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Sequence Analysis - 2-16-16 (1)

1. Sequence analysis was performed on DNA from Glycine max (soybean) cultured cells using T3 and T7 primers, and the resulting sequences were edited and aligned. 2. The edited sequences showed high similarity, with some discrepancies, and contained restriction enzyme cut sites for EcoRI and XhoI as well as a polyA tail. 3. Multiple sequence alignments identified differences between the T3 and T7 sequences, with the greatest similarity from positions 51-900.

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1 Sequence Analysis of an Isolated Culture, Pertaining to Glycine Max By: Alexander Ward CMB 426 - Dr. Tsou 3/15/2016
2 Original/Unedited Sequences3 – T3 Primer – GAACTCCGCGGGTGCGGCCGCTCTGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGGCACGAGCGGCAACAGCAGCTGCCA CCTCGTCCTTTATGGGGACGCGTCTCCTGGAGGCTCACTCCGGAGCGGGGCGAGTGCACGCCCGATTCGGCTTCGGCAAGAAA AAGGCTCCCGCCCCAAAGAAAGCCTCCAGGGGATCGGGCCGAGACACCGACAGACCCCTTTGGTATCCGGGCGCCAAAGCGC CCGAATACCTCGATGGGAGTCTTGTCGGAGACTACGGGTTCGATCCGTTTGGGCTAGGGAAGCCCGCGGAGTACCTGCAGTTC GAGCTGGACTCGCTGGACCAGAACCTTGCGAAGAACGTGGCTGGGGACATCATTGGAACCAGGACCGAGCTCGCGGACGTGA AGTCCACGCCGTTTCAGCCCTACAGCGAGGTGTTTGGGCTCCAGAGGTTCCGTGAGTGCGAACTCATCCATGGAAGGTGGGCC ATGCTCGCCACTCTCGGAGCTCTCACTGTTGAGTGGCTCACTGGTGTTACATGGCAAGACGCCGGAAAGGTGGAGCTAGTAGA AGGGTCATCATACCTTGGGCAACCACTTCCATTCTCAATCACCACACTGATCTGGATCGAGGTACTCGTAATTGGCTACATAGA GTTCCAGAGGAATGCAGAGCTCGACCCAGAGAAGAGGTTGTACCCAGGTGGCAGTTACTTCGACCCATTGGGCCTGGCCTCAG ACCCAGAGAAGAAAGCCACCCTTCAATTGGCGGAGATCAAGCACGCCCGTCTTGCCATGGTGGGCTTCTTGGGCTTTGC AGTC CAAGCCGCCGCCACCGGCAAGGGTCCGCTCAACAACTGGGCCACCCACTTGAGTGACCCACTCCACACAACCATCATTGACAC CTTCTCATCCTCCTCTTAAGAAGAAGAGTCTTTCTTGTGCCTCGTCACTATTACTAGCATATTGTAAAAGTCTTTTCTTCTTCGGCT TTTGGTTGTAATTAAAACATTTTCACTTANTTGGAATNAGTAAATACTTGTGAAAAAACTTNGTAAACGTGGAAANTNGGANAG GCTAAACCAAAAATGGCTTTCTGCTTNAATGTTAAAAAAAAAAAAAAAAAAAAAAAATTCGAGGGGGGGNCCCGGGTAACCC NAATTNCCCCCNTAAAAGNGGNGTTCNAAATTAAAAANTTCACTGGGCCNGTTCGTTTTTNAAAACGTTCNTNAACGGGGGN AAAAACCCCTGGGGGGTTTACCCCAANTTTAAATTCCCCCTTTNGGAAANAAAATTCCCCCTTTTTNCCCNANGNTGGGGGNT NAAANAACNAAAAAANNGCCCCCCCCACCAAATTNNCCCNTTTTCCAAAAAANTTTNCCCAACCNTNAAAAGGGNNAAAN GGNAAAA T7 Primer – Slight Taq slip following poly-T CGGGCCCCCTGCGTTTTTTTTTTTTTTTTTTTTACATCAAGCAGAAGCATTTCTGCTTAGCCTCTCCAATTTCACGTTACAAGTTTTT CACAAGTACTACTAATCCAACTAAGTGAAAATGTTCTAATTACAACCAAAAGCCGAAGAAGAAAAGACTTTTACAATATGCTAG TAATAGTGACGAGGCACAAGAAAGACTCTTCTTCTTAAGAGGAGGATGAGAAGGTGTCAATGATGGTTGTGTGGAGTGGGTCA CTCAAGTGGGTGGCCCAGTTGTTGAGCGGACCCTTGCCGGTGGCGGCGGCTTGGACTGCAAAGCCCAAGAAGCCCACCATGG CAAGACGGGCGTGCTTGATCTCCGCCAATTGAAGGGTGGCTTTCTTCTCTGGGTCTGAGGCCAGGCCCAATGGGTCGAAGTAA CTGCCACCTGGGTACAACCTCTTCTCTGGGTCGAGCTCTGCATTCCTCTGGAACTCTATGTAGCCAATTACGAGTACCTCGATCC AGATCAGTGTGGTGATTGAGAATGGAAGTGGTTGCCCAAGGTATGATGAC CCTTCTACTAGCTCCACCTTTCCGGCGTCTTGCC ATGTAACACCAGTGAGCCACTCAACAGTGAGAGCTCCGAGAGTGGCGAGCATGGCCCACCTTCCATGGATGAGTTCGCACTCA CGGAACCTCTGGAGCCCAAACACCTCGCTGTAGGGCTGAAACGGCGTGGACTTCACGTCCGCGAGCTCGGTCCTGGTTCCAAT GATGTCCCCAGCCACGTTCTTCGCAAGGTTCTGGTCCAGCGAGTCCAGCTCGAACTGCAGGTACTCCGCGGGCTTCCCTAGCCC AAACGGGATCGAACCCGTAGTCTCCGACAAGACTCCCCATCGAGGTATTTCGGGGCGCTTTTGGCGCCCGGGATACCAAAGGG GGTCTGTCGGGTGGTCTCCGGCCCCGAATCCCCTGGGAGGGCTTTTCTTTTGGGGGGCGGGAAGCCTTTTTTTCTTGCCGGAAG CCCGAAAATCGGGGGCGGTGGNACTNCGCCCCCCGGNTTCNGGGANGTGAAGCCCTTCCCNAGGAANAAACNNNNGTTCC CCCCCNATAAAAANGGANCNAAANGGTGGGCNANCCCTTGCCTTTTTTGCCCCNCCTTCCGTNGNCCCAAAAATTCCNTGGC CANNCCCCCGGGGGGGGAAATCCCCCCTTAANNTTTNTTAAAAAAGCGGGGCCCCCCCCNCCCNCCCNGGGNGGGNAAAC CTTCCCNACCCNTTTTTTGNTTTCCCCCCNTTTTANNGGGGAN
3 Edited T3 Primer – GAATTCGGCACGAGCGGCAACAGCAGCTGCCACCTCGTCCTTTATGGGGACGCGTCTCCTGGAGGCTCACTCCGGAGCGGGG CGAGTGCACGCCCGATTCGGCTTCGGCAAGAAAAAGGCTCCCGCCCCAAAGAAAGCCTCCAGGGGATCGGGCCGAGACACC GACAGACCCCTTTGGTATCCGGGCGCCAAAGCGCCCGAATACCTCGATGGGAGTCTTGTCGGAGACTACGGGTTCGATCCGTT TGGGCTAGGGAAGCCCGCGGAGTACCTGCAGTTCGAGCTGGACTCGCTGGACCAGAACCTTGCGAAGAACGTGGCTGGGGAC ATCATTGGAACCAGGACCGAGCTCGCGGACGTGAAGTCCACGCCGTTTCAGCCCTACAGCGAGGTGTTTGGGCTCCAGAGGTT CCGTGAGTGCGAACTCATCCATGGAAGGTGGGCCATGCTCGCCACTCTCGGAGCTCTCACTGTTGAGTGGCTCACTGGTGTTAC ATGGCAAGACGCCGGAAAGGTGGAGCTAGTAGAAGGGTCATCATACCTTGGGCAACCACTTCCATTCTCAATCACCACACTGA TCTGGATCGAGGTACTCGTAATTGGCTACATAGAGTTCCAGAGGAATGCAGAGCTCGACCCAGAGAAGAGGTTGTACCCAGGT GGCAGTTACTTCGACCCATTGGGCCTGGCCTCAGACCCAGAGAAGAAAGCCACCCTTCAATTGGCGGAGATCAAGCACGCCCG TCTTGCCATGGTGGGCTTCTTGGGCTTTGCAGTCCAAGCCGCCGCCACCGGCAAGGGTCCGCTCAACAACTGGGCCACCCACTT GAGTGACCCACTCCACACAACCATCATTGACACCTTCTCATCCTCCTCTTAAGAAGAAGAGTCTTTCTTGTGCCTCGTCACTATTA CTAGCATATTGTAAAAGTCTTTTCTTCTTCGGCTTTTGGTTGTAATTAAAACATTTTCACTTA Edited T7 Primer – ACATCAAGCAGAAGCATTTCTGCTTAGCCTCTCCAATTTCACGTTACAAGTTTTTCACAAGTACTACTAATCCAACTAAGTGAAA ATGTTCTAATTACAACCAAAAGCCGAAGAAGAAAAGACTTTTACAATATGCTAGTAATAGTGACGAGGCACAAGAAAGACTCTT CTTCTTAAGAGGAGGATGAGAAGGTGTCAATGATGGTTGTGTGGAGTGGGTCACTCAAGTGGGTGGCCCAGTTGTTGAGCGGA CCCTTGCCGGTGGCGGCGGCTTGGACTGCAAAGCCCAAGAAGCCCACCATGGCAAGACGGGCGTGCTTGATCTCCGCCAATT GAAGGGTGGCTTTCTTCTCTGGGTCTGAGGCCAGGCCCAATGGGTCGAAGTAACTGCCACCTGGGTACAACCTCTTCTCTGGGT CGAGCTCTGCATTCCTCTGGAACTCTATGTAGCCAATTACGAGTACCTCGATCCAGATCAGTGTGGTGATTGAGAATGGAAGTG GTTGCCCAAGGTATGATGACCCTTCTACTAGCTCCACCTTTCCGGCGTCTTGCCATGTAACACCAGTGAGCCACTCAACAGTGA GAGCTCCGAGAGTGGCGAGCATGGCCCACCTTCCATGGATGAGTTCGCACTCACGGAACCTCTGGAGCCCAAACACCTCGCTG TAGGGCTGAAACGGCGTGGACTTCACGTCCGCGAGCTCGGTCCTGGTTCCAATGATGTCCCCAGCCACGTTCTTCGCAAGGTTC TGGTCCAGCGAGTCCAGCTCGAACTGCAGGTACTCCGCGGGCTTCCCTAGCCCAAACGGGATCGAACCCGTAGTCTCCGACAA GACTCCCCATCGAGGTATTTCGGGGCGCTTTTGGCGCCCGGGATACCAAAGGGGGTCTGTCGGGTGGTCTCCGGCCCCGAATC CCCTGGGAGGGCTTTTCTTTTGGGGGGCGGGAAGCCTTTTTTTCTTGCCGGAAGCCCGAAAATCGGGGGCGGTGG Green = EcoRI Cut Site, Red = XhoI Cut Site, Teal = PolyA Tail, Purple = Sequence discrepancies; Multiple cut sites were not observed.
4 Align 2 or more sequences using CLUSTALW2 – PILEMSF Format 1 50 T3_edited GAATTCGGCA CGAGCGGCAA CAGCAGCTGC CACCTCGTCC TTTATGGGGA T7_edited__Reve .......... .......... .......... .......... .......... 51 100 T3_edited CGCGTCTCCT GGAGGCTCAC TCCGGAGCGG GGCGAGTGCA CGCCCGATTC T7_edited__Reve .......... .......... .......... ..CCACCGCC CCCGATTTTC 101 150 T3_edited GG..CTTCGG CAAGAAAAAG G...CTCCCG CCCC...AAA GAAAG...CC T7_edited__Reve GGGCTTCCGG CAAGAAAAAA AGGCTTCCCG CCCCCCAAAA GAAAAGCCCT 151 200 T3_edited TCCAGGGGAT C..GGGCCG. AGACA..CCG ACAGACCCC. TTTGGTATCC T7_edited__Reve CCCAGGGGAT TCGGGGCCGG AGACCACCCG ACAGACCCCC TTTGGTATCC 201 250 T3_edited .GGGCGCCAA A.GCGCCC.. GAATACCTCG ATGGG.AGTC TTGTCGGAGA T7_edited__Reve CGGGCGCCAA AAGCGCCCCG AAATACCTCG ATGGGGAGTC TTGTCGGAGA 251 300 T3_edited CTACGGGTTC GATCC.GTTT GGGCTAGGGA AGCCCGCGGA GTACCTGCAG T7_edited__Reve CTACGGGTTC GATCCCGTTT GGGCTAGGGA AGCCCGCGGA GTACCTGCAG 301 350 T3_edited TTCGAGCTGG ACTCGCTGGA CCAGAACCTT GCGAAGAACG TGGCTGGGGA T7_edited__Reve TTCGAGCTGG ACTCGCTGGA CCAGAACCTT GCGAAGAACG TGGCTGGGGA 351 400 T3_edited CATCATTGGA ACCAGGACCG AGCTCGCGGA CGTGAAGTCC ACGCCGTTTC T7_edited__Reve CATCATTGGA ACCAGGACCG AGCTCGCGGA CGTGAAGTCC ACGCCGTTTC 401 450 T3_edited AGCCCTACAG CGAGGTGTTT GGGCTCCAGA GGTTCCGTGA GTGCGAACTC T7_edited__Reve AGCCCTACAG CGAGGTGTTT GGGCTCCAGA GGTTCCGTGA GTGCGAACTC 451 500 T3_edited ATCCATGGAA GGTGGGCCAT GCTCGCCACT CTCGGAGCTC TCACTGTTGA T7_edited__Reve ATCCATGGAA GGTGGGCCAT GCTCGCCACT CTCGGAGCTC TCACTGTTGA 501 550 T3_edited GTGGCTCACT GGTGTTACAT GGCAAGACGC CGGAAAGGTG GAGCTAGTAG T7_edited__Reve GTGGCTCACT GGTGTTACAT GGCAAGACGC CGGAAAGGTG GAGCTAGTAG 551 600 T3_edited AAGGGTCATC ATACCTTGGG CAACCACTTC CATTCTCAAT CACCACACTG T7_edited__Reve AAGGGTCATC ATACCTTGGG CAACCACTTC CATTCTCAAT CACCACACTG 601 650 T3_edited ATCTGGATCG AGGTACTCGT AATTGGCTAC ATAGAGTTCC AGAGGAATGC T7_edited__Reve ATCTGGATCG AGGTACTCGT AATTGGCTAC ATAGAGTTCC AGAGGAATGC 651 700 T3_edited AGAGCTCGAC CCAGAGAAGA GGTTGTACCC AGGTGGCAGT TACTTCGACC T7_edited__Reve AGAGCTCGAC CCAGAGAAGA GGTTGTACCC AGGTGGCAGT TACTTCGACC
5 701 750 T3_edited CATTGGGCCT GGCCTCAGAC CCAGAGAAGA AAGCCACCCT TCAATTGGCG T7_edited__Reve CATTGGGCCT GGCCTCAGAC CCAGAGAAGA AAGCCACCCT TCAATTGGCG 751 800 T3_edited GAGATCAAGC ACGCCCGTCT TGCCATGGTG GGCTTCTTGG GCTTTGCAGT T7_edited__Reve GAGATCAAGC ACGCCCGTCT TGCCATGGTG GGCTTCTTGG GCTTTGCAGT 801 850 T3_edited CCAAGCCGCC GCCACCGGCA AGGGTCCGCT CAACAACTGG GCCACCCACT T7_edited__Reve CCAAGCCGCC GCCACCGGCA AGGGTCCGCT CAACAACTGG GCCACCCACT 851 900 T3_edited TGAGTGACCC ACTCCACACA ACCATCATTG ACACCTTCTC ATCCTCCTCT T7_edited__Reve TGAGTGACCC ACTCCACACA ACCATCATTG ACACCTTCTC ATCCTCCTCT 901 950 T3_edited TAAGAAGAAG AGTCTTTCTT GTGCCTCGTC ACTATTACTA GCATATTGTA T7_edited__Reve TAAGAAGAAG AGTCTTTCTT GTGCCTCGTC ACTATTACTA GCATATTGTA 951 1000 T3_edited AAAGTCTTTT CTTCTTCGGC TTTTGGTTGT AATTAAAACA TTTTCACTTA T7_edited__Reve AAAGTCTTTT CTTCTTCGGC TTTTGGTTGT AATTAGAACA TTTTCACTTA 1001 1050 T3_edited .......... .......... .......... .......... .......... T7_edited__Reve GTTGGATTAG TAGTACTTGT GAAAAACTTG TAACGTGAAA TTGGAGAGGC 1051 1076 T3_edited .......... .......... ...... T7_edited__Reve TAAGCAGAAA TGCTTCTGCT TGATGT 76 at the end and 82 at the beginning are the only base pairs not overlapping, 152 total, indicating an overlap of 924 bp.
6 Glycine max chlorophyll a-b binding protein CP29.2, chloroplastic-like (LOC100785415), mRNA Sequence for BLAST input, entire T3 cDNA insert sequence – TTCTTAAGAGGAGGATGAGAAGGTGTCAATGATGGTTGTGTGGAGTGGGTCACTCAAGTGGGTGGCCCAGTTGTTGAGCGGAC CCTTGCCGGTGGCGGCGGCTTGGACTGCAAAGCCCAAGAAGCCCACCATGGCAAGACGGGCGTGCTTGATCTCCGCCAATTG AAGGGTGGCTTTCTTCTCTGGGTCTGAGGCCAGGCCCAATGGGTCGAAGTAACTGCCACCTGGGTACAACCTCTTCTCTGGGTC GAGCTCTGCATTCCTCTGGAACTCTATGTAGCCAATTACGAGTACCTCGATCCAGATCAGTGTGGTGATTGAGAATGGAAGTGG TTGCCCAAGGTATGATGACCCTTCTACTAGCTCCACCTTTCCGGCGTCTTGCCATGTAACACCAGTGAGCCACTCAACAGTGAG AGCTCCGAGAGTGGCGAGCATGGCCCACCTTCCATGGATGAGTTCGCACTCACGGAACCTCTGGAGCCCAAACACCTCGCTGT AGGGCTGAAACGGCGTGGACTTCACGTCCGCGAGCTCGGTCCTGGTTCCAATGATGTCCCCAGCCACGTTCTTCGCAAGGTTCT GGTCCAGCGAGTCCAGCTCGAACTGCAGGTACTCCGCGGGCTTCCCTAGCCCAAACGG Top 20 BLAST hits1 – 1. Glycine max cDNA, clone: GMFL01-17-I17 (99% ID) 2. Glycine max chlorophyll a-b binding protein CP29.2, chloroplastic-like (LOC100785415), mRNA (99% ID) 3. PREDICTED: Glycine max chlorophyll a-b binding protein CP29.2, chloroplastic-like (LOC100785180), mRNA (99% ID) 4. Glycine max cDNA, clone: GMFL01-03-O02 (96% ID) 5. Vigna radiata chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC106774260), mRNA (90% ID) 6. Phaseolus vulgaris hypothetical protein (PHAVU_010G002100g) mRNA, complete cds (90% ID) 7. Morus notabilis hypothetical protein partial mRNA (Too small) 8. Glycine max strain Williams 82 clone GM_WBb0012I20, complete sequence (Too large) 9. PREDICTED: Oryza brachyantha chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC102700159), mRNA (83% ID) 10. Phyllostachys edulis chloroplast chlorophyll a/b binding protein cab-PhE7 mRNA, complete cds; nuclear gene for chloroplast product (82%) 11. Pyrus x bretschneideri clone 915 a-b binding protein mRNA, complete cds (83% ID) 12. Pyrus x bretschneideri chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC103936594), mRNA (Too small) 13. Phyllostachys edulis cDNA clone: bphyem211d01, full insert sequence (82% ID) 14. PREDICTED: Malus x domestica chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC103450616), mRNA (83% ID) 15. PREDICTED: Oryza sativa Japonica Group chlorophyll a-b binding protein CP29.1, chloroplastic (LOC4343583), mRNA (82% ID) 16. Oryza sativa (indica cultivar-group) cDNA clone:OSIGCSN014K09, full insert sequence (82% ID) 17. Oryza sativa Japonica Group cDNA clone:001-204-B02, full insert sequence (82% ID) 18. Oryza sativa Japonica Group cDNA clone:006-309-F11, full insert sequence (82% ID) 19. Oryza sativa Japonica Group cDNA clone:001-013-C12, full insert sequence (82% ID) 20. Oryza sativa Japonica Group cDNA clone:J013098H13, full insert sequence (82% ID) Top 5 Protein BLAST hits1 – 1. chlorophyll a-b binding protein CP29.2, chloroplastic-like [Glycine max] (Length = 290) (100% ID) 2. hypothetical protein GLYMA_03G060300 [Glycine max] (Length = 290) (99% ID)
7 3. PREDICTED: chlorophyll a-b binding protein CP29.2, chloroplastic-like [Glycine max] (Length = 290) (97% ID) 4. PREDICTED: chlorophyll a-b binding protein CP29.3, chloroplastic-like [Glycine max] (Length = 278) (73% ID) 5. PREDICTED: chlorophyll a-b binding protein CP29.3, chloroplastic-like [Glycine max] (Length = 282) (72% ID) Of the Light Harvesting Complex (LHC), two molecules which interact with visible light, chlorophyll a and chlorophyll b (Chl-a and Chl-b respectively). The protein which is coded by the gene of interest is chlorophyll a/b binding protein, which acts as an antenna for photosystems 1 and 2, a peripheral light- harvesting protein. ORF Analysis2 – F S H T R R T L A Q T I M A T A T A A A 2 ttttctcacaccaggcgcacgttagcacaaactatcatggccacggcaacagcagctgcc 61 T S S F M G T R L L E A H S G A G R V H 62 acctcgtcctttatggggacgcgtctcctggaggctcactccggggcggggcgagtgcac 121 A R F G F G K K K A P A Q K K A S R G S 122 gcccgattcggcttcggcaagaaaaaggctcccgcccaaaagaaagcctccaggggatcg 181 G R D T V R P L W Y P G A K A P E Y L D 182 ggccgagacaccgtcagacccctttggtatccgggcgccaaagcgcccgaatacctcgat 241 G S L V G D Y G F D P F G L G K P A E Y 242 gggagtcttgtcggagactacgggttcgatccgtttgggctagggaagcccgcggagtac 301 L Q F E L D S L D Q N L A K N V A G D I 302 ctgcagttcgagctggactcgctggaccagaaccttgcgaagaacgtggctggggacatc 361 I G T R T E L A D V K S T P F Q P Y S E 362 attggaaccaggaccgagcttgcggacgtgaagtccacgccgtttcagccctacagcgag 421 V F G L Q R F R E C E L I H G R W A M L 422 gtgtttgggctccagaggttccgtgagtgcgaactcatccatggaaggtgggccatgctc 481 A T L G A L T V E W L T G V T W Q D A G 482 gccactctcggagctctcactgttgagtggctcactggtgttacatggcaagacgccgga 541 K V E L V E G S S Y L G Q P L P F S I T 542 aaggtggagctagtagaagggtcatcataccttgggcaaccacttccattctcaatcacc 601 T L I W I E A L V I G Y I E F Q R N A E 602 acactgatctggatcgaggcactcgtaattggctacatagagttccagaggaatgcagag 661 L D P E K R L Y P G G S Y F D P L G L A 662 ctcgacccagagaagaggttgtacccaggtggcagttacttcgacccattgggcctggcc 721 S D P E K K A T L Q L A E I K H A R L A 722 tcagacccagagaagaaagccacccttcaattggcggagatcaagcacgcccgtcttgcc 781 M V G F L G F A V Q A A V T G K G P L N 782 atggtgggcttcttgggctttgcagtccaagccgccgtcaccggcaagggcccgctcaac 841 N W A T H L S D P L H T T I I D T F S S 842 aactgggccacccacttgagtgacccactccacacaaccatcattgacaccttctcatcc 901 S S * E E E S F L C L V T I T S I L * K 902 tcctcttaagaagaagagtctttcttgtgcctcgtcactattactagcatattgtaaaag 961 S F L L R L L V V I R T F S L S W I S S 962 tcttttcttcttcggcttttggttgtaattagaacattttcacttagttggattagtagt 1021 T C E K L V T * N W R G * A E M L L L D 1022 acttgtgaaaaacttgtaacgtgaaattggagaggctaagcagaaatgcttctgcttgat 1081 V K C S P V N V Y Y A R N R E G M T R V 1082 gttaagtgttctcctgtaaatgtttattatgcacggaatcgagagggaatgacgagggta 1141 K K Y * * N * D H Y E R * W Q G I G L R 1142 aaaaaatactgataaaattgagatcactacgaaaggtaatggcagggaattggattgagg 1201 P K K K K K K K K 1202 ccaaaaaaaaaaaaaaaaaaaaaaaaaaa 1230
8 Start codon – Methionine highlighted in yellow. Stop codons are red asterisks (8 total), the first observed around the beginning of line 902. Using SIXFRAME from the Biology Workbench, this frame had the longest ORF, and least stop codons. Protein Sequence1 – MATATAAATSSFMGTRLLEAHSGAGRVHARFGFGKKKAPAQKKASRGSGRDTVRPLWYPGAKAPEYLDGSLVGDYGFDPFGLGKP AEYLQFELDSLDQNLAKNVAGDIIGTRTELADVKSTPFQPYSEVFGLQRFRECELIHGRWAMLATLGALTVEWLTGVTWQDAGKVELV EGSSYLGQPLPFSITTLIWIEALVIGYIEFQRNAELDPEKRLYPGGSYFDPLGLASDPEKKATLQLAEIKHARLAMVGFLGFAVQAAVTGK GPLNNWATHLSDPLHTTIIDTFSSSS  The first ATG (Methionine) is the start codon in the ORF analysis. I believe to have 100% of the cDNA.  The cDNA insert has a LAMP motif,2 which codes for proteins responsible for interacting with light. Most photosynthetic proteins are coded by DNA with a LAMP motif, as well as proteins in ocular tissues.  A hydropathy plot determines the hydrophobicity or hydrophilicity of a protein based on the amino acid sequence.
9 The window size of the hydropathy plot has been set to 9.4 Above the line indicates regions of hydrophobicity, most likely transmembrane or hydrophobic pockets within the protein structure. Most of the protein appears hydrophilic however, and is most likely in an aqueous state.
10 References (1) Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb Miller (2000), "A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(1-2):203-14. (2) "SDSC Biology Workbench." SDSC Biology Workbench. Web. 14 Mar. 2016. <http://seqtool.sdsc.edu/>. (3) "Software Developer of Next Gen Sequencing DNA Genetic Analysis and LIMS." Software Developer of Next Gen Sequencing DNA Genetic Analysis and LIMS. Perkin Elmer, n.d. Web. 25 Feb. 2016. <http://www.geospiza.com/>. (4) "Genomics and Bioinformatics @ Davidson College." Genomics and Bioinformatics @ Davidson College. Web. 15 Mar. 2016. <http://gcat.davidson.edu/>.

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Sequence Analysis - 2-16-16 (1)

  • 1.
    1 Sequence Analysis ofan Isolated Culture, Pertaining to Glycine Max By: Alexander Ward CMB 426 - Dr. Tsou 3/15/2016
  • 2.
    2 Original/Unedited Sequences3 – T3 Primer– GAACTCCGCGGGTGCGGCCGCTCTGAACTAGTGGATCCCCCGGGCTGCAGGAATTCGGCACGAGCGGCAACAGCAGCTGCCA CCTCGTCCTTTATGGGGACGCGTCTCCTGGAGGCTCACTCCGGAGCGGGGCGAGTGCACGCCCGATTCGGCTTCGGCAAGAAA AAGGCTCCCGCCCCAAAGAAAGCCTCCAGGGGATCGGGCCGAGACACCGACAGACCCCTTTGGTATCCGGGCGCCAAAGCGC CCGAATACCTCGATGGGAGTCTTGTCGGAGACTACGGGTTCGATCCGTTTGGGCTAGGGAAGCCCGCGGAGTACCTGCAGTTC GAGCTGGACTCGCTGGACCAGAACCTTGCGAAGAACGTGGCTGGGGACATCATTGGAACCAGGACCGAGCTCGCGGACGTGA AGTCCACGCCGTTTCAGCCCTACAGCGAGGTGTTTGGGCTCCAGAGGTTCCGTGAGTGCGAACTCATCCATGGAAGGTGGGCC ATGCTCGCCACTCTCGGAGCTCTCACTGTTGAGTGGCTCACTGGTGTTACATGGCAAGACGCCGGAAAGGTGGAGCTAGTAGA AGGGTCATCATACCTTGGGCAACCACTTCCATTCTCAATCACCACACTGATCTGGATCGAGGTACTCGTAATTGGCTACATAGA GTTCCAGAGGAATGCAGAGCTCGACCCAGAGAAGAGGTTGTACCCAGGTGGCAGTTACTTCGACCCATTGGGCCTGGCCTCAG ACCCAGAGAAGAAAGCCACCCTTCAATTGGCGGAGATCAAGCACGCCCGTCTTGCCATGGTGGGCTTCTTGGGCTTTGC AGTC CAAGCCGCCGCCACCGGCAAGGGTCCGCTCAACAACTGGGCCACCCACTTGAGTGACCCACTCCACACAACCATCATTGACAC CTTCTCATCCTCCTCTTAAGAAGAAGAGTCTTTCTTGTGCCTCGTCACTATTACTAGCATATTGTAAAAGTCTTTTCTTCTTCGGCT TTTGGTTGTAATTAAAACATTTTCACTTANTTGGAATNAGTAAATACTTGTGAAAAAACTTNGTAAACGTGGAAANTNGGANAG GCTAAACCAAAAATGGCTTTCTGCTTNAATGTTAAAAAAAAAAAAAAAAAAAAAAAATTCGAGGGGGGGNCCCGGGTAACCC NAATTNCCCCCNTAAAAGNGGNGTTCNAAATTAAAAANTTCACTGGGCCNGTTCGTTTTTNAAAACGTTCNTNAACGGGGGN AAAAACCCCTGGGGGGTTTACCCCAANTTTAAATTCCCCCTTTNGGAAANAAAATTCCCCCTTTTTNCCCNANGNTGGGGGNT NAAANAACNAAAAAANNGCCCCCCCCACCAAATTNNCCCNTTTTCCAAAAAANTTTNCCCAACCNTNAAAAGGGNNAAAN GGNAAAA T7 Primer – Slight Taq slip following poly-T CGGGCCCCCTGCGTTTTTTTTTTTTTTTTTTTTACATCAAGCAGAAGCATTTCTGCTTAGCCTCTCCAATTTCACGTTACAAGTTTTT CACAAGTACTACTAATCCAACTAAGTGAAAATGTTCTAATTACAACCAAAAGCCGAAGAAGAAAAGACTTTTACAATATGCTAG TAATAGTGACGAGGCACAAGAAAGACTCTTCTTCTTAAGAGGAGGATGAGAAGGTGTCAATGATGGTTGTGTGGAGTGGGTCA CTCAAGTGGGTGGCCCAGTTGTTGAGCGGACCCTTGCCGGTGGCGGCGGCTTGGACTGCAAAGCCCAAGAAGCCCACCATGG CAAGACGGGCGTGCTTGATCTCCGCCAATTGAAGGGTGGCTTTCTTCTCTGGGTCTGAGGCCAGGCCCAATGGGTCGAAGTAA CTGCCACCTGGGTACAACCTCTTCTCTGGGTCGAGCTCTGCATTCCTCTGGAACTCTATGTAGCCAATTACGAGTACCTCGATCC AGATCAGTGTGGTGATTGAGAATGGAAGTGGTTGCCCAAGGTATGATGAC CCTTCTACTAGCTCCACCTTTCCGGCGTCTTGCC ATGTAACACCAGTGAGCCACTCAACAGTGAGAGCTCCGAGAGTGGCGAGCATGGCCCACCTTCCATGGATGAGTTCGCACTCA CGGAACCTCTGGAGCCCAAACACCTCGCTGTAGGGCTGAAACGGCGTGGACTTCACGTCCGCGAGCTCGGTCCTGGTTCCAAT GATGTCCCCAGCCACGTTCTTCGCAAGGTTCTGGTCCAGCGAGTCCAGCTCGAACTGCAGGTACTCCGCGGGCTTCCCTAGCCC AAACGGGATCGAACCCGTAGTCTCCGACAAGACTCCCCATCGAGGTATTTCGGGGCGCTTTTGGCGCCCGGGATACCAAAGGG GGTCTGTCGGGTGGTCTCCGGCCCCGAATCCCCTGGGAGGGCTTTTCTTTTGGGGGGCGGGAAGCCTTTTTTTCTTGCCGGAAG CCCGAAAATCGGGGGCGGTGGNACTNCGCCCCCCGGNTTCNGGGANGTGAAGCCCTTCCCNAGGAANAAACNNNNGTTCC CCCCCNATAAAAANGGANCNAAANGGTGGGCNANCCCTTGCCTTTTTTGCCCCNCCTTCCGTNGNCCCAAAAATTCCNTGGC CANNCCCCCGGGGGGGGAAATCCCCCCTTAANNTTTNTTAAAAAAGCGGGGCCCCCCCCNCCCNCCCNGGGNGGGNAAAC CTTCCCNACCCNTTTTTTGNTTTCCCCCCNTTTTANNGGGGAN
  • 3.
    3 Edited T3 Primer– GAATTCGGCACGAGCGGCAACAGCAGCTGCCACCTCGTCCTTTATGGGGACGCGTCTCCTGGAGGCTCACTCCGGAGCGGGG CGAGTGCACGCCCGATTCGGCTTCGGCAAGAAAAAGGCTCCCGCCCCAAAGAAAGCCTCCAGGGGATCGGGCCGAGACACC GACAGACCCCTTTGGTATCCGGGCGCCAAAGCGCCCGAATACCTCGATGGGAGTCTTGTCGGAGACTACGGGTTCGATCCGTT TGGGCTAGGGAAGCCCGCGGAGTACCTGCAGTTCGAGCTGGACTCGCTGGACCAGAACCTTGCGAAGAACGTGGCTGGGGAC ATCATTGGAACCAGGACCGAGCTCGCGGACGTGAAGTCCACGCCGTTTCAGCCCTACAGCGAGGTGTTTGGGCTCCAGAGGTT CCGTGAGTGCGAACTCATCCATGGAAGGTGGGCCATGCTCGCCACTCTCGGAGCTCTCACTGTTGAGTGGCTCACTGGTGTTAC ATGGCAAGACGCCGGAAAGGTGGAGCTAGTAGAAGGGTCATCATACCTTGGGCAACCACTTCCATTCTCAATCACCACACTGA TCTGGATCGAGGTACTCGTAATTGGCTACATAGAGTTCCAGAGGAATGCAGAGCTCGACCCAGAGAAGAGGTTGTACCCAGGT GGCAGTTACTTCGACCCATTGGGCCTGGCCTCAGACCCAGAGAAGAAAGCCACCCTTCAATTGGCGGAGATCAAGCACGCCCG TCTTGCCATGGTGGGCTTCTTGGGCTTTGCAGTCCAAGCCGCCGCCACCGGCAAGGGTCCGCTCAACAACTGGGCCACCCACTT GAGTGACCCACTCCACACAACCATCATTGACACCTTCTCATCCTCCTCTTAAGAAGAAGAGTCTTTCTTGTGCCTCGTCACTATTA CTAGCATATTGTAAAAGTCTTTTCTTCTTCGGCTTTTGGTTGTAATTAAAACATTTTCACTTA Edited T7 Primer – ACATCAAGCAGAAGCATTTCTGCTTAGCCTCTCCAATTTCACGTTACAAGTTTTTCACAAGTACTACTAATCCAACTAAGTGAAA ATGTTCTAATTACAACCAAAAGCCGAAGAAGAAAAGACTTTTACAATATGCTAGTAATAGTGACGAGGCACAAGAAAGACTCTT CTTCTTAAGAGGAGGATGAGAAGGTGTCAATGATGGTTGTGTGGAGTGGGTCACTCAAGTGGGTGGCCCAGTTGTTGAGCGGA CCCTTGCCGGTGGCGGCGGCTTGGACTGCAAAGCCCAAGAAGCCCACCATGGCAAGACGGGCGTGCTTGATCTCCGCCAATT GAAGGGTGGCTTTCTTCTCTGGGTCTGAGGCCAGGCCCAATGGGTCGAAGTAACTGCCACCTGGGTACAACCTCTTCTCTGGGT CGAGCTCTGCATTCCTCTGGAACTCTATGTAGCCAATTACGAGTACCTCGATCCAGATCAGTGTGGTGATTGAGAATGGAAGTG GTTGCCCAAGGTATGATGACCCTTCTACTAGCTCCACCTTTCCGGCGTCTTGCCATGTAACACCAGTGAGCCACTCAACAGTGA GAGCTCCGAGAGTGGCGAGCATGGCCCACCTTCCATGGATGAGTTCGCACTCACGGAACCTCTGGAGCCCAAACACCTCGCTG TAGGGCTGAAACGGCGTGGACTTCACGTCCGCGAGCTCGGTCCTGGTTCCAATGATGTCCCCAGCCACGTTCTTCGCAAGGTTC TGGTCCAGCGAGTCCAGCTCGAACTGCAGGTACTCCGCGGGCTTCCCTAGCCCAAACGGGATCGAACCCGTAGTCTCCGACAA GACTCCCCATCGAGGTATTTCGGGGCGCTTTTGGCGCCCGGGATACCAAAGGGGGTCTGTCGGGTGGTCTCCGGCCCCGAATC CCCTGGGAGGGCTTTTCTTTTGGGGGGCGGGAAGCCTTTTTTTCTTGCCGGAAGCCCGAAAATCGGGGGCGGTGG Green = EcoRI Cut Site, Red = XhoI Cut Site, Teal = PolyA Tail, Purple = Sequence discrepancies; Multiple cut sites were not observed.
  • 4.
    4 Align 2 ormore sequences using CLUSTALW2 – PILEMSF Format 1 50 T3_edited GAATTCGGCA CGAGCGGCAA CAGCAGCTGC CACCTCGTCC TTTATGGGGA T7_edited__Reve .......... .......... .......... .......... .......... 51 100 T3_edited CGCGTCTCCT GGAGGCTCAC TCCGGAGCGG GGCGAGTGCA CGCCCGATTC T7_edited__Reve .......... .......... .......... ..CCACCGCC CCCGATTTTC 101 150 T3_edited GG..CTTCGG CAAGAAAAAG G...CTCCCG CCCC...AAA GAAAG...CC T7_edited__Reve GGGCTTCCGG CAAGAAAAAA AGGCTTCCCG CCCCCCAAAA GAAAAGCCCT 151 200 T3_edited TCCAGGGGAT C..GGGCCG. AGACA..CCG ACAGACCCC. TTTGGTATCC T7_edited__Reve CCCAGGGGAT TCGGGGCCGG AGACCACCCG ACAGACCCCC TTTGGTATCC 201 250 T3_edited .GGGCGCCAA A.GCGCCC.. GAATACCTCG ATGGG.AGTC TTGTCGGAGA T7_edited__Reve CGGGCGCCAA AAGCGCCCCG AAATACCTCG ATGGGGAGTC TTGTCGGAGA 251 300 T3_edited CTACGGGTTC GATCC.GTTT GGGCTAGGGA AGCCCGCGGA GTACCTGCAG T7_edited__Reve CTACGGGTTC GATCCCGTTT GGGCTAGGGA AGCCCGCGGA GTACCTGCAG 301 350 T3_edited TTCGAGCTGG ACTCGCTGGA CCAGAACCTT GCGAAGAACG TGGCTGGGGA T7_edited__Reve TTCGAGCTGG ACTCGCTGGA CCAGAACCTT GCGAAGAACG TGGCTGGGGA 351 400 T3_edited CATCATTGGA ACCAGGACCG AGCTCGCGGA CGTGAAGTCC ACGCCGTTTC T7_edited__Reve CATCATTGGA ACCAGGACCG AGCTCGCGGA CGTGAAGTCC ACGCCGTTTC 401 450 T3_edited AGCCCTACAG CGAGGTGTTT GGGCTCCAGA GGTTCCGTGA GTGCGAACTC T7_edited__Reve AGCCCTACAG CGAGGTGTTT GGGCTCCAGA GGTTCCGTGA GTGCGAACTC 451 500 T3_edited ATCCATGGAA GGTGGGCCAT GCTCGCCACT CTCGGAGCTC TCACTGTTGA T7_edited__Reve ATCCATGGAA GGTGGGCCAT GCTCGCCACT CTCGGAGCTC TCACTGTTGA 501 550 T3_edited GTGGCTCACT GGTGTTACAT GGCAAGACGC CGGAAAGGTG GAGCTAGTAG T7_edited__Reve GTGGCTCACT GGTGTTACAT GGCAAGACGC CGGAAAGGTG GAGCTAGTAG 551 600 T3_edited AAGGGTCATC ATACCTTGGG CAACCACTTC CATTCTCAAT CACCACACTG T7_edited__Reve AAGGGTCATC ATACCTTGGG CAACCACTTC CATTCTCAAT CACCACACTG 601 650 T3_edited ATCTGGATCG AGGTACTCGT AATTGGCTAC ATAGAGTTCC AGAGGAATGC T7_edited__Reve ATCTGGATCG AGGTACTCGT AATTGGCTAC ATAGAGTTCC AGAGGAATGC 651 700 T3_edited AGAGCTCGAC CCAGAGAAGA GGTTGTACCC AGGTGGCAGT TACTTCGACC T7_edited__Reve AGAGCTCGAC CCAGAGAAGA GGTTGTACCC AGGTGGCAGT TACTTCGACC
  • 5.
    5 701 750 T3_edited CATTGGGCCTGGCCTCAGAC CCAGAGAAGA AAGCCACCCT TCAATTGGCG T7_edited__Reve CATTGGGCCT GGCCTCAGAC CCAGAGAAGA AAGCCACCCT TCAATTGGCG 751 800 T3_edited GAGATCAAGC ACGCCCGTCT TGCCATGGTG GGCTTCTTGG GCTTTGCAGT T7_edited__Reve GAGATCAAGC ACGCCCGTCT TGCCATGGTG GGCTTCTTGG GCTTTGCAGT 801 850 T3_edited CCAAGCCGCC GCCACCGGCA AGGGTCCGCT CAACAACTGG GCCACCCACT T7_edited__Reve CCAAGCCGCC GCCACCGGCA AGGGTCCGCT CAACAACTGG GCCACCCACT 851 900 T3_edited TGAGTGACCC ACTCCACACA ACCATCATTG ACACCTTCTC ATCCTCCTCT T7_edited__Reve TGAGTGACCC ACTCCACACA ACCATCATTG ACACCTTCTC ATCCTCCTCT 901 950 T3_edited TAAGAAGAAG AGTCTTTCTT GTGCCTCGTC ACTATTACTA GCATATTGTA T7_edited__Reve TAAGAAGAAG AGTCTTTCTT GTGCCTCGTC ACTATTACTA GCATATTGTA 951 1000 T3_edited AAAGTCTTTT CTTCTTCGGC TTTTGGTTGT AATTAAAACA TTTTCACTTA T7_edited__Reve AAAGTCTTTT CTTCTTCGGC TTTTGGTTGT AATTAGAACA TTTTCACTTA 1001 1050 T3_edited .......... .......... .......... .......... .......... T7_edited__Reve GTTGGATTAG TAGTACTTGT GAAAAACTTG TAACGTGAAA TTGGAGAGGC 1051 1076 T3_edited .......... .......... ...... T7_edited__Reve TAAGCAGAAA TGCTTCTGCT TGATGT 76 at the end and 82 at the beginning are the only base pairs not overlapping, 152 total, indicating an overlap of 924 bp.
  • 6.
    6 Glycine max chlorophylla-b binding protein CP29.2, chloroplastic-like (LOC100785415), mRNA Sequence for BLAST input, entire T3 cDNA insert sequence – TTCTTAAGAGGAGGATGAGAAGGTGTCAATGATGGTTGTGTGGAGTGGGTCACTCAAGTGGGTGGCCCAGTTGTTGAGCGGAC CCTTGCCGGTGGCGGCGGCTTGGACTGCAAAGCCCAAGAAGCCCACCATGGCAAGACGGGCGTGCTTGATCTCCGCCAATTG AAGGGTGGCTTTCTTCTCTGGGTCTGAGGCCAGGCCCAATGGGTCGAAGTAACTGCCACCTGGGTACAACCTCTTCTCTGGGTC GAGCTCTGCATTCCTCTGGAACTCTATGTAGCCAATTACGAGTACCTCGATCCAGATCAGTGTGGTGATTGAGAATGGAAGTGG TTGCCCAAGGTATGATGACCCTTCTACTAGCTCCACCTTTCCGGCGTCTTGCCATGTAACACCAGTGAGCCACTCAACAGTGAG AGCTCCGAGAGTGGCGAGCATGGCCCACCTTCCATGGATGAGTTCGCACTCACGGAACCTCTGGAGCCCAAACACCTCGCTGT AGGGCTGAAACGGCGTGGACTTCACGTCCGCGAGCTCGGTCCTGGTTCCAATGATGTCCCCAGCCACGTTCTTCGCAAGGTTCT GGTCCAGCGAGTCCAGCTCGAACTGCAGGTACTCCGCGGGCTTCCCTAGCCCAAACGG Top 20 BLAST hits1 – 1. Glycine max cDNA, clone: GMFL01-17-I17 (99% ID) 2. Glycine max chlorophyll a-b binding protein CP29.2, chloroplastic-like (LOC100785415), mRNA (99% ID) 3. PREDICTED: Glycine max chlorophyll a-b binding protein CP29.2, chloroplastic-like (LOC100785180), mRNA (99% ID) 4. Glycine max cDNA, clone: GMFL01-03-O02 (96% ID) 5. Vigna radiata chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC106774260), mRNA (90% ID) 6. Phaseolus vulgaris hypothetical protein (PHAVU_010G002100g) mRNA, complete cds (90% ID) 7. Morus notabilis hypothetical protein partial mRNA (Too small) 8. Glycine max strain Williams 82 clone GM_WBb0012I20, complete sequence (Too large) 9. PREDICTED: Oryza brachyantha chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC102700159), mRNA (83% ID) 10. Phyllostachys edulis chloroplast chlorophyll a/b binding protein cab-PhE7 mRNA, complete cds; nuclear gene for chloroplast product (82%) 11. Pyrus x bretschneideri clone 915 a-b binding protein mRNA, complete cds (83% ID) 12. Pyrus x bretschneideri chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC103936594), mRNA (Too small) 13. Phyllostachys edulis cDNA clone: bphyem211d01, full insert sequence (82% ID) 14. PREDICTED: Malus x domestica chlorophyll a-b binding protein CP29.1, chloroplastic-like (LOC103450616), mRNA (83% ID) 15. PREDICTED: Oryza sativa Japonica Group chlorophyll a-b binding protein CP29.1, chloroplastic (LOC4343583), mRNA (82% ID) 16. Oryza sativa (indica cultivar-group) cDNA clone:OSIGCSN014K09, full insert sequence (82% ID) 17. Oryza sativa Japonica Group cDNA clone:001-204-B02, full insert sequence (82% ID) 18. Oryza sativa Japonica Group cDNA clone:006-309-F11, full insert sequence (82% ID) 19. Oryza sativa Japonica Group cDNA clone:001-013-C12, full insert sequence (82% ID) 20. Oryza sativa Japonica Group cDNA clone:J013098H13, full insert sequence (82% ID) Top 5 Protein BLAST hits1 – 1. chlorophyll a-b binding protein CP29.2, chloroplastic-like [Glycine max] (Length = 290) (100% ID) 2. hypothetical protein GLYMA_03G060300 [Glycine max] (Length = 290) (99% ID)
  • 7.
    7 3. PREDICTED: chlorophylla-b binding protein CP29.2, chloroplastic-like [Glycine max] (Length = 290) (97% ID) 4. PREDICTED: chlorophyll a-b binding protein CP29.3, chloroplastic-like [Glycine max] (Length = 278) (73% ID) 5. PREDICTED: chlorophyll a-b binding protein CP29.3, chloroplastic-like [Glycine max] (Length = 282) (72% ID) Of the Light Harvesting Complex (LHC), two molecules which interact with visible light, chlorophyll a and chlorophyll b (Chl-a and Chl-b respectively). The protein which is coded by the gene of interest is chlorophyll a/b binding protein, which acts as an antenna for photosystems 1 and 2, a peripheral light- harvesting protein. ORF Analysis2 – F S H T R R T L A Q T I M A T A T A A A 2 ttttctcacaccaggcgcacgttagcacaaactatcatggccacggcaacagcagctgcc 61 T S S F M G T R L L E A H S G A G R V H 62 acctcgtcctttatggggacgcgtctcctggaggctcactccggggcggggcgagtgcac 121 A R F G F G K K K A P A Q K K A S R G S 122 gcccgattcggcttcggcaagaaaaaggctcccgcccaaaagaaagcctccaggggatcg 181 G R D T V R P L W Y P G A K A P E Y L D 182 ggccgagacaccgtcagacccctttggtatccgggcgccaaagcgcccgaatacctcgat 241 G S L V G D Y G F D P F G L G K P A E Y 242 gggagtcttgtcggagactacgggttcgatccgtttgggctagggaagcccgcggagtac 301 L Q F E L D S L D Q N L A K N V A G D I 302 ctgcagttcgagctggactcgctggaccagaaccttgcgaagaacgtggctggggacatc 361 I G T R T E L A D V K S T P F Q P Y S E 362 attggaaccaggaccgagcttgcggacgtgaagtccacgccgtttcagccctacagcgag 421 V F G L Q R F R E C E L I H G R W A M L 422 gtgtttgggctccagaggttccgtgagtgcgaactcatccatggaaggtgggccatgctc 481 A T L G A L T V E W L T G V T W Q D A G 482 gccactctcggagctctcactgttgagtggctcactggtgttacatggcaagacgccgga 541 K V E L V E G S S Y L G Q P L P F S I T 542 aaggtggagctagtagaagggtcatcataccttgggcaaccacttccattctcaatcacc 601 T L I W I E A L V I G Y I E F Q R N A E 602 acactgatctggatcgaggcactcgtaattggctacatagagttccagaggaatgcagag 661 L D P E K R L Y P G G S Y F D P L G L A 662 ctcgacccagagaagaggttgtacccaggtggcagttacttcgacccattgggcctggcc 721 S D P E K K A T L Q L A E I K H A R L A 722 tcagacccagagaagaaagccacccttcaattggcggagatcaagcacgcccgtcttgcc 781 M V G F L G F A V Q A A V T G K G P L N 782 atggtgggcttcttgggctttgcagtccaagccgccgtcaccggcaagggcccgctcaac 841 N W A T H L S D P L H T T I I D T F S S 842 aactgggccacccacttgagtgacccactccacacaaccatcattgacaccttctcatcc 901 S S * E E E S F L C L V T I T S I L * K 902 tcctcttaagaagaagagtctttcttgtgcctcgtcactattactagcatattgtaaaag 961 S F L L R L L V V I R T F S L S W I S S 962 tcttttcttcttcggcttttggttgtaattagaacattttcacttagttggattagtagt 1021 T C E K L V T * N W R G * A E M L L L D 1022 acttgtgaaaaacttgtaacgtgaaattggagaggctaagcagaaatgcttctgcttgat 1081 V K C S P V N V Y Y A R N R E G M T R V 1082 gttaagtgttctcctgtaaatgtttattatgcacggaatcgagagggaatgacgagggta 1141 K K Y * * N * D H Y E R * W Q G I G L R 1142 aaaaaatactgataaaattgagatcactacgaaaggtaatggcagggaattggattgagg 1201 P K K K K K K K K 1202 ccaaaaaaaaaaaaaaaaaaaaaaaaaaa 1230
  • 8.
    8 Start codon –Methionine highlighted in yellow. Stop codons are red asterisks (8 total), the first observed around the beginning of line 902. Using SIXFRAME from the Biology Workbench, this frame had the longest ORF, and least stop codons. Protein Sequence1 – MATATAAATSSFMGTRLLEAHSGAGRVHARFGFGKKKAPAQKKASRGSGRDTVRPLWYPGAKAPEYLDGSLVGDYGFDPFGLGKP AEYLQFELDSLDQNLAKNVAGDIIGTRTELADVKSTPFQPYSEVFGLQRFRECELIHGRWAMLATLGALTVEWLTGVTWQDAGKVELV EGSSYLGQPLPFSITTLIWIEALVIGYIEFQRNAELDPEKRLYPGGSYFDPLGLASDPEKKATLQLAEIKHARLAMVGFLGFAVQAAVTGK GPLNNWATHLSDPLHTTIIDTFSSSS  The first ATG (Methionine) is the start codon in the ORF analysis. I believe to have 100% of the cDNA.  The cDNA insert has a LAMP motif,2 which codes for proteins responsible for interacting with light. Most photosynthetic proteins are coded by DNA with a LAMP motif, as well as proteins in ocular tissues.  A hydropathy plot determines the hydrophobicity or hydrophilicity of a protein based on the amino acid sequence.
  • 9.
    9 The window sizeof the hydropathy plot has been set to 9.4 Above the line indicates regions of hydrophobicity, most likely transmembrane or hydrophobic pockets within the protein structure. Most of the protein appears hydrophilic however, and is most likely in an aqueous state.
  • 10.
    10 References (1) Zheng Zhang,Scott Schwartz, Lukas Wagner, and Webb Miller (2000), "A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(1-2):203-14. (2) "SDSC Biology Workbench." SDSC Biology Workbench. Web. 14 Mar. 2016. <http://seqtool.sdsc.edu/>. (3) "Software Developer of Next Gen Sequencing DNA Genetic Analysis and LIMS." Software Developer of Next Gen Sequencing DNA Genetic Analysis and LIMS. Perkin Elmer, n.d. Web. 25 Feb. 2016. <http://www.geospiza.com/>. (4) "Genomics and Bioinformatics @ Davidson College." Genomics and Bioinformatics @ Davidson College. Web. 15 Mar. 2016. <http://gcat.davidson.edu/>.