Bacteria (and therefore chloroplasts) do have ribosome binding sites,
but they are called "Shine Dalgarno" rather than "Kozak" consensus
sequences. Depending on the species they can be ~6-10n upstream of the
actual start codon; a variance of 1-2n won't kill expression but there
is an ideal gap.

The shine dalgarno sequence at least partially matches the 3' end of the
16S rRNA ribosomal subunit, so if you don't have an example of a
chloroplast shine-dalgarno sequence, you can cheat by copying the end of
a 16S transcript..but that can backfire, because a shine-dalgarno that's
too strong can actually prevent translation.

You should be able to find literature on chloroplast shine-dalgarnos,
I'd expect.

On 21/11/12 20:20, Andreas Sturm wrote:
> Yeah, thank you... Figured that out, should have beed ds-DNA view then I'd
> have seen it earlier.
>
> Well, AFAIK, the polymerase moves over the DNA strand until  - no you're
> correct. the RBS should have a certain distance from the ATG. However, in
> chloroplasts that doesn't matter because they seem not to need RBS   :D
>
> For pUC19 insertion that may matter. pVIB is based on pBR322, so you've got
> 20 copies per cell. With pUC19 you'll get 100 copies per cell (IIRC).
> Immagine that glow ;)
>
>
>
>
>
> On Wed, Nov 21, 2012 at 8:48 PM, Cathal Garvey <cathalgarvey@gmail.com>wrote:
>
>> Hey Nathan, Mega..
>> Not sure that the frame of the promoter matters so much; it's OK to have
>> some 5' bits of variable length AFAIK. Rather, the frame of the start
>> codon (and to a lesser extent the Kozak/Shine-Dalgarno sequence) that
>> matters.
>>
>> I'd err on slightly more distance from promoter to start codon than
>> less, as transcription start points aren't always immediately at the
>> promoter itself, and may be poorly characterised. Perhaps even variable,
>> I can't claim to know..
>>
>> On 21/11/12 17:04, Nathan McCorkle wrote:
>>> Mega, why not just rotate the image in a photoshop-type program, then it
>>> will be in the normal orientation! It really is that simple though,
>> you're
>>> just looking at things rotated around! Design your primers so either end
>>> has a different restriction sequence, then when you PCR the promoter just
>>> use the same restriction site for the end of the promoter.
>>>
>>> Also make sure the ATG of the gene remains in the correct coding frame as
>>> the promoter, the distance from the start of the promoter to the start of
>>> the gene should be evenly divisible by 3
>>>
>>>
>>> On Wed, Nov 21, 2012 at 3:42 AM, Mega <masterstorm123@gmail.com> wrote:
>>>
>>>> Hi guys,
>>>>
>>>> When I do a PCR and then get an operon which is reversly orientated (see
>>>> picture http://www.ncbi.nlm.nih.gov/gene/3280765 LuxA-G-fre needed)
>> and I
>>>> need to clone it next to a normally oriented promoter, what can I do to
>>>> turn it around?
>>>>
>>>> I thouht about cutting with restriction enzymes, which will cut it in a
>>>> way that they just fit together if correctly inserted. But none of the
>> RE
>>>> have restriction sites that I immagine would fit together...
>>>>
>>>>
>>>> Has anyone experience with this?
>>>>
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>>>>
>>>
>>>
>>>
>>
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