The HMA3 Sequence is with 2619 bp quite big which make it a little bit difficult for us to actually isolate this sequence out of the A. halleri. That is the reason why we decided to get it synthesized by IDT.

First option was to use the whole sequence (2631 bp). A CaMV 35S (universal promoter, Cauliflower Mosaic Virus) is used as a promoter with a length of 345 bp.

For our Biobrick we decided to use the Vector called pSB1C3 with a chloramphenicol resistance. By including the sequence of the restriction sites of EcoRI at the beginning and PstI at the end of our sequence, which are the same restriction sites for the plasmide, we can insert our sequence into our targeted vector. In our specific case EcoRI would cut about two times into our Gene HMA3 for example. To make sure the restriction enzyme will not cut several times into our sequence we had to change some codons in the original sequence of our Gene HMA3 (you can see the changes of the codons below in red).

Sequence	Length [bp]	Color
CaMV 35S promoter	345	letters
AhHMA3 cDNA	2274	highlighted
Restriction site of EcoRI (G/AATTC)	6	highlighted
restriction site of PstI (CTGCA/G)	6	highlighted
whole sequence	2613

Promoter and gene sequence

In the following the whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3 is shown.
GAGTTC = changed codon to make sure that EcoRI will not cut several times

2nd idea
- Separating the sequence

The second option is to separate our whole promoter and gene sequence into 4 fragment parts. You can see the parts in the table to the right-hand side.

Sequence	Length [bp]	beginning of the sequence	end of the sequence
		Restriction site at the
IDT 1. Part sequence	614	EcorRI: G/AATTC	HindIII: A/AGCTT
IDT 2. Part sequence	593	HindIII: A/AGCTT	SmaI: CCC/GGG; blunt end
IDT 3. Part sequence	556	SmaI: CCC/GGG; blunt end	EcoRII: -/CCWGG; W=A/T
IDT 4. Part sequence	885	EcoRII: -/CCWGG; W=A/T	PstI: CTGCA/G
Total length of the synthesized fragments	2.648
Length of the sequence after the ligation	2.613

The following part shows the 4 fragments, which were synthesized by the IDT. The black letters indicates the promoter sequence and the green letters the HMA3 gene sequence. The restriction sites for each restriction enzyme are highlighted in different colors to clearly differentiate inbetween them. To ligate these fragments into the vector pSB1C3, the first fragment needs to have the restriction site of EcoRI at the beginning as well as the fourth fragment needs to end with the restriction site of PstI. For this sequence design, we also have to changed a few codons to make sure that EcoRI and HindIII will not cut several times in the sequence. All four fragments will bind together because of the different restriction sites. For example, the end of the first fragment and the beginning of the second fragment have the same restriction site. This means only one end fits the other end.

1st fragment

Length: 614 bp

Restriction enzymes
EcoRI: G/AATTC
HindIII: A/AGCTT
GAGTTC = changed codon to make sure that EcoRI will not cut several times

2nd fragment

Length: 593 bp

Restriction enzymes
HindIII: A/AGCTT
SmaI: CCC/GGG; blunt end

3rd fragment

Length: 556 bp

Restriction enzymes
SmaI: CCC/GGG; blunt end
EcoRII: -/CCWGG; W=A/T
AAGCT = changed codon at the restriction site to make sure that HindIII will not cut several times

4th fragment

Length: 885 bp

Restriction enzymes
EcoRII: -/CCWGG; W=A/T
PstI: CTGCA/G

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Team:HSHL/Design

Design

1st idea - Working with the whole sequence

Promoter and gene sequence

2nd idea - Separating the sequence

1st idea
- Working with the whole sequence

2nd idea
- Separating the sequence