Sequence of p35S-GFP revealed

[Mega]

Good news, everyone!


I got the sequences of the 35S-GFP-nos_terminator plasmid (expression casettte for GFP in plants)
that is delivered with agrobacterium Ti plasmid pGreenII  ( http://www.pgreen.ac.uk/a_pls_fr.htm )

35S-GFP sequence

GATATCGTACCCCTACTCCAAAAATGTCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATTTCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCATTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGACATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACAGCCCAAGCTGATCCAAGGAGATATAACAATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCTGAGGGATCTGTGCAGGAGAGGACCATCTTCTTCAAGGACGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAAGGGAATCGATTTCAAGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCCCACAACGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGCGAATTAATTCGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCGATATC

I called the primers "after GFP" - it seqeuces the last 200 bp of the terminator and wht comes after; and pre GFP - sequences the first 200 bp of the promoter and the stuff before.... 

After GFP: AAYTTTCTCTCTCWTTTTCTCATAAATAATGTGTGAGTAGTTTCCCGATAAGGGAAATTA
GGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTG
TATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTA
AAATCCAGATCGATATCGATCTAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTG
AAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGC
CTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTT
CCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGG
CGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT
TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC
AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA
AAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA
TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC
CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC
CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG
TTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGA
CCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC

Pre-GFP 

CCATCGAACGCATGATGGCATTTGTAGGAGCCACCTTCCTTTTCTACTGTCCTTTCGATG
AAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGAAATTACCCTTTGTTGA
AAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGACATTTTTGGAGTAGGGG
TACGATATCGTACCGCGAATTAGCTTGGCACTGGCAGGTGGCACTTTTCGGGGAAATGTG
CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGA
CAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACAT
TTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCA
GAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATC
GAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCA
ATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG
CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCA
GTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATA
ACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAG
CTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCG
GAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCA
ACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG 

[Mega]

bad news, no restriction sites in the sequence???

According to NCBI, it seems to be  pSat-6 http://www.stonybrook.edu/biochem/citovsky/33.pdf

But they didn't give the name of it?  Didn't they know the plasmid? Or was it not totally legal?

[Dakota Hamill]

Genome Compiler was the only desktop icon I had so I opened that up, figured I'd put it through it's paces since I hadn't used it for a while.

Copy and pasted the sequence you gave in the .pdf into it, it gave a CDS bar, and I switched to a nucleotide view and did a cut sequence display.

http://gyazo.com/a93729f4264ceee40cb85b3d038037e5

http://gyazo.com/795b5fc856ba15ee19651dbbda78d960

There are EcoRV cut-sites right where you'd want them at either end of the CDS.

I also pasted your raw sequence above into text document and used Ctrl+F to do a sequence find for EcoRV, and that showed up at either end as well.

[Mega]

Problem is, i don't like ecorv.

Hoped for other sites being around...

[Jeswin]

On Fri, Jul 19, 2013 at 3:15 AM, Mega <masters...@gmail.com> wrote:
> Problem is, i don't like ecorv.
>
> Hoped for other sites being around...
>

What's wrong with EcorV?

[Mega]

Blunt-ends....

[Koeng]

PCR it! Don't be lazy

On Friday, July 19, 2013 7:29:22 AM UTC-7, Mega wrote:

Blunt-ends....

[Andreas Sturm]

Gotta see to get my lux PCR done first... Still doesn't work yet... Although the new primers without much hertogenous bases (only 2 per primer)

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[Andreas Sturm]

*heterogenous

[Cathal Garvey (Phone)]

I think you need special phosphorylated primers to ligate straight from pcr? IIRC you can get by with just one strand phosphorylated though so perhaps not a big deal..

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[Andreas Sturm]

There's a restriction site in each primer, though each primer has only 2 nucleotides exchanged for heterologous ones. So they actaully should work. Also annealing temperature is ~52°C and homodimers are weak.... Getting no product, or only very very weak...

No need to directly ligate the PCR product, which would need special primers then.

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[Dakota Hamill]

https://www.neb.com/products/m2200-quick-ligation-kit#tabselect2

same thing about, fast ligation kit, but from a German copmany

http://www.mobitec.com/de/products/bio/02_genomics/index.php?turbolig.html

Also, how come sometimes you show up as Mega and sometimes as Andreas Sturm.  I thought I was going crazy because I kept thinking to myself...these two people sound a lot a like, then I noticed the email.

[Andreas Sturm]

I have no idea, why... It may be different if I write from gmail or form google groups. I'll look how I can change that...

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