Open reading frame of insert - (Mar/20/2006 )

freinds to check the reading frame with vector sequence how much sequence of vector is taken before gene 5 prime end.

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generally, i use 50bases before 5' end of the region of interest
you're welcome

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I just check about 20 bases but I am doing it for the first time. My protein is not giving expression.
I want to confirm from some one else me that either my protein is in reading frame or not.
Here is my gene seuence

CGCAGGAGCATCTTCGATGCTGTGGCTCAGAGTAACTGCCAGGAGCTGGAGAGCCTGCTGCCCTTCCTGCAGAGGAGCAA
GAAGCGCCTGACTGACAGCGAGTTCAAAGACCCAGAGACAGGAAAGACCTGTCTGCTAAAAGCCATGCTCAATCTGCACAA
TGGGCAGAATGACACCATCGCTCTGCTCCTGGACGTTGCCCGGAAGACAGACAGCCTGAAGCAGTTTGTCAATGCCAGCTA
CACAGACAGCTACTACAAGGGCCAGACAGCACTGCACATTGCCATTGAACGGCGGAACATGACGCTGGTGACCCTCTTGGT
GGAGAATGGAGCAGATGTCCAGGCTGCGGCTAACGGGGACTTCTTCAAGAAAACCAAAGGGAGGCCTGGCTTCTACTTTGG
TGAGCTGCCCCTGTCCCTGGCTGCGTGCACCAACCAGCTGGCCATTGTGAAGTTCCTGCTGCAGAACTCCTGGCAGCCTGC
AGACATCAGCGCCCGGGACTCAGTGGGCAACACGGTGCTTCATGCCCTGGTGGAGGTGGCAGATAACACAGTTGACAACAC
CAAGTTCGTGACAAGCATGTACAACGAGATCTTGATCCTGGGGGCCAAACTCCACCCCACGCTGAAGCTGGAAGAGATCAC
CAACAGGAAGGGGCTCACGCCACTGGCTCTGGCTGCTAGCAGTGGGAAGATCGGGGTCTTGGCCTACATTCTCCAGAGGGA
GATCCATGAACCCGAGTGCCGACACCTATCCAGGAAGTTCACCGAA

its reading frame with out vector sequence

5'3' Frame 1
R R S I F D A V A Q S N C Q E L E S L L P F L Q R S K K R L T D S E F K D P E T G K T C L L K A Met L N L H N G Q N D T I A L L L D V A R K T D S L K Q F V N A S Y T D S Y Y K G Q T A L H I A I E R R N Met T L V T L L V E N G A D V Q A A A N G D F F K K T K G R P G F Y F G E L P L S L A A C T N Q L A I V K F L L Q N S W Q P A D I S A R D S V G N T V L H A L V E V A D N T V D N T K F V T S Met Y N E I L I L G A K L H P T L K L E E I T N R K G L T P L A L A A S S G K I G V L A Y I L Q R E I H E P E C R H L S R K F T E

I am trying to express it in pET32 a (+) vector.
Please confirm it either its right or wrong sequence to express.

best regards

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My gene is between Eco R5 and Hind III restriction site.

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hi
i've attahced the construction i made by your given data. Seems you have a frameshift in your construction...

what i've done is : i downloaded the PET32a(+) seq from novagen.
I virtually digest that vector and your fragment by EcoRV and HindIII and ligate them (all by pDRAW32)
and i analyzed the sequences i obtained.

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Actually i did not understand the whole logic. My Protein is 28kd and 774 bp gene sequence.
I made primers with ECRO R5 AND hind III site and amplifed and cloned into vector.
I am taking vector sequece multiple of 3 otherwise vector sequence itself not in reading frame.
Suppose Take folloing vector sequence

TATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTAT
CCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTT
CCTCGACGCTAACCTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGG
TTCTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGA
CAAGGCCATGGCT " GATATC "

which includes stag, enterokinase and EcoRV sequence enclose in commos

Its reading frame is as follows

YQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPR GSGMKETAAAKFERQHMDSPDLGTDDDDKAMADI

Now my sequence

CGCAGGAGCATCTTCGATGCTGTGGCTCAGAGTAACTGCCAGGAGCTGGAGAGCCTGCTGCCCTTCCTGCAGAGGAGCAA
GAAGCGCCTGACTGACAGCGAGTTCAAAGACCCAGAGACAGGAAAGACCTGTCTGCTAAAAGCCATGCTCAATCTGCACAA
TGGGCAGAATGACACCATCGCTCTGCTCCTGGACGTTGCCCGGAAGACAGACAGCCTGAAGCAGTTTGTCAATGCCAGCTA
CACAGACAGCTACTACAAGGGCCAGACAGCACTGCACATTGCCATTGAACGGCGGAACATGACGCTGGTGACCCTCTTGGT
GGAGAATGGAGCAGATGTCCAGGCTGCGGCTAACGGGGACTTCTTCAAGAAAACCAAAGGGAGGCCTGGCTTCTACTTTGG
TGAGCTGCCCCTGTCCCTGGCTGCGTGCACCAACCAGCTGGCCATTGTGAAGTTCCTGCTGCAGAACTCCTGGCAGCCTGC
AGACATCAGCGCCCGGGACTCAGTGGGCAACACGGTGCTTCATGCCCTGGTGGAGGTGGCAGATAACACAGTTGACAACAC
CAAGTTCGTGACAAGCATGTACAACGAGATCTTGATCCTGGGGGCCAAACTCCACCCCACGCTGAAGCTGGAAGAGATCAC
CAACAGGAAGGGGCTCACGCCACTGGCTCTGGCTGCTAGCAGTGGGAAGATCGGGGTCTTGGCCTACATTCTCCAGAGGGA
GATCCATGAACCCGAGTGCCGACACCTATCCAGGAAGTTCACCGAA

when I combine both of them and make its reading frame through DNA Star and Edit Sequence its completely in reading frame

Here is amino acid sequence with out stops
YQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPR
GSGMKETAAAKFERQHMDSPDLGTDDDDKAMADIRRSIFDAVAQSNCQELESLLPFLQRSKKRLTDSEFKDPETGKTCLLK
AMLNLHNGQNDTIALLLDVARKTDSLKQFVNASYTDSYYKGQTALHIAIERRNMTLVTLLVENGADVQAAANGDFFKKTKG
RPGFYFGELPLSLAACTNQLAIVKFLLQNSWQPADISARDSVGNTVLHALVEVADNTVDNTKFVTSMYNEILILGAKLHPT
LKLEEITNRKGLTPLALAASSGKIGVLAYILQREIHEPECRHLSRKFTE


I have check it through expasy tools as well.

Y Q G K L T V A K L N I D Q N P G T A P K Y G I R G I P T L L L F K N G E V A A T K V G A L S K G Q L K E F L D A N L A G S G S G H Met H H H H H H S S G L V P R G S G Met K E T A A A K F E R Q H Met D S P D L G T D D D D K A Met A D I R R S I F D A V A Q S N C Q E L E S L L P F L Q R S K K R L T D S E F K D P E T G K T C L L K A Met L N L H N G Q N D T I A L L L D V A R K T D S L K Q F V N A S Y T D S Y Y K G Q T A L H I A I E R R N Met T L V T L L V E N G A D V Q A A A N G D F F K K T K G R P G F Y F G E L P L S L A A C T N Q L A I V K F L L Q N S W Q P A D I S A R D S V G N T V L H A L V E V A D N T V D N T K F V T S Met Y N E I L I L G A K L H P T L K L E E I T N R K G L T P L A L A A S S G K I G V L A Y I L Q R E I H E P E C R H L S R K F T E

Vector sequence (342bp) + gene sequence (774bp) = 1116bp and 372 amino acid sequence.

If there is frame shift which I am not able to determine it its because of my fault of construct or natural problem.
According to you I have a stop codon inside my sequence. Please explain me what will be the solution for this problem.

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hi well can you give me the sequence of primers you use?

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Here is my forward and Reverse Primer

Forward Primer with EcoR5 5’ AAAAAA GATATC CGCAGGAGCATCTTCGAT 3’

Reverse Primer with Hind III 5’ AAAAAA AAGCTT TTA TTA TTCGGTGAACTTCCTGGA 3’

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hi
i finally get all the stuff.
Seems your seq is in frame.
I've attached the map i've constructed for you, with restriction sites at 5' 3' overhangs and blunted ends (limited by max cuts = 3).
I got some pbs to construct it as i needed to inverse the sequence you gave.
Anyway, restriction analysis will tell you if you've cloned it properly.
But if cloned correctly (regarding Eco/hind) it's definitely in frame.

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yes i check it through restriction and sequencing its in right orientation but no expression at all.
Its a membrane protein it can form inculsion body but i could not find any band of 45 kd.
28 kd of my protein and 17 kd of tag proteia. instead i am getting band round about 25 kd.
very strange thing now i am using different strains of bacteria to get expression.
may be it will work. but thanks for your service and time you spent me on my problem.
Zour constrcut is looking very beautiful beofer this post it did not used that software but now i have one more good software and its also very good and i show it to my lab fellows they also like it very much. thanks very much for clone and i will take fruther advice for taking expression.
best regards

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