 AGAAAGCTCAAGCGCCACCATAAGCTGAC Forward 272 66 AGTGCTTACCTCGCTTGTCCTCAAGCGAAAAC 679 Reverse 950 65 CGTCGGCTCATAAGGTCGTGACAGGAATGA Forward 272 66 AGTGCTTACCTCGCTTGTCCTCAAGCGAAAAC 68 Reverse 339 65 TTCGTCCCCAAACATGTGAACATGTTTGTGGA Forward 311 64 CAAACATGTTCACATGTTTGGGGACGAAGGT 709 Reverse 1019 64 GTCGGCTCATAAGGTCGCGACAGG Forward 311 64 CAAACATGTTCACATGTTTGGGGACGAAGGT 671 Reverse 981 64 AAAGCTCAAGCGCCACCATAGATGATGGTT Forward 311 64 CAAACATGTTCACATGTTTGGGGACGAAGGT 386 Reverse 696 64 TGACTTTACCCTTGAGGTAAACCGTTGGGAG Forward 784 65 GGTCATCTATTTTGGAACGGATGTCGCGACC 356 Reverse 1139 65 AAGATGATGGTTTCGTCGGTCGCTGCT Forward 784 65 GGTCATCTATTTTGGAACGGATGTCGCGACC 198 Reverse 981 64 AAAGCTCAAGCGCCACCATAGATGATGGTT Forward 784 65 GGTCATCTATTTTGGAACGGATGTCGCGACC 167 Reverse 950 65 CGTCGGCTCATAAGGTCGTGACAGGAATGA Forward 784 65 GGTCATCTATTTTGGAACGGATGTCGCGACC -444 Reverse 339 65 TTCGTCCCCAAACATGTGAACATGTTTGTGGA Forward 784 65 GGTCATCTATTTTGGAACGGATGTCGCGACC -496 Reverse 287 64 AGCGAGGTAAGCACTTGGTTTTCGCTT Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG 324 Reverse 1139 65 AAGATGATGGTTTCGTCGGTCGCTGCT Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG 204 Reverse 1019 64 GTCGGCTCATAAGGTCGCGACAGG Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG 166 Reverse 981 64 AAAGCTCAAGCGCCACCATAGATGATGGTT Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG 135 Reverse 950 65 CGTCGGCTCATAAGGTCGTGACAGGAATGA Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG 38 Reverse 853 64 AAGCGCCACCCTAAGATGATGGTTTCGT Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG -119 Reverse 696 64 TGACTTTACCCTTGAGGTAAACCGTTGGGAG Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG -476 Reverse 339 65 TTCGTCCCCAAACATGTGAACATGTTTGTGGA Forward 816 65 TATGAGCCGACGAAACCATCATCTTAGGGTGG -528 Reverse 287 64 AGCGAGGTAAGCACTTGGTTTTCGCTT Forward 921 65 CATTCCTGTCACGACCTTATGAGCCGACGA 99 Reverse 1019 64 GTCGGCTCATAAGGTCGCGACAGG Forward 921 65 CATTCCTGTCACGACCTTATGAGCCGACGA 30 Reverse 950 65 CGTCGGCTCATAAGGTCGTGACAGGAATGA Forward 921 65 CATTCCTGTCACGACCTTATGAGCCGACGA -224 Reverse 696 64 TGACTTTACCCTTGAGGTAAACCGTTGGGAG Forward 921 65 CATTCCTGTCACGACCTTATGAGCCGACGA -581 Reverse 339 65 TTCGTCCCCAAACATGTGAACATGTTTGTGGA Forward 947 64 ACGAAACCATCATCTATGGTGGCGCTTGA 4 Reverse 950 65 CGTCGGCTCATAAGGTCGTGACAGGAATGA Forward 947 64 ACGAAACCATCATCTATGGTGGCGCTTGA -250 Reverse 696 64 TGACTTTACCCTTGAGGTAAACCGTTGGGAG Forward 947 64 ACGAAACCATCATCTATGGTGGCGCTTGA -607 Reverse 339 65 TTCGTCCCCAAACATGTGAACATGTTTGTGGA Forward 995 66 CCTGTCGCGACCTTATGAGCCGACGAA 60 Reverse 1054 65 AGAAAGCTCAAGCGCCACCATAAGCTGAC Forward 995 66 CCTGTCGCGACCTTATGAGCCGACGAA 25 Reverse 1019 64 GTCGGCTCATAAGGTCGCGACAGG Forward 995 66 CCTGTCGCGACCTTATGAGCCGACGAA -44 Reverse 950 65 CGTCGGCTCATAAGGTCGTGACAGGAATGA Forward 995 66 CCTGTCGCGACCTTATGAGCCGACGAA -298 Reverse 696 64 TGACTTTACCCTTGAGGTAAACCGTTGGGAG Forward 995 66 CCTGTCGCGACCTTATGAGCCGACGAA -655 Reverse 339 65 TTCGTCCCCAAACATGTGAACATGTTTGTGGA Forward 1025 65 GTCAGCTTATGGTGGCGCTTGAGCTTTCT 115 Reverse 1139 65 AAGATGATGGTTTCGTCGGTCGCTGCT Forward 1025 65 GTCAGCTTATGGTGGCGCTTGAGCTTTCT 30 Reverse 1054 65 AGAAAGCTCAAGCGCCACCATAAGCTGAC Forward 1025 65 GTCAGCTTATGGTGGCGCTTGAGCTTTCT -171 Reverse 853 64 AAGCGCCACCCTAAGATGATGGTTTCGT Forward 1025 65 GTCAGCTTATGGTGGCGCTTGAGCTTTCT -737 Reverse 287 64 AGCGAGGTAAGCACTTGGTTTTCGCTT