1PRIMER PICKING RESULTS FOR boundary_test_00 2 3No mispriming library specified 4No internal oligo mishyb library specified 5Using 1-based sequence positions 6OLIGO start len tm gc% any 3' seq 7LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 8RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 9INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 10SEQUENCE SIZE: 84 11INCLUDED REGION SIZE: 84 12 13PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 14 15 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 16 >>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 17 18 61 CACTGTCATTTCCAAACAATTCNN 19 <<<<<<<<<<<<<<<<<<<<<< 20 21KEYS (in order of precedence): 22>>>>>> left primer 23<<<<<< right primer 24^^^^^^ internal oligo 25 26 27Statistics 28 con too in in not no tm tm high high high 29 sid many tar excl ok bad GC too too any 3' poly end 30 ered Ns get reg reg GC% clamp low high compl compl X stab ok 31Left 35 33 0 0 0 0 0 0 0 0 0 0 0 2 32Right 35 33 0 0 0 0 0 0 0 0 0 0 0 2 33Intl 60 57 0 0 0 0 0 0 0 0 0 0 0 3 34Pair Stats: 35considered 4, unacceptable product size 1, no internal oligo 2, ok 1 36primer3 release 2.0.0 37 38 39PRIMER PICKING RESULTS FOR boundary_test_01 40 41No mispriming library specified 42No internal oligo mishyb library specified 43Using 1-based sequence positions 44OLIGO start len tm gc% any 3' seq 45LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 46RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 47INTERNAL OLIGO 30 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 48SEQUENCE SIZE: 84 49INCLUDED REGION SIZE: 84 50 51PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 52 53 1 NNCACGTCATCTCATCAAATTTGATTGNNCACGTCATCTCATCAAATTTGATTGNNNACT 54 >>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 55 56 61 CACTGTCATTTCCAAACAATTCNN 57 <<<<<<<<<<<<<<<<<<<<<< 58 59KEYS (in order of precedence): 60>>>>>> left primer 61<<<<<< right primer 62^^^^^^ internal oligo 63 64 65Statistics 66 con too in in not no tm tm high high high 67 sid many tar excl ok bad GC too too any 3' poly end 68 ered Ns get reg reg GC% clamp low high compl compl X stab ok 69Left 30 28 0 0 0 0 0 0 0 0 0 0 0 2 70Right 30 29 0 0 0 0 0 0 0 0 0 0 0 1 71Intl 60 57 0 0 0 0 0 0 0 0 0 0 0 3 72Pair Stats: 73considered 2, unacceptable product size 1, ok 1 74primer3 release 2.0.0 75 76 77PRIMER PICKING RESULTS FOR boundary_test_02 78 79No mispriming library specified 80No internal oligo mishyb library specified 81Using 1-based sequence positions 82OLIGO start len tm gc% any 3' seq 83LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 84RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 85INTERNAL OLIGO 30 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 86SEQUENCE SIZE: 84 87INCLUDED REGION SIZE: 84 88 89PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 90 91 1 NNCACGTCATCTCATCAAATTTGATTGNNCACGTCATCTCATCAAATTTGATTGNNNACT 92 >>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 93 94 61 CACTGTCATTTCCAAACAATTCNN 95 <<<<<<<<<<<<<<<<<<<<<< 96 97KEYS (in order of precedence): 98>>>>>> left primer 99<<<<<< right primer 100^^^^^^ internal oligo 101 102 103Statistics 104 con too in in not no tm tm high high high 105 sid many tar excl ok bad GC too too any 3' poly end 106 ered Ns get reg reg GC% clamp low high compl compl X stab ok 107Left 5 4 0 0 0 0 0 0 0 0 0 0 0 1 108Right 5 4 0 0 0 0 0 0 0 0 0 0 0 1 109Intl 60 57 0 0 0 0 0 0 0 0 0 0 0 3 110Pair Stats: 111considered 1, ok 1 112primer3 release 2.0.0 113 114 115PRIMER PICKING RESULTS FOR boundary_test_05 116 117No mispriming library specified 118No internal oligo mishyb library specified 119Using 1-based sequence positions 120OLIGO start len tm gc% any 3' seq 121LEFT PRIMER 3 24 60.24 33.33 12.00 10.00 CACGTCATCTCATCAAATTTGATT 122RIGHT PRIMER 82 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 123INTERNAL OLIGO 30 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 124SEQUENCE SIZE: 84 125INCLUDED REGION SIZE: 84 126 127PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 2.00 128 129 1 NNCACGTCATCTCATCAAATTTGATTGNNCACGTCATCTCATCAAATTTGATTGNNNACT 130 >>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ << 131 132 61 CACTGTCATTTCCAAACAATTCNN 133 <<<<<<<<<<<<<<<<<<<<<< 134 135KEYS (in order of precedence): 136>>>>>> left primer 137<<<<<< right primer 138^^^^^^ internal oligo 139 140 141Statistics 142 con too in in not no tm tm high high high 143 sid many tar excl ok bad GC too too any 3' poly end 144 ered Ns get reg reg GC% clamp low high compl compl X stab ok 145Left 5 3 0 0 0 0 0 0 0 0 0 0 0 2 146Right 5 3 0 0 0 0 0 0 0 0 0 0 0 2 147Intl 60 57 0 0 0 0 0 0 0 0 0 0 0 3 148Pair Stats: 149considered 4, unacceptable product size 3, ok 1 150primer3 release 2.0.0 151 152 153PRIMER PICKING RESULTS FOR boundary_test_11 154 155No mispriming library specified 156No internal oligo mishyb library specified 157Using 1-based sequence positions 158NO PRIMERS FOUND 159 160SEQUENCE SIZE: 84 161INCLUDED REGION SIZE: 84 162 163 164 1 NNCACGTCATCTNATCAAATTTGATTGNNCACGTCATCTNATCAAATTTGATTGNNNACT 165 61 CACTGTCATTTCCAAACAATTCNN 166 167 168Statistics 169 con too in in not no tm tm high high high 170 sid many tar excl ok bad GC too too any 3' poly end 171 ered Ns get reg reg GC% clamp low high compl compl X stab ok 172Left 5 3 0 0 0 0 0 0 0 0 0 0 0 2 173Right 5 1 0 0 0 0 0 0 0 0 0 0 0 4 174Intl 60 59 0 0 0 0 0 0 0 0 0 0 0 1 175Pair Stats: 176considered 8, unacceptable product size 6, no internal oligo 2, ok 0 177primer3 release 2.0.0 178 179 180PRIMER PICKING RESULTS FOR boundary_test_11 181 182No mispriming library specified 183No internal oligo mishyb library specified 184Using 1-based sequence positions 185OLIGO start len tm gc% any 3' seq 186LEFT PRIMER 3 24 60.81 31.82 12.00 11.50 CACGTCATCNNATCAAATTTGATT 187RIGHT PRIMER 82 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 188INTERNAL OLIGO 30 25 63.22 34.78 12.00 11.00 CACGTCATCNNATCAAATTTGATTG 189SEQUENCE SIZE: 84 190INCLUDED REGION SIZE: 84 191 192PRODUCT SIZE: 80, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 2.00 193 194 1 NNCACGTCATCNNATCAAATTTGATTGNNCACGTCATCNNATCAAATTTGATTGNNNACT 195 >>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ << 196 197 61 CACTGTCATTTCCAAACAATTCNN 198 <<<<<<<<<<<<<<<<<<<<<< 199 200KEYS (in order of precedence): 201>>>>>> left primer 202<<<<<< right primer 203^^^^^^ internal oligo 204 205ADDITIONAL OLIGOS 206 start len tm gc% any 3' seq 207 208 1 LEFT PRIMER 4 24 60.81 31.82 12.00 11.00 ACGTCATCNNATCAAATTTGATTG 209 RIGHT PRIMER 83 24 61.74 34.78 4.00 3.00 NGAATTGTTTGGAAATGACAGTGA 210 INTERNAL OLIGO 30 25 63.22 34.78 12.00 11.00 CACGTCATCNNATCAAATTTGATTG 211 PRODUCT SIZE: 80, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00 212 213Statistics 214 con too in in not no tm tm high high high 215 sid many tar excl ok bad GC too too any 3' poly end 216 ered Ns get reg reg GC% clamp low high compl compl X stab ok 217Left 5 3 0 0 0 0 0 0 0 0 0 0 0 2 218Right 5 0 0 0 0 0 0 0 0 0 0 0 0 5 219Intl 60 52 0 0 0 0 0 0 0 0 0 0 0 8 220Pair Stats: 221considered 10, unacceptable product size 8, ok 2 222primer3 release 2.0.0 223 224 225PRIMER PICKING RESULTS FOR boundary_test_12 226 227No mispriming library specified 228No internal oligo mishyb library specified 229Using 1-based sequence positions 230OLIGO start len tm gc% any 3' seq 231LEFT PRIMER 3 24 60.81 31.82 12.00 11.50 CACGTCATCNNATCAAATTTGATT 232RIGHT PRIMER 82 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 233INTERNAL OLIGO 30 25 63.22 34.78 12.00 11.00 CACGTCATCNNATCAAATTTGATTG 234SEQUENCE SIZE: 84 235INCLUDED REGION SIZE: 84 236 237PRODUCT SIZE: 80, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 2.00 238 239 1 NNCACGTCATCNNATCAAATTTGATTGNNCACGTCATCNNATCAAATTTGATTGNNNACT 240 >>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ << 241 242 61 CACTGTCATTTCCAAACAATTCNN 243 <<<<<<<<<<<<<<<<<<<<<< 244 245KEYS (in order of precedence): 246>>>>>> left primer 247<<<<<< right primer 248^^^^^^ internal oligo 249 250ADDITIONAL OLIGOS 251 start len tm gc% any 3' seq 252 253 1 LEFT PRIMER 4 24 60.81 31.82 12.00 11.00 ACGTCATCNNATCAAATTTGATTG 254 RIGHT PRIMER 83 24 61.74 34.78 4.00 3.00 NGAATTGTTTGGAAATGACAGTGA 255 INTERNAL OLIGO 30 25 63.22 34.78 12.00 11.00 CACGTCATCNNATCAAATTTGATTG 256 PRODUCT SIZE: 80, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 2.00 257 258Statistics 259 con too in in not no tm tm high high high 260 sid many tar excl ok bad GC too too any 3' poly end 261 ered Ns get reg reg GC% clamp low high compl compl X stab ok 262Left 5 3 0 0 0 0 0 0 0 0 0 0 0 2 263Right 5 0 0 0 0 0 0 0 0 0 0 0 0 5 264Intl 60 52 0 0 0 0 0 0 0 0 0 0 0 8 265Pair Stats: 266considered 10, unacceptable product size 8, ok 2 267primer3 release 2.0.0 268 269 270PRIMER PICKING RESULTS FOR boundary_test_13 271 272No mispriming library specified 273No internal oligo mishyb library specified 274Using 1-based sequence positions 275OLIGO start len tm gc% any 3' seq 276LEFT PRIMER 3 24 54.69 0.00 0.00 0.00 AAAAAAAAAAAAAAAAAAAAAAAA 277RIGHT PRIMER 82 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 278INTERNAL OLIGO 32 25 56.20 0.00 0.00 0.00 TTTTTTTTTTTTTTTTTTTTTTTNN 279SEQUENCE SIZE: 84 280INCLUDED REGION SIZE: 84 281 282PRODUCT SIZE: 80, PAIR ANY COMPL: 4.00, PAIR 3' COMPL: 0.00 283 284 1 NNAAAAAAAAAAAAAAAAAAAAAAAAANNTTTTTTTTTTTTTTTTTTTTTTTTTNNNACT 285 >>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ << 286 287 61 CACTGTCATTTCCAAACAATTCNN 288 <<<<<<<<<<<<<<<<<<<<<< 289 290KEYS (in order of precedence): 291>>>>>> left primer 292<<<<<< right primer 293^^^^^^ internal oligo 294 295 296Statistics 297 con too in in not no tm tm high high high 298 sid many tar excl ok bad GC too too any 3' poly end 299 ered Ns get reg reg GC% clamp low high compl compl X stab ok 300Left 5 3 0 0 0 0 0 0 0 0 0 0 0 2 301Right 5 3 0 0 0 0 0 0 0 0 0 0 0 2 302Intl 60 23 0 0 0 0 0 0 0 0 0 0 0 37 303Pair Stats: 304considered 4, unacceptable product size 3, ok 1 305primer3 release 2.0.0 306 307 308PRIMER PICKING RESULTS FOR boundary_test_14 309 310No mispriming library specified 311No internal oligo mishyb library specified 312Using 1-based sequence positions 313OLIGO start len tm gc% any 3' seq 314LEFT PRIMER 3 24 55.24 4.17 0.00 0.00 AAAAAAAAAAAAAAAAACAAAAAA 315RIGHT PRIMER 82 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 316INTERNAL OLIGO 30 25 56.20 0.00 0.00 0.00 TTTTTTTTTTTTTTTTTTTTTTTNN 317SEQUENCE SIZE: 84 318INCLUDED REGION SIZE: 84 319 320PRODUCT SIZE: 80, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 0.00 321 322 1 NNAAAAAAAAAAAAAAAAACAAAAAAATTTTTTTTTTTTTTTTTTTTTTTTTNNNNNACT 323 >>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ << 324 325 61 CACTGTCATTTCCAAACAATTCNN 326 <<<<<<<<<<<<<<<<<<<<<< 327 328KEYS (in order of precedence): 329>>>>>> left primer 330<<<<<< right primer 331^^^^^^ internal oligo 332 333 334Statistics 335 con too in in not no tm tm high high high 336 sid many tar excl ok bad GC too too any 3' poly end 337 ered Ns get reg reg GC% clamp low high compl compl X stab ok 338Left 5 2 0 0 0 0 0 0 0 0 0 0 0 3 339Right 5 3 0 0 0 0 0 0 0 0 0 0 0 2 340Intl 60 25 0 0 0 0 0 0 0 0 0 0 0 35 341Pair Stats: 342considered 6, unacceptable product size 5, ok 1 343primer3 release 2.0.0 344 345 346PRIMER PICKING RESULTS FOR boundary_test_15 347 348No mispriming library specified 349No internal oligo mishyb library specified 350Using 1-based sequence positions 351OLIGO start len tm gc% any 3' seq 352LEFT PRIMER 3 24 89.31 100.00 0.00 0.00 GGGGGGGGGGGGGGGGGGGGGGGG 353RIGHT PRIMER 82 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 354INTERNAL OLIGO 33 25 55.84 0.00 0.00 0.00 TTTTTTTTTTTTTTTTTTTTTTTTT 355SEQUENCE SIZE: 84 356INCLUDED REGION SIZE: 84 357 358PRODUCT SIZE: 80, PAIR ANY COMPL: 1.00, PAIR 3' COMPL: 0.00 359 360 1 NNGGGGGGGGGGGGGGGGGGGGGGGGGNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTACT 361 >>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ << 362 363 61 CACTGTCATTTCCAAACAATTCNN 364 <<<<<<<<<<<<<<<<<<<<<< 365 366KEYS (in order of precedence): 367>>>>>> left primer 368<<<<<< right primer 369^^^^^^ internal oligo 370 371 372Statistics 373 con too in in not no tm tm high high high 374 sid many tar excl ok bad GC too too any 3' poly end 375 ered Ns get reg reg GC% clamp low high compl compl X stab ok 376Left 5 3 0 0 0 0 0 0 0 0 0 0 0 2 377Right 5 2 0 0 0 0 0 0 0 0 0 0 0 3 378Intl 60 25 0 0 0 0 0 0 0 0 0 0 0 35 379Pair Stats: 380considered 6, unacceptable product size 5, ok 1 381primer3 release 2.0.0 382 383 384PRIMER PICKING RESULTS FOR boundary_test_16 385 386No mispriming library specified 387No internal oligo mishyb library specified 388Using 1-based sequence positions 389NO PRIMERS FOUND 390 391SEQUENCE SIZE: 59 392INCLUDED REGION SIZE: 59 393 394 395 1 NNGGGGGGGGGGGGGGGGGGGGGGGGGNNNNNACTCACTGTCATTTCCAAACAATTCNN 396 397 398Statistics 399 con too in in not no tm tm high high high 400 sid many tar excl ok bad GC too too any 3' poly end 401 ered Ns get reg reg GC% clamp low high compl compl X stab ok 402Left 10 8 0 0 0 0 0 0 0 0 0 0 0 2 403Right 10 8 0 0 0 0 0 0 0 0 0 0 0 2 404Intl 35 0 0 0 0 0 0 0 0 0 0 0 0 35 405Pair Stats: 406considered 4, no internal oligo 4, ok 0 407primer3 release 2.0.0 408 409 410PRIMER PICKING RESULTS FOR boundary_test_17 411 412No mispriming library specified 413No internal oligo mishyb library specified 414Using 1-based sequence positions 415NO PRIMERS FOUND 416 417SEQUENCE SIZE: 84 418INCLUDED REGION SIZE: 84 419 420 421 1 NNCACGTCATCTCATCAAATTTGATTGNNTTTTTTTTTTTTTTTTTTTTTTTTGNNNACT 422 61 CACTGTCATTTCCAAACAATTCNN 423 424 425Statistics 426 con too in in not no tm tm high high high 427 sid many tar excl ok bad GC too too any 3' poly end 428 ered Ns get reg reg GC% clamp low high compl compl X stab ok 429Left 35 31 0 0 0 0 0 0 0 0 0 0 0 4 430Right 35 31 0 0 0 0 0 0 0 0 0 0 0 4 431Intl 60 0 0 0 0 29 0 0 0 0 0 0 0 31 432Pair Stats: 433considered 16, unacceptable product size 4, no internal oligo 12, ok 0 434primer3 release 2.0.0 435 436 437PRIMER PICKING RESULTS FOR boundary_test_18 438 439No mispriming library specified 440No internal oligo mishyb library specified 441Using 1-based sequence positions 442NO PRIMERS FOUND 443 444SEQUENCE SIZE: 111 445INCLUDED REGION SIZE: 111 446 447INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 11,50 448 449 1 NNCACGTCATCTCATCAAATTTGATTGNNACCGCTATTGGGCCATTTGCCCATTGCATGN 450 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 451 452 61 NNNNNNNNNNNNNNNNNNNNNNNNACTCACTGTCATTTCCAAACAATTCNN 453 454 455KEYS (in order of precedence): 456xxxxxx excluded region for internal oligo 457 458 459Statistics 460 con too in in not no tm tm high high high 461 sid many tar excl ok bad GC too too any 3' poly end 462 ered Ns get reg reg GC% clamp low high compl compl X stab ok 463Left 62 53 0 0 0 0 0 0 0 0 0 0 0 9 464Right 62 53 0 0 0 0 0 0 0 0 0 0 0 9 465Intl 87 39 0 40 0 0 0 0 0 0 0 0 0 8 466Pair Stats: 467considered 81, unacceptable product size 49, no internal oligo 32, ok 0 468primer3 release 2.0.0 469 470 471PRIMER PICKING RESULTS FOR boundary_test_19 472 473No mispriming library specified 474No internal oligo mishyb library specified 475Using 1-based sequence positions 476OLIGO start len tm gc% any 3' seq 477LEFT PRIMER 4 24 60.24 33.33 12.00 10.00 ACGTCATCTCATCAAATTTGATTG 478RIGHT PRIMER 113 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 479INTERNAL OLIGO 61 25 65.58 52.00 6.00 1.00 CCGTTAGGTGCTAGCCGTGTAGATT 480SEQUENCE SIZE: 115 481INCLUDED REGION SIZE: 115 482 483PRODUCT SIZE: 110, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00 484INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 11,50 485 486 1 NNCACGTCATCTCATCAAATTTGATTGNNAAGCTAAGCTTGCCCGTATCCCCTGGATNNN 487 >>>>>>>xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 488 489 61 CCGTTAGGTGCTAGCCGTGTAGATTNNNACTCACTGTCATTTCCAAACAATTCNN 490 ^^^^^^^^^^^^^^^^^^^^^^^^^ <<<<<<<<<<<<<<<<<<<<<<<< 491 492KEYS (in order of precedence): 493xxxxxx excluded region for internal oligo 494>>>>>> left primer 495<<<<<< right primer 496^^^^^^ internal oligo 497 498ADDITIONAL OLIGOS 499 start len tm gc% any 3' seq 500 501 1 LEFT PRIMER 3 24 60.24 33.33 12.00 10.00 CACGTCATCTCATCAAATTTGATT 502 RIGHT PRIMER 113 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 503 INTERNAL OLIGO 61 25 65.58 52.00 6.00 1.00 CCGTTAGGTGCTAGCCGTGTAGATT 504 PRODUCT SIZE: 111, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 2.00 505 506 2 LEFT PRIMER 4 24 60.24 33.33 12.00 10.00 ACGTCATCTCATCAAATTTGATTG 507 RIGHT PRIMER 112 24 58.98 33.33 4.00 3.00 AATTGTTTGGAAATGACAGTGAGT 508 INTERNAL OLIGO 61 25 65.58 52.00 6.00 1.00 CCGTTAGGTGCTAGCCGTGTAGATT 509 PRODUCT SIZE: 109, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00 510 511 3 LEFT PRIMER 3 24 60.24 33.33 12.00 10.00 CACGTCATCTCATCAAATTTGATT 512 RIGHT PRIMER 112 24 58.98 33.33 4.00 3.00 AATTGTTTGGAAATGACAGTGAGT 513 INTERNAL OLIGO 61 25 65.58 52.00 6.00 1.00 CCGTTAGGTGCTAGCCGTGTAGATT 514 PRODUCT SIZE: 110, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 2.00 515 516 4 LEFT PRIMER 30 24 67.92 54.17 9.00 0.00 AAGCTAAGCTTGCCCGTATCCCCT 517 RIGHT PRIMER 113 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 518 INTERNAL OLIGO 61 25 65.58 52.00 6.00 1.00 CCGTTAGGTGCTAGCCGTGTAGATT 519 PRODUCT SIZE: 84, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 2.00 520 521Statistics 522 con too in in not no tm tm high high high 523 sid many tar excl ok bad GC too too any 3' poly end 524 ered Ns get reg reg GC% clamp low high compl compl X stab ok 525Left 66 57 0 0 0 0 0 0 0 0 0 0 0 9 526Right 66 57 0 0 0 0 0 0 0 0 0 0 0 9 527Intl 91 84 0 5 0 0 0 0 0 0 0 0 0 2 528Pair Stats: 529considered 16, unacceptable product size 3, no internal oligo 8, ok 5 530primer3 release 2.0.0 531 532 533PRIMER PICKING RESULTS FOR boundary_test_20 534 535No mispriming library specified 536No internal oligo mishyb library specified 537Using 1-based sequence positions 538OLIGO start len tm gc% any 3' seq 539LEFT PRIMER 4 24 60.24 33.33 12.00 10.00 ACGTCATCTCATCAAATTTGATTG 540RIGHT PRIMER 252 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 541INTERNAL OLIGO 76 25 63.81 52.00 12.00 12.00 CTGGTCAAAGCTAGCGGTAGCTTAG 542SEQUENCE SIZE: 291 543INCLUDED REGION SIZE: 250 544 545PRODUCT SIZE: 249, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00 546INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 6,50 61,15 547 548 1 NNCACGTCATCTCATCAAATTTGATTGNNAAGCTAAGCTGTCGATGCGGTGGAGACGATA 549 .. >>xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 550 551 61 CGGTAAGCTAGCTAACTGGTCAAAGCTAGCGGTAGCTTAGCTNNNNNNNNNNNNNNNNNN 552 xxxxxxxxxxxxxxx^^^^^^^^^^^^^^^^^^^^^^^^^ 553 554 121 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 555 556 557 181 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNACTCACTGTCATT 558 <<<<<<<<<<<< 559 560 241 TCCAAACAATTCATCTCTCTCTCATATATATATATATATATATATATATAT 561 <<<<<<<<<<<<....................................... 562 563KEYS (in order of precedence): 564...... vector sequence 565xxxxxx excluded region for internal oligo 566>>>>>> left primer 567<<<<<< right primer 568^^^^^^ internal oligo 569 570ADDITIONAL OLIGOS 571 start len tm gc% any 3' seq 572 573 1 LEFT PRIMER 3 24 60.24 33.33 12.00 10.00 CACGTCATCTCATCAAATTTGATT 574 RIGHT PRIMER 252 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 575 INTERNAL OLIGO 76 25 63.81 52.00 12.00 12.00 CTGGTCAAAGCTAGCGGTAGCTTAG 576 PRODUCT SIZE: 250, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 2.00 577 578 2 LEFT PRIMER 51 24 60.30 50.00 8.00 6.00 GGAGACGATACGGTAAGCTAGCTA 579 RIGHT PRIMER 252 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 580 INTERNAL OLIGO 76 25 63.81 52.00 12.00 12.00 CTGGTCAAAGCTAGCGGTAGCTTAG 581 PRODUCT SIZE: 202, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00 582 583 3 LEFT PRIMER 4 24 60.24 33.33 12.00 10.00 ACGTCATCTCATCAAATTTGATTG 584 RIGHT PRIMER 251 24 58.98 33.33 4.00 3.00 AATTGTTTGGAAATGACAGTGAGT 585 INTERNAL OLIGO 76 25 63.81 52.00 12.00 12.00 CTGGTCAAAGCTAGCGGTAGCTTAG 586 PRODUCT SIZE: 248, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00 587 588 4 LEFT PRIMER 3 24 60.24 33.33 12.00 10.00 CACGTCATCTCATCAAATTTGATT 589 RIGHT PRIMER 251 24 58.98 33.33 4.00 3.00 AATTGTTTGGAAATGACAGTGAGT 590 INTERNAL OLIGO 76 25 63.81 52.00 12.00 12.00 CTGGTCAAAGCTAGCGGTAGCTTAG 591 PRODUCT SIZE: 249, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 2.00 592 593Statistics 594 con too in in not no tm tm high high high 595 sid many tar excl ok bad GC too too any 3' poly end 596 ered Ns get reg reg GC% clamp low high compl compl X stab ok 597Left 151 99 0 0 0 0 0 0 0 0 0 0 0 52 598Right 151 148 0 0 0 0 0 0 0 0 0 0 0 3 599Intl 226 175 0 47 0 0 0 0 0 0 0 0 0 4 600Pair Stats: 601considered 14, no internal oligo 8, ok 6 602primer3 release 2.0.0 603 604 605PRIMER PICKING RESULTS FOR boundary_test_21 606 607No mispriming library specified 608No internal oligo mishyb library specified 609Using 1-based sequence positions 610OLIGO start len tm gc% any 3' seq 611LEFT PRIMER 4 24 60.24 33.33 12.00 10.00 ACGTCATCTCATCAAATTTGATTG 612RIGHT PRIMER 174 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 613INTERNAL OLIGO 62 25 55.84 0.00 0.00 0.00 AAAAAAAAAAAAAAAAAAAAAAAAA 614SEQUENCE SIZE: 176 615INCLUDED REGION SIZE: 176 616 617PRODUCT SIZE: 171, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00 618 619 1 NNCACGTCATCTCATCAAATTTGATTGNNGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG 620 >>>>>>>>>>>>>>>>>>>>>>>> 621 622 61 GAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG 623 ^^^^^^^^^^^^^^^^^^^^^^^^^ 624 625 121 GGGGGGGGGGGGGGGGGGGGGGGGGGNNNACTCACTGTCATTTCCAAACAATTCNN 626 <<<<<<<<<<<<<<<<<<<<<<<< 627 628KEYS (in order of precedence): 629>>>>>> left primer 630<<<<<< right primer 631^^^^^^ internal oligo 632 633ADDITIONAL OLIGOS 634 start len tm gc% any 3' seq 635 636 1 LEFT PRIMER 3 24 60.24 33.33 12.00 10.00 CACGTCATCTCATCAAATTTGATT 637 RIGHT PRIMER 174 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 638 INTERNAL OLIGO 62 25 55.84 0.00 0.00 0.00 AAAAAAAAAAAAAAAAAAAAAAAAA 639 PRODUCT SIZE: 172, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 2.00 640 641 2 LEFT PRIMER 4 24 60.24 33.33 12.00 10.00 ACGTCATCTCATCAAATTTGATTG 642 RIGHT PRIMER 173 24 58.98 33.33 4.00 3.00 AATTGTTTGGAAATGACAGTGAGT 643 INTERNAL OLIGO 62 25 55.84 0.00 0.00 0.00 AAAAAAAAAAAAAAAAAAAAAAAAA 644 PRODUCT SIZE: 170, PAIR ANY COMPL: 6.00, PAIR 3' COMPL: 1.00 645 646 3 LEFT PRIMER 3 24 60.24 33.33 12.00 10.00 CACGTCATCTCATCAAATTTGATT 647 RIGHT PRIMER 173 24 58.98 33.33 4.00 3.00 AATTGTTTGGAAATGACAGTGAGT 648 INTERNAL OLIGO 62 25 55.84 0.00 0.00 0.00 AAAAAAAAAAAAAAAAAAAAAAAAA 649 PRODUCT SIZE: 171, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 2.00 650 651 4 LEFT PRIMER 38 24 89.31 100.00 0.00 0.00 GGGGGGGGGGGGGGGGGGGGGGGG 652 RIGHT PRIMER 174 24 59.91 37.50 4.00 1.00 GAATTGTTTGGAAATGACAGTGAG 653 INTERNAL OLIGO 62 25 55.84 0.00 0.00 0.00 AAAAAAAAAAAAAAAAAAAAAAAAA 654 PRODUCT SIZE: 137, PAIR ANY COMPL: 1.00, PAIR 3' COMPL: 0.00 655 656Statistics 657 con too in in not no tm tm high high high 658 sid many tar excl ok bad GC too too any 3' poly end 659 ered Ns get reg reg GC% clamp low high compl compl X stab ok 660Left 77 27 0 0 0 0 0 0 0 0 0 0 0 50 661Right 77 28 0 0 0 0 0 0 0 0 0 0 0 49 662Intl 152 57 0 0 0 0 0 1 93 0 0 0 0 1 663Pair Stats: 664considered 323, unacceptable product size 223, no internal oligo 87, ok 13 665primer3 release 2.0.0 666 667 668PRIMER PICKING RESULTS FOR boundary_test_22 669 670No mispriming library specified 671No internal oligo mishyb library specified 672Using 1-based sequence positions 673OLIGO start len tm gc% any 3' seq 674LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 675RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 676INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 677SEQUENCE SIZE: 84 678INCLUDED REGION SIZE: 84 679 680PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 681 682 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 683 >>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 684 685 61 CACTGTCATTTCCAAACAATTCNN 686 <<<<<<<<<<<<<<<<<<<<<< 687 688KEYS (in order of precedence): 689>>>>>> left primer 690<<<<<< right primer 691^^^^^^ internal oligo 692 693 694Statistics 695 con too in in not no tm tm high high high 696 sid many tar excl ok bad GC too too any 3' poly end 697 ered Ns get reg reg GC% clamp low high compl compl X stab ok 698Left 5 4 0 0 0 0 0 0 0 0 0 0 0 1 699Right 5 4 0 0 0 0 0 0 0 0 0 0 0 1 700Intl 60 57 0 0 0 0 0 0 0 0 0 0 0 3 701Pair Stats: 702considered 1, ok 1 703primer3 release 2.0.0 704 705 706PRIMER PICKING RESULTS FOR boundary_test_23 707 708No mispriming library specified 709No internal oligo mishyb library specified 710Using 1-based sequence positions 711WARNING: PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION outside of INCLUDED_REGION 712 713OLIGO start len tm gc% any 3' seq 714LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 715RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 716INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 717SEQUENCE SIZE: 84 718INCLUDED REGION SIZE: 80 719 720PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 721INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 2,1 722 723 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 724 ..>>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 725 726 61 CACTGTCATTTCCAAACAATTCNN 727 <<<<<<<<<<<<<<<<<<<<<<.. 728 729KEYS (in order of precedence): 730...... vector sequence 731xxxxxx excluded region for internal oligo 732>>>>>> left primer 733<<<<<< right primer 734^^^^^^ internal oligo 735 736 737Statistics 738 con too in in not no tm tm high high high 739 sid many tar excl ok bad GC too too any 3' poly end 740 ered Ns get reg reg GC% clamp low high compl compl X stab ok 741Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 742Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 743Intl 56 53 0 0 0 0 0 0 0 0 0 0 0 3 744Pair Stats: 745considered 1, ok 1 746primer3 release 2.0.0 747 748 749PRIMER PICKING RESULTS FOR boundary_test_24 750 751No mispriming library specified 752No internal oligo mishyb library specified 753Using 1-based sequence positions 754OLIGO start len tm gc% any 3' seq 755LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 756RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 757INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 758SEQUENCE SIZE: 84 759INCLUDED REGION SIZE: 80 760 761PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 762INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 3,1 763 764 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 765 ..x>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 766 767 61 CACTGTCATTTCCAAACAATTCNN 768 <<<<<<<<<<<<<<<<<<<<<<.. 769 770KEYS (in order of precedence): 771...... vector sequence 772xxxxxx excluded region for internal oligo 773>>>>>> left primer 774<<<<<< right primer 775^^^^^^ internal oligo 776 777 778Statistics 779 con too in in not no tm tm high high high 780 sid many tar excl ok bad GC too too any 3' poly end 781 ered Ns get reg reg GC% clamp low high compl compl X stab ok 782Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 783Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 784Intl 56 53 0 1 0 0 0 0 0 0 0 0 0 2 785Pair Stats: 786considered 1, ok 1 787primer3 release 2.0.0 788 789 790PRIMER PICKING RESULTS FOR boundary_test_25 791 792No mispriming library specified 793No internal oligo mishyb library specified 794Using 1-based sequence positions 795WARNING: PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION outside of INCLUDED_REGION 796 797OLIGO start len tm gc% any 3' seq 798LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 799RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 800INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 801SEQUENCE SIZE: 84 802INCLUDED REGION SIZE: 80 803 804PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 805INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 1,3 806 807 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 808 ..x>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 809 810 61 CACTGTCATTTCCAAACAATTCNN 811 <<<<<<<<<<<<<<<<<<<<<<.. 812 813KEYS (in order of precedence): 814...... vector sequence 815xxxxxx excluded region for internal oligo 816>>>>>> left primer 817<<<<<< right primer 818^^^^^^ internal oligo 819 820 821Statistics 822 con too in in not no tm tm high high high 823 sid many tar excl ok bad GC too too any 3' poly end 824 ered Ns get reg reg GC% clamp low high compl compl X stab ok 825Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 826Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 827Intl 56 53 0 1 0 0 0 0 0 0 0 0 0 2 828Pair Stats: 829considered 1, ok 1 830primer3 release 2.0.0 831 832 833PRIMER PICKING RESULTS FOR boundary_test_26 834 835No mispriming library specified 836No internal oligo mishyb library specified 837Using 1-based sequence positions 838OLIGO start len tm gc% any 3' seq 839LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 840RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 841INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 842SEQUENCE SIZE: 84 843INCLUDED REGION SIZE: 80 844 845PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 846INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 30,1 847 848 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 849 ..>>>>>>>>>>>>>>>>>>>>>>>>> x^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 850 851 61 CACTGTCATTTCCAAACAATTCNN 852 <<<<<<<<<<<<<<<<<<<<<<.. 853 854KEYS (in order of precedence): 855...... vector sequence 856xxxxxx excluded region for internal oligo 857>>>>>> left primer 858<<<<<< right primer 859^^^^^^ internal oligo 860 861 862Statistics 863 con too in in not no tm tm high high high 864 sid many tar excl ok bad GC too too any 3' poly end 865 ered Ns get reg reg GC% clamp low high compl compl X stab ok 866Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 867Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 868Intl 56 53 0 0 0 0 0 0 0 0 0 0 0 3 869Pair Stats: 870considered 1, ok 1 871primer3 release 2.0.0 872 873 874PRIMER PICKING RESULTS FOR boundary_test_27 875 876No mispriming library specified 877No internal oligo mishyb library specified 878Using 1-based sequence positions 879NO PRIMERS FOUND 880 881SEQUENCE SIZE: 84 882INCLUDED REGION SIZE: 80 883 884INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 31,1 885 886 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 887 .. x 888 889 61 CACTGTCATTTCCAAACAATTCNN 890 .. 891 892KEYS (in order of precedence): 893...... vector sequence 894xxxxxx excluded region for internal oligo 895 896 897Statistics 898 con too in in not no tm tm high high high 899 sid many tar excl ok bad GC too too any 3' poly end 900 ered Ns get reg reg GC% clamp low high compl compl X stab ok 901Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 902Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 903Intl 56 53 0 1 0 0 0 0 0 0 0 0 0 2 904Pair Stats: 905considered 1, no internal oligo 1, ok 0 906primer3 release 2.0.0 907 908 909PRIMER PICKING RESULTS FOR boundary_test_28 910 911No mispriming library specified 912No internal oligo mishyb library specified 913Using 1-based sequence positions 914NO PRIMERS FOUND 915 916SEQUENCE SIZE: 84 917INCLUDED REGION SIZE: 80 918 919INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 55,1 920 921 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 922 .. x 923 924 61 CACTGTCATTTCCAAACAATTCNN 925 .. 926 927KEYS (in order of precedence): 928...... vector sequence 929xxxxxx excluded region for internal oligo 930 931 932Statistics 933 con too in in not no tm tm high high high 934 sid many tar excl ok bad GC too too any 3' poly end 935 ered Ns get reg reg GC% clamp low high compl compl X stab ok 936Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 937Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 938Intl 56 53 0 1 0 0 0 0 0 0 0 0 0 2 939Pair Stats: 940considered 1, no internal oligo 1, ok 0 941primer3 release 2.0.0 942 943 944PRIMER PICKING RESULTS FOR boundary_test_29 945 946No mispriming library specified 947No internal oligo mishyb library specified 948Using 1-based sequence positions 949OLIGO start len tm gc% any 3' seq 950LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 951RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 952INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 953SEQUENCE SIZE: 84 954INCLUDED REGION SIZE: 80 955 956PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 957INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 56,1 958 959 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 960 ..>>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^x <<< 961 962 61 CACTGTCATTTCCAAACAATTCNN 963 <<<<<<<<<<<<<<<<<<<<<<.. 964 965KEYS (in order of precedence): 966...... vector sequence 967xxxxxx excluded region for internal oligo 968>>>>>> left primer 969<<<<<< right primer 970^^^^^^ internal oligo 971 972 973Statistics 974 con too in in not no tm tm high high high 975 sid many tar excl ok bad GC too too any 3' poly end 976 ered Ns get reg reg GC% clamp low high compl compl X stab ok 977Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 978Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 979Intl 56 53 0 0 0 0 0 0 0 0 0 0 0 3 980Pair Stats: 981considered 1, ok 1 982primer3 release 2.0.0 983 984 985PRIMER PICKING RESULTS FOR boundary_test_30 986 987No mispriming library specified 988No internal oligo mishyb library specified 989Using 1-based sequence positions 990OLIGO start len tm gc% any 3' seq 991LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 992RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 993INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 994SEQUENCE SIZE: 84 995INCLUDED REGION SIZE: 80 996 997PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 998INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 82,1 999 1000 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 1001 ..>>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 1002 1003 61 CACTGTCATTTCCAAACAATTCNN 1004 <<<<<<<<<<<<<<<<<<<<<x.. 1005 1006KEYS (in order of precedence): 1007...... vector sequence 1008xxxxxx excluded region for internal oligo 1009>>>>>> left primer 1010<<<<<< right primer 1011^^^^^^ internal oligo 1012 1013 1014Statistics 1015 con too in in not no tm tm high high high 1016 sid many tar excl ok bad GC too too any 3' poly end 1017 ered Ns get reg reg GC% clamp low high compl compl X stab ok 1018Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1019Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1020Intl 56 53 0 1 0 0 0 0 0 0 0 0 0 2 1021Pair Stats: 1022considered 1, ok 1 1023primer3 release 2.0.0 1024 1025 1026PRIMER PICKING RESULTS FOR boundary_test_31 1027 1028No mispriming library specified 1029No internal oligo mishyb library specified 1030Using 1-based sequence positions 1031WARNING: PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION outside of INCLUDED_REGION 1032 1033OLIGO start len tm gc% any 3' seq 1034LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 1035RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 1036INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 1037SEQUENCE SIZE: 84 1038INCLUDED REGION SIZE: 80 1039 1040PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 1041INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 83,1 1042 1043 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 1044 ..>>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 1045 1046 61 CACTGTCATTTCCAAACAATTCNN 1047 <<<<<<<<<<<<<<<<<<<<<<.. 1048 1049KEYS (in order of precedence): 1050...... vector sequence 1051xxxxxx excluded region for internal oligo 1052>>>>>> left primer 1053<<<<<< right primer 1054^^^^^^ internal oligo 1055 1056 1057Statistics 1058 con too in in not no tm tm high high high 1059 sid many tar excl ok bad GC too too any 3' poly end 1060 ered Ns get reg reg GC% clamp low high compl compl X stab ok 1061Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1062Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1063Intl 56 53 0 0 0 0 0 0 0 0 0 0 0 3 1064Pair Stats: 1065considered 1, ok 1 1066primer3 release 2.0.0 1067 1068 1069PRIMER PICKING RESULTS FOR boundary_test_32 1070 1071No mispriming library specified 1072No internal oligo mishyb library specified 1073Using 1-based sequence positions 1074WARNING: PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION outside of INCLUDED_REGION 1075 1076OLIGO start len tm gc% any 3' seq 1077LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 1078RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 1079INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 1080SEQUENCE SIZE: 84 1081INCLUDED REGION SIZE: 80 1082 1083PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 1084INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 82,2 1085 1086 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 1087 ..>>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 1088 1089 61 CACTGTCATTTCCAAACAATTCNN 1090 <<<<<<<<<<<<<<<<<<<<<x.. 1091 1092KEYS (in order of precedence): 1093...... vector sequence 1094xxxxxx excluded region for internal oligo 1095>>>>>> left primer 1096<<<<<< right primer 1097^^^^^^ internal oligo 1098 1099 1100Statistics 1101 con too in in not no tm tm high high high 1102 sid many tar excl ok bad GC too too any 3' poly end 1103 ered Ns get reg reg GC% clamp low high compl compl X stab ok 1104Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1105Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1106Intl 56 53 0 1 0 0 0 0 0 0 0 0 0 2 1107Pair Stats: 1108considered 1, ok 1 1109primer3 release 2.0.0 1110 1111 1112PRIMER PICKING RESULTS FOR boundary_test_33 1113 1114No mispriming library specified 1115No internal oligo mishyb library specified 1116Using 1-based sequence positions 1117OLIGO start len tm gc% any 3' seq 1118LEFT PRIMER 3 25 62.73 36.00 12.00 10.00 CACGTCATCTCATCAAATTTGATTG 1119RIGHT PRIMER 82 25 60.65 36.00 4.00 3.00 GAATTGTTTGGAAATGACAGTGAGT 1120INTERNAL OLIGO 31 25 71.50 56.00 12.00 8.00 ACGTACGTACGTTGCAGTCGACGCA 1121SEQUENCE SIZE: 84 1122INCLUDED REGION SIZE: 80 1123 1124PRODUCT SIZE: 80, PAIR ANY COMPL: 7.00, PAIR 3' COMPL: 1.00 1125INTERNAL OLIGO EXCLUDED REGIONS (start, len)*: 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 82,1 1126 1127 1 NNCACGTCATCTCATCAAATTTGATTGNNNACGTACGTACGTTGCAGTCGACGCANNACT 1128 ..>>>>>>>>>>>>>>>>>>>>>>>>> ^^^^^^^^^^^^^^^^^^^^^^^^^ <<< 1129 1130 61 CACTGTCATTTCCAAACAATTCNN 1131 <<<<<<<<<<<<<<<<<<<<<x.. 1132 1133KEYS (in order of precedence): 1134...... vector sequence 1135xxxxxx excluded region for internal oligo 1136>>>>>> left primer 1137<<<<<< right primer 1138^^^^^^ internal oligo 1139 1140 1141Statistics 1142 con too in in not no tm tm high high high 1143 sid many tar excl ok bad GC too too any 3' poly end 1144 ered Ns get reg reg GC% clamp low high compl compl X stab ok 1145Left 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1146Right 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1147Intl 56 53 0 1 0 0 0 0 0 0 0 0 0 2 1148Pair Stats: 1149considered 1, ok 1 1150primer3 release 2.0.0 1151 1152 1153PRIMER PICKING RESULTS FOR boundary_test_34 1154 1155INPUT PROBLEM: Too many elements for tag SEQUENCE_INTERNAL_EXCLUDED_REGION 1156 1157