Clone Sottrattivo

Primer utilizzati durante la costruzione di banche sottrattiva con la tecnica SSH
>N2R
agcgtggtcgcggccgaggt
>N1
tcgagcggccgcccgggcaggt
>CN2R
Acctcggccgcgaccacgct
>CN1
Acctgcccgggcggccgctcga
> AG10_SP6 -- 13..1007 of sequence

AAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCT
TAGCGTGGTCGCGGCCGAGGT
ACTCCTGGCAGAATGTTGCTCCTTTCTGCTTGGCATCCATTACCAACCC
CTCAATGAAGTTTGAGGATGATTCTGACACAACCGGGGTGATATCACAGTCATCCTCTGG
TGGTCCAACGGTCAATTTCGCTACTTTGGCCTTAACTTTCTCAACAAGAGCATCAGCTAC
AGACTCCATCACCAAGACAACCTTGACGGCGGTGCATCTTTGACCACTGTAAGAGAAGCC
TCCTTTTATGATGTTTGCTGCAACTAAATCCAAATCAGCATCTTCAAGCACAATGCATGC
ATCTTTCCCACCTAGTTCCATTTGAAGAGGGATCATGCCTGCCTTCTTTGAGATTGCAAT
TCCAGTGTCTCCACCGGTAAAACTTATACAGTTCACACCAGGATGCATTGTGAGGAAATC
TCCAATCTCGGAGCCTTTTCCAGTGATGCAACTAATTAGGCCTTTGGGAAAACCAGCCAA
ATGAAAGCAATGCACCATGTGGAGAGCAGCTACAGCACCCTGAGTTGGAGGTTTGAGGAC
AAGGGAGTTTCCAGCAATCAGAGCAGGAGCAATTTTTGAGACAGCTAGGTTGACAGGATA
GTTGAAAGGTGGGATTGCTAAAATGACCCCAAGTGGAATCTTAGAAGAAAGGCAATATTT
GGTCCTTTCGTTCCCTGGAAAGCTATCAGACACTA
AAGGAGCAACATTCTGCCAGGAGT
ORA MANDIAMO A SEQUENZIARE DA T7 PER OTTENERE LA SEQUENZA COMPLETA DEL NOSTRO

CLONE
> AG10_T7 -- 26..815 of sequence
TCTGCAGAATTCGCCCTTACCTGCCCGGGCGGCCGCTCGAACGCGGGGCGGAGCTCCTTC
ACAAGGCAGCTGCGATCCTGAAAGAACACAAGGTCCCCATTGCAGAGTGCCTGGTCAAAG
AGATCGCAAAACCCGCCAAGGACGCAGTCACTGAGGTCGTTAGGTCTGGAGATCTGGTGT
CTTACTGTGCCGAAGAAGGCGTTAGGATTCTGGGAGAGGGGAAGTTCCTAGTGTCTGATA
GCTTTCCAGGGAACGAAAGGACCAAATATTGCCTTTCTTCTAAGATTCCACTTGGGGTCA
TTTTAGCAATCCCACCTTTCAACTATCCTGTCAACCTAGCTGTCTCAAAAATTGCTCCTG
CTCTGATTGCTGGAAACTCCATTTTCCACAAACCTCCAAC
UTILIZZIAMO IL PROGRAMMA Multiple Sequence Alignment by CLUSTALW PER VEDERE

LE ZONE DI SOVRAPPOSIZIONE AL FINE DI AVERE LA SEQUENZA COMPLETA DEL CLONE
T GTGCCGAAGAAGGCGTTAGGATTCTGGGAGAGGGGAAGTTCCTAGTGTCTGATAGCTTTC
S ------------------------------------------TAGTGTCTGATAGCTTTC
******************
T CAGGGAACGAAAGGACCAAATATTGCCTTTCTTCTAAGATTCCACTTGGGGTCATTTTAG
S CAGGGAACGAAAGGACCAAATATTGCCTTTCTTCTAAGATTCCACTTGGGGTCATTTTAG
************************************************************
T CAATCCCACCTTTCAACTATCCTGTCAACCTAGCTGTCTCAAAAATTGCTCCTGCTCTGA
S CAATCCCACCTTTCAACTATCCTGTCAACCTAGCTGTCTCAAAAATTGCTCCTGCTCTGA
************************************************************
T TTGCTGGAAACTCCATTTTCCACAAACCTCCAAC--------------------------
S TTGCTGGAAACTCCCTTGTCCTCAAACCTCCAACTCAGGGTGCTGTAGCTGCTCTCCACA
************** ** *** ************
T ------------------------------------------------------------
S TGGTGCATTGCTTTCATTTGGCTGGTTTTCCCAAAGGCCTAATTAGTTGCATCACTGGAA
SEQUENZA COMPLETA DEL CLONE
ACGCGGGGCGGAGCTCCTTCACAAGGCAGCTGCGATCCTGAAAGAACACAAGGTCCCCATT
GCAGAGTGCCTGGTCAAAGAGATCGCAAAACCCGCCAAGGACGCAGTCACTGAGGTCGTTA
GGTCTGGAGATCTGGTGTCTTACTGTGCCGAAGAAGGCGTTAGGATTCTGGGAGAGGGGAA
GTTCCTAGTGTCTGATAGCTTTCCAGGGAACGAAAGGACCAAATATTGCCTTTCTTCTAAG
ATTCCACTTGGGGTCATTTTAGCAATCCCACCTTTCAACTATCCTGTCAACCTAGCTGTCT
CAAAAATTGCTCCTGCTCTGATTGCTGGAAACTCCCTTGTCCTCAAACCTCCAACTCAGGG
TGCTGTAGCTGCTCTCCACATGGTGCATTGCTTTCATTTGGCTGGTTTTCCCAAAGGCCTA
ATTAGTTGCATCACTGGAAAAGGCTCCGAGATTGGAGATTTCCTCACAATGCATCCTGGTG
TGAACTGTATAAGTTTTACCGGTGGAGACACTGGAATTGCAATCTCAAAGAAGGCAGGCAT
GATCCCTCTTCAAATGGAACTAGGTGGGAAAGATGCATGCATTGTGCTTGAAGATGCTGAT
TTGGATTTAGTTGCAGCAAACATCATAAAAGGAGGCTTCTCTTACAGTGGTCAAAGATGCA
CCGCCGTCAAGGTTGTCTTGGTGATGGAGTCTGTAGCTGATGCTCTTGTTGAGAAAGTTAA
GGCCAAAGTAGCGAAATTGACCGTTGGACCACCAGAGGATGACTGTGATATCACCCCGGTT
GTGTCAGAATCATCCTCAAACTTCATTGAGGGGTTGGTAATGGATGCCAAGCAGAAAGGAG
CAACATTCTGCCAGGAGT
Controllare con BLASTN o FASTA

Possiamo utilizzare il programma BLASTX
> emb|CAQ16337.1| NADP-dependent glyceraldehyde-3-phosphate dehydrogenase
[Platanus
x acerifolia]
Length=290
Score = 542 bits (1397), Expect = 1e-152

Identities = 290/290 (100%), Positives = 290/290 (100%), Gaps = 0/290 (0%)
Frame = +2
Query 2 RGAELLHKAAAILKEHKVPIAECLVKEIAKPAKDAVTEVVRSGDLVSYCAEEGVRILGEG 181

RGAELLHKAAAILKEHKVPIAECLVKEIAKPAKDAVTEVVRSGDLVSYCAEEGVRILGEG
Sbjct 1 RGAELLHKAAAILKEHKVPIAECLVKEIAKPAKDAVTEVVRSGDLVSYCAEEGVRILGEG 60
Query 182 KFLVSDSFPGNERTKYCLSSKIPLGVILAIPPFNYPVNLAVSKIAPALIAGNSLVLKPPT 361

KFLVSDSFPGNERTKYCLSSKIPLGVILAIPPFNYPVNLAVSKIAPALIAGNSLVLKPPT
Sbjct 61 KFLVSDSFPGNERTKYCLSSKIPLGVILAIPPFNYPVNLAVSKIAPALIAGNSLVLKPPT 120
Query 362 QGAVAALHMVHCFHLAGFPKGLISCITGKGSEIGDFLTMHPGVNCISFTGGDTGIAISKK 541

QGAVAALHMVHCFHLAGFPKGLISCITGKGSEIGDFLTMHPGVNCISFTGGDTGIAISKK
Sbjct 121 QGAVAALHMVHCFHLAGFPKGLISCITGKGSEIGDFLTMHPGVNCISFTGGDTGIAISKK 180
Query 542 AGMIPLQMELGGKDACIVLEDADLDLVAANIIKGGFSYSGQRCTAVKVVLVMESvadalv 721

AGMIPLQMELGGKDACIVLEDADLDLVAANIIKGGFSYSGQRCTAVKVVLVMESVADALV
Sbjct 181 AGMIPLQMELGGKDACIVLEDADLDLVAANIIKGGFSYSGQRCTAVKVVLVMESVADALV 240
Query 722 ekvkakvakLTVGPPEDDCDITPVVSESSSNFIEGLVMDAKQKGATFCQE 871

EKVKAKVAKLTVGPPEDDCDITPVVSESSSNFIEGLVMDAKQKGATFCQE
Sbjct 241 EKVKAKVAKLTVGPPEDDCDITPVVSESSSNFIEGLVMDAKQKGATFCQE 290
UTILIZZIAMO il Programma Expasy Translate tool

Translate is a tool which allows the translation of a nucleotide (DNA/RNA) sequence to a protein sequence.
Tenendo presente dal risultato di BLATX che il frame giusto è il +2
5'3' Frame 2
RGAELLHKAAAILKEHKVPIAECLVKEIAKPAKD
AVTEVVRSGDLVSYCAEEGVRILGEGKFLVSDSF
PGNERTKYCLSSKIPLGVILAIPPFNYPVNLAVSK
I A P A L I A G N S L V L K P P T Q G A V A A L H Met V H C F H L A
G F P K G L I S C I T G K G S E I G D F L T Met H P G V N C I S F T G
G D T G I A I S K K A G Met I P L Q Met E L G G K D A C I V L E D A
D L D L V A A N I I K G G F S Y S G Q R C T A V K V V L V Met E S V
ADALVEKVKAKVAKLTVGPPEDDCDITPVVSESS
S N F I E G L V Met D A K Q K G A T F C Q E

Clone Sottrattivo

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Clone Sottrattivo

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Primer utilizzati durante la costruzione di banche sottrattiva con la tecnica SSH

> AG10_SP6 -- 13..1007 of sequence

ORA MANDIAMO A SEQUENZIARE DA T7 PER OTTENERE LA SEQUENZA COMPLETA DEL NOSTRO

UTILIZZIAMO IL PROGRAMMA Multiple Sequence Alignment by CLUSTALW PER VEDERE

SEQUENZA COMPLETA DEL CLONE

Controllare con BLASTN o FASTA

Score = 542 bits (1397), Expect = 1e-152

Query 2 RGAELLHKAAAILKEHKVPIAECLVKEIAKPAKDAVTEVVRSGDLVSYCAEEGVRILGEG 181

Query 182 KFLVSDSFPGNERTKYCLSSKIPLGVILAIPPFNYPVNLAVSKIAPALIAGNSLVLKPPT 361

Query 362 QGAVAALHMVHCFHLAGFPKGLISCITGKGSEIGDFLTMHPGVNCISFTGGDTGIAISKK 541

Query 542 AGMIPLQMELGGKDACIVLEDADLDLVAANIIKGGFSYSGQRCTAVKVVLVMESvadalv 721

Query 722 ekvkakvakLTVGPPEDDCDITPVVSESSSNFIEGLVMDAKQKGATFCQE 871

UTILIZZIAMO il Programma Expasy Translate tool

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