Universidad de La Sabana

Faculty of Engineering
Chemical engineering program
Biotechnology

Professor. Jorge Eduardo Cortzar

Chia, Cundinamarca
19, April / 2017

Valentina Quevedo Salazar, 201413445, Valentinaqusa@unisabana.edu.co

_________________________________________________________________________

Central dogma of molecular biology-WORK SECOND CUT

Introduction

This study will assess the following points regarding protein Phospholipase and the
micro-organism Streptomyces clavuligerus: transcription and cloning in the
expression of given vector pet29a. Additionally seeks a being of eukaryote which
summarizes the same protein as it was Oryctolagus cuniculus, which assessed the
work of mutation and expression of vector given pet29a cloning.

Expression vector
pet29a

String assigned
GGAGAGACGGAGTCTTCTCACGGCCGCCGCGGCCCTGACCGCGACCGCCCTCATCCCCCCGCCCGGCGCGCTCGCGGCCCCCCGCCGCGCCCGCGCCCCCCAGGACTGGATG
GCGGCCCATGCCGACACCACCCCGCTCCAGCGGCTGACCATCCCCGGAACCCACAACTCCGGCGCCCGTTTCGGCGGCCTCTGGGCCCAGTGCCAGACCACCACGATCGCCGA
GCAGCTCGGCAGCGGCATCCGTTTCCTGGACATCCGCTGCCGGATCACCGGGGACGCGTACGCCATCCACCACGGGCCCGCGTACCAGAACGCGATGTTCGGCGACGTCCTG
ACCGCCTGCCGGGACTTCCTGGCCGCGCACCCCTCCGAGACGGTGCTGATGCGCGTCAGACAGGAGTACTCGGAGGAGTCCGACGCCGCCTTCCGCCGGATCTTCGACCTCTA
TCTGGACGGGAAGGGCTGGCGCTCCCTCTTCCGCATCGGCGACGGCGTCCCCCGCCTCGGCGAGGCCCGCGGCCGGGTGGTCCTCCTCGCCGACAACGGTGGCCTGCCCGG
GGTGCGGTACGGGGACGGGGCCCTCTTCGACATCCAGGACGAGTGGAACGCGGCGCCCGCGGCCAAGTACCCGCGGATCGAGGCGCACTTCCGCAGAGCGGTGGAACAGC
CGGGGCGGCTCTTCGTCAACTATGTCTCCACCGCCGCGTACCTCCCGCCGCGCTGGAACGCCGACAACCTGAACCCGCGGGTCCACTCCTTCCTGGACGGGGCCGCGGGCGG
CTGGCGGGGGCTCGGGATCGTTCCGCTGGACTTCCCGGCGACCAGGGCGGGGCTGGTGGAGTCGCTGCTGCGGCACAACTGA

Phospholipase [Streptomyces clavuligerus]

Protein: Phospholipase
A phospholipase is an enzyme that hydrolyzes phospholipids into fatty acids and other
lipophilic substances

Organism: Streptomyces clavuligerus

These bacteria are widely distributed in nature, Streptomyces clavuligerus

especially in the soil. The characteristic earthy Scientific classification
smell of freshly plowed soil is attributed to the Kingdom: Bacteria
aromatic torpedoed geosmin produced by
Phylum: Actinobacteria
species of Streptomyces. There are currently
 364 known species of this genus, many of which Order: Actinomycetales
are the most important industrial producers of Family: Streptomycetaceae
antibiotics and other secondary metabolites of Genus: Streptomyces
antibacterial, antifungal, antiviral, and
Species: S. clavuligerus
antitumor nature, as well as
immunosuppressants, Binomial name
antihypercholesterolemics, etc. Streptomyces clavuligerus
Higgens & Kastner 1971

Streptomycetes are crucial in the soil environment because:

Their diverse metabolism allows them to degrade the insoluble remains of other
organisms, including recalcitrant compounds such as lignocelluloses and chitin.
Produce both substrate and aerial mycelium.
The presence of type-I peptidoglycan in the cell walls that contains characteristic
interpeptide glycine bridges.
They contain very large (~8 million base pairs which is about twice the size of most
bacterial genomes) linear chromosomes with distinct telomeres.
Streptomyces clavuligerus produces the beta-lactamase inhibitor clavulanic acid and the
beta-lactam antibiotic cephamycin C. Cephamycin C is a methoxylated cephalosporin.
Methoxylation increases the effectiveness of the antibiotic on bacterial cell wall synthesis
and reduces the inactivation of the antibiotic by 3-lactamases. Cephamycin C and other
methoxylated cephalosporins are therefore effective against many cephalosporin-resistant
bacteria.
(Ncbi.nlm.nih.gov, n.d.)

1. Define which sequence is involved (which sequence of protein produces) and to

which living being belongs.

First, you must translate the sent sequence, for this we use the help of the website
ExPASy, once the name is published in the search engine, click on Translate tool. As can
be seen in figure 1.
 Figure 1.
Once the ExPASy page is open, paste the DNA sequence and click on Translate
Sequence, as we can see in figure 2.

Figure 2.

When the page has loaded, we will see open reading frames highlighted in red, as shown in
Figure 3; You must select one of the following boxes to retrieve the sequence of our amino
acid. To select the correct box, the most highlighted in red is chosen so that when analyzed
in BLAST 100% match the protein and the living being that corresponds to it. For the above,
choose 5'3'Frame 2, all is selected and copied.
 Figure 3.
When the translation is ready, we go to the search engine to open the BLAST page, once
you have given the results, click on Protein BLAST. As we can see in the figure 4.

Figure 4.

When the BLAST page has been opened, a window will appear as shown in Figure 5, in the
blank the selected amino acid sequence is pasted, in our case the sequence 5'3'Frame 2,
when we have pasted the Sequence, we click on the BLAST button and wait for the page to
load (it takes a long time, we must have patience).
 Figure 5.

ERRSLLTAAAALTATALIPPPGALAAPRRARAPQDWMetAAHADTTPLQRLTIPGTHNSGARFGGLWAQCQTTTIAEQLGSGIRFLDIRCRITGDAYAIHHGPAYQNAMetFGDVLTAC
RDFLAAHPSETVLMetRVRQEYSEESDAAFRRIFDLYLDGKGWRSLFRIGDGVPRLGEARGRVVLLADNGGLPGVRYGDGALFDIQDEWNAAPAAKYPRIEAHFRRAVEQPGRLFVNY
VSTAAYLPPRWNADNLNPRVHSFLDGAAGGWRGLGIVPLDFPATRAGLVESLLRHN

Amino acid sequence

When the page has loaded, we will see a window like the one shown in Figure 6, where we
see a red box almost full, we are indicating that the section of translation we chose is 100%,
below this table, can observe a list of possible living beings and proteins to which the DNA
sequence belongs. It must be chosen very well, for this must be considered that being alive
has more coincidence. Therefore, it is decided that Phospholipase [Streptomyces clavuligerus]
belongs to the given DNA sequence.
 Figure 6.

2. Cloning into the assigned expression vector the complete given DNA sequence.
Plasmit
Cloning in pet29a
In the first place, we should know the plasmid belonging to pet29a, for this we place in the
searcher "pet29a" as shown in figure 7, and we look for a pdf where we show the linear
sequence of the plasmid.

Figure 7.
When we open the pdf, we will find the plasmid in a linear way as shown in figure 8. In the
sequence landmarks it is possible to observe the enzymes that cut in the given vector, as
they are: Xho I(158) Not I(166) Eag I(166) Hind III(173) Sal I(179) Sac I(190) EcoR I(192) BamH
I(198) EcoR V(206) Nco I(212) Kpn I(238) Bgl II(241) Nsp V(268) Nde I(295) .

Figure 8.

Now, it is possible that several of these enzymes cut into other vectors something we do not
want, so we must look for restrictive enzymes, that is enzymes that do not cut into another
vector and only cut. For this we go to the restrictionmapper page, when accessing this page,
we find a window as shown in figure 9. In the blank space, you should place the given DNA
sequence and click on Mapsites.
 Figure 9.
Once the page loads, the window shown in Figure 10 will open, this window shows the
restriction enzymes (enzymes that do not cut into another vector).

Figure 10.

Now in the pET-29a-c (+) cloning / expression region two enzymes are chosen that do not
cut in another vector to make the first forward and backward. EcoR V and EcoR I, enzymes
belonging to this plasmid are chosen as shown in Figure 11, these enzymes do not cut into
other vectors as shown in Figure 10 in highlighted portions.
 Figure 11.
Once the restriction enzymes have been chosen, the places where they cut these enzymes
are searched with the help of the NEB page, the results are shown below in Figures 12 and
13.

Figure 12. Figure 13.

Now I make the first Fordward and backward for which the first 20 letters belonging to the
given DNA are chosen, those 20 letters will be the Fordward and the backward will be these
same letters backwards but exchanging them, knowing that G = C and T = A. For the forward
is placed the sequence that shows the plasmid of the first enzyme selected before and for
the Backward the given sequence of the enzyme is placed in the end. This sequence is viewed
and selected from the plasmid, as shown in Figure 14.

Figure 14.

GATATC GGAGAGACGGAGTCTTCTCA TGAGAAGACTCCGTCTCTCC GAATTC

Fordward Backward
When we have already made the fordward and backward we added it to the original AND, the Fordward
at the beginning and the backward at the end, as shown below:

GATATCGGAGAGACGGAGTCTTCTCAGGAGAGACGGAGTCTTCTCACGGCCGCCGCGGCCCTGACCGCGACCGCCCTCATCCCCCCGCCCGGCGCGCTCGCGGCCCCCCGCCGCGCCCGCGCCCCCCA
GGACTGGATGGCGGCCCATGCCGACACCACCCCGCTCCAGCGGCTGACCATCCCCGGAACCCACAACTCCGGCGCCCGTTTCGGCGGCCTCTGGGCCCAGTGCCAGACCACCACGATCGCCGAGCAGC
TCGGCAGCGGCATCCGTTTCCTGGACATCCGCTGCCGGATCACCGGGGACGCGTACGCCATCCACCACGGGCCCGCGTACCAGAACGCGATGTTCGGCGACGTCCTGACCGCCTGCCGGGACTTCCTG
GCCGCGCACCCCTCCGAGACGGTGCTGATGCGCGTCAGACAGGAGTACTCGGAGGAGTCCGACGCCGCCTTCCGCCGGATCTTCGACCTCTATCTGGACGGGAAGGGCTGGCGCTCCCTCTTCCGCAT
CGGCGACGGCGTCCCCCGCCTCGGCGAGGCCCGCGGCCGGGTGGTCCTCCTCGCCGACAACGGTGGCCTGCCCGGGGTGCGGTACGGGGACGGGGCCCTCTTCGACATCCAGGACGAGTGGAACGC
GGCGCCCGCGGCCAAGTACCCGCGGATCGAGGCGCACTTCCGCAGAGCGGTGGAACAGCCGGGGCGGCTCTTCGTCAACTATGTCTCCACCGCCGCGTACCTCCCGCCGCGCTGGAACGCCGACAACC
TGAACCCGCGGGTCCACTCCTTCCTGGACGGGGCCGCGGGCGGCTGGCGGGGGCTCGGGATCGTTCCGCTGGACTTCCCGGCGACCAGGGCGGGGCTGGTGGAGTCGCTGCTGCGGCACAACTGAT
GAGAAGACTCCGTCTCTCC GAATTC

Pre-Mutated DNA.

The sequence of the plasmid is now taken and the portion where the two previously selected enzymes
are cut off as shown in Figure 15.

Figure 15.
In the plasmid, the part where the enzymes are cut is selected and in that place the pre-mutated DNA.

AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAAGAAACCGCTGCTG
CTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCAC
CACCACCACTGAGATCCGGCTGCTAACAAAGCCC

Plasmid

AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAAGAAACCGCTGCTG
CTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCCATGGCTGATGATATCGGAGAGACGGAGTCTTCTCAGGAGAGACGGAGTCTTCTCACGGCCGCCGCG
GCCCTGACCGCGACCGCCCTCATCCCCCCGCCCGGCGCGCTCGCGGCCCCCCGCCGCGCCCGCGCCCCCCAGGACTGGATGGCGGCCCATGCCGACACCACCCCGCTCCAGCGGCTGACCATCCCCGG
AACCCACAACTCCGGCGCCCGTTTCGGCGGCCTCTGGGCCCAGTGCCAGACCACCACGATCGCCGAGCAGCTCGGCAGCGGCATCCGTTTCCTGGACATCCGCTGCCGGATCACCGGGGACGCGTACG
CCATCCACCACGGGCCCGCGTACCAGAACGCGATGTTCGGCGACGTCCTGACCGCCTGCCGGGACTTCCTGGCCGCGCACCCCTCCGAGACGGTGCTGATGCGCGTCAGACAGGAGTACTCGGAGGAG
TCCGACGCCGCCTTCCGCCGGATCTTCGACCTCTATCTGGACGGGAAGGGCTGGCGCTCCCTCTTCCGCATCGGCGACGGCGTCCCCCGCCTCGGCGAGGCCCGCGGCCGGGTGGTCCTCCTCGCCGAC
AACGGTGGCCTGCCCGGGGTGCGGTACGGGGACGGGGCCCTCTTCGACATCCAGGACGAGTGGAACGCGGCGCCCGCGGCCAAGTACCCGCGGATCGAGGCGCACTTCCGCAGAGCGGTGGAACAG
CCGGGGCGGCTCTTCGTCAACTATGTCTCCACCGCCGCGTACCTCCCGCCGCGCTGGAACGCCGACAACCTGAACCCGCGGGTCCACTCCTTCCTGGACGGGGCCGCGGGCGGCTGGCGGGGGCTCGG
GATCGTTCCGCTGGACTTCCCGGCGACCAGGGCGGGGCTGGTGGAGTCGCTGCTGCGGCACAACTGATGAGAAGACTCCGTCTCTCCGAATTCAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTC
GAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC

Mutated DNA.

Now the mutated DNA must be compared to the original DNA, for this the sequence must be passed to
amino acid sequence. In the original amino acid sequence, we can see that the selection we made is 5'3
'Frame 2, where most are selected in red (figure 16), the part that does not appear in red if it is not pink
is the part that must appear in the mutated amino acid sequence BUT in the red part (Figure 17). Since it
should be in the proper open reading frame. Once this is accomplished, we will have the desired mutation.

Figure 16

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

Original Amino acid sequence

Figure 17

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

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

Mutated Amino acid sequence

 3. Perform the same procedure to express a protein in the same assigned plasmid, where to
express protein must be of a eukaryote organism.

First, we should look for a living Eukaryote that produces or synthesizes the same protein, in our case
must produce Phospholipase. For this search the NCBI page is used, which will be placed in the search
engine as shown in figure 18 and we click on the first option.

Figure 18.

Then, it is necessary to pass from AllDatabases to Protein, and in the searcher, the Phospholipase protein
is placed. The species are filtered only by animals, to obtain 100% eukaryote data. The procedures and
places to be clicked are shown in Figure 19.
When you get results, I choose Phospholipase Oryctolagus cuniculus for phospholipase and give click.

Figure 19.
By clicking we get the definition and the organism which produces the same protein, confirming that it is
Eukaryote. As show in Figure 20. Then, DBSOURCE is looked and we give click in the link that us appears
in this section. This section is very important since it shows us the organism that synthesizes the same
protein.
 Figure 20.

DEFINITION Phospholipase [Oryctolagus cuniculus].

SOURCE Oryctolagus cuniculus (rabbit)
ORGANISM Oryctolagus cuniculus
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata;
Euteleostomi;
Mammalia; Eutheria; Euarchontoglires; Glires; Lagomorpha;
Leporidae; Oryctolagus.

The organization that also produces this protein is Oryctolagus cuniculus (European rabbit, or the
common rabbit is a species of lagomorph mammal in the Leporidae family, and the only current Member
of the genus cuniculus. It is included in the list of 100 of the most damaging invasive alien species of the
world of the International Union for the conservation of nature.)
When we have given click in the link that appears in DBSOURCE, a window will appear as it the figure
shows 21, in this part we give click in CDS.
 Figure 21.

On having given click in CDS it us will appear a sequence of amino acids where the origin can observer
where the protein synthesizes, the sequence codification of the protein begins in the nucleotide 1 and
finishes in 4381 like it we can see later:
ORIGIN
1 ccgaggcacc tgcagcgtcc aggcatggct ctgtggccga gcgtcttcct cctggggctg
61 ctgccgctcc tggggcgagg tgcagatcag atccaaacct cttctggaaa gaacacattg
121 gaagggcagc tctggccaga gagcctgaag acttttccat tcccatgcga cccaaagacg
181 ttagcagaga gcgtgccctc tgagtcagtt cactctctga ggccttctga cattaaattt
241 gttgcagcca ttggcaatgt ggaaactgct ccagactcag gggcggacga cctagaggag
301 caagacggga cggagaagag gccagagcag gcgtgcatgg gcgtggtgac cgtcctttca
361 gacatcatcg gccgtttcag tccttctgct ctgatgccgc tgtgtcctga gacaagactt
421 gtacctcgtg gaggtgctga agacttgtgg atgcaggcta cagagctggt gagaagcatg
481 agagagaacc cgcaacttga ctttgaacat gactggaagc tcatcaacgt gttcttcagt
541 aacacaagcc agtgtttccc gtgtccctct gctcaacaga aggggctcgt gctggggggc
601 atggacaagc tgacaaggac cctggactac ctgcagcagg aggtccccaa agcgttcgtc
661 aacctggtgg atctctcgga gttggcagca ttctctcgct ggcgtcaggg ggctcagctc
721 agcccagcag ccgagccctg caggtgctta agggaaacct cccagctgac caaggtcctg
781 acgcagtggt cctacctgga agcctgggac agcctcctgg cgtccagcaa gtacaacaca
841 caggagtcct tcgcggtggt cttccagcct ttcttctacg agagctcgct gtcggcgctg
901 ttggcggagc ccccactcca ggaccccacc acactggccc tgagtctctg gaacagaatg
961 atggagccca tagggcgcaa agaagagccg ttcagtgaga aggagaggaa gccactgaga
1021 tgccccacac aggagagccc ctacctattc acctacagga acagcggcca gctgaccagg
1081 gtgtcacagc cccaggggaa gctcgaggtg agagaaggga cggaaatccg ctgtcccgac
1141 aaggacccct ccgactcggt tcccacctca gttcacaggc tgaagcccgc cgacatcaag
1201 gtcataggtg ccatgggtga ctccctcacg gcaggcaacg gggcggggtc ccagcctggg
1261 aacatcctgg atgtccttac tcagtaccga ggcctctcct ggagcgtcgg gggcgatcag
1321 aacatcagca cggtcaccac cctggccaac atcctccggg aattcaaccc ttccctgcag
1381 ggcttctccg tcggcaccgg gagagagacc acttcccaag ccttcttcaa ccaggcagtg
1441 gcgggcgccc gagccgatgg gttaattccc caggcccagc ggctggtggc cctgatgaag
1501 aatgacacga ggataaattt tcaggaggat tggaagataa taaccgtgtt cattggaggc
1561 aacgacctct gtgatttctg caatgacccg gtgcgctact ccccccagaa cttcacggac
1621 aacatcggga cggccctgga catcctccac gctgagattc cccgggcctt tgtgaacctg
 1681 gtgaaggtgc ttgagatctc caagctgaga gagctgtacc aggagactaa agtgagctgc
1741 cccaggatga tcctcaggtc tctgtgtccc tgtgtcctga agttcgatga caactccaca
1801 gaaattgcct ccctcatcga aacgatcaag gagtatcagg agaggaccca gcagctgatt
1861 gacagcgggc gatacgacac gagggacgac ttcacggtgg ttttgcagcc gttttttgaa
1921 aaagtgaaca tgccaaagac ccaggacggg ctacccgaca actctttctt cgcgcccgac
1981 tgcttccact tcagcagcaa ggcccacgcg cacgcagcta gcgccctctg gaacaacatg
2041 ctggaacctg ttggccagaa gacaacacac aatgattttg aaggagcagt caacattacg
2101 tgtccaaacc aggtctggcc gtttctgagt acctacaaga acagcgtgca gggttttggg
2161 acctggctgc cgtgcagaga cagatccccg tctgcctcgc cccccacctc agtgcacgcc
2221 ctgagacccg cagatatcca agttgtggct gctctgggag attctctaac tgctggcatt
2281 ggaattggct ccaaaccaaa tgacctctca gatggcacca ctcagtatcg gggactgtca
2341 tatagttcag gaggggacgg ctccctggac aacgtgacca ccttgcctaa tatcctccgg
2401 cagttcaaca gcaacctcat gggcttcgcc gtgggcactg gtgatgccag tggcaccaat
2461 gctttcttca accaggctgt tcccggagca aaggcgaggg atcttatgag ccaagtgcag
2521 actctggttc agaggatgaa ggacgaccat agagtaaatt tccaagaaga ttggaaggtc
2581 atcacggtgc agatcggagc cagtgacttg tgtgactact gcacggattc gaacctgtat
2641 tctgcagcca acttttacga ccatctccga gacgccttgg acgccctcca cagagaggtg
2701 cccagagctc tggtcaacct cgtggacttc atgaacccaa gtgtcacacg acaggtgttc
2761 ctggggaacc ccgacaagtg cccagtgcag caggccagcg ccctgtgtaa ctgcgtcttg
2821 agcccccggg agaactccta tgagctggcc cggttggagg ccctcgcaca agcctaccag
2881 agcagcctgc gggagctggt ggagtccggc cgctacgaca cgcgggagga cttctccgtg
2941 gtgctgcagc ccttcttcca cagcatccag ctccccgtcc tgcaggacgg gcgcctggac
3001 acgtctttct tcgccccaga ctgcgtccac ccaaatcaga agttccactc ccagctctcc
3061 agagccctct ggcgcaacat gctggagccc ctgggaggga aaacggatgc actggacctg
3121 acggcagcca tcaccctcac ctgccccact cagaacgagc ctttcctgag aaccttccgg
3181 aacagtgact acacctaccc cagcaggcca gccgtcgaga actggggcag tgacttcctg
3241 tgcaccgcgt ggaatgcttc caggggtgtt ccgaactcag tccatgagct ccaaccagga
3301 gatatcaaag tcgtagctgc cctgggtgac tcgctgacgc tggccatggg cgctcgacca
3361 agcaactcca gtgacccacc catgttctgg aggggcctct cctggagcat tggtggggac
3421 ggggccttgg agactcacac cacactgccc aacattctga agaagttcaa cccttccatc
3481 ctggggtttt ctactggaac cttggaggga accatgggac tcaacgtagc agtgcaaggg
3541 gccagagctc aggacatgcc agcccaagcc cgggacctgg tggagcgaat gagaaacagc
3601 cctgagatcg acctggagaa ggactggaag ctggtcacac tcttcgtggg gggcaatgac
3661 ctgtgtcact tctgcgagaa tccggagggc agctccgagg gggagtacgt ccagcacatc
3721 cagcaggccc tggacgtcct gtacgaggag cttccgagga cttttgtcaa tgtggtggag
3781 gtcatggagc tggccggcct gcaccaggac caaggcggga gatgtgccac actgctggca
3841 gctcagagcc actgcacctg cttcaaatac tctcagagct cagtggagat gcaagaactg
3901 aagaaagtga actggaacct ccagagcggc ctctccaggc tctcctactc acaccagtac
3961 gtgcagcgtg aggacttcgc cgtcgttgtg cagcctttct tccagaacac tctcgtccca
4021 ctgaacggga gaggggacac tgacctcacc ttcttctctg acgactgctt ccacttctca
4081 gagcgcggtc atgccgagat ggccattgcc ctctggaaca acatgctgga gccagtgggc
4141 cacaagacaa cctccaacaa cttcacctac agccgaacca aactcaagtg cccctctccc
4201 gacagcccgt acctctacac cctccggaac agccggctac tccccgacca ggctgaggca
4261 gaccccacgg tgctctactg ggcagtgccg gtggcagcag gagccggcct tctcatcggc
4321 atcctcgcca tggtggcagg gagaggcatg cgctgtcgcc cgcgggagga ccccccgctg
4381 agcctgagca caggcctcta ggcccatgtg tgtggtcctc acccggaagg tcccggccac
4441 actccaacct ggcccttcta cctcagccac ctgtggccac cacgggcaga acctgcttct
4501 acacctgaga cacagcagcc aaagggacag tcaggacttc ttggggcctg ggcttctccc
4561 aggtcaatgc tcctgggatt ggcaaactta aataaaaagc cgaacatgtt ct
//

Origin.
Once we see this sequence, we got to see the name, it is important to appear mRNA in the title and
then will know that if you have a sequence of DNA. Once confirmed, give click in FASTA, where the new
DNA will be to analyze.

Figure 22.

CCGAGGCACCTGCAGCGTCCAGGCATGGCTCTGTGGCCGAGCGTCTTCCTCCTGGGGCTGCTGCCGCTCCTGGGGCGAGGTGCAGATCAGATCCAAACCTCTTCTGGAAAGAACACATTGGAAGGGCA
GCTCTGGCCAGAGAGCCTGAAGACTTTTCCATTCCCATGCGACCCAAAGACGTTAGCAGAGAGCGTGCCCTCTGAGTCAGTTCACTCTCTGAGGCCTTCTGACATTAAATTTGTTGCAGCCATTGGCAAT
GTGGAAACTGCTCCAGACTCAGGGGCGGACGACCTAGAGGAGCAAGACGGGACGGAGAAGAGGCCAGAGCAGGCGTGCATGGGCGTGGTGACCGTCCTTTCAGACATCATCGGCCGTTTCAGTCCTT
CTGCTCTGATGCCGCTGTGTCCTGAGACAAGACTTGTACCTCGTGGAGGTGCTGAAGACTTGTGGATGCAGGCTACAGAGCTGGTGAGAAGCATGAGAGAGAACCCGCAACTTGACTTTGAACATGACT
GGAAGCTCATCAACGTGTTCTTCAGTAACACAAGCCAGTGTTTCCCGTGTCCCTCTGCTCAACAGAAGGGGCTCGTGCTGGGGGGCATGGACAAGCTGACAAGGACCCTGGACTACCTGCAGCAGGAG
GTCCCCAAAGCGTTCGTCAACCTGGTGGATCTCTCGGAGTTGGCAGCATTCTCTCGCTGGCGTCAGGGGGCTCAGCTCAGCCCAGCAGCCGAGCCCTGCAGGTGCTTAAGGGAAACCTCCCAGCTGACC
AAGGTCCTGACGCAGTGGTCCTACCTGGAAGCCTGGGACAGCCTCCTGGCGTCCAGCAAGTACAACACACAGGAGTCCTTCGCGGTGGTCTTCCAGCCTTTCTTCTACGAGAGCTCGCTGTCGGCGCTG
TTGGCGGAGCCCCCACTCCAGGACCCCACCACACTGGCCCTGAGTCTCTGGAACAGAATGATGGAGCCCATAGGGCGCAAAGAAGAGCCGTTCAGTGAGAAGGAGAGGAAGCCACTGAGATGCCCCA
CACAGGAGAGCCCCTACCTATTCACCTACAGGAACAGCGGCCAGCTGACCAGGGTGTCACAGCCCCAGGGGAAGCTCGAGGTGAGAGAAGGGACGGAAATCCGCTGTCCCGACAAGGACCCCTCCGA
CTCGGTTCCCACCTCAGTTCACAGGCTGAAGCCCGCCGACATCAAGGTCATAGGTGCCATGGGTGACTCCCTCACGGCAGGCAACGGGGCGGGGTCCCAGCCTGGGAACATCCTGGATGTCCTTACTCA
GTACCGAGGCCTCTCCTGGAGCGTCGGGGGCGATCAGAACATCAGCACGGTCACCACCCTGGCCAACATCCTCCGGGAATTCAACCCTTCCCTGCAGGGCTTCTCCGTCGGCACCGGGAGAGAGACCA
CTTCCCAAGCCTTCTTCAACCAGGCAGTGGCGGGCGCCCGAGCCGATGGGTTAATTCCCCAGGCCCAGCGGCTGGTGGCCCTGATGAAGAATGACACGAGGATAAATTTTCAGGAGGATTGGAAGATA
ATAACCGTGTTCATTGGAGGCAACGACCTCTGTGATTTCTGCAATGACCCGGTGCGCTACTCCCCCCAGAACTTCACGGACAACATCGGGACGGCCCTGGACATCCTCCACGCTGAGATTCCCCGGGCCT
TTGTGAACCTGGTGAAGGTGCTTGAGATCTCCAAGCTGAGAGAGCTGTACCAGGAGACTAAAGTGAGCTGCCCCAGGATGATCCTCAGGTCTCTGTGTCCCTGTGTCCTGAAGTTCGATGACAACTCCA
CAGAAATTGCCTCCCTCATCGAAACGATCAAGGAGTATCAGGAGAGGACCCAGCAGCTGATTGACAGCGGGCGATACGACACGAGGGACGACTTCACGGTGGTTTTGCAGCCGTTTTTTGAAAAAGTG
AACATGCCAAAGACCCAGGACGGGCTACCCGACAACTCTTTCTTCGCGCCCGACTGCTTCCACTTCAGCAGCAAGGCCCACGCGCACGCAGCTAGCGCCCTCTGGAACAACATGCTGGAACCTGTTGGC
CAGAAGACAACACACAATGATTTTGAAGGAGCAGTCAACATTACGTGTCCAAACCAGGTCTGGCCGTTTCTGAGTACCTACAAGAACAGCGTGCAGGGTTTTGGGACCTGGCTGCCGTGCAGAGACAGA
TCCCCGTCTGCCTCGCCCCCCACCTCAGTGCACGCCCTGAGACCCGCAGATATCCAAGTTGTGGCTGCTCTGGGAGATTCTCTAACTGCTGGCATTGGAATTGGCTCCAAACCAAATGACCTCTCAGATG
GCACCACTCAGTATCGGGGACTGTCATATAGTTCAGGAGGGGACGGCTCCCTGGACAACGTGACCACCTTGCCTAATATCCTCCGGCAGTTCAACAGCAACCTCATGGGCTTCGCCGTGGGCACTGGTG
ATGCCAGTGGCACCAATGCTTTCTTCAACCAGGCTGTTCCCGGAGCAAAGGCGAGGGATCTTATGAGCCAAGTGCAGACTCTGGTTCAGAGGATGAAGGACGACCATAGAGTAAATTTCCAAGAAGATT
GGAAGGTCATCACGGTGCAGATCGGAGCCAGTGACTTGTGTGACTACTGCACGGATTCGAACCTGTATTCTGCAGCCAACTTTTACGACCATCTCCGAGACGCCTTGGACGCCCTCCACAGAGAGGTGC
CCAGAGCTCTGGTCAACCTCGTGGACTTCATGAACCCAAGTGTCACACGACAGGTGTTCCTGGGGAACCCCGACAAGTGCCCAGTGCAGCAGGCCAGCGCCCTGTGTAACTGCGTCTTGAGCCCCCGGG
AGAACTCCTATGAGCTGGCCCGGTTGGAGGCCCTCGCACAAGCCTACCAGAGCAGCCTGCGGGAGCTGGTGGAGTCCGGCCGCTACGACACGCGGGAGGACTTCTCCGTGGTGCTGCAGCCCTTCTTC
CACAGCATCCAGCTCCCCGTCCTGCAGGACGGGCGCCTGGACACGTCTTTCTTCGCCCCAGACTGCGTCCACCCAAATCAGAAGTTCCACTCCCAGCTCTCCAGAGCCCTCTGGCGCAACATGCTGGAGC
CCCTGGGAGGGAAAACGGATGCACTGGACCTGACGGCAGCCATCACCCTCACCTGCCCCACTCAGAACGAGCCTTTCCTGAGAACCTTCCGGAACAGTGACTACACCTACCCCAGCAGGCCAGCCGTCG
AGAACTGGGGCAGTGACTTCCTGTGCACCGCGTGGAATGCTTCCAGGGGTGTTCCGAACTCAGTCCATGAGCTCCAACCAGGAGATATCAAAGTCGTAGCTGCCCTGGGTGACTCGCTGACGCTGGCC
ATGGGCGCTCGACCAAGCAACTCCAGTGACCCACCCATGTTCTGGAGGGGCCTCTCCTGGAGCATTGGTGGGGACGGGGCCTTGGAGACTCACACCACACTGCCCAACATTCTGAAGAAGTTCAACCCT
TCCATCCTGGGGTTTTCTACTGGAACCTTGGAGGGAACCATGGGACTCAACGTAGCAGTGCAAGGGGCCAGAGCTCAGGACATGCCAGCCCAAGCCCGGGACCTGGTGGAGCGAATGAGAAACAGCC
CTGAGATCGACCTGGAGAAGGACTGGAAGCTGGTCACACTCTTCGTGGGGGGCAATGACCTGTGTCACTTCTGCGAGAATCCGGAGGGCAGCTCCGAGGGGGAGTACGTCCAGCACATCCAGCAGGC
CCTGGACGTCCTGTACGAGGAGCTTCCGAGGACTTTTGTCAATGTGGTGGAGGTCATGGAGCTGGCCGGCCTGCACCAGGACCAAGGCGGGAGATGTGCCACACTGCTGGCAGCTCAGAGCCACTGC
ACCTGCTTCAAATACTCTCAGAGCTCAGTGGAGATGCAAGAACTGAAGAAAGTGAACTGGAACCTCCAGAGCGGCCTCTCCAGGCTCTCCTACTCACACCAGTACGTGCAGCGTGAGGACTTCGCCGTC
GTTGTGCAGCCTTTCTTCCAGAACACTCTCGTCCCACTGAACGGGAGAGGGGACACTGACCTCACCTTCTTCTCTGACGACTGCTTCCACTTCTCAGAGCGCGGTCATGCCGAGATGGCCATTGCCCTCTG
GAACAACATGCTGGAGCCAGTGGGCCACAAGACAACCTCCAACAACTTCACCTACAGCCGAACCAAACTCAAGTGCCCCTCTCCCGACAGCCCGTACCTCTACACCCTCCGGAACAGCCGGCTACTCCCC
GACCAGGCTGAGGCAGACCCCACGGTGCTCTACTGGGCAGTGCCGGTGGCAGCAGGAGCCGGCCTTCTCATCGGCATCCTCGCCATGGTGGCAGGGAGAGGCATGCGCTGTCGCCCGCGGGAGGAC
CCCCGCTGAGCCTGAGCACAGGCCTCTAGGCCCATGTGTGTGGTCCTCACCCGGAAGGTCCCGGCCACACTCCAACCTGGCCCTTCTACCTCAGCCACCTGTGGCCACCACGGGCAGAACCTGCTTCTAA
CCTGAGACACAGCAGCCAAAGGGACAGTCAGGACTTCTTGGGGCCTGGGCTTCTCCCAGGTCAATGCTCCTGGGATTGGCAAACTTAAATAAAAAGCCGAACATGTTCT

Phospholipase [Oryctolagus cuniculus] DNA

You must translate the sent sequence, for this we use the help of the website ExPASy, once the name is
published in the search engine, click on Translate tool. As can be seen in procedure in the point 2 and
now you can see the amino acid sequence.
MetALWPSVFLLGLLPLLGRGADQIQTSSGKNTLEGQLWPESLKTFPFPCDPKTLAESVPSESVHSLRPSDIKFVAAIGNVETAPDSGADDLEEQDGTEKRPEQACMetGVVTVLSDIIGRFSPSALMetPLCPETRLV
PRGGAEDLWMetQATELVRSMetRENPQLDFEHDWKLINVFFSNTSQCFPCPSAQQKGLVLGGMetDKLTRTLDYLQQEVPKAFVNLVDLSELAAFSRWRQGAQLSPAAEPCRCLRETSQLTKVLTQWSYLEA
WDSLLASSKYNTQESFAVVFQPFFYESSLSALLAEPPLQDPTTLALSLWNRMetMetEPIGRKEE P F S E K E R K P L R C P T Q E S P Y L F T Y R N S G Q L T R V S Q P Q G K L E V R E G T E I R C P D K
D P S D S V P T S V H R L K P A D I K V I G A Met G D S L T A G N G A G S Q P G N I L D V L T Q Y R G L S W S V G G D Q N I S T V T T L A N I L R E F N P S L Q G F S V G T G R E T T S Q A F F
N Q A V A G A R A D G L I P Q A Q R L V A L Met K N D T R I N F Q E D W K I I T V F I G G N D L C D F C N D P V R Y S P Q N F T D N I G T A L D I L H A E I P R A F V N L V K V L E I S K L R E
L Y Q E T K V S C P R Met I L R S L C P C V L K F D D N S T E I A S L I E T I K E Y Q E R T Q Q L I D S G R Y D T R D D F T V V L Q P F F E K V N Met P K T Q D G L P D N S F F A P D C F H F S S
K A H A H A A S A L W N N Met L E P V G Q K T T H N D F E G A V N I T C P N Q V W P F L S T Y K N S V Q G F G T W L P C R D R S P S A S P P T S V H A L R P A D I Q V V A A L G D S L T A
G I G I G S K P N D L S D G T T Q Y R G L S Y S S G G D G S L D N V T T L P N I L R Q F N S N L Met G F A V G T G D A S G T N A F F N Q A V P G A K A R D L Met S Q V Q T L V Q R Met K D
D H R V N F Q E D W K V I T V Q I G A S D L C D Y C T D S N L Y S A A N F Y D H L R D A L D A L H R E V P R A L V N L V D F Met N P S V T R Q V F L G N P D K C P V Q Q A S A L C N C V L S
P R E N S Y E L A R L E A L A Q A Y Q S S L R E L V E S G R Y D T R E D F S V V L Q P F F H S I Q L P V L Q D G R L D T S F F A P D C V H P N Q K F H S Q L S R A L W R N Met L E P L G G K T D
A L D L T A A I T L T C P T Q N E P F L R T F R N S D Y T Y P S R P A V E N W G S D F L C T A W N A S R G V P N S V H E L Q P G D I K V V A A L G D S L T L A Met G A R P S N S S D P P Met F
W R G L S W S I G G D G A L E T H T T L P N I L K K F N P S I L G F S T G T L E G T Met G L N V A V Q G A R A Q D Met P A Q A R D L V E R Met R N S P E I D L E K D W K L V T L F V G G N D
L C H F C E N P E G S S E G E Y V Q H I Q Q A L D V L Y E E L P R T F V N V V E V Met E L A G L H Q D Q G G R C A T L L A A Q S H C T C F K Y S Q S S V E Met Q E L K K V N W N L Q S G L S
R L S Y S H Q Y V Q R E D F A V V V Q P F F Q N T L V P L N G R G D T D L T F F S D D C F H F S E R G H A E Met A I A L W N N Met L E P V G H K T T S N N F T Y S R T K L K C P S P D S P Y L
Y T L R N S R L L P D Q A E A D P T V L Y W A V P V A A G A G L L I G I L A Met V A G R G Met R C R P R E D P P L S L S T G L

Aminoacid sequence

Now, it is possible that several of these enzymes cut into other vectors something we do not want, so we
must look for restrictive enzymes, that is enzymes that do not cut into another vector and only cut. For
this we go to the restrictionmapper page and see the restriction enzymes (enzymes that do not cut into
another vector), that you can see in the figure 24.

Figure 24.
Once the restriction enzymes have been chosen, the places where they cut these enzymes are searched
with the help of the NEB page, the results are shown below in Figures 25 and 26.

Figure 25. Figure 26.

Now I make the first Fordward and backward for which the first 20 letters belonging to the given DNA are
chosen, those 20 letters will be the Fordward and the backward will be these same letters backwards but
exchanging them, knowing that G = C and T = A. For the forward is placed the sequence that shows the
plasmid of the first enzyme selected before and for the Backward the given sequence of the enzyme is
placed in the end. This sequence is viewed and selected from the plasmid, as shown in Figure 27.

Figure 27.
 GGATCCCCGAGGCACCTGCAGCGTCC GGACGCTGCAGGTGCCTCGGGAATTC

Fordward Backward

When we have already made the fordward and backward we added it to the original AND,
the Fordward at the beginning and the backward at the end, as shown below:

GGATCCCCGAGGCACCTGCAGCGTCCCCGAGGCACCTGCAGCGTCCAGGCATGGCTCTGTGGCCGAGCGTCTTCCTCCTGGGGCTGCTGCCGCTCCTG
GGGCGAGGTGCAGATCAGATCCAAACCTCTTCTGGAAAGAACACATTGGAAGGGCAGCTCTGGCCAGAGAGCCTGAAGACTTTTCCATTCCCATGCGA
CCCAAAGACGTTAGCAGAGAGCGTGCCCTCTGAGTCAGTTCACTCTCTGAGGCCTTCTGACATTAAATTTGTTGCAGCCATTGGCAATGTGGAAACTGCT
CCAGACTCAGGGGCGGACGACCTAGAGGAGCAAGACGGGACGGAGAAGAGGCCAGAGCAGGCGTGCATGGGCGTGGTGACCGTCCTTTCAGACATC
ATCGGCCGTTTCAGTCCTTCTGCTCTGATGCCGCTGTGTCCTGAGACAAGACTTGTACCTCGTGGAGGTGCTGAAGACTTGTGGATGCAGGCTACAGAG
CTGGTGAGAAGCATGAGAGAGAACCCGCAACTTGACTTTGAACATGACTGGAAGCTCATCAACGTGTTCTTCAGTAACACAAGCCAGTGTTTCCCGTGT
CCCTCTGCTCAACAGAAGGGGCTCGTGCTGGGGGGCATGGACAAGCTGACAAGGACCCTGGACTACCTGCAGCAGGAGGTCCCCAAAGCGTTCGTCAA
CCTGGTGGATCTCTCGGAGTTGGCAGCATTCTCTCGCTGGCGTCAGGGGGCTCAGCTCAGCCCAGCAGCCGAGCCCTGCAGGTGCTTAAGGGAAACCT
CCCAGCTGACCAAGGTCCTGACGCAGTGGTCCTACCTGGAAGCCTGGGACAGCCTCCTGGCGTCCAGCAAGTACAACACACAGGAGTCCTTCGCGGTG
GTCTTCCAGCCTTTCTTCTACGAGAGCTCGCTGTCGGCGCTGTTGGCGGAGCCCCCACTCCAGGACCCCACCACACTGGCCCTGAGTCTCTGGAACAGAA
TGATGGAGCCCATAGGGCGCAAAGAAGAGCCGTTCAGTGAGAAGGAGAGGAAGCCACTGAGATGCCCCACACAGGAGAGCCCCTACCTATTCACCTAC
AGGAACAGCGGCCAGCTGACCAGGGTGTCACAGCCCCAGGGGAAGCTCGAGGTGAGAGAAGGGACGGAAATCCGCTGTCCCGACAAGGACCCCTCC
GACTCGGTTCCCACCTCAGTTCACAGGCTGAAGCCCGCCGACATCAAGGTCATAGGTGCCATGGGTGACTCCCTCACGGCAGGCAACGGGGCGGGGTC
CCAGCCTGGGAACATCCTGGATGTCCTTACTCAGTACCGAGGCCTCTCCTGGAGCGTCGGGGGCGATCAGAACATCAGCACGGTCACCACCCTGGCCAA
CATCCTCCGGGAATTCAACCCTTCCCTGCAGGGCTTCTCCGTCGGCACCGGGAGAGAGACCACTTCCCAAGCCTTCTTCAACCAGGCAGTGGCGGGCGC
CCGAGCCGATGGGTTAATTCCCCAGGCCCAGCGGCTGGTGGCCCTGATGAAGAATGACACGAGGATAAATTTTCAGGAGGATTGGAAGATAATAACCG
TGTTCATTGGAGGCAACGACCTCTGTGATTTCTGCAATGACCCGGTGCGCTACTCCCCCCAGAACTTCACGGACAACATCGGGACGGCCCTGGACATCCT
CCACGCTGAGATTCCCCGGGCCTTTGTGAACCTGGTGAAGGTGCTTGAGATCTCCAAGCTGAGAGAGCTGTACCAGGAGACTAAAGTGAGCTGCCCCA
GGATGATCCTCAGGTCTCTGTGTCCCTGTGTCCTGAAGTTCGATGACAACTCCACAGAAATTGCCTCCCTCATCGAAACGATCAAGGAGTATCAGGAGAG
GACCCAGCAGCTGATTGACAGCGGGCGATACGACACGAGGGACGACTTCACGGTGGTTTTGCAGCCGTTTTTTGAAAAAGTGAACATGCCAAAGACCC
AGGACGGGCTACCCGACAACTCTTTCTTCGCGCCCGACTGCTTCCACTTCAGCAGCAAGGCCCACGCGCACGCAGCTAGCGCCCTCTGGAACAACATGC
TGGAACCTGTTGGCCAGAAGACAACACACAATGATTTTGAAGGAGCAGTCAACATTACGTGTCCAAACCAGGTCTGGCCGTTTCTGAGTACCTACAAGA
ACAGCGTGCAGGGTTTTGGGACCTGGCTGCCGTGCAGAGACAGATCCCCGTCTGCCTCGCCCCCCACCTCAGTGCACGCCCTGAGACCCGCAGATATCC
AAGTTGTGGCTGCTCTGGGAGATTCTCTAACTGCTGGCATTGGAATTGGCTCCAAACCAAATGACCTCTCAGATGGCACCACTCAGTATCGGGGACTGTC
ATATAGTTCAGGAGGGGACGGCTCCCTGGACAACGTGACCACCTTGCCTAATATCCTCCGGCAGTTCAACAGCAACCTCATGGGCTTCGCCGTGGGCAC
TGGTGATGCCAGTGGCACCAATGCTTTCTTCAACCAGGCTGTTCCCGGAGCAAAGGCGAGGGATCTTATGAGCCAAGTGCAGACTCTGGTTCAGAGGAT
GAAGGACGACCATAGAGTAAATTTCCAAGAAGATTGGAAGGTCATCACGGTGCAGATCGGAGCCAGTGACTTGTGTGACTACTGCACGGATTCGAACC
TGTATTCTGCAGCCAACTTTTACGACCATCTCCGAGACGCCTTGGACGCCCTCCACAGAGAGGTGCCCAGAGCTCTGGTCAACCTCGTGGACTTCATGAA
CCCAAGTGTCACACGACAGGTGTTCCTGGGGAACCCCGACAAGTGCCCAGTGCAGCAGGCCAGCGCCCTGTGTAACTGCGTCTTGAGCCCCCGGGAGA
ACTCCTATGAGCTGGCCCGGTTGGAGGCCCTCGCACAAGCCTACCAGAGCAGCCTGCGGGAGCTGGTGGAGTCCGGCCGCTACGACACGCGGGAGGA
CTTCTCCGTGGTGCTGCAGCCCTTCTTCCACAGCATCCAGCTCCCCGTCCTGCAGGACGGGCGCCTGGACACGTCTTTCTTCGCCCCAGACTGCGTCCAC
CCAAATCAGAAGTTCCACTCCCAGCTCTCCAGAGCCCTCTGGCGCAACATGCTGGAGCCCCTGGGAGGGAAAACGGATGCACTGGACCTGACGGCAGC
CATCACCCTCACCTGCCCCACTCAGAACGAGCCTTTCCTGAGAACCTTCCGGAACAGTGACTACACCTACCCCAGCAGGCCAGCCGTCGAGAACTGGGG
CAGTGACTTCCTGTGCACCGCGTGGAATGCTTCCAGGGGTGTTCCGAACTCAGTCCATGAGCTCCAACCAGGAGATATCAAAGTCGTAGCTGCCCTGGG
TGACTCGCTGACGCTGGCCATGGGCGCTCGACCAAGCAACTCCAGTGACCCACCCATGTTCTGGAGGGGCCTCTCCTGGAGCATTGGTGGGGACGGGG
CCTTGGAGACTCACACCACACTGCCCAACATTCTGAAGAAGTTCAACCCTTCCATCCTGGGGTTTTCTACTGGAACCTTGGAGGGAACCATGGGACTCAA
CGTAGCAGTGCAAGGGGCCAGAGCTCAGGACATGCCAGCCCAAGCCCGGGACCTGGTGGAGCGAATGAGAAACAGCCCTGAGATCGACCTGGAGAA
GGACTGGAAGCTGGTCACACTCTTCGTGGGGGGCAATGACCTGTGTCACTTCTGCGAGAATCCGGAGGGCAGCTCCGAGGGGGAGTACGTCCAGCAC
ATCCAGCAGGCCCTGGACGTCCTGTACGAGGAGCTTCCGAGGACTTTTGTCAATGTGGTGGAGGTCATGGAGCTGGCCGGCCTGCACCAGGACCAAGG
CGGGAGATGTGCCACACTGCTGGCAGCTCAGAGCCACTGCACCTGCTTCAAATACTCTCAGAGCTCAGTGGAGATGCAAGAACTGAAGAAAGTGAACT
GGAACCTCCAGAGCGGCCTCTCCAGGCTCTCCTACTCACACCAGTACGTGCAGCGTGAGGACTTCGCCGTCGTTGTGCAGCCTTTCTTCCAGAACACTCT
CGTCCCACTGAACGGGAGAGGGGACACTGACCTCACCTTCTTCTCTGACGACTGCTTCCACTTCTCAGAGCGCGGTCATGCCGAGATGGCCATTGCCCTC
TGGAACAACATGCTGGAGCCAGTGGGCCACAAGACAACCTCCAACAACTTCACCTACAGCCGAACCAAACTCAAGTGCCCCTCTCCCGACAGCCCGTAC
CTCTACACCCTCCGGAACAGCCGGCTACTCCCCGACCAGGCTGAGGCAGACCCCACGGTGCTCTACTGGGCAGTGCCGGTGGCAGCAGGAGCCGGCCT
TCTCATCGGCATCCTCGCCATGGTGGCAGGGAGAGGCATGCGCTGTCGCCCGCGGGAGGACCCCCCGCTGAGCCTGAGCACAGGCCTCTAGGCCCATG
TGTGTGGTCCTCACCCGGAAGGTCCCGGCCACACTCCAACCTGGCCCTTCTACCTCAGCCACCTGTGGCCACCACGGGCAGAACCTGCTTCTACACCTGA
GACACAGCAGCCAAAGGGACAGTCAGGACTTCTTGGGGCCTGGGCTTCTCCCAGGTCAATGCTCCTGGGATTGGCAAACTTAAATAAAAAGCCGAACAT
GTTCTGGACGCTGCAGGTGCCTCGGGAATTC
Pre-Mutated DNA.

In the plasmid, the part where the enzymes are cut is selected and in that place the pre-
mutated DNA.
AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA
GGAGATATACATATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCCATGGCT
GATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC
Plasmid
AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAA
GGAGATATACATATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCCATGGCT
GATATCGGGATCCCCGAGGCACCTGCAGCGTCCCCGAGGCACCTGCAGCGTCCAGGCATGGCTCTGTGGCCGAGCGTCTTCCTCCTGGGGCTGCTGCC
GCTCCTGGGGCGAGGTGCAGATCAGATCCAAACCTCTTCTGGAAAGAACACATTGGAAGGGCAGCTCTGGCCAGAGAGCCTGAAGACTTTTCCATTCCC
ATGCGACCCAAAGACGTTAGCAGAGAGCGTGCCCTCTGAGTCAGTTCACTCTCTGAGGCCTTCTGACATTAAATTTGTTGCAGCCATTGGCAATGTGGAA
ACTGCTCCAGACTCAGGGGCGGACGACCTAGAGGAGCAAGACGGGACGGAGAAGAGGCCAGAGCAGGCGTGCATGGGCGTGGTGACCGTCCTTTCA
GACATCATCGGCCGTTTCAGTCCTTCTGCTCTGATGCCGCTGTGTCCTGAGACAAGACTTGTACCTCGTGGAGGTGCTGAAGACTTGTGGATGCAGGCT
ACAGAGCTGGTGAGAAGCATGAGAGAGAACCCGCAACTTGACTTTGAACATGACTGGAAGCTCATCAACGTGTTCTTCAGTAACACAAGCCAGTGTTTC
CCGTGTCCCTCTGCTCAACAGAAGGGGCTCGTGCTGGGGGGCATGGACAAGCTGACAAGGACCCTGGACTACCTGCAGCAGGAGGTCCCCAAAGCGTT
CGTCAACCTGGTGGATCTCTCGGAGTTGGCAGCATTCTCTCGCTGGCGTCAGGGGGCTCAGCTCAGCCCAGCAGCCGAGCCCTGCAGGTGCTTAAGGG
AAACCTCCCAGCTGACCAAGGTCCTGACGCAGTGGTCCTACCTGGAAGCCTGGGACAGCCTCCTGGCGTCCAGCAAGTACAACACACAGGAGTCCTTCG
CGGTGGTCTTCCAGCCTTTCTTCTACGAGAGCTCGCTGTCGGCGCTGTTGGCGGAGCCCCCACTCCAGGACCCCACCACACTGGCCCTGAGTCTCTGGA
ACAGAATGATGGAGCCCATAGGGCGCAAAGAAGAGCCGTTCAGTGAGAAGGAGAGGAAGCCACTGAGATGCCCCACACAGGAGAGCCCCTACCTATT
CACCTACAGGAACAGCGGCCAGCTGACCAGGGTGTCACAGCCCCAGGGGAAGCTCGAGGTGAGAGAAGGGACGGAAATCCGCTGTCCCGACAAGGA
CCCCTCCGACTCGGTTCCCACCTCAGTTCACAGGCTGAAGCCCGCCGACATCAAGGTCATAGGTGCCATGGGTGACTCCCTCACGGCAGGCAACGGGGC
GGGGTCCCAGCCTGGGAACATCCTGGATGTCCTTACTCAGTACCGAGGCCTCTCCTGGAGCGTCGGGGGCGATCAGAACATCAGCACGGTCACCACCC
TGGCCAACATCCTCCGGGAATTCAACCCTTCCCTGCAGGGCTTCTCCGTCGGCACCGGGAGAGAGACCACTTCCCAAGCCTTCTTCAACCAGGCAGTGG
CGGGCGCCCGAGCCGATGGGTTAATTCCCCAGGCCCAGCGGCTGGTGGCCCTGATGAAGAATGACACGAGGATAAATTTTCAGGAGGATTGGAAGAT
AATAACCGTGTTCATTGGAGGCAACGACCTCTGTGATTTCTGCAATGACCCGGTGCGCTACTCCCCCCAGAACTTCACGGACAACATCGGGACGGCCCT
GGACATCCTCCACGCTGAGATTCCCCGGGCCTTTGTGAACCTGGTGAAGGTGCTTGAGATCTCCAAGCTGAGAGAGCTGTACCAGGAGACTAAAGTGA
GCTGCCCCAGGATGATCCTCAGGTCTCTGTGTCCCTGTGTCCTGAAGTTCGATGACAACTCCACAGAAATTGCCTCCCTCATCGAAACGATCAAGGAGTA
TCAGGAGAGGACCCAGCAGCTGATTGACAGCGGGCGATACGACACGAGGGACGACTTCACGGTGGTTTTGCAGCCGTTTTTTGAAAAAGTGAACATGC
CAAAGACCCAGGACGGGCTACCCGACAACTCTTTCTTCGCGCCCGACTGCTTCCACTTCAGCAGCAAGGCCCACGCGCACGCAGCTAGCGCCCTCTGGA
ACAACATGCTGGAACCTGTTGGCCAGAAGACAACACACAATGATTTTGAAGGAGCAGTCAACATTACGTGTCCAAACCAGGTCTGGCCGTTTCTGAGTA
CCTACAAGAACAGCGTGCAGGGTTTTGGGACCTGGCTGCCGTGCAGAGACAGATCCCCGTCTGCCTCGCCCCCCACCTCAGTGCACGCCCTGAGACCCG
CAGATATCCAAGTTGTGGCTGCTCTGGGAGATTCTCTAACTGCTGGCATTGGAATTGGCTCCAAACCAAATGACCTCTCAGATGGCACCACTCAGTATCG
GGGACTGTCATATAGTTCAGGAGGGGACGGCTCCCTGGACAACGTGACCACCTTGCCTAATATCCTCCGGCAGTTCAACAGCAACCTCATGGGCTTCGC
CGTGGGCACTGGTGATGCCAGTGGCACCAATGCTTTCTTCAACCAGGCTGTTCCCGGAGCAAAGGCGAGGGATCTTATGAGCCAAGTGCAGACTCTGG
TTCAGAGGATGAAGGACGACCATAGAGTAAATTTCCAAGAAGATTGGAAGGTCATCACGGTGCAGATCGGAGCCAGTGACTTGTGTGACTACTGCACG
GATTCGAACCTGTATTCTGCAGCCAACTTTTACGACCATCTCCGAGACGCCTTGGACGCCCTCCACAGAGAGGTGCCCAGAGCTCTGGTCAACCTCGTGG
ACTTCATGAACCCAAGTGTCACACGACAGGTGTTCCTGGGGAACCCCGACAAGTGCCCAGTGCAGCAGGCCAGCGCCCTGTGTAACTGCGTCTTGAGCC
CCCGGGAGAACTCCTATGAGCTGGCCCGGTTGGAGGCCCTCGCACAAGCCTACCAGAGCAGCCTGCGGGAGCTGGTGGAGTCCGGCCGCTACGACAC
GCGGGAGGACTTCTCCGTGGTGCTGCAGCCCTTCTTCCACAGCATCCAGCTCCCCGTCCTGCAGGACGGGCGCCTGGACACGTCTTTCTTCGCCCCAGA
CTGCGTCCACCCAAATCAGAAGTTCCACTCCCAGCTCTCCAGAGCCCTCTGGCGCAACATGCTGGAGCCCCTGGGAGGGAAAACGGATGCACTGGACCT
GACGGCAGCCATCACCCTCACCTGCCCCACTCAGAACGAGCCTTTCCTGAGAACCTTCCGGAACAGTGACTACACCTACCCCAGCAGGCCAGCCGTCGA
GAACTGGGGCAGTGACTTCCTGTGCACCGCGTGGAATGCTTCCAGGGGTGTTCCGAACTCAGTCCATGAGCTCCAACCAGGAGATATCAAAGTCGTAGC
TGCCCTGGGTGACTCGCTGACGCTGGCCATGGGCGCTCGACCAAGCAACTCCAGTGACCCACCCATGTTCTGGAGGGGCCTCTCCTGGAGCATTGGTG
GGGACGGGGCCTTGGAGACTCACACCACACTGCCCAACATTCTGAAGAAGTTCAACCCTTCCATCCTGGGGTTTTCTACTGGAACCTTGGAGGGAACCA
TGGGACTCAACGTAGCAGTGCAAGGGGCCAGAGCTCAGGACATGCCAGCCCAAGCCCGGGACCTGGTGGAGCGAATGAGAAACAGCCCTGAGATCGA
CCTGGAGAAGGACTGGAAGCTGGTCACACTCTTCGTGGGGGGCAATGACCTGTGTCACTTCTGCGAGAATCCGGAGGGCAGCTCCGAGGGGGAGTAC
GTCCAGCACATCCAGCAGGCCCTGGACGTCCTGTACGAGGAGCTTCCGAGGACTTTTGTCAATGTGGTGGAGGTCATGGAGCTGGCCGGCCTGCACCA
GGACCAAGGCGGGAGATGTGCCACACTGCTGGCAGCTCAGAGCCACTGCACCTGCTTCAAATACTCTCAGAGCTCAGTGGAGATGCAAGAACTGAAGA
AAGTGAACTGGAACCTCCAGAGCGGCCTCTCCAGGCTCTCCTACTCACACCAGTACGTGCAGCGTGAGGACTTCGCCGTCGTTGTGCAGCCTTTCTTCCA
GAACACTCTCGTCCCACTGAACGGGAGAGGGGACACTGACCTCACCTTCTTCTCTGACGACTGCTTCCACTTCTCAGAGCGCGGTCATGCCGAGATGGC
CATTGCCCTCTGGAACAACATGCTGGAGCCAGTGGGCCACAAGACAACCTCCAACAACTTCACCTACAGCCGAACCAAACTCAAGTGCCCCTCTCCCGAC
AGCCCGTACCTCTACACCCTCCGGAACAGCCGGCTACTCCCCGACCAGGCTGAGGCAGACCCCACGGTGCTCTACTGGGCAGTGCCGGTGGCAGCAGG
AGCCGGCCTTCTCATCGGCATCCTCGCCATGGTGGCAGGGAGAGGCATGCGCTGTCGCCCGCGGGAGGACCCCCCGCTGAGCCTGAGCACAGGCCTCT
AGGCCCATGTGTGTGGTCCTCACCCGGAAGGTCCCGGCCACACTCCAACCTGGCCCTTCTACCTCAGCCACCTGTGGCCACCACGGGCAGAACCTGCTT
CTACACCTGAGACACAGCAGCCAAAGGGACAGTCAGGACTTCTTGGGGCCTGGGCTTCTCCCAGGTCAATGCTCCTGGGATTGGCAAACTTAAATAAAA
AGCCGAACATGTTCTGGACGCTGCAGGTGCCTCGGGAATTCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC
Mutated DNA.

Now the mutated DNA must be compared to the original DNA, for this the sequence must
be passed to amino acid sequence. In the original amino acid sequence, we can see that
the selection we made is 5'3 'Frame 1, where most are selected in red (figure 28), the part
that does not appear in red if it is not pink is the part that must appear in the mutated
amino acid sequence BUT in the red part (Figure 29). Since it should be in the proper open
reading frame. Once this is accomplished, we will have the desired mutation

Figure 28.
P R H L Q R P G Met A L W P S V F L L G L L P L L G R G A D Q I Q T S S G K N T L E G Q L W P E S L K T F P F P C D P K T L A E S V P S E S V H S L
R P S D I K F V A A I G N V E T A P D S G A D D L E E Q D G T E K R P E Q A C Met G V V T V L S D I I G R F S P S A L Met P L C P E T R L V P R G G
A E D L W Met Q A T E L V R S Met R E N P Q L D F E H D W K L I N V F F S N T S Q C F P C P S A Q Q K G L V L G G Met D K L T R T L D Y L Q Q
EVPKAFVNLVDLSELAAFSRWRQGAQLSPAAEPCRCLRETSQLTKVLTQWSYLEAWDSLLASSKYNTQESFAV
V F Q P F F Y E S S L S A L L A E P P L Q D P T T L A L S L W N R Met Met E P I G R K E E P F S E K E R K P L R C P T Q E S P Y L F T Y R N S G Q L T
R V S Q P Q G K L E V R E G T E I R C P D K D P S D S V P T S V H R L K P A D I K V I G A Met G D S L T A G N G A G S Q P G N I L D V L T Q Y R G
L S W S V G G D Q N I S T V T T L A N I L R E F N P S L Q G F S V G T G R E T T S Q A F F N Q A V A G A R A D G L I P Q A Q R L V A L Met K N D T
RINFQEDWKIITVFIGGNDLCDFCNDPVRYSPQNFTDNIGTALDILHAEIPRAFVNLVKVLEISKLRELYQETKVS
C P R Met I L R S L C P C V L K F D D N S T E I A S L I E T I K E Y Q E R T Q Q L I D S G R Y D T R D D F T V V L Q P F F E K V N Met P K T Q D G L P
D N S F F A P D C F H F S S K A H A H A A S A L W N N Met L E P V G Q K T T H N D F E G A V N I T C P N Q V W P F L S T Y K N S V Q G F G T W
LPCRDRSPSASPPTSVHALRPADIQVVAALGDSLTAGIGIGSKPNDLSDGTTQYRGLSYSSGGDGSLDNVTTLP
N I L R Q F N S N L Met G F A V G T G D A S G T N A F F N Q A V P G A K A R D L Met S Q V Q T L V Q R Met K D D H R V N F Q E D W K V I T V
Q I G A S D L C D Y C T D S N L Y S A A N F Y D H L R D A L D A L H R E V P R A L V N L V D F Met N P S V T R Q V F L G N P D K C P V Q Q A S A
LCNCVLSPRENSYELARLEALAQAYQSSLRELVESGRYDTREDFSVVLQPFFHSIQLPVLQDGRLDTSFFAPDCV
H P N Q K F H S Q L S R A L W R N Met L E P L G G K T D A L D L T A A I T L T C P T Q N E P F L R T F R N S D Y T Y P S R P A V E N W G S D F L C
T A W N A S R G V P N S V H E L Q P G D I K V V A A L G D S L T L A Met G A R P S N S S D P P Met F W R G L S W S I G G D G A L E T H T T L P
N I L K K F N P S I L G F S T G T L E G T Met G L N V A V Q G A R A Q D Met P A Q A R D L V E R Met R N S P E I D L E K D W K L V T L F V G G N
D L C H F C E N P E G S S E G E Y V Q H I Q Q A L D V L Y E E L P R T F V N V V E V Met E L A G L H Q D Q G G R C A T L L A A Q S H C T C F K Y S
Q S S V E Met Q E L K K V N W N L Q S G L S R L S Y S H Q Y V Q R E D F A V V V Q P F F Q N T L V P L N G R G D T D L T F F S D D C F H F S E R
G H A E Met A I A L W N N Met L E P V G H K T T S N N F T Y S R T K L K C P S P D S P Y L Y T L R N S R L L P D Q A E A D P T V L Y W A V P V A
A G A G L L I G I L A Met V A G R G Met R C R P R E D P P L S L S T G L

Original Amino acid sequence.

 Figure 28.

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

D I G I P E A P A A S P R H L Q R P G Met A L W P S V F L L G L L P L L G R G A D Q I Q T S S G K N T L E G Q L W P E S L K T F P F P C D P K T L A
E S V P S E S V H S L R P S D I K F V A A I G N V E T A P D S G A D D L E E Q D G T E K R P E Q A C Met G V V T V L S D I I G R F S P S A L Met P L
C P E T R L V P R G G A E D L W Met Q A T E L V R S Met R E N P Q L D F E H D W K L I N V F F S N T S Q C F P C P S A Q Q K G L V L G G Met D
KLTRTLDYLQQEVPKAFVNLVDLSELAAFSRWRQGAQLSPAAEPCRCLRETSQLTKVLTQWSYLEAWDSLLAS
S K Y N T Q E S F A V V F Q P F F Y E S S L S A L L A E P P L Q D P T T L A L S L W N R Met Met E P I G R K E E P F S E K E R K P L R C P T Q E S P Y
L F T Y R N S G Q L T R V S Q P Q G K L E V R E G T E I R C P D K D P S D S V P T S V H R L K P A D I K V I G A Met G D S L T A G N G A G S Q P G
NILDVLTQYRGLSWSVGGDQNISTVTTLANILREFNPSLQGFSVGTGRETTSQAFFNQAVAGARADGLIPQAQ
R L V A L Met K N D T R I N F Q E D W K I I T V F I G G N D L C D F C N D P V R Y S P Q N F T D N I G T A L D I L H A E I P R A F V N L V K V L E I S
K L R E L Y Q E T K V S C P R Met I L R S L C P C V L K F D D N S T E I A S L I E T I K E Y Q E R T Q Q L I D S G R Y D T R D D F T V V L Q P F F E K V
N Met P K T Q D G L P D N S F F A P D C F H F S S K A H A H A A S A L W N NMet L E P V G Q K T T H N D F E G A V N I T C P N Q V W P F L S T
YKNSVQGFGTWLPCRDRSPSASPPTSVHALRPADIQVVAALGDSLTAGIGIGSKPNDLSDGTTQYRGLSYSSGG
D G S L D N V T T L P N I L R Q F N S N L Met G F A V G T G D A S G T N A F F N Q A V P G A K A R D L Met S Q V Q T L V Q R Met K D D H R V
N F Q E D W K V I T V Q I G A S D L C D Y C T D S N L Y S A A N F Y D H L R D A L D A L H R E V P R A L V N L V D F Met N P S V T R Q V F L G N
PDKCPVQQASALCNCVLSPRENSYELARLEALAQAYQSSLRELVESGRYDTREDFSVVLQPFFHSIQLPVLQDG
R L D T S F F A P D C V H P N Q K F H S Q L S R A L W R N Met L E P L G G K T D A L D L T A A I T L T C P T Q N E P F L R T F R N S D Y T Y P S R P
A V E N W G S D F L C T A W N A S R G V P N S V H E L Q P G D I K V V A A L G D S L T L A Met G A R P S N S S D P P Met F W R G L S W S I G G
D G A L E T H T T L P N I L K K F N P S I L G F S T G T L E G T Met G L N V A V Q G A R A Q D Met P A Q A R D L V E R Met R N S P E I D L E K D
W K L V T L F V G G N D L C H F C E N P E G S S E G E Y V Q H I Q Q A L D V L Y E E L P R T F V N V V E V Met E L A G L H Q D Q G G R C A T L L
A A Q S H C T C F K Y S Q S S V E Met Q E L K K V N W N L Q S G L S R L S Y S H Q Y V Q R E D F A V V V Q P F F Q N T L V P L N G R G D T D L T
F F S D D C F H F S E R G H A E Met A I A L W N N Met L E P V G H K T T S N N F T Y S R T K L K C P S P D S P Y L Y T L R N S R L L P D Q A E A D
P T V L Y W A V P V A A G A G L L I G I L A Met V A G R G Met R C R P R E D P P L S L S T G L
Mutated Amino acid sequence.

4. On the sequence of nucleotides Eukaryote to make each a tenth of the sequence

point mutations such as nitrogenous bases Exchange.

CCGAGGCACCTGCAGCGTCCAGGCATGGCTCTGTGGCCGAGCGTCTTCCTCCTGGGGCTGCTGCCGCTCCTGGGGCGAGGTGCAGATCAGATCCAAAC
CTCTTCTGGAAAGAACACATTGGAAGGGCAGCTCTGGCCAGAGAGCCTGAAGACTTTTCCATTCCCATGCGACCCAAAGACGTTAGCAGAGAGCGTGCC
CTCTGAGTCAGTTCACTCTCTGAGGCCTTCTGACATTAAATTTGTTGCAGCCATTGGCAATGTGGAAACTGCTCCAGACTCAGGGGCGGACGACCTAGAG
GAGCAAGACGGGACGGAGAAGAGGCCAGAGCAGGCGTGCATGGGCGTGGTGACCGTCCTTTCAGACATCATCGGCCGTTTCAGTCCTTCTGCTCTGAT
GCCGCTGTGTCCTGAGACAAGACTTGTACCTCGTGGAGGTGCTGAAGACTTGTGGATGCAGGCTA
CAGAGCTGGTGAGAAGCATGAGAGAGAACCCGCAACTTGACTTTGAACATGACTGGAAGCTCATCAACGTGTTCTTCAGTAACACAAGCCAGTGTTTCC
CGTGTCCCTCTGCTCAACAGAAGGGGCTCGTGCTGGGGGGCATGGACAAGCTGACAAGGACCCTGGACTACCTGCAGCAGGAGGTCCCCAAAGCGTTC
GTCAACCTGGTGGATCTCTCGGAGTTGGCAGCATTCTCTCGCTGGCGTCAGGGGGCTCAGCTCAGCCCAGCAGCCGAGCCCTGCAGGTGCTTAAGGGA
AACCTCCCAGCTGACCAAGGTCCTGACGCAGTGGTCCTACCTGGAAGCCTGGGACAGCCTCCTGGCGTCCAGCAAGTACAACACACAGGAGTCCTTCGC
GGTGGTCTTCCAGCCTTTCTTCTACGAGAGCTCGCTGTCGGCGCTGTTGGCGGAGCCCCCACTCCA
GGACCCCACCACACTGGCCCTGAGTCTCTGGAACAGAATGATGGAGCCCATAGGGCGCAAAGAAGAGCCGTTCAGTGAGAAGGAGAGGAAGCCACTG
AGATGCCCCACACAGGAGAGCCCCTACCTATTCACCTACAGGAACAGCGGCCAGCTGACCAGGGTGTCACAGCCCCAGGGGAAGCTCGAGGTGAGAG
AAGGGACGGAAATCCGCTGTCCCGACAAGGACCCCTCCGACTCGGTTCCCACCTCAGTTCACAGGCTGAAGCCCGCCGACATCAAGGTCATAGGTGCCA
TGGGTGACTCCCTCACGGCAGGCAACGGGGCGGGGTCCCAGCCTGGGAACATCCTGGATGTCCTTACTCAGTACCGAGGCCTCTCCTGGAGCGTCGGG
GGCGATCAGAACATCAGCACGGTCACCACCCTGGCCAACATCCTCCGGGAATTCAACCCTTCCCTGCAG
GGCTTCTCCGTCGGCACCGGGAGAGAGACCACTTCCCAAGCCTTCTTCAACCAGGCAGTGGCGGGCGCCCGAGCCGATGGGTTAATTCCCCAGGCCCA
GCGGCTGGTGGCCCTGATGAAGAATGACACGAGGATAAATTTTCAGGAGGATTGGAAGATAATAACCGTGTTCATTGGAGGCAACGACCTCTGTGATT
TCTGCAATGACCCGGTGCGCTACTCCCCCCAGAACTTCACGGACAACATCGGGACGGCCCTGGACATCCTCCACGCTGAGATTCCCCGGGCCTTTGTGA
ACCTGGTGAAGGTGCTTGAGATCTCCAAGCTGAGAGAGCTGTACCAGGAGACTAAAGTGAGCTGCCCCAGGATGATCCTCAGGTCTCTGTGTCCCTGTG
TCCTGAAGTTCGATGACAACTCCACAGAAATTGCCTCCCTCATCGAAACGATCAAGGAGTATCAGG
AGAGGACCCAGCAGCTGATTGACAGCGGGCGATACGACACGAGGGACGACTTCACGGTGGTTTTGCAGCCGTTTTTTGAAAAAGTGAACATGCCAAAG
ACCCAGGACGGGCTACCCGACAACTCTTTCTTCGCGCCCGACTGCTTCCACTTCAGCAGCAAGGCCCACGCGCACGCAGCTAGCGCCCTCTGGAACAAC
ATGCTGGAACCTGTTGGCCAGAAGACAACACACAATGATTTTGAAGGAGCAGTCAACATTACGTGTCCAAACCAGGTCTGGCCGTTTCTGAGTACCTAC
AAGAACAGCGTGCAGGGTTTTGGGACCTGGCTGCCGTGCAGAGACAGATCCCCGTCTGCCTCGCCCCCCACCTCAGTGCACGCCCTGAGACCCGCAGA
TATCCAAGTTGTGGCTGCTCTGGGAGATTCTCTAACTGCTGGCATTGGAATTGGCTCCAAACCAAA
TGACCTCTCAGATGGCACCACTCAGTATCGGGGACTGTCATATAGTTCAGGAGGGGACGGCTCCCTGGACAACGTGACCACCTTGCCTAATATCCTCCG
GCAGTTCAACAGCAACCTCATGGGCTTCGCCGTGGGCACTGGTGATGCCAGTGGCACCAATGCTTTCTTCAACCAGGCTGTTCCCGGAGCAAAGGCGAG
GGATCTTATGAGCCAAGTGCAGACTCTGGTTCAGAGGATGAAGGACGACCATAGAGTAAATTTCCAAGAAGATTGGAAGGTCATCACGGTGCAGATCG
GAGCCAGTGACTTGTGTGACTACTGCACGGATTCGAACCTGTATTCTGCAGCCAACTTTTACGACCATCTCCGAGACGCCTTGGACGCCCTCCACAGAGA
GGTGCCCAGAGCTCTGGTCAACCTCGTGGACTTCATGAACCCAAGTGTCACACGACAGGTGTTC
CTGGGGAACCCCGACAAGTGCCCAGTGCAGCAGGCCAGCGCCCTGTGTAACTGCGTCTTGAGCCCCCGGGAGAACTCCTATGAGCTGGCCCGGTTGGA
GGCCCTCGCACAAGCCTACCAGAGCAGCCTGCGGGAGCTGGTGGAGTCCGGCCGCTACGACACGCGGGAGGACTTCTCCGTGGTGCTGCAGCCCTTCT
TCCACAGCATCCAGCTCCCCGTCCTGCAGGACGGGCGCCTGGACACGTCTTTCTTCGCCCCAGACTGCGTCCACCCAAATCAGAAGTTCCACTCCCAGCT
CTCCAGAGCCCTCTGGCGCAACATGCTGGAGCCCCTGGGAGGGAAAACGGATGCACTGGACCTGACGGCAGCCATCACCCTCACCTGCCCCACTCAGA
ACGAGCCTTTCCTGAGAACCTTCCGGAACAGTGACTACACCTACCCCAGCAGGCCAGCCGTCGAGA
ACTGGGGCAGTGACTTCCTGTGCACCGCGTGGAATGCTTCCAGGGGTGTTCCGAACTCAGTCCATGAGCTCCAACCAGGAGATATCAAAGTCGTAGCTG
CCCTGGGTGACTCGCTGACGCTGGCCATGGGCGCTCGACCAAGCAACTCCAGTGACCCACCCATGTTCTGGAGGGGCCTCTCCTGGAGCATTGGTGGG
GACGGGGCCTTGGAGACTCACACCACACTGCCCAACATTCTGAAGAAGTTCAACCCTTCCATCCTGGGGTTTTCTACTGGAACCTTGGAGGGAACCATG
GGACTCAACGTAGCAGTGCAAGGGGCCAGAGCTCAGGACATGCCAGCCCAAGCCCGGGACCTGGTGGAGCGAATGAGAAACAGCCCTGAGATCGACC
TGGAGAAGGACTGGAAGCTGGTCACACTCTTCGTGGGGGGCAATGACCTGTGTCACTTCTGCGAGAA
TCCGGAGGGCAGCTCCGAGGGGGAGTACGTCCAGCACATCCAGCAGGCCCTGGACGTCCTGTACGAGGAGCTTCCGAGGACTTTTGTCAATGTGGTGG
AGGTCATGGAGCTGGCCGGCCTGCACCAGGACCAAGGCGGGAGATGTGCCACACTGCTGGCAGCTCAGAGCCACTGCACCTGCTTCAAATACTCTCAG
AGCTCAGTGGAGATGCAAGAACTGAAGAAAGTGAACTGGAACCTCCAGAGCGGCCTCTCCAGGCTCTCCTACTCACACCAGTACGTGCAGCGTGAGGA
CTTCGCCGTCGTTGTGCAGCCTTTCTTCCAGAACACTCTCGTCCCACTGAACGGGAGAGGGGACACTGACCTCACCTTCTTCTCTGACGACTGCTTCCACT
TCTCAGAGCGCGGTCATGCCGAGATGGCCATTGCCCTCTGGAACAACATGCTGGAGCCAGTGGGC
CACAAGACAACCTCCAACAACTTCACCTACAGCCGAACCAAACTCAAGTGCCCCTCTCCCGACAGCCCGTACCTCTACACCCTCCGGAACAGCCGGCTAC
TCCCCGACCAGGCTGAGGCAGACCCCACGGTGCTCTACTGGGCAGTGCCGGTGGCAGCAGGAGCCGGCCTTCTCATCGGCATCCTCGCCATGGTGGCA
GGGAGAGGCATGCGCTGTCGCCCGCGGGAGGACCCCCCGCTGAGCCTGAGCACAGGCCTCTAGGCCCATGTGTGTGGTCCTCACCCGGAAGGTCCCG
GCCACACTCCAACCTGGCCCTTCTACCTCAGCCACCTGTGGCCACCACGGGCAGAACCTGCTTCTACACCTGAGACACAGCAGCCAAAGGGACAGTCAG
GACTTCTTGGGGCCTGGGCTTCTCCCAGGTCAATGCTCCTGGGATTGGCAAACTTAAATAAAAAGC
CGAACATGTTCT

Phospholipase [Oryctolagus cuniculus] DNA

It has a 4612-nucleotide sequence, each 460 nucleotides will be a change. Mutations are
changing the lyric where US 460 nucleotides, this change can be done in a random way and
as you want, remember that only I could place letters like G C A or T.

I. A are mutated by T
II. A are mutated by G
III. G are mutated by T
IV. G are mutated by C
V. A are mutated by T
VI. C are mutated by G
VII. A are mutated by T
VIII. A are mutated by G
IX. C are mutated by T
X. C are mutated by G
CCGAGGCACCTGCAGCGTCCAGGCATGGCTCTGTGGCCGAGCGTCTTCCTCCTGGGGCTGCTGCCGCTCCTGGGGCGAGGTGCAGATCAGATCCAAAC
CTCTTCTGGAAAGAACACATTGGAAGGGCAGCTCTGGCCAGAGAGCCTGAAGACTTTTCCATTCCCATGCGACCCAAAGACGTTAGCAGAGAGCGTGCC
CTCTGAGTCAGTTCACTCTCTGAGGCCTTCTGACATTAAATTTGTTGCAGCCATTGGCAATGTGGAAACTGCTCCAGACTCAGGGGCGGACGACCTAGAG
GAGCAAGACGGGACGGAGAAGAGGCCAGAGCAGGCGTGCATGGGCGTGGTGACCGTCCTTTCAGACATCATCGGCCGTTTCAGTCCTTCTGCTCTGAT
GCCGCTGTGTCCTGAGACAAGACTTGTACCTCGTGGAGGTGCTGAAGACTTGTGGATGCAGGCTTCAGAGCTGGTGAGAAGCATGAGAGAGAACCCGC
AACTTGACTTTGAACATGACTGGAAGCTCATCAACGTGTTCTTCAGTAACACAAGCCAGTGTTTCCCGTGTCCCTCTGCTCAACAGAAGGGGCTCGTGCT
GGGGGGCATGGACAAGCTGACAAGGACCCTGGACTACCTGCAGCAGGAGGTCCCCAAAGCGTTCGTCAACCTGGTGGATCTCTCGGAGTTGGCAGCAT
TCTCTCGCTGGCGTCAGGGGGCTCAGCTCAGCCCAGCAGCCGAGCCCTGCAGGTGCTTAAGGGAAACCTCCCAGCTGACCAAGGTCCTGACGCAGTGG
TCCTACCTGGAAGCCTGGGACAGCCTCCTGGCGTCCAGCAAGTACAACACACAGGAGTCCTTCGCGGTGGTCTTCCAGCCTTTCTTCTACGAGAGCTCGC
TGTCGGCGCTGTTGGCGGAGCCCCCACTCCGGGACCCCACCACACTGGCCCTGAGTCTCTGGAACAGAATGATGGAGCCCATAGGGCGCAAAGAAGAG
CCGTTCAGTGAGAAGGAGAGGAAGCCACTGAGATGCCCCACACAGGAGAGCCCCTACCTATTCACCTACAGGAACAGCGGCCAGCTGACCAGGGTGTC
ACAGCCCCAGGGGAAGCTCGAGGTGAGAGAAGGGACGGAAATCCGCTGTCCCGACAAGGACCCCTCCGACTCGGTTCCCACCTCAGTTCACAGGCTGA
AGCCCGCCGACATCAAGGTCATAGGTGCCATGGGTGACTCCCTCACGGCAGGCAACGGGGCGGGGTCCCAGCCTGGGAACATCCTGGATGTCCTTACT
CAGTACCGAGGCCTCTCCTGGAGCGTCGGGGGCGATCAGAACATCAGCACGGTCACCACCCTGGCCAACATCCTCCGGGAATTCAACCCTTCCCTGCAT
GGCTTCTCCGTCGGCACCGGGAGAGAGACCACTTCCCAAGCCTTCTTCAACCAGGCAGTGGCGGGCGCCCGAGCCGATGGGTTAATTCCCCAGGCCCA
GCGGCTGGTGGCCCTGATGAAGAATGACACGAGGATAAATTTTCAGGAGGATTGGAAGATAATAACCGTGTTCATTGGAGGCAACGACCTCTGTGATT
TCTGCAATGACCCGGTGCGCTACTCCCCCCAGAACTTCACGGACAACATCGGGACGGCCCTGGACATCCTCCACGCTGAGATTCCCCGGGCCTTTGTGA
ACCTGGTGAAGGTGCTTGAGATCTCCAAGCTGAGAGAGCTGTACCAGGAGACTAAAGTGAGCTGCCCCAGGATGATCCTCAGGTCTCTGTGTCCCTGTG
TCCTGAAGTTCGATGACAACTCCACAGAAATTGCCTCCCTCATCGAAACGATCAAGGAGTATCAGCAGAGGACCCAGCAGCTGATTGACAGCGGGCGAT
ACGACACGAGGGACGACTTCACGGTGGTTTTGCAGCCGTTTTTTGAAAAAGTGAACATGCCAAAGACCCAGGACGGGCTACCCGACAACTCTTTCTTCG
CGCCCGACTGCTTCCACTTCAGCAGCAAGGCCCACGCGCACGCAGCTAGCGCCCTCTGGAACAACATGCTGGAACCTGTTGGCCAGAAGACAACACACA
ATGATTTTGAAGGAGCAGTCAACATTACGTGTCCAAACCAGGTCTGGCCGTTTCTGAGTACCTACAAGAACAGCGTGCAGGGTTTTGGGACCTGGCTGC
CGTGCAGAGACAGATCCCCGTCTGCCTCGCCCCCCACCTCAGTGCACGCCCTGAGACCCGCAGATATCCAAGTTGTGGCTGCTCTGGGAGATTCTCTAAC
TGCTGGCATTGGAATTGGCTCCAAACCAATTGACCTCTCAGATGGCACCACTCAGTATCGGGGACTGTCATATAGTTCAGGAGGGGACGGCTCCCTGGA
CAACGTGACCACCTTGCCTAATATCCTCCGGCAGTTCAACAGCAACCTCATGGGCTTCGCCGTGGGCACTGGTGATGCCAGTGGCACCAATGCTTTCTTC
AACCAGGCTGTTCCCGGAGCAAAGGCGAGGGATCTTATGAGCCAAGTGCAGACTCTGGTTCAGAGGATGAAGGACGACCATAGAGTAAATTTCCAAGA
AGATTGGAAGGTCATCACGGTGCAGATCGGAGCCAGTGACTTGTGTGACTACTGCACGGATTCGAACCTGTATTCTGCAGCCAACTTTTACGACCATCTC
CGAGACGCCTTGGACGCCCTCCACAGAGAGGTGCCCAGAGCTCTGGTCAACCTCGTGGACTTCATGAACCCAAGTGTCACACGACAGGTGTTGCTGGG
GAACCCCGACAAGTGCCCAGTGCAGCAGGCCAGCGCCCTGTGTAACTGCGTCTTGAGCCCCCGGGAGAACTCCTATGAGCTGGCCCGGTTGGAGGCCC
TCGCACAAGCCTACCAGAGCAGCCTGCGGGAGCTGGTGGAGTCCGGCCGCTACGACACGCGGGAGGACTTCTCCGTGGTGCTGCAGCCCTTCTTCCAC
AGCATCCAGCTCCCCGTCCTGCAGGACGGGCGCCTGGACACGTCTTTCTTCGCCCCAGACTGCGTCCACCCAAATCAGAAGTTCCACTCCCAGCTCTCCA
GAGCCCTCTGGCGCAACATGCTGGAGCCCCTGGGAGGGAAAACGGATGCACTGGACCTGACGGCAGCCATCACCCTCACCTGCCCCACTCAGAACGAG
CCTTTCCTGAGAACCTTCCGGAACAGTGACTACACCTACCCCAGCAGGCCAGCCGTCGAGTACTGGGGCAGTGACTTCCTGTGCACCGCGTGGAATGCT
TCCAGGGGTGTTCCGAACTCAGTCCATGAGCTCCAACCAGGAGATATCAAAGTCGTAGCTGCCCTGGGTGACTCGCTGACGCTGGCCATGGGCGCTCG
ACCAAGCAACTCCAGTGACCCACCCATGTTCTGGAGGGGCCTCTCCTGGAGCATTGGTGGGGACGGGGCCTTGGAGACTCACACCACACTGCCCAACAT
TCTGAAGAAGTTCAACCCTTCCATCCTGGGGTTTTCTACTGGAACCTTGGAGGGAACCATGGGACTCAACGTAGCAGTGCAAGGGGCCAGAGCTCAGG
ACATGCCAGCCCAAGCCCGGGACCTGGTGGAGCGAATGAGAAACAGCCCTGAGATCGACCTGGAGAAGGACTGGAAGCTGGTCACACTCTTCGTGGG
GGGCAATGACCTGTGTCACTTCTGCGAGAGTCCGGAGGGCAGCTCCGAGGGGGAGTACGTCCAGCACATCCAGCAGGCCCTGGACGTCCTGTACGAG
GAGCTTCCGAGGACTTTTGTCAATGTGGTGGAGGTCATGGAGCTGGCCGGCCTGCACCAGGACCAAGGCGGGAGATGTGCCACACTGCTGGCAGCTCA
GAGCCACTGCACCTGCTTCAAATACTCTCAGAGCTCAGTGGAGATGCAAGAACTGAAGAAAGTGAACTGGAACCTCCAGAGCGGCCTCTCCAGGCTCTC
CTACTCACACCAGTACGTGCAGCGTGAGGACTTCGCCGTCGTTGTGCAGCCTTTCTTCCAGAACACTCTCGTCCCACTGAACGGGAGAGGGGACACTGA
CCTCACCTTCTTCTCTGACGACTGCTTCCACTTCTCAGAGCGCGGTCATGCCGAGATGGCCATTGCCCTCTGGAACAACATGCTGGAGCCAGTGGGTCAC
AAGACAACCTCCAACAACTTCACCTACAGCCGAACCAAACTCAAGTGCCCCTCTCCCGACAGCCCGTACCTCTACACCCTCCGGAACAGCCGGCTACTCC
CCGACCAGGCTGAGGCAGACCCCACGGTGCTCTACTGGGCAGTGCCGGTGGCAGCAGGAGCCGGCCTTCTCATCGGCATCCTCGCCATGGTGGCAGG
GAGAGGCATGCGCTGTCGCCCGCGGGAGGACCCCCCGCTGAGCCTGAGCACAGGCCTCTAGGCCCATGTGTGTGGTCCTCACCCGGAAGGTCCCGGC
CACACTCCAACCTGGCCCTTCTACCTCAGCCACCTGTGGCCACCACGGGCAGAACCTGCTTCTACACCTGAGACACAGCAGCCAAAGGGACAGTCAGGA
CTTCTTGGGGCCTGGGCTTCTCCCAGGTCAATGCTCCTGGGATTGGCAAACTTAAATAAAAAGGCGAACATGTTCT
Mutated Phospholipase [Oryctolagus cuniculus] DNA

When the DNA sequence follows the transcript for the amino acid sequence with the help
of the ExPASy platform, the frame was taken up with more red color. In our case, we
obtained the 5'3 ' Frame1 as shown in Figure 29.
 Figure 29.
Met A L W P S V F L L G L L P L L G R G A D Q I Q T S S G K N T L E G Q L W P E S L K T F P F P C D P K T L A E S V P S E S V H S L R P S D I K F V
A A I G N V E T A P D S G A D D L E E Q D G T E K R P E Q A C Met G V V T V L S D I I G R F S P S A L Met P L C P E T R L V P R G G A E D L
W Met Q A S E L V R S Met R E N P Q L D F E H D W K L I N V F F S N T S Q C F P C P S A Q Q K G L V L G G Met D K L T R T L D Y L Q Q E V P K
AFVNLVDLSELAAFSRWRQGAQLSPAAEPCRCLRETSQLTKVLTQWSYLEAWDSLLASSKYNTQESFAVVFQP
F F Y E S S L S A L L A E P P L R D P T T L A L S L W N R Met Met E P I G R K E E P F S E K E R K P L R C P T Q E S P Y L F T Y R N S G Q L T R V S
Q P Q G K L E V R E G T E I R C P D K D P S D S V P T S V H R L K P A D I K V I G A Met G D S L T A G N G A G S Q P G N I L D V L T Q Y R G L S
W S V G G D Q N I S T V T T L A N I L R E F N P S L H G F S V G T G R E T T S Q A F F N Q A V A G A R A D G L I P Q A Q R L V A L Met K N D T R
INFQEDWKIITVFIGGNDLCDFCNDPVRYSPQNFTDNIGTALDILHAEIPRAFVNLVKVLEISKLRELYQETKVS
C P R Met I L R S L C P C V L K F D D N S T E I A S L I E T I K E Y Q Q R T Q Q L I D S G R Y D T R D D F T V V L Q P F F E K V N Met P K T Q D G L
P D N S F F A P D C F H F S S K A H A H A A S A L W N N Met L E P V G Q K T T H N D F E G A V N I T C P N Q V W P F L S T Y K N S V Q G F G T
WLPCRDRSPSASPPTSVHALRPADIQVVAALGDSLTAGIGIGSKPIDLSDGTTQYRGLSYSSGGDGSLDNVTTL
P N I L R Q F N S N L Met G F A V G T G D A S G T N A F F N Q A V P G A K A R D L Met S Q V Q T L V Q R Met K D D H R V N F Q E D W K V I T
V Q I G A S D L C D Y C T D S N L Y S A A N F Y D H L R D A L D A L H R E V P R A L V N L V D F Met N P S V T R Q V L L G N P D K C P V Q Q A S
ALCNCVLSPRENSYELARLEALAQAYQSSLRELVESGRYDTREDFSVVLQPFFHSIQLPVLQDGRLDTSFFAPD
C V H P N Q K F H S Q L S R A L W R N Met L E P L G G K T D A L D L T A A I T L T C P T Q N E P F L R T F R N S D Y T Y P S R P A V E Y W G S D F
L C T A W N A S R G V P N S V H E L Q P G D I K V V A A L G D S L T L A Met G A R P S N S S D P P Met F W R G L S W S I G G D G A L E T H T T
L P N I L K K F N P S I L G F S T G T L E G T Met G L N V A V Q G A R A Q D Met P A Q A R D L V E R Met R N S P E I D L E K D W K L V T L F V G
G N D L C H F C E S P E G S S E G E Y V Q H I Q Q A L D V L Y E E L P R T F V N V V E V Met E L A G L H Q D Q G G R C A T L L A A Q S H C T C F
K Y S Q S S V E Met Q E L K K V N W N L Q S G L S R L S Y S H Q Y V Q R E D F A V V V Q P F F Q N T L V P L N G R G D T D L T F F S D D C F H F
S E R G H A E Met A I A L W N N Met L E P V G H K T T S N N F T Y S R T K L K C P S P D S P Y L Y T L R N S R L L P D Q A E A D P T V L Y W A V
P V A A G A G L L I G I L A Met V A G R G Met R C R P R E D P P L S L S T G L
Mutated Aminoacid sequence

Once obtained the amino acid sequence, we headed for the EMBOSS Neeedle platform,
which will help us to see that type of mutations were performed. Open EMBOSS Needle you
can see a window as shown in Figure 30. STEP 1 should contain the amino acid sequence
obtained on ExPASy's original DNA; While in STEP 2 place the amino acid, sequence obtained
in the DNA with the 10 mutations ExPASy and finally click on Submit.
 Figure 30.
A window like the one obtained in Figure 31.

Figure 31.
Then we see the comparison with amino acids and it is highlighted in the part where
presented mutations or changes.

EMBOSS_001 1 MetALWPSVFLLGLLPLLGRGADQIQTSSGKNTLEGQLWPESLKTFPFPC 50
||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1 MetALWPSVFLLGLLPLLGRGADQIQTSSGKNTLEGQLWPESLKTFPFPC 50

EMBOSS_001 51 DPKTLAESVPSESVHSLRPSDIKFVAAIGNVETAPDSGADDLEEQDGTEK 100

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 51 DPKTLAESVPSESVHSLRPSDIKFVAAIGNVETAPDSGADDLEEQDGTEK 100

EMBOSS_001 101 RPEQACMetGVVTVLSDIIGRFSPSALMetPLCPETRLVPRGGAEDLWMe 150

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 101 RPEQACMetGVVTVLSDIIGRFSPSALMetPLCPETRLVPRGGAEDLWMe 150

EMBOSS_001 151 tQATELVRSMetRENPQLDFEHDWKLINVFFSNTSQCFPCPSAQQKGLVL 200

|||:||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 151 tQASELVRSMetRENPQLDFEHDWKLINVFFSNTSQCFPCPSAQQKGLVL 200

EMBOSS_001 201 GGMetDKLTRTLDYLQQEVPKAFVNLVDLSELAAFSRWRQGAQLSPAAEP 250

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 201 GGMetDKLTRTLDYLQQEVPKAFVNLVDLSELAAFSRWRQGAQLSPAAEP 250

EMBOSS_001 251 CRCLRETSQLTKVLTQWSYLEAWDSLLASSKYNTQESFAVVFQPFFYESS 300

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 251 CRCLRETSQLTKVLTQWSYLEAWDSLLASSKYNTQESFAVVFQPFFYESS 300

EMBOSS_001 301 LSALLAEPPLQDPTTLALSLWNRMetMetEPIGRKEEPFSEKERKPLRCP 350

||||||||||:|||||||||||||||||||||||||||||||||||||||
EMBOSS_001 301 LSALLAEPPLRDPTTLALSLWNRMetMetEPIGRKEEPFSEKERKPLRCP 350

EMBOSS_001 351 TQESPYLFTYRNSGQLTRVSQPQGKLEVREGTEIRCPDKDPSDSVPTSVH 400

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 351 TQESPYLFTYRNSGQLTRVSQPQGKLEVREGTEIRCPDKDPSDSVPTSVH 400

EMBOSS_001 401 RLKPADIKVIGAMetGDSLTAGNGAGSQPGNILDVLTQYRGLSWSVGGDQ 450

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 401 RLKPADIKVIGAMetGDSLTAGNGAGSQPGNILDVLTQYRGLSWSVGGDQ 450

EMBOSS_001 451 NISTVTTLANILREFNPSLQGFSVGTGRETTSQAFFNQAVAGARADGLIP 500

|||||||||||||||||||.||||||||||||||||||||||||||||||
EMBOSS_001 451 NISTVTTLANILREFNPSLHGFSVGTGRETTSQAFFNQAVAGARADGLIP 500

EMBOSS_001 501 QAQRLVALMetKNDTRINFQEDWKIITVFIGGNDLCDFCNDPVRYSPQNF 550

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 501 QAQRLVALMetKNDTRINFQEDWKIITVFIGGNDLCDFCNDPVRYSPQNF 550

EMBOSS_001 551 TDNIGTALDILHAEIPRAFVNLVKVLEISKLRELYQETKVSCPRMetILR 600

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 551 TDNIGTALDILHAEIPRAFVNLVKVLEISKLRELYQETKVSCPRMetILR 600

EMBOSS_001 601 SLCPCVLKFDDNSTEIASLIETIKEYQERTQQLIDSGRYDTRDDFTVVLQ 650

|||||||||||||||||||||||||||:||||||||||||||||||||||
EMBOSS_001 601 SLCPCVLKFDDNSTEIASLIETIKEYQQRTQQLIDSGRYDTRDDFTVVLQ 650

EMBOSS_001 651 PFFEKVNMetPKTQDGLPDNSFFAPDCFHFSSKAHAHAASALWNNMetLE 700

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 651 PFFEKVNMetPKTQDGLPDNSFFAPDCFHFSSKAHAHAASALWNNMetLE 700
EMBOSS_001 701 PVGQKTTHNDFEGAVNITCPNQVWPFLSTYKNSVQGFGTWLPCRDRSPSA 750
||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 701 PVGQKTTHNDFEGAVNITCPNQVWPFLSTYKNSVQGFGTWLPCRDRSPSA 750

EMBOSS_001 751 SPPTSVHALRPADIQVVAALGDSLTAGIGIGSKPNDLSDGTTQYRGLSYS 800

||||||||||||||||||||||||||||||||||.|||||||||||||||
EMBOSS_001 751 SPPTSVHALRPADIQVVAALGDSLTAGIGIGSKPIDLSDGTTQYRGLSYS 800

EMBOSS_001 801 SGGDGSLDNVTTLPNILRQFNSNLMetGFAVGTGDASGTNAFFNQAVPGA 850

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 801 SGGDGSLDNVTTLPNILRQFNSNLMetGFAVGTGDASGTNAFFNQAVPGA 850

EMBOSS_001 851 KARDLMetSQVQTLVQRMetKDDHRVNFQEDWKVITVQIGASDLCDYCTD 900

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 851 KARDLMetSQVQTLVQRMetKDDHRVNFQEDWKVITVQIGASDLCDYCTD 900

EMBOSS_001 901 SNLYSAANFYDHLRDALDALHREVPRALVNLVDFMetNPSVTRQVFLGNP 950

|||||||||||||||||||||||||||||||||||||||||||||.||||
EMBOSS_001 901 SNLYSAANFYDHLRDALDALHREVPRALVNLVDFMetNPSVTRQVLLGNP 950

EMBOSS_001 951 DKCPVQQASALCNCVLSPRENSYELARLEALAQAYQSSLRELVESGRYDT 1000

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 951 DKCPVQQASALCNCVLSPRENSYELARLEALAQAYQSSLRELVESGRYDT 1000

EMBOSS_001 1001 REDFSVVLQPFFHSIQLPVLQDGRLDTSFFAPDCVHPNQKFHSQLSRALW 1050

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1001 REDFSVVLQPFFHSIQLPVLQDGRLDTSFFAPDCVHPNQKFHSQLSRALW 1050

EMBOSS_001 1051 RNMetLEPLGGKTDALDLTAAITLTCPTQNEPFLRTFRNSDYTYPSRPAV 1100

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1051 RNMetLEPLGGKTDALDLTAAITLTCPTQNEPFLRTFRNSDYTYPSRPAV 1100

EMBOSS_001 1101 ENWGSDFLCTAWNASRGVPNSVHELQPGDIKVVAALGDSLTLAMetGARP 1150

|.||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1101 EYWGSDFLCTAWNASRGVPNSVHELQPGDIKVVAALGDSLTLAMetGARP 1150

EMBOSS_001 1151 SNSSDPPMetFWRGLSWSIGGDGALETHTTLPNILKKFNPSILGFSTGTL 1200

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1151 SNSSDPPMetFWRGLSWSIGGDGALETHTTLPNILKKFNPSILGFSTGTL 1200

EMBOSS_001 1201 EGTMetGLNVAVQGARAQDMetPAQARDLVERMetRNSPEIDLEKDWKLV 1250

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1201 EGTMetGLNVAVQGARAQDMetPAQARDLVERMetRNSPEIDLEKDWKLV 1250

EMBOSS_001 1251 TLFVGGNDLCHFCENPEGSSEGEYVQHIQQALDVLYEELPRTFVNVVEVM 1300

||||||||||||||:|||||||||||||||||||||||||||||||||||
EMBOSS_001 1251 TLFVGGNDLCHFCESPEGSSEGEYVQHIQQALDVLYEELPRTFVNVVEVM 1300

EMBOSS_001 1301 etELAGLHQDQGGRCATLLAAQSHCTCFKYSQSSVEMetQELKKVNWNLQ 1350

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1301 etELAGLHQDQGGRCATLLAAQSHCTCFKYSQSSVEMetQELKKVNWNLQ 1350

EMBOSS_001 1351 SGLSRLSYSHQYVQREDFAVVVQPFFQNTLVPLNGRGDTDLTFFSDDCFH 1400

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1351 SGLSRLSYSHQYVQREDFAVVVQPFFQNTLVPLNGRGDTDLTFFSDDCFH 1400

EMBOSS_001 1401 FSERGHAEMetAIALWNNMetLEPVGHKTTSNNFTYSRTKLKCPSPDSPY 1450

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1401 FSERGHAEMetAIALWNNMetLEPVGHKTTSNNFTYSRTKLKCPSPDSPY 1450

EMBOSS_001 1451 LYTLRNSRLLPDQAEADPTVLYWAVPVAAGAGLLIGILAMetVAGRGMet 1500

||||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1451 LYTLRNSRLLPDQAEADPTVLYWAVPVAAGAGLLIGILAMetVAGRGMet 1500
EMBOSS_001 1501 RCRPREDPPLSLSTGL 1516
||||||||||||||||
EMBOSS_001 1501 RCRPREDPPLSLSTGL 1516

#---------------------------------------
#---------------------------------------

Show 8 mutations, which means that my mutant DNA sequence has 8 not silent mutations
(1-8 mutations) and silent 2 (9 and 10) mutation, not silent mutations will be analyzed one
by one then with the help of Figure 32:

Figure 32

Before you begin, you should keep in mind that when we find : or . means that the mutation
is not silent, is say that it changes of protein, where we do not find points means that in those
places the protein does not change, i.e. remains the same. A mutation is conservative when
it changes the protein, but not its character and not conservative is that changes the protein
and changes the character. Normally assume that: means that it is conservative and. It is not
conservative; however, you should confirm so.
I. EMBOSS_001 151 tQATELVRSMetRENPQLDFEHDWKLINVFFSNTSQCFPCPSAQQKGLVL 200
|||:||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 151 tQASELVRSMetRENPQLDFEHDWKLINVFFSNTSQCFPCPSAQQKGLVL 200

The first mutation found is Non-Silent - Conservative because we can see that:
Original Mutated
Abbreviated Amino Acid Encoded by the codon Abbreviated Amino Acid Encoded by the codon

Threonine UCA
T ACA S Serine
Where U=T
It is not silent as you can be seen, there is the same protein and is conservative since the
Threonine to serine, changes the protein, but does not change the character of the
proteins which are polar amino acids with no charged side chains.
II. EMBOSS_001 301 LSALLAEPPLQDPTTLALSLWNRMetMetEPIGRKEEPFSEKERKPLRCP 350
||||||||||:|||||||||||||||||||||||||||||||||||||||
EMBOSS_001 301 LSALLAEPPLRDPTTLALSLWNRMetMetEPIGRKEEPFSEKERKPLRCP 350

The second mutation found is Non-Silent Non-Conservative because we can see that:
Original Mutated
Abbreviated Amino Acid Encoded by the codon Abbreviated Amino Acid Encoded by the codon
Q Glutamine CAG/CAG R Arginine CGG

It is not silent as shown, there is the same protein and is not conservative because it changes
from Glutamine to Arginine, it changes the protein, and changes the nature of proteins
because the Glutamine is an amino acid, Polar, the Arginine is a basic amino acid and in terms
of its structure, the Glutamine has a polar side chain not loaded and the Arginine has a side
chain with positive Group (Basic).
III. EMBOSS_001 451 NISTVTTLANILREFNPSLQGFSVGTGRETTSQAFFNQAVAGARADGLIP 500
|||||||||||||||||||.||||||||||||||||||||||||||||||
EMBOSS_001 451 NISTVTTLANILREFNPSLHGFSVGTGRETTSQAFFNQAVAGARADGLIP 500

The third mutation found is Non-Silent Non-Conservative because we can see that:
Original Mutated
Abbreviated Amino Acid Encoded by the codon Abbreviated Amino Acid Encoded by the codon
Q Glutamine CAG H Histidine CAU

It is not silent as shown, there is the same protein and is not conservative because it changes
from Glutamine to Histidine protein changes, and changes the nature of proteins because
the Glutamine is an amino acid, Polar, the Histidine is a basic amino acid and in terms of its
structure, the Glutamine has a polar side chain not loaded and the Histidine has a side chain
with positive Group (Basic).
IV. EMBOSS_001 601 SLCPCVLKFDDNSTEIASLIETIKEYQERTQQLIDSGRYDTRDDFTVVLQ 650
|||||||||||||||||||||||||||:||||||||||||||||||||||
EMBOSS_001 601 SLCPCVLKFDDNSTEIASLIETIKEYQQRTQQLIDSGRYDTRDDFTVVLQ 650

The fourth mutation found is Non-Silent Conservative because we can see that:
Original Mutated
Abbreviated Amino Acid Encoded by the codon Abbreviated Amino Acid Encoded by the codon
Glutaminic
E Acid GAG Q Glutamine CAG

It is not silent as shown, there is the same protein and is conservative since it changes from
Glutamine Acid Glutamine to, changing the protein, and does not change the nature of
proteins since the Glutamine Acid is an amino acid, Polar and the Glutamine is also a polar
amino acid. In terms of its structure the Glutamine Acid has a side chain with negative group
(acid) and the Glutamine has a non-charged side chain.
V. EMBOSS_001 751 SPPTSVHALRPADIQVVAALGDSLTAGIGIGSKPNDLSDGTTQYRGLSYS 800
||||||||||||||||||||||||||||||||||.|||||||||||||||
 EMBOSS_001 751 SPPTSVHALRPADIQVVAALGDSLTAGIGIGSKPIDLSDGTTQYRGLSYS 800

The fifth mutation found is Non-Silent Conservative because we can see that:
Original Mutated
Abbreviated Amino Acid Encoded by the codon Abbreviated Amino Acid Encoded by the codon
N Asparagine AAT T Threonine ACT

It is not silent as shown, there is the same protein and is not conservative because it changes
from Asparagine to Threonine, changes the protein, but does not change the nature of
proteins since the Asparagine is a neutral Polar amino acid and Threonine is a neutral polar
amino acid. In terms of its structure the Asparagine has a non-charged side chain and the
Threonine also has a non-charged side chain.
VI. EMBOSS_001 901 SNLYSAANFYDHLRDALDALHREVPRALVNLVDFMetNPSVTRQVFLGNP 950
|||||||||||||||||||||||||||||||||||||||||||||.||||
EMBOSS_001 901 SNLYSAANFYDHLRDALDALHREVPRALVNLVDFMetNPSVTRQVLLGNP 950

The sixth mutation found is Non-Silent Conservative because we can see that:
Original Mutated
Encoded by the Encoded by the
Abbreviated Amino Acid Abbreviated Amino Acid
codon codon
F Phenylalanine TTC L Leucine TTG

It is not silent as shown, there is the same protein and is conservative change of
Phenylalanine to Leucine, it changes the protein, but does not change the nature of proteins
the Phenylalanine is a non-polar amino acid and Leucine is a non-polar amino acid. In terms
of its structure the Phenylalanine has a non-polar side chain and the Leucine also has a non-
polar side chain.
VII. EMBOSS_001 1101 ENWGSDFLCTAWNASRGVPNSVHELQPGDIKVVAALGDSLTLAMetGARP 1150
|.||||||||||||||||||||||||||||||||||||||||||||||||
EMBOSS_001 1101 EYWGSDFLCTAWNASRGVPNSVHELQPGDIKVVAALGDSLTLAMetGARP 1150

The seventh mutation found is Non-Silent Conservative because we can see that:
Original Mutated
Abbreviated Amino Acid Encoded by the codon Abbreviated Amino Acid Encoded by the codon
N Asparagine AAC Y Tyrosine TAC

It is not silent as you can be seen, there is the same protein but is conservative because it
changes from Asparagine to Tyrosine, changes the protein, but it does not change the nature
of proteins since the Asparagine is a Polar amino acid and Tyrosine is a polar amino acid. In
terms of its structure the Asparagine has a polar side chain not loaded and the Tyrosine also
has a polar side chain not loaded.
VIII. EMBOSS_001 1251 TLFVGGNDLCHFCENPEGSSEGEYVQHIQQALDVLYEELPRTFVNVVEVM 1300
||||||||||||||:|||||||||||||||||||||||||||||||||||
EMBOSS_001 1251 TLFVGGNDLCHFCESPEGSSEGEYVQHIQQALDVLYEELPRTFVNVVEVM 1300

The eighth mutation found is Non-Silent Conservative because we can see that:
Original Mutated
Abbreviated Amino Acid Encoded by the codon Abbreviated Amino Acid Encoded by the codon
N Asparagine AAT S Serine AGT
It is not silent as as you can be seen, there is the same protein but is conservative because it
changes from Asparagine to Tyrosine, changes the protein, but it does not change the nature
of proteins since the Asparagine is a Polar amino acid and Tyrosine is a polar amino acid. In
terms of its structure the Asparagine has a polar side chain not loaded and the Tyrosine also
has a polar side chain not loaded.

Ncbi.nlm.nih.gov. (n.d.). Streptomyces clavuligerus (ID 834) - Genome - NCBI. [online] Available at:
https://www.ncbi.nlm.nih.gov/genome/?term=Streptomyces%20clavuligerus [Accessed 18 Apr.
2017].
https://www.staff.ncl.ac.uk/p.dean/Lab_Resources/pET-29a.pdf

http://www.restrictionmapper.org/cgi-bin/sitefind3.pl

https://www.neb.com/products/r0101-ecori