Dear all , my job is to clone part of the gene already carried on pJC40 vector .i sent this vector for sequencing . i made also squence alignment, i have found only 97% identity . this mean about five nuclutides are not matching with original sequence . when i made protein blast i also found 4 amino acid were altered .
this is sequencing result 10285313.seq - ID: 130912 Amr-T7 on 2012/9/17-4:14:28 automatically edited with PhredPhrap, start with base no.: 10 Internal Params: Windowsize: 20, Goodqual: 19, Badqual: 10, Minseqlength: 50, nbadelimit: 1 tnaaaTaTTTtgTTTtaCTTtAGanngagaTATACCatggGGcCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAgGTCGTCATATGATACCCCAAcgtCGCCAACAACAACAACAACAACAACAGCAACAACAACgtGatgAACGTgGAATACCACCATTTTCGGAAGgtGCACCACCAAgtgTTATCGATGAATTTTATAATTTGCTAAAAACTGATGAAAATAAAACTGATCAACAAACtgAAGCTGATGTTGAAGCATTCATCAATAGACTTGGTGGCAGTTACAAGGTTCGATTTACTCAATTCATGGAAGAAGTAAAGAAAGCTAGAGCTGATTATGAAAGAATCCATCAGCAGGCAGTAGCAAGATTTTCGCCAGCAGCAAAAGATGCTGACGCAAGGATGTCCGCTATTGCTGGTTCGCCGCATCTAACTACGCGACAAAAATCGCAGCAAATTCAAGCCATCATGGATTCATTATCTGAGAGCGTTCGAGGagaGATCATTAAGGCATTGAGCCCACAAGAATAAGGATCCCGGGCCCTAGCTAACTGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATtTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCcCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAaGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC this is DNA alignment..... gb|U00693.1|OVU00693 Onchocerca volvulus Rainforest immunodominant hypodermal antigen mRNA, complete cds Length=880 Score = 798 bits (432), Expect = 0.0 Identities = 442/447 (99%), Gaps = 0/447 (0%) Strand=Plus/Plus Query 48 ATACCCCAACGTCGCcaacaacaacaacaacaacaacagcaacaacaacGTGATGAACGT 107 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 117 ATACCCCAACGTCGCCAACAACAACAACAACAACAACAGCAACAACAACGTGATGAACGT 176 Query 108 GGAATACCACCATTTTCGGAAGGTGCACCACCAAGTGTTATCGATGAATTTTATAATTTG 167 | |||||||||||||| ||||||||||||||||||||||||||||||||||||||||||| Sbjct 177 GAAATACCACCATTTTTGGAAGGTGCACCACCAAGTGTTATCGATGAATTTTATAATTTG 236 Query 168 CTAAAAACTGATGAAAATAAAACTGATCAACAAACTGAAGCTGATGTTGAAGCATTCATC 227 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 237 CTAAAAACTGATGAAAATAAAACTGATCAACAAACTGAAGCTGATGTTGAAGCATTCATC 296 Query 228 AATAGACTTGGTGGCAGTTACAAGGTTCGATTTACTCAATTCATGGAAGAAGTAAAGAAA 287 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 297 AATAGACTTGGTGGCAGTTACAAGGTTCGATTTACTCAATTCATGGAAGAAGTAAAGAAA 356 Query 288 GCTAGAGCTGATTATGAAAGAATCCATCAGCAGGCAGTAGCAAGATTTTCGCCAGCAGCA 347 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 357 GCTAGAGCTGATTATGAAAGAATCCATCAGCAGGCAGTAGCAAGATTTTCGCCAGCAGCA 416 Query 348 AAAGATGCTGACGCAAGGATGTCCGCTATTGCTGGTTCGCCGCATCTAACTACGCGACAA 407 |||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||| Sbjct 417 AAAGATGCTGACGCAAGGATGTCCGCTATTGCTGATTCGCCGCATCTAACTACGCGACAA 476 Query 408 AAATCGCAGCAAATTCAAGCCATCATGGATTCATTATCTGAGAGCGTTCGAGGAGAGATC 467 ||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||| Sbjct 477 AAATCGCAGCAAATTCAAGCCATCATGGATTCATTATCTGAGAGCGTTCGAAGAGAGATC 536 Query 468 ATTAAGGCATTGAGCCCACAAGAATAA 494 ||||| ||||||||||||||||||||| Sbjct 537 ATTAATGCATTGAGCCCACAAGAATAA 563 and this is amino acid sequence alignment . lcl|8325 unnamed protein product Length=148 Score = 256 bits (655), Expect = 7e-93, Method: Compositional matrix adjust. Identities = 143/148 (97%), Positives = 143/148 (97%), Gaps = 0/148 (0%) Query 1 IPQRRQQQQQQQQQQQRDEREIPPFLEGAPPSVIDEFYNLLKTDENKTDQQTEADVEAFI 60 IPQRRQQQQQQQQQQQRDER IPPF EGAPPSVIDEFYNLLKTDENKTDQQTEADVEAFI Sbjct 1 IPQRRQQQQQQQQQQQRDERGIPPFSEGAPPSVIDEFYNLLKTDENKTDQQTEADVEAFI 60 Query 61 NRLGGSYKVRFTQFMEEVKKARADYERIHQQAVARFSPAAKDADARMSAIADSPHLTTRQ 120 NRLGGSYKVRFTQFMEEVKKARADYERIHQQAVARFSPAAKDADARMSAIA SPHLTTRQ Sbjct 61 NRLGGSYKVRFTQFMEEVKKARADYERIHQQAVARFSPAAKDADARMSAIAGSPHLTTRQ 120 Query 121 KSQQIQAIMDSLSESVRREIINALSPQE 148 KSQQIQAIMDSLSESVR EII ALSPQE Sbjct 121 KSQQIQAIMDSLSESVRGEIIKALSPQE 148. now what should i do? continue and design primers or repeat the sequencing again or begin every thing from zero point . best regards Amr
