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Molecular Cloning Part 2: Plasmids, Restriction Enzymes & Gene Fusions
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Section 1.5 Molecular Cloning Part 2 Learning Objectives By the end of this lecture students should be able to: 1. Design a cloning experiment. Keeping in mind order, reading frame, and start/stop codons 2. Describe key elements of a plasmids and their role in a cloning experiment 3. Understand the difference between translational and transcriptional fusions Rules to manipulate and modify DNA sequences • DNA can only be synthesized 5’-3’ – DNA replication • Gene expression is driven by promoters - Transcription • Alternative splice variants can be important – Posttranscriptional processing • Ribosomes translate open reading frames. You can fuse two genes together as long as there is only one stop codon. Translation Tools to manipulate DNA sequences • isolate/amplify precise sequences such as genes - PCR • cut and paste DNA sequences – restriction enzymes and ligase • amplify large DNA sequences • transform our new sequences into an organism • verify that we’ve done it correctly - sequencing Amplifying large pieces of DNA Escherichia coli • Gram-negative bacteria that is pathogenic to humans. • Using genetics it is possible to disarm E.coli of its pathogenesis features for safe use in a laboratory setting. • Like most bacteria, E.coli contains both chromosomal DNA and smaller circular plasmid DNA. Plasmids replicate independently and are easy to work with because they are relatively small molecules. • Because E.coli replicates every 20 minutes, it is possible to obtain massive amounts of DNA in a short time. For this reason, it has been an important cloning vehicle since the 1970s. • Most laboratory strains contain three methylases that methylate only specific sequences and only interfere with a few restriction sites Methylases in common lab strains of E. coli In prokaryotes, Methyltransferases have most often been identified as elements of restriction/modification systems that act to protect host DNA from cleavage by the corresponding restriction endonuclease. Most laboratory strains of E. coli contain three site-specific DNA methylases: •Dam methylase–methylation at the N6 position of the adenine in the sequence GATC. •Dcm methyltransferases–methylation at the C5 position of the second cytosine in the sequences CCAGG and CCTGG. •EcoKI methylase–methylation of adenine in the sequences AAC(N6)GTGC and GCAC(N6)GTT. If the methylase site overlaps an endonuclease recognition site it may prevent cleavage if the DNA was extracted from strains expressing the Dam or Dcm methylases. For example, plasmid DNA isolated from dam+ E. coli is completely resistant to cleavage by BclI, which cleaves at GATC sites. Anatomy of a cloning plasmid What do bacterial chromosomes needs to replicate? Anatomy of a cloning plasmid Plasmid vectors contain: • a replication origin (ORI) sequence where DNA replication can be initiated by host-cell enzymes • a selectable gene, such as ampr encoding the enzyme β-lactamase, which confers resistance to ampicillin. This allows us to tell the difference between cells that have our plasmid vs those that do not. • a synthetic polylinker region containing single copies of several restriction enzyme recognition sequences, into which an exogenous DNA fragment can be ligated **** restriction sites occur at other regions of the plasmid **** Promoter driving antibiotic resistance gene Multiple Cloning Site (MCS) a.k.a the polylinker region Antibiotic resistance gene Origin of Replication Note that there are restriction sites are not confined to the MCS Reporter gene for quick assessment of plasmid reassembly From the Lac operon Will produce a blue color in the presence of substrate What do you think would happen to the LacZ gene if you cut the plasmid and integrate new sequences at the MCS? Blue/white screening 16 hr growth at 37C • Each colony represents a unique transformation event. • Always start cultures from a single colony. • Cells within a single colonies are clones because bacteria replicate by mitosis DNA can be amplified by PCR for use in cloning • PCR: • Primer sequences that are unique to target flanking regions are synthesized to include restriction enzyme recognition sequences not in the target sequence: • Primer 1 – contains a BamHI recognition sequence • Primer 2 – contains a HindIII recognition sequence • PCR for 20 cycles [Note: only one of the two strands is shown for simplicity.] • Remember that in PCR reactions the primer becomes part of the newly generated DNA sequence. This means that we can add sequences at the end of the fragment. • Cloning: • PCR-amplified sequences are cut with BamHI and HindIII, generating sticky ends. • Fragments are ligated into polylinker region of a similarly cut plasmid. • Recombinant vector is transformed into E. coli. of interest e en Cloning example G of interest e en Cloning example G BamHI GGATCC CCTAGG HindIII AAGCTT TTCGAA Cloning example – Step 1 cut the vector HindIII AGCTT A P lasmid GATCC G BamHI BamHI GGATCC CCTAGG HindIII AAGCTT TTCGAA Cloning example – Step 2 cut the fragment GOI HindIII A TTCGA AAGCTT TTCGAA\ G CCTAG BamHI We will talk about how we introduce cut sites to DNA fragments in a few slides Cloning example – Step 3 mix and ligate G CCTAG GATCC G BamHI BamHI A AGCTT TTCGA A P P GOI AGCTT A GOI A TTCGA G GATCC CCTAGG We have now made recombinant DNA! DNA, proteins, cells, or organisms that are made by combining genetic material from two different sources 1. We can create a circular DNA sequence from two linear pieces 2. There is directionality in that there is only one way that the fragments can fit together 3. The restriction sites are recreated following ligations lasmid HindIII lasmid HindIII Bacteria naturally have plasmids • Lab strains of E.coli have had natural plasmids removed. • Unnatural, engineered plasmids with unique features are then transformed into E.coli and selected for based on the antibiotic resistance marker. • E.coli then does what it does normally - divides. Each round of cell division involves DNA replication. → Exponential amplification of the plasmid. Transforming plasmids into E.coli ThermoFisher Scientific Now that we know how to • isolate/amplify precise sequences such as genes - PCR • cut and paste DNA sequences – restriction enzymes and ligase • transform our new sequences into an organism • amplify large DNA sequences What is it good for? Translational fusions → tracking proteins • This type of fusion is used to understand a gene product (protein) • This system lets you infer where a protein localizes within a cell (i.e., cytoplasm, ER, Golgi, nucleus, plasma membrane). • Or where it localizes within an organism (i.e., in what cell types, after stress, etc). • You can also learn about the regulation of the protein and determine the levels of that protein Translational fusions Noam Morris Transcriptional fusions → to learn how the expression of a gene is regulated promoter reporter • This type of fusion is used to understand promoter function and regulation • From this you can infer where the gene that normally sits downstream of that promoter is expressed (i.e., in what cell types, after stress, etc). • You can also infer what elements control the expression driving by this promoter Transcriptional Fusions These results tell us that the PR gene is upregulated after a range of stresses. These results tell us that the TF gene is expressed in a specific cell type – in this case, plant trichomes. Reporters commonly included in recombinant clones GFP GUS Common ‘reporter’ genes • • • • • B-glucoronidase (GUS) B-galactosidase (GAL) GFP (and a lot of other engineered FPs) Firefly luciferase Epitope tags – HA, His, FLAG, etc Luciferase So-called because they ‘report’ on gene expression, protein localization or protein levels Let’s create a translational fusion Generating recombinant clones FLS2 = Flagellin Sensing 2 • Immune receptor that localizes to the plasma membrane. It binds bacterial ligans on the outside of the cell and contains a kinase in the inside of the cell. • Use PCR to amplify the gene encoding FLS2 from Arabidopsis • Be sure to include a promoter to drive FLS2 expression • Check what restriction sites it has to determine what we can use for cloning GFP = Green Fluorescence Protein • Use PCR to amplify the gene encoding GFP from jellyfish • Check what restriction sites it has to determine what we can use for cloning Let’s create a translational fusion to determine where FLS2 localized in the cell ? pFLS2 FLS2 FLS2 GFP GFP Promoter that works in the organism of interest * Endogenous (the one that normally regulates FLS2) * Other promoters (if we want to give it new properties e.g. inducible) Promoter choice depends on the organism you want to express your engineered protein • A promoter that drives expression in E.coli probably won’t drive expression in a plant or a mouse • It is important that the antibioticR gene product (an enzyme to enable growth on media with that antibiotic) be expressed by E.coli – so it will have a bacterial promoter • But is it not important that E.coli express pFLS2:FLS2-GFP – only that it replicate it. The ORI takes care of that. • Eventually, we want to express pFLS2:FLS2-GFP in a plant through transformation – so it is important that we include a plant promoter. DNA Ligase BamHI EcoRI KpnI SalI pFLS2 pFLS2 FLS2 FLS2 GFP plasmid backbone (pCAMBIA2300) GFP The backbone will contain the origin of replication and selection marker How do you know which restriction enzymes to use? https://nc3.neb.com/NEBcutter/ NEB cutter output GFP ORF >GFP ORF ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTG TCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAG AGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGA ATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAAC ATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACC ATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGC TGGGATTACACATGGCATGGATGAACTATACAAATAG // >> use NEB cutter to determine what cut sites you can use: SalI no sites – OK to use BamHI no sites – OK to use KpnI no sites – OK to use NcoI 1 site – CANNOT use Etc… CCATGG is the NCOI cut site GFP ORF >GFP ORF ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTG TCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAG AGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGA ATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAAC ATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACC ATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGC TGGGATTACACATGGCATGGATGAACTATACAAATAG // >> use NEB cutter to determine what cut sites you can use: SalI no sites – OK to use BamHI no sites – OK to use KpnI no sites – OK to use NcoI 1 site – CANNOT use Etc… CCATGG is the NCOI cut site Repeat this for every piece you want to join. You want to select a RE that exist on the plasmid (so you can cut it open) but that does not exist on any of other DNA fragments we want to bring together (so that they stay intact) DNA Ligase BamHI EcoRI KpnI SalI pFLS2 pFLS2 FLS2 FLS2 GFP plasmid backbone (pCAMBIA2300) GFP The backbone will contain the origin of replication and selection marker Very rarely do pieces that we want to stitch together have the desired restriction site in the locations we need them… so we have to introduce them I oR Ec DNA Ligase BamHI EcoRI I mH Ba pFLS2 pFLS2 KpnI SalI FLS2 GFP mH Ba I l Sa FLS2 I plasmid backbone (pCAMBIA2300) lI Sa nI Kp GFP The backbone will contain the origin of replication and selection marker pFLS2 >FLS2 promoter tttgactcctttgttcatctttattttaaaatcaatataaacccttttcggacattctaaatattttgacccaacctaatgatat tcattatttgtctaacgggtcaactttcaagtcttcaagtaaacatgatatggaattatatgggtcaactccaagtaagaacagt gcatgcatcttgaagacaaaattgatttttttttttgtttttaattaatggaaattttgaaatactaatgggtcaagatgaggtt gtccaaatcaactgatatcatatatgaccagattcctcaatagtcattagtcaatacaaaatccataatcttttttaaaaatata atctttttattttaaatcgcctatttttttccattcatttgtccaatgtgaaaagccataaatcatatattaagaagttgtgaat tgtgatcataggagtaatatttctatctgaacaaaaattgaattttgttttgctttttgttgaaagataaataaaatgtttggca gattgtttgtacagaacacaacttgttgataaaacaaattccatcaagacagctaatgagcttttgatagtcagagctttgaaga cgtgttaatgaattaaactgattagacctaaaaccaaatatttagaaagaaaaaaatatgataattgttatgttatttaatcaat agtcaaatactatgttgagtaccaatcatttcagtttcaacgattgctactcttttgaccaataacaaaaaggtttttatttttc atttctcaaactccatttttgtttgtttttagcataaaaacagctagaatgtgagtttcatatgtattatatactataagaaatc atataaattaacgactagaacatattgttttcattctagtggtatatctgagtttgactagaacgtttggaggcagattaaagtg atttcgccttgtcaaaactactttgtatatgttatcaatcaatcgctcaaaactaaatcggaaaaaaactacataaataagatat ggaaaatgatactaacacaaactagtaagtaagtagtcactacatattctggtcccatgaatatggcttacatagtacatacatt gtgagattaaatgaatatgatatataatacaagcatataagcatgtgcatgactatgaagtgggaatacatttagtgcctttata tgcaacttgacagaattaccaattatgtgaatagtgaaacttatattgcgtcttggccttttcacatccgtgacatgatccctca aatctatatcttcttgcggcatatgcttatatataaacatatgacttcgtctatagatttatatccagataattaattctctcat aatatcttctccacaatttcaactcttctaaagtctaaaccATG // Lower case letters… the ATG just indicates where the start codon would go >FLS2 promoter – simple version 5’-tttgactcctttgttcatctttattttaaaatcaatataaacccttttcg………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaacc-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttgg-5’ If we consider the bottom strand 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttgg-5’ The primer would be as follows: EcoRI 5’-GAATTCtttgactcctttgttcatct-3’ primer During PCR the primer and the bottom strand would anneal as follows: EcoRI Free 3’OH 5’-GAATTCtttgactcctttgttcatct-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttgg-5’ After the 1st round of PCR you get the following 5’-GAATTCtttgactcctttgttcatctttattttaaaatcaatataaacccttttcg………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaacc-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttgg-5’ >FLS2 promoter – simple version 5’-tttgactcctttgttcatctttattttaaaatcaatataaacccttttcg………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaacc-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttgg-5’ If we consider the top strand 5’-tttgactcctttgttcatctttattttaaaatcaatataaacccttttcg………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaacc-3’ The primer would be as follows: BamH1 5’-GGATCCggtttagactttagaagagt-3’ primer During PCR the primer and the top strand would anneal as follows: 5’-tttgactcctttgttcatctttattttaaaatcaatataaacccttttcg………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaacc-3’ 3’-tgagaagatttcagatttggCCTAGG-5’ Free 3’OH BamH1 After the 1st round of PCR you get the following 5’-tttgactcctttgttcatctttattttaaaatcaatataaacccttttcg………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaacc-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttggCCTAGG-5’ >FLS2 promoter – simple version ** second round of PCR ** 5’-tttgactcctttgttcatctttattttaaaatcaatataaacccttttcg………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaacc-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttggCCTAGG-5’ If we consider the bottom strand 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgggaaaagc………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttggCCTAGG-5’ The primer would behave as follows: EcoRI 5’-GAATTCtttgactcctttgttcatct-3’ primer During PCR the primer and the top strand would anneal as follows: 5’-GAATTCtttgactcctttgttcatct-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgg………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttggCCTAGG-5’ After the 1st round of PCR you get the following 5’-GAATTCtttgactcctttgttcatctttattttaaaatcaatataaacc………………………………attaattctctcataatatcttctccacaatttcaactcttctaaagtctaaaccGGATCC-3’ 3’-aaactgaggaaacaagtagaaataaaattttagttatatttgg………………………………taattaagagagtattatagaagaggtgttaaagttgagaagatttcagatttggCCTAGG-5’ >FLS2 promoter tttgactcctttgttcatctttattttaaaatcaatataaacccttttcggacattctaaatattttgacccaacctaatgatat tcattatttgtctaacgggtcaactttcaagtcttcaagtaaacatgatatggaattatatgggtcaactccaagtaagaacagt gcatgcatcttgaagacaaaattgatttttttttttgtttttaattaatggaaattttgaaatactaatgggtcaagatgaggtt gtccaaatcaactgatatcatatatgaccagattcctcaatagtcattagtcaatacaaaatccataatcttttttaaaaatata atctttttattttaaatcgcctatttttttccattcatttgtccaatgtgaaaagccataaatcatatattaagaagttgtgaat tgtgatcataggagtaatatttctatctgaacaaaaattgaattttgttttgctttttgttgaaagataaataaaatgtttggca gattgtttgtacagaacacaacttgttgataaaacaaattccatcaagacagctaatgagcttttgatagtcagagctttgaaga cgtgttaatgaattaaactgattagacctaaaaccaaatatttagaaagaaaaaaatatgataattgttatgttatttaatcaat agtcaaatactatgttgagtaccaatcatttcagtttcaacgattgctactcttttgaccaataacaaaaaggtttttatttttc atttctcaaactccatttttgtttgtttttagcataaaaacagctagaatgtgagtttcatatgtattatatactataagaaatc atataaattaacgactagaacatattgttttcattctagtggtatatctgagtttgactagaacgtttggaggcagattaaagtg atttcgccttgtcaaaactactttgtatatgttatcaatcaatcgctcaaaactaaatcggaaaaaaactacataaataagatat ggaaaatgatactaacacaaactagtaagtaagtagtcactacatattctggtcccatgaatatggcttacatagtacatacatt gtgagattaaatgaatatgatatataatacaagcatataagcatgtgcatgactatgaagtgggaatacatttagtgcctttata tgcaacttgacagaattaccaattatgtgaatagtgaaacttatattgcgtcttggccttttcacatccgtgacatgatccctca aatctatatcttcttgcggcatatgcttatatataaacatatgacttcgtctatagatttatatccagataattaattctctcat aatatcttctccacaatttcaactcttctaaagtctaaaccATG // >FLS2 ORF ATGAAGTTACTCTCAAAGACC TTTTTGATATTAACTCTCACCTTCTTCTTCTTTGGCATTGCACTAGCGAAACAGAGCTTTGAACCAGAGATCGAAGCTTTGAAATCCTTCAAGAATGGTATTTCC AACGACCCTTTAGGAGTATTA TCAGATTGGACCATCATCGGTTCGTTACGACACTGTAATTGGACCGGAATCACCTGCGATAGTACCGGACATGTAGTCTCGGTTTCCTTGCTGGAGAAGCAACTT GAAGGTGTTCTGTCTCCAGCC ATAGCGAATCTCACCTATCTCCAGGTTCTTGATCTCACTTCAAATAGTTTTACCGGCAAAATACCGGCTGAAATAGGAAAGTTAACCGAGCTTAACCAGCTTATT CTGTACCTAAACTATTTCTCT GGTTCGATTCCTTCTGGAATCTGGGAGCTTAAGAATATTTTCTATCTTGATCTTAGAAATAATTTGTTGTCCGGTGATGTTCCTGAGGAAATCTGCAAAACCAGT TCTTTGGTATTGATTGGGTTT GATTACAACAACTTAACCGGGAAAATACCAGAATGCTTAGGAGATTTGGTTCATCTCCAAATGTTTGTAGCAGCTGGTAACCATTTAACTGGTTCGATTCCGGTA TCAATTGGTACTCTGGCTAAT TTAACGGATTTAGACCTGAGTGGTAACCAGTTAACCGGAAAAATACCGAGAGATTTTGGAAATCTCTTGAACTTACAGTCTCTCGTTTTAACTGAAAACTTGTTG GAAGGAGATATACCAGCTGAG ATCGGAAACTGCTCGAGCTTGGTCCAACTTGAGCTTTACGATAACCAGTTAACCGGGAAAATACCAGCTGAATTAGGGAATTTGGTTCAGCTGCAAGCACTCCGG ATATACAAGAACAAACTTACT TCTTCAATTCCATCTTCATTGTTCCGGTTAACTCAGTTAACCCATTTGGGGTTATCAGAAAACCATTTGGTTGGACCGATATCAGAAGAAATCGGTTTTCTTGAG TCACTTGAAGTCCTCACACTT CATTCCAACAACTTCACAGGAGAGTTTCCACAGTCCATCACAAACTTGAGGAACTTGACAGTCCTAACGGTGGGGTTCAATAATATTTCCGGTGAGCTCCCGGCG GATCTAGGGCTTCTTACAAAC CTTCGGAACCTTTCAGCGCACGACAATCTTCTTACCGGACCAATACCTTCCAGCATAAGTAACTGCACCGGTCTTAAACTCCTGGACCTGTCTCACAACCAAATG ACTGGCGAGATCCCGCGGGGT TTCGGAAGGATGAATCTTACGTTCATTTCTATTGGGAGGAATCATTTCACCGGTGAAATTCCAGATGATATCTTCAACTGTTCAAACTTGGAAACTCTTAGTGTG GCAGATAACAACTTAACAGGA ACTCTCAAGCCATTAATTGGGAAGCTTCAAAAACTCAGGATTTTGCAAGTTTCATATAACTCTCTCACTGGACCGATTCCTCGAGAAATCGGGAATCTGAAAGAT TTGAATATCTTGTACCTTCAC TCTAATGGTTTCACAGGGAGAATCCCGAGAGAGATGTCGAATCTCACTCTCCTCCAGGGGCTAAGGATGTATTCAAATGATCTTGAAGGTCCAATTCCTGAAGAA ATGTTTGATATGAAGCTACTC TCAGTTCTTGATCTTTCCAACAACAAATTCTCAGGTCAAATTCCTGCCTTGTTCTCCAAGCTTGAATCGCTTACCTACTTGAGTCTTCAAGGAAACAAATTCAAC GGGTCTATCCCTGCAAGCCTT AAGTCGCTTTCGCTTCTCAACACATTCGATATCTCCGACAATCTTCTCACTGGAACCATCCCTGGAGAGCTGTTAGCTTCTTTGAAAAACATGCAGCTTTACCTC AACTTCTCAAACAACTTGTTG ACTGGAACCATCCCAAAGGAGCTTGGAAAGCTTGAAATGGTTCAAGAAATCGACCTTTCAAACAATCTCTTTTCTGGGTCTATTCCAAGATCTTTACAGGCCTGC AAAAATGTGTTCACACTGGAT TTTTCGCAGAACAATCTCTCGGGTCATATACCAGATGAAGTCTTCCAAGGCATGGATATGATCATAAGCTTGAACCTTTCAAGGAACAGTTTCTCTGGAGAAATC CCTCAGAGCTTCGGGAACATG ACGCATTTGGTCTCCTTGGATCTCTCTAGTAACAATCTCACTGGTGAAATTCCAGAGAGTCTCGCCAATCTTTCGACTCTGAAACATCTCAAACTAGCTTCAAAC AACCTCAAAGGCCATGTTCCT GAATCCGGGGTGTTCAAAAACATCAACGCCTCTGATCTAATGGGAAACACAGATCTCTGTGGTAGCAAGAAGCCTCTCAAGCCATGTACGATCAAGCAGAAGTCG AGCCACTTCTCGAAGAGAACC AGAGTCATCCTGATTATTCTTGGATCAGCCGCGGCTCTTCTTCTTGTCCTGCTTCTTGTTCTGATTCTAACCTGTTGCAAGAAAAAAGAAAAAAAGATTGAAAAT TCATCAGAGTCCTCATTACCG GATTTGGATTCAGCTCTGAAACTGAAGAGATTTGAACCAAAAGAGTTGGAGCAAGCAACAGATTCATTCAACAGTGCCAACATCATTGGCTCAAGCAGCTTAAGC ACAGTGTACAAAGGTCAGCTA GAAGATGGGACAGTGATTGCAGTAAAAGTATTGAATCTAAAGGAATTCTCTGCAGAATCAGACAAGTGGTTCTACACAGAAGCTAAAACATTGAGCCAACTAAAA CATCGAAACCTGGTGAAGATC TTAGGGTTTGCGTGGGAAAGCGGCAAAACGAAAGCTTTAGTGCTTCCATTTATGGAGAATGGAAACTTGGAGGACACCATTCACGGCTCTGCAGCACCGATTGGG TCGCTTTTAGAAAAAATCGAT CTTTGTGTTCATATCGCAAGCGGAATCGATTATCTTCATTCTGGATATGGTTTTCCCATCGTTCATTGTGATCTGAAGCCAGCTAATATACTCCTTGACAGTGAC CGCGTTGCTCACGTAAGCGAT TTTGGAACTGCTCGGATTCTAGGTTTCCGCGAAGATGGAAGCACCACAGCTTCAACATCAGCCTTCGAGGGTACAATTGGATACTTAGCTCCAGAGTTTGCTTAT ATGAGGAAAGTGACAACAAAA GCCGATGTATTCAGCTTCGGGATCATAATGATGGAGCTGATGACGAAACAGAGACCAACTTCGTTGAATGATGAAGATTCACAAGACATGACTTTGCGCCAATTG GTGGAGAAATCGATTGGAAAT GGAAGAAAAGGGATGGTTAGGGTTCTTGATATGGAACTCGGGGACTCTATTGTTTCTCTGAAACAGGAAGAGGCTATTGAAGACTTTCTGAAGCTTTGTTTGTTC TGTACAAGCTCTAGACCTGAA GATCGACCTGATATGAACGAGATTCTTACACATCTGATGAAACTTAGAGGCAAAGCGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG// Remember an important rule: Ribosomes translate open reading frames. You can fuse two genes together – like FLS2 and GFP - as long as there is only one stop codon. >GFP ORF ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTG TCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAG AGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGA ATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAAC ATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACC ATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGC TGGGATTACACATGGCATGGATGAACTATACAAATAG // >FLS2 promoter tttgactcctttgttcatctttattttaaaatcaatataaacccttttcggacattctaaatattttgacccaacctaatgatat tcattatttgtctaacgggtcaactttcaagtcttcaagtaaacatgatatggaattatatgggtcaactccaagtaagaacagt gcatgcatcttgaagacaaaattgatttttttttttgtttttaattaatggaaattttgaaatactaatgggtcaagatgaggtt gtccaaatcaactgatatcatatatgaccagattcctcaatagtcattagtcaatacaaaatccataatcttttttaaaaatata atctttttattttaaatcgcctatttttttccattcatttgtccaatgtgaaaagccataaatcatatattaagaagttgtgaat tgtgatcataggagtaatatttctatctgaacaaaaattgaattttgttttgctttttgttgaaagataaataaaatgtttggca gattgtttgtacagaacacaacttgttgataaaacaaattccatcaagacagctaatgagcttttgatagtcagagctttgaaga cgtgttaatgaattaaactgattagacctaaaaccaaatatttagaaagaaaaaaatatgataattgttatgttatttaatcaat agtcaaatactatgttgagtaccaatcatttcagtttcaacgattgctactcttttgaccaataacaaaaaggtttttatttttc atttctcaaactccatttttgtttgtttttagcataaaaacagctagaatgtgagtttcatatgtattatatactataagaaatc atataaattaacgactagaacatattgttttcattctagtggtatatctgagtttgactagaacgtttggaggcagattaaagtg atttcgccttgtcaaaactactttgtatatgttatcaatcaatcgctcaaaactaaatcggaaaaaaactacataaataagatat ggaaaatgatactaacacaaactagtaagtaagtagtcactacatattctggtcccatgaatatggcttacatagtacatacatt gtgagattaaatgaatatgatatataatacaagcatataagcatgtgcatgactatgaagtgggaatacatttagtgcctttata tgcaacttgacagaattaccaattatgtgaatagtgaaacttatattgcgtcttggccttttcacatccgtgacatgatccctca aatctatatcttcttgcggcatatgcttatatataaacatatgacttcgtctatagatttatatccagataattaattctctcat aatatcttctccacaatttcaactcttctaaagtctaaaccATG // AAA CTT AGA GGC AAA GCG AAT TCA TTT CGA GAA GAT CGT AAC GAG GAT CGA GAA GTT TAG GTC CGA CAT GAG TAA AGG AGA stop >FLS2 ORF ATGAAGTTACTCTCAAAGACC TTTTTGATATTAACTCTCACCTTCTTCTTCTTTGGCATTGCACTAGCGAAACAGAGCTTTGAACCAGAGATCGAAGCTTTGAAATCCTTCAAGAATGGTATTTCC AACGACCCTTTAGGAGTATTA TCAGATTGGACCATCATCGGTTCGTTACGACACTGTAATTGGACCGGAATCACCTGCGATAGTACCGGACATGTAGTCTCGGTTTCCTTGCTGGAGAAGCAACTT GAAGGTGTTCTGTCTCCAGCC ATAGCGAATCTCACCTATCTCCAGGTTCTTGATCTCACTTCAAATAGTTTTACCGGCAAAATACCGGCTGAAATAGGAAAGTTAACCGAGCTTAACCAGCTTATT CTGTACCTAAACTATTTCTCT GGTTCGATTCCTTCTGGAATCTGGGAGCTTAAGAATATTTTCTATCTTGATCTTAGAAATAATTTGTTGTCCGGTGATGTTCCTGAGGAAATCTGCAAAACCAGT TCTTTGGTATTGATTGGGTTT GATTACAACAACTTAACCGGGAAAATACCAGAATGCTTAGGAGATTTGGTTCATCTCCAAATGTTTGTAGCAGCTGGTAACCATTTAACTGGTTCGATTCCGGTA TCAATTGGTACTCTGGCTAAT TTAACGGATTTAGACCTGAGTGGTAACCAGTTAACCGGAAAAATACCGAGAGATTTTGGAAATCTCTTGAACTTACAGTCTCTCGTTTTAACTGAAAACTTGTTG GAAGGAGATATACCAGCTGAG ATCGGAAACTGCTCGAGCTTGGTCCAACTTGAGCTTTACGATAACCAGTTAACCGGGAAAATACCAGCTGAATTAGGGAATTTGGTTCAGCTGCAAGCACTCCGG ATATACAAGAACAAACTTACT TCTTCAATTCCATCTTCATTGTTCCGGTTAACTCAGTTAACCCATTTGGGGTTATCAGAAAACCATTTGGTTGGACCGATATCAGAAGAAATCGGTTTTCTTGAG TCACTTGAAGTCCTCACACTT CATTCCAACAACTTCACAGGAGAGTTTCCACAGTCCATCACAAACTTGAGGAACTTGACAGTCCTAACGGTGGGGTTCAATAATATTTCCGGTGAGCTCCCGGCG GATCTAGGGCTTCTTACAAAC CTTCGGAACCTTTCAGCGCACGACAATCTTCTTACCGGACCAATACCTTCCAGCATAAGTAACTGCACCGGTCTTAAACTCCTGGACCTGTCTCACAACCAAATG ACTGGCGAGATCCCGCGGGGT TTCGGAAGGATGAATCTTACGTTCATTTCTATTGGGAGGAATCATTTCACCGGTGAAATTCCAGATGATATCTTCAACTGTTCAAACTTGGAAACTCTTAGTGTG GCAGATAACAACTTAACAGGA ACTCTCAAGCCATTAATTGGGAAGCTTCAAAAACTCAGGATTTTGCAAGTTTCATATAACTCTCTCACTGGACCGATTCCTCGAGAAATCGGGAATCTGAAAGAT TTGAATATCTTGTACCTTCAC TCTAATGGTTTCACAGGGAGAATCCCGAGAGAGATGTCGAATCTCACTCTCCTCCAGGGGCTAAGGATGTATTCAAATGATCTTGAAGGTCCAATTCCTGAAGAA ATGTTTGATATGAAGCTACTC TCAGTTCTTGATCTTTCCAACAACAAATTCTCAGGTCAAATTCCTGCCTTGTTCTCCAAGCTTGAATCGCTTACCTACTTGAGTCTTCAAGGAAACAAATTCAAC GGGTCTATCCCTGCAAGCCTT AAGTCGCTTTCGCTTCTCAACACATTCGATATCTCCGACAATCTTCTCACTGGAACCATCCCTGGAGAGCTGTTAGCTTCTTTGAAAAACATGCAGCTTTACCTC AACTTCTCAAACAACTTGTTG ACTGGAACCATCCCAAAGGAGCTTGGAAAGCTTGAAATGGTTCAAGAAATCGACCTTTCAAACAATCTCTTTTCTGGGTCTATTCCAAGATCTTTACAGGCCTGC AAAAATGTGTTCACACTGGAT TTTTCGCAGAACAATCTCTCGGGTCATATACCAGATGAAGTCTTCCAAGGCATGGATATGATCATAAGCTTGAACCTTTCAAGGAACAGTTTCTCTGGAGAAATC CCTCAGAGCTTCGGGAACATG ACGCATTTGGTCTCCTTGGATCTCTCTAGTAACAATCTCACTGGTGAAATTCCAGAGAGTCTCGCCAATCTTTCGACTCTGAAACATCTCAAACTAGCTTCAAAC AACCTCAAAGGCCATGTTCCT GAATCCGGGGTGTTCAAAAACATCAACGCCTCTGATCTAATGGGAAACACAGATCTCTGTGGTAGCAAGAAGCCTCTCAAGCCATGTACGATCAAGCAGAAGTCG AGCCACTTCTCGAAGAGAACC AGAGTCATCCTGATTATTCTTGGATCAGCCGCGGCTCTTCTTCTTGTCCTGCTTCTTGTTCTGATTCTAACCTGTTGCAAGAAAAAAGAAAAAAAGATTGAAAAT TCATCAGAGTCCTCATTACCG GATTTGGATTCAGCTCTGAAACTGAAGAGATTTGAACCAAAAGAGTTGGAGCAAGCAACAGATTCATTCAACAGTGCCAACATCATTGGCTCAAGCAGCTTAAGC ACAGTGTACAAAGGTCAGCTA GAAGATGGGACAGTGATTGCAGTAAAAGTATTGAATCTAAAGGAATTCTCTGCAGAATCAGACAAGTGGTTCTACACAGAAGCTAAAACATTGAGCCAACTAAAA CATCGAAACCTGGTGAAGATC TTAGGGTTTGCGTGGGAAAGCGGCAAAACGAAAGCTTTAGTGCTTCCATTTATGGAGAATGGAAACTTGGAGGACACCATTCACGGCTCTGCAGCACCGATTGGG TCGCTTTTAGAAAAAATCGAT CTTTGTGTTCATATCGCAAGCGGAATCGATTATCTTCATTCTGGATATGGTTTTCCCATCGTTCATTGTGATCTGAAGCCAGCTAATATACTCCTTGACAGTGAC CGCGTTGCTCACGTAAGCGAT TTTGGAACTGCTCGGATTCTAGGTTTCCGCGAAGATGGAAGCACCACAGCTTCAACATCAGCCTTCGAGGGTACAATTGGATACTTAGCTCCAGAGTTTGCTTAT ATGAGGAAAGTGACAACAAAA GCCGATGTATTCAGCTTCGGGATCATAATGATGGAGCTGATGACGAAACAGAGACCAACTTCGTTGAATGATGAAGATTCACAAGACATGACTTTGCGCCAATTG GTGGAGAAATCGATTGGAAAT GGAAGAAAAGGGATGGTTAGGGTTCTTGATATGGAACTCGGGGACTCTATTGTTTCTCTGAAACAGGAAGAGGCTATTGAAGACTTTCTGAAGCTTTGTTTGTTC TGTACAAGCTCTAGACCTGAA GATCGACCTGATATGAACGAGATTCTTACACATCTGATGAAACTTAGAGGCAAAGCGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG// We must remove the stop codon to create a fusion protein, a protein with a reporter (GFP) attached to it. We ultimately want the FLS2 protein to glow! >GFP ORF ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTG TCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAG AGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGA ATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAAC ATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACC ATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGC TGGGATTACACATGGCATGGATGAACTATACAAATAG // >FLS2 promoter tttgactcctttgttcatctttattttaaaatcaatataaacccttttcggacattctaaatattttgacccaacctaatgatat tcattatttgtctaacgggtcaactttcaagtcttcaagtaaacatgatatggaattatatgggtcaactccaagtaagaacagt gcatgcatcttgaagacaaaattgatttttttttttgtttttaattaatggaaattttgaaatactaatgggtcaagatgaggtt gtccaaatcaactgatatcatatatgaccagattcctcaatagtcattagtcaatacaaaatccataatcttttttaaaaatata atctttttattttaaatcgcctatttttttccattcatttgtccaatgtgaaaagccataaatcatatattaagaagttgtgaat tgtgatcataggagtaatatttctatctgaacaaaaattgaattttgttttgctttttgttgaaagataaataaaatgtttggca gattgtttgtacagaacacaacttgttgataaaacaaattccatcaagacagctaatgagcttttgatagtcagagctttgaaga cgtgttaatgaattaaactgattagacctaaaaccaaatatttagaaagaaaaaaatatgataattgttatgttatttaatcaat agtcaaatactatgttgagtaccaatcatttcagtttcaacgattgctactcttttgaccaataacaaaaaggtttttatttttc atttctcaaactccatttttgtttgtttttagcataaaaacagctagaatgtgagtttcatatgtattatatactataagaaatc atataaattaacgactagaacatattgttttcattctagtggtatatctgagtttgactagaacgtttggaggcagattaaagtg atttcgccttgtcaaaactactttgtatatgttatcaatcaatcgctcaaaactaaatcggaaaaaaactacataaataagatat ggaaaatgatactaacacaaactagtaagtaagtagtcactacatattctggtcccatgaatatggcttacatagtacatacatt gtgagattaaatgaatatgatatataatacaagcatataagcatgtgcatgactatgaagtgggaatacatttagtgcctttata tgcaacttgacagaattaccaattatgtgaatagtgaaacttatattgcgtcttggccttttcacatccgtgacatgatccctca aatctatatcttcttgcggcatatgcttatatataaacatatgacttcgtctatagatttatatccagataattaattctctcat aatatcttctccacaatttcaactcttctaaagtctaaaccATG // AAA CTT AGA GGC AAA GCG AAT TCA TTT CGA GAA GAT CGT AAC GAG GAT CGA GAA GTT GTC CGA CAT GAG TAA AGG AGA Val Arg His Glu Stop >FLS2 ORF ATGAAGTTACTCTCAAAGACC TTTTTGATATTAACTCTCACCTTCTTCTTCTTTGGCATTGCACTAGCGAAACAGAGCTTTGAACCAGAGATCGAAGCTTTGAAATCCTTCAAGAATGGTATTTCC AACGACCCTTTAGGAGTATTA TCAGATTGGACCATCATCGGTTCGTTACGACACTGTAATTGGACCGGAATCACCTGCGATAGTACCGGACATGTAGTCTCGGTTTCCTTGCTGGAGAAGCAACTT GAAGGTGTTCTGTCTCCAGCC ATAGCGAATCTCACCTATCTCCAGGTTCTTGATCTCACTTCAAATAGTTTTACCGGCAAAATACCGGCTGAAATAGGAAAGTTAACCGAGCTTAACCAGCTTATT CTGTACCTAAACTATTTCTCT GGTTCGATTCCTTCTGGAATCTGGGAGCTTAAGAATATTTTCTATCTTGATCTTAGAAATAATTTGTTGTCCGGTGATGTTCCTGAGGAAATCTGCAAAACCAGT TCTTTGGTATTGATTGGGTTT GATTACAACAACTTAACCGGGAAAATACCAGAATGCTTAGGAGATTTGGTTCATCTCCAAATGTTTGTAGCAGCTGGTAACCATTTAACTGGTTCGATTCCGGTA TCAATTGGTACTCTGGCTAAT TTAACGGATTTAGACCTGAGTGGTAACCAGTTAACCGGAAAAATACCGAGAGATTTTGGAAATCTCTTGAACTTACAGTCTCTCGTTTTAACTGAAAACTTGTTG GAAGGAGATATACCAGCTGAG ATCGGAAACTGCTCGAGCTTGGTCCAACTTGAGCTTTACGATAACCAGTTAACCGGGAAAATACCAGCTGAATTAGGGAATTTGGTTCAGCTGCAAGCACTCCGG ATATACAAGAACAAACTTACT TCTTCAATTCCATCTTCATTGTTCCGGTTAACTCAGTTAACCCATTTGGGGTTATCAGAAAACCATTTGGTTGGACCGATATCAGAAGAAATCGGTTTTCTTGAG TCACTTGAAGTCCTCACACTT CATTCCAACAACTTCACAGGAGAGTTTCCACAGTCCATCACAAACTTGAGGAACTTGACAGTCCTAACGGTGGGGTTCAATAATATTTCCGGTGAGCTCCCGGCG GATCTAGGGCTTCTTACAAAC CTTCGGAACCTTTCAGCGCACGACAATCTTCTTACCGGACCAATACCTTCCAGCATAAGTAACTGCACCGGTCTTAAACTCCTGGACCTGTCTCACAACCAAATG ACTGGCGAGATCCCGCGGGGT TTCGGAAGGATGAATCTTACGTTCATTTCTATTGGGAGGAATCATTTCACCGGTGAAATTCCAGATGATATCTTCAACTGTTCAAACTTGGAAACTCTTAGTGTG GCAGATAACAACTTAACAGGA ACTCTCAAGCCATTAATTGGGAAGCTTCAAAAACTCAGGATTTTGCAAGTTTCATATAACTCTCTCACTGGACCGATTCCTCGAGAAATCGGGAATCTGAAAGAT TTGAATATCTTGTACCTTCAC TCTAATGGTTTCACAGGGAGAATCCCGAGAGAGATGTCGAATCTCACTCTCCTCCAGGGGCTAAGGATGTATTCAAATGATCTTGAAGGTCCAATTCCTGAAGAA ATGTTTGATATGAAGCTACTC TCAGTTCTTGATCTTTCCAACAACAAATTCTCAGGTCAAATTCCTGCCTTGTTCTCCAAGCTTGAATCGCTTACCTACTTGAGTCTTCAAGGAAACAAATTCAAC GGGTCTATCCCTGCAAGCCTT AAGTCGCTTTCGCTTCTCAACACATTCGATATCTCCGACAATCTTCTCACTGGAACCATCCCTGGAGAGCTGTTAGCTTCTTTGAAAAACATGCAGCTTTACCTC AACTTCTCAAACAACTTGTTG ACTGGAACCATCCCAAAGGAGCTTGGAAAGCTTGAAATGGTTCAAGAAATCGACCTTTCAAACAATCTCTTTTCTGGGTCTATTCCAAGATCTTTACAGGCCTGC AAAAATGTGTTCACACTGGAT TTTTCGCAGAACAATCTCTCGGGTCATATACCAGATGAAGTCTTCCAAGGCATGGATATGATCATAAGCTTGAACCTTTCAAGGAACAGTTTCTCTGGAGAAATC CCTCAGAGCTTCGGGAACATG ACGCATTTGGTCTCCTTGGATCTCTCTAGTAACAATCTCACTGGTGAAATTCCAGAGAGTCTCGCCAATCTTTCGACTCTGAAACATCTCAAACTAGCTTCAAAC AACCTCAAAGGCCATGTTCCT GAATCCGGGGTGTTCAAAAACATCAACGCCTCTGATCTAATGGGAAACACAGATCTCTGTGGTAGCAAGAAGCCTCTCAAGCCATGTACGATCAAGCAGAAGTCG AGCCACTTCTCGAAGAGAACC AGAGTCATCCTGATTATTCTTGGATCAGCCGCGGCTCTTCTTCTTGTCCTGCTTCTTGTTCTGATTCTAACCTGTTGCAAGAAAAAAGAAAAAAAGATTGAAAAT TCATCAGAGTCCTCATTACCG GATTTGGATTCAGCTCTGAAACTGAAGAGATTTGAACCAAAAGAGTTGGAGCAAGCAACAGATTCATTCAACAGTGCCAACATCATTGGCTCAAGCAGCTTAAGC ACAGTGTACAAAGGTCAGCTA GAAGATGGGACAGTGATTGCAGTAAAAGTATTGAATCTAAAGGAATTCTCTGCAGAATCAGACAAGTGGTTCTACACAGAAGCTAAAACATTGAGCCAACTAAAA CATCGAAACCTGGTGAAGATC TTAGGGTTTGCGTGGGAAAGCGGCAAAACGAAAGCTTTAGTGCTTCCATTTATGGAGAATGGAAACTTGGAGGACACCATTCACGGCTCTGCAGCACCGATTGGG TCGCTTTTAGAAAAAATCGAT CTTTGTGTTCATATCGCAAGCGGAATCGATTATCTTCATTCTGGATATGGTTTTCCCATCGTTCATTGTGATCTGAAGCCAGCTAATATACTCCTTGACAGTGAC CGCGTTGCTCACGTAAGCGAT TTTGGAACTGCTCGGATTCTAGGTTTCCGCGAAGATGGAAGCACCACAGCTTCAACATCAGCCTTCGAGGGTACAATTGGATACTTAGCTCCAGAGTTTGCTTAT ATGAGGAAAGTGACAACAAAA GCCGATGTATTCAGCTTCGGGATCATAATGATGGAGCTGATGACGAAACAGAGACCAACTTCGTTGAATGATGAAGATTCACAAGACATGACTTTGCGCCAATTG GTGGAGAAATCGATTGGAAAT GGAAGAAAAGGGATGGTTAGGGTTCTTGATATGGAACTCGGGGACTCTATTGTTTCTCTGAAACAGGAAGAGGCTATTGAAGACTTTCTGAAGCTTTGTTTGTTC TGTACAAGCTCTAGACCTGAA GATCGACCTGATATGAACGAGATTCTTACACATCTGATGAAACTTAGAGGCAAAGCGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG// The actual GFP aa sequence is… ATG AGT AAA GGA GAA GAA CTT … Met Ser Lys Gly Glu Glu Leu We must make sure to preserve the reading frame >GFP ORF ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTG TCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAG AGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGA ATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAAC ATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACC ATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGC TGGGATTACACATGGCATGGATGAACTATACAAATAG // Adding restriction sites add sequence Be careful not to engineer stop codons or frame-shifts GGATCCATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTCTCACCTTCTTCTTCTTTGGCATTGCACTAGCGAAACAGAGCTTTGAACCAGAGATCGAAGCTTTGAAATCCTTCAAGAATGGTATTTCCAACGAC CCTTTAGGAGTATTATCAGATTGGACCATCATCGGTTCGTTACGACACTGTAATTGGACCGGAATCACCTGCGATAGTACCGGACATGTAGTCTCGGTTTCCTTGCTGGAGAAGCAACTTGAAGGTGTTCTGTCTCCA GCCATAGCGAATCTCACCTATCTCCAGGTTCTTGATCTCACTTCAAATAGTTTTACCGGCAAAATACCGGCTGAAATAGGAAAGTTAACCGAGCTTAACCAGCTTATTCTGTACCTAAACTATTTCTCTGGTTCGATT CCTTCTGGAATCTGGGAGCTTAAGAATATTTTCTATCTTGATCTTAGAAATAATTTGTTGTCCGGTGATGTTCCTGAGGAAATCTGCAAAACCAGTTCTTTGGTATTGATTGGGTTTGATTACAACAACTTAACCGGG AAAATACCAGAATGCTTAGGAGATTTGGTTCATCTCCAAATGTTTGTAGCAGCTGGTAACCATTTAACTGGTTCGATTCCGGTATCAATTGGTACTCTGGCTAATTTAACGGATTTAGACCTGAGTGGTAACCAGTTA ACCGGAAAAATACCGAGAGATTTTGGAAATCTCTTGAACTTACAGTCTCTCGTTTTAACTGAAAACTTGTTGGAAGGAGATATACCAGCTGAGATCGGAAACTGCTCGAGCTTGGTCCAACTTGAGCTTTACGATAAC CAGTTAACCGGGAAAATACCAGCTGAATTAGGGAATTTGGTTCAGCTGCAAGCACTCCGGATATACAAGAACAAACTTACTTCTTCAATTCCATCTTCATTGTTCCGGTTAACTCAGTTAACCCATTTGGGGTTATCA GAAAACCATTTGGTTGGACCGATATCAGAAGAAATCGGTTTTCTTGAGTCACTTGAAGTCCTCACACTTCATTCCAACAACTTCACAGGAGAGTTTCCACAGTCCATCACAAACTTGAGGAACTTGACAGTCCTAACG GTGGGGTTCAATAATATTTCCGGTGAGCTCCCGGCGGATCTAGGGCTTCTTACAAACCTTCGGAACCTTTCAGCGCACGACAATCTTCTTACCGGACCAATACCTTCCAGCATAAGTAACTGCACCGGTCTTAAACTC CTGGACCTGTCTCACAACCAAATGACTGGCGAGATCCCGCGGGGTTTCGGAAGGATGAATCTTACGTTCATTTCTATTGGGAGGAATCATTTCACCGGTGAAATTCCAGATGATATCTTCAACTGTTCAAACTTGGAA ACTCTTAGTGTGGCAGATAACAACTTAACAGGAACTCTCAAGCCATTAATTGGGAAGCTTCAAAAACTCAGGATTTTGCAAGTTTCATATAACTCTCTCACTGGACCGATTCCTCGAGAAATCGGGAATCTGAAAGAT TTGAATATCTTGTACCTTCACTCTAATGGTTTCACAGGGAGAATCCCGAGAGAGATGTCGAATCTCACTCTCCTCCAGGGGCTAAGGATGTATTCAAATGATCTTGAAGGTCCAATTCCTGAAGAAATGTTTGATATG AAGCTACTCTCAGTTCTTGATCTTTCCAACAACAAATTCTCAGGTCAAATTCCTGCCTTGTTCTCCAAGCTTGAATCGCTTACCTACTTGAGTCTTCAAGGAAACAAATTCAACGGGTCTATCCCTGCAAGCCTTAAG TCGCTTTCGCTTCTCAACACATTCGATATCTCCGACAATCTTCTCACTGGAACCATCCCTGGAGAGCTGTTAGCTTCTTTGAAAAACATGCAGCTTTACCTCAACTTCTCAAACAACTTGTTGACTGGAACCATCCCA AAGGAGCTTGGAAAGCTTGAAATGGTTCAAGAAATCGACCTTTCAAACAATCTCTTTTCTGGGTCTATTCCAAGATCTTTACAGGCCTGCAAAAATGTGTTCACACTGGATTTTTCGCAGAACAATCTCTCGGGTCAT ATACCAGATGAAGTCTTCCAAGGCATGGATATGATCATAAGCTTGAACCTTTCAAGGAACAGTTTCTCTGGAGAAATCCCTCAGAGCTTCGGGAACATGACGCATTTGGTCTCCTTGGATCTCTCTAGTAACAATCTC ACTGGTGAAATTCCAGAGAGTCTCGCCAATCTTTCGACTCTGAAACATCTCAAACTAGCTTCAAACAACCTCAAAGGCCATGTTCCTGAATCCGGGGTGTTCAAAAACATCAACGCCTCTGATCTAATGGGAAACACA GATCTCTGTGGTAGCAAGAAGCCTCTCAAGCCATGTACGATCAAGCAGAAGTCGAGCCACTTCTCGAAGAGAACCAGAGTCATCCTGATTATTCTTGGATCAGCCGCGGCTCTTCTTCTTGTCCTGCTTCTTGTTCTG ATTCTAACCTGTTGCAAGAAAAAAGAAAAAAAGATTGAAAATTCATCAGAGTCCTCATTACCGGATTTGGATTCAGCTCTGAAACTGAAGAGATTTGAACCAAAAGAGTTGGAGCAAGCAACAGATTCATTCAACAGT GCCAACATCATTGGCTCAAGCAGCTTAAGCACAGTGTACAAAGGTCAGCTAGAAGATGGGACAGTGATTGCAGTAAAAGTATTGAATCTAAAGGAATTCTCTGCAGAATCAGACAAGTGGTTCTACACAGAAGCTAAA ACATTGAGCCAACTAAAACATCGAAACCTGGTGAAGATCTTAGGGTTTGCGTGGGAAAGCGGCAAAACGAAAGCTTTAGTGCTTCCATTTATGGAGAATGGAAACTTGGAGGACACCATTCACGGCTCTGCAGCACCG ATTGGGTCGCTTTTAGAAAAAATCGATCTTTGTGTTCATATCGCAAGCGGAATCGATTATCTTCATTCTGGATATGGTTTTCCCATCGTTCATTGTGATCTGAAGCCAGCTAATATACTCCTTGACAGTGACCGCGTT GCTCACGTAAGCGATTTTGGAACTGCTCGGATTCTAGGTTTCCGCGAAGATGGAAGCACCACAGCTTCAACATCAGCCTTCGAGGGTACAATTGGATACTTAGCTCCAGAGTTTGCTTATATGAGGAAAGTGACAACA AAAGCCGATGTATTCAGCTTCGGGATCATAATGATGGAGCTGATGACGAAACAGAGACCAACTTCGTTGAATGATGAAGATTCACAAGACATGACTTTGCGCCAATTGGTGGAGAAATCGATTGGAAATGGAAGAAAA GGGATGGTTAGGGTTCTTGATATGGAACTCGGGGACTCTATTGTTTCTCTGAAACAGGAAGAGGCTATTGAAGACTTTCTGAAGCTTTGTTTGTTCTGTACAAGCTCTAGACCTGAAGATCGACCTGATATGAACGAG ATTCTTACACATCTGATGAAACTTAGAGGCAAAGCGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTGTCGACATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCT TATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAA GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAA AAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACA CAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGGGTACC >FLS2 ORF 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAA ATC-5’ If we consider the bottom strand 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAA ATC-5’ The primer would be as follows BamH1 5’-GGATCCATGAAGTTACTCTCAAAGAC-3’ primer During PCR the primer and the bottom strand would anneal as follows: 5’-GGATCCATGAAGTTACTCTCAAAGAC-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAAATC-5’ >FLS2 ORF 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAA ATC-5’ If we consider the bottom strand 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAA ATC-5’ The primer would be as follows BamH1 5’-GGATCCATGAAGTTACTCTCAAAGAC-3’ primer During PCR the primer and the bottom strand would anneal as follows: 5’-GGATCCATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAAATC-5’ >FLS2 ORF 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAA ATC-5’ If we consider the top strand 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTT TAG-3’ The primer would be as follows SalI primer 5’-GTCCGACCCAACTTCTCGATCCTCGTTAC-3’ To keep the reading frame During PCR the primer and the bottom strand would anneal as follows: 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAACCCAGCCTG-5’ >FLS2 ORF 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAA ATC-5’ If we consider the top strand 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTT TAG-3’ The primer would be as follows SalI primer 5’-GTCCGACCCAACTTCTCGATCCTCGTTAC-3’ To keep the reading frame Removing the TAG stop codon During PCR the primer and the bottom strand would anneal as follows: 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-CATTGCTCCTAGCTCTTCAACCCAGCCTG-5’ Nucleotides added to maintain the reading frame must be added the side that will not be removed by the digestion reaction 3’-CATTGCTCCTAGCTCTTCAACCCAGCCTG-5 Added to keep the reading frame 3’-CATTGCTCCTAGCTCTTCAACAGCCTGCC-5 >FLS2 ORF – simple version ** second round of PCR ** 5’-ATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTTAG-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAACCCAGCCTG-5’ If we consider the bottom strand 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAACCCAGCCTG-5’ The primer would behave as follows: BamH1 5’-GGATCCATGAAGTTACTCTCAAAGAC-3’ primer During PCR the primer and the top strand would anneal as follows: 5’-GGATCCATGAAGTTACTCTCAAAGAC-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAACCCAGCCTG-5’ After the 1 st round of PCR you get the following 5’-GGATCCATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTGGGTCGGAC-3’ 3’-TACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAACCCAGCCTG-5’ This is the PCR fragment you generated 5’-GGATCCATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTGGGTCGGAC-3’ 3’-CCTAGGTACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAACCCAGCCTG-5’ When you perform the restriction digestion reaction with BamH1 and SalI (we can digest with two enzymes at once) 5’-G 3’-CCTAG 5’-GATCCATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTC…………………CGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTGGG-3’ 3’-GTACTTCAATGAGAGTTTCTGGAAAAACTATAATTGAG…………………GCTTAAGTAAAGCTCTTCTAGCATTGCTCCTAGCTCTTCAACCCAGCCT-5’ 5’-TCGGAC-3’ 3’-G-5’ You end up with 3 fragments – two are very small and one is large – the ORG Note that the nucleotides we added to maintain the frame are retained (not cut away by SalI) SalI KpnI GFP G TCGGAC CAGCCT G G GTACC CCATG G Fwd 5’-GTCGGACATGAGTAAAGGAGAAGAACT-3’ Rev 5’-GGTACCCTATTTGTATAGTTCATCC-3’ Only need to fix the frame once >GFP ORF ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTG TCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATG GCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAG AGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGT TTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGA ATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAAC ATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACC ATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGC TGGGATTACACATGGCATGGATGAACTATACAAATAG // GTAACGAGGATCGAGAAGTTGGGTCGGACATGAGTAAAGGAGAAGAACT CATTGCTCCTAGCTCTTCAACCCAGCCTGTACTCATTTCCTCTTCTTGA Did we maintain a single frame? ...GTAACGAGGATCGAGAAGTTGGGTCGGACATGAGTAAAGGAGAAGAACT... FLS2 SalI site GFP ...GT AAC GAG GAT CGA GAA GTT GGG TCG GAC ATG AGT AAA GGA GAA GAA CT... ... N E FLS2 D R E V G S D ‘Linker’ M S K G GFP Added sequence Sometime that linker region is engineered to have specific properties E E ... GAATTCtttgactcctttgttcatctttattttaaaatcaatataaacccttttcggacattctaaatattttgacccaacctaatgatattcattatttgtctaacgggtcaactttcaagtcttcaagt aaacatgatatggaat tatatgggtcaactccaagtaagaacagtgcatgcatcttgaagacaaaattgatttttttttttgtttttaattaatggaaattttgaaatactaatgggtcaagatgaggttgtccaaatcaa ctgatatcatatatgaccagat tcctcaatagtcattagtcaatacaaaatccataatcttttttaaaaatataatctttttattttaaatcgcctatttttttccattcatttgtccaatgtgaaaagccataaatcatatattaa gaagttgtgaattgtgatcata ggagtaatatttctatctgaacaaaaattgaattttgttttgctttttgttgaaagataaataaaatgtttggcagattgtttgtacagaacacaacttgttgataaaacaaattccatcaagac agctaatgagcttttgatagtc agagctttgaagacgtgttaatgaattaaactgattagacctaaaaccaaatatttagaaagaaaaaaatatgataattgttatgttatttaatcaatagtcaaatactatgttgagtaccaatc atttcagtttcaacgattgcta ctcttttgaccaataacaaaaaggtttttatttttcatttctcaaactccatttttgtttgtttttagcataaaaacagctagaatgtgagtttcatatgtattatatactataagaaatcatat aaattaacgactagaacatatt gttttcattctagtggtatatctgagtttgactagaacgtttggaggcagattaaagtgatttcgccttgtcaaaactactttgtatatgttatcaatcaatcgctcaaaactaaatcggaaaaa aactacataaataagatatgga aaatgatactaacacaaactagtaagtaagtagtcactacatattctggtcccatgaatatggcttacatagtacatacattgtgagattaaatgaatatgatatataatacaagcatataagca tgtgcatgactatgaagtggga atacatttagtgcctttatatgcaacttgacagaattaccaattatgtgaatagtgaaacttatattgcgtcttggccttttcacatccgtgacatgatccctcaaatctatatcttcttgcggc atatgcttatatataaacatat gacttcgtctatagatttatatccagataattaattctctcataatatcttctccacaatttcaactcttctaaagtctaaaccGGATCCATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTCTCACCTTCTTCTTCTTTGGC ATTGCACTAGCGAAACAGAGCTTTGAACCAGAGATCGAAGCTTTGAAATCCTTCAAGAATGGTATTTCCAACGACCCTTTAGGAGTATTATCAGATTGGACCATCATCGGTTCGTTACGACACTG TAATTGGACCGGAATCACCTGC GATAGTACCGGACATGTAGTCTCGGTTTCCTTGCTGGAGAAGCAACTTGAAGGTGTTCTGTCTCCAGCCATAGCGAATCTCACCTATCTCCAGGTTCTTGATCTCACTTCAAATAGTTTTACCGG CAAAATACCGGCTGAAATAGGA AAGTTAACCGAGCTTAACCAGCTTATTCTGTACCTAAACTATTTCTCTGGTTCGATTCCTTCTGGAATCTGGGAGCTTAAGAATATTTTCTATCTTGATCTTAGAAATAATTTGTTGTCCGGTGA TGTTCCTGAGGAAATCTGCAAA ACCAGTTCTTTGGTATTGATTGGGTTTGATTACAACAACTTAACCGGGAAAATACCAGAATGCTTAGGAGATTTGGTTCATCTCCAAATGTTTGTAGCAGCTGGTAACCATTTAACTGGTTCGAT TCCGGTATCAATTGGTACTCTG GCTAATTTAACGGATTTAGACCTGAGTGGTAACCAGTTAACCGGAAAAATACCGAGAGATTTTGGAAATCTCTTGAACTTACAGTCTCTCGTTTTAACTGAAAACTTGTTGGAAGGAGATATACC AGCTGAGATCGGAAACTGCTCG AGCTTGGTCCAACTTGAGCTTTACGATAACCAGTTAACCGGGAAAATACCAGCTGAATTAGGGAATTTGGTTCAGCTGCAAGCACTCCGGATATACAAGAACAAACTTACTTCTTCAATTCCATC TTCATTGTTCCGGTTAACTCAG TTAACCCATTTGGGGTTATCAGAAAACCATTTGGTTGGACCGATATCAGAAGAAATCGGTTTTCTTGAGTCACTTGAAGTCCTCACACTTCATTCCAACAACTTCACAGGAGAGTTTCCACAGTC CATCACAAACTTGAGGAACTTG ACAGTCCTAACGGTGGGGTTCAATAATATTTCCGGTGAGCTCCCGGCGGATCTAGGGCTTCTTACAAACCTTCGGAACCTTTCAGCGCACGACAATCTTCTTACCGGACCAATACCTTCCAGCAT AAGTAACTGCACCGGTCTTAAA CTCCTGGACCTGTCTCACAACCAAATGACTGGCGAGATCCCGCGGGGTTTCGGAAGGATGAATCTTACGTTCATTTCTATTGGGAGGAATCATTTCACCGGTGAAATTCCAGATGATATCTTCAA CTGTTCAAACTTGGAAACTCTT AGTGTGGCAGATAACAACTTAACAGGAACTCTCAAGCCATTAATTGGGAAGCTTCAAAAACTCAGGATTTTGCAAGTTTCATATAACTCTCTCACTGGACCGATTCCTCGAGAAATCGGGAATCT GAAAGATTTGAATATCTTGTAC CTTCACTCTAATGGTTTCACAGGGAGAATCCCGAGAGAGATGTCGAATCTCACTCTCCTCCAGGGGCTAAGGATGTATTCAAATGATCTTGAAGGTCCAATTCCTGAAGAAATGTTTGATATGAA GCTACTCTCAGTTCTTGATCTT TCCAACAACAAATTCTCAGGTCAAATTCCTGCCTTGTTCTCCAAGCTTGAATCGCTTACCTACTTGAGTCTTCAAGGAAACAAATTCAACGGGTCTATCCCTGCAAGCCTTAAGTCGCTTTCGCT TCTCAACACATTCGATATCTCC GACAATCTTCTCACTGGAACCATCCCTGGAGAGCTGTTAGCTTCTTTGAAAAACATGCAGCTTTACCTCAACTTCTCAAACAACTTGTTGACTGGAACCATCCCAAAGGAGCTTGGAAAGCTTGA AATGGTTCAAGAAATCGACCTT TCAAACAATCTCTTTTCTGGGTCTATTCCAAGATCTTTACAGGCCTGCAAAAATGTGTTCACACTGGATTTTTCGCAGAACAATCTCTCGGGTCATATACCAGATGAAGTCTTCCAAGGCATGGA TATGATCATAAGCTTGAACCTT TCAAGGAACAGTTTCTCTGGAGAAATCCCTCAGAGCTTCGGGAACATGACGCATTTGGTCTCCTTGGATCTCTCTAGTAACAATCTCACTGGTGAAATTCCAGAGAGTCTCGCCAATCTTTCGAC TCTGAAACATCTCAAACTAGCT TCAAACAACCTCAAAGGCCATGTTCCTGAATCCGGGGTGTTCAAAAACATCAACGCCTCTGATCTAATGGGAAACACAGATCTCTGTGGTAGCAAGAAGCCTCTCAAGCCATGTACGATCAAGCA GAAGTCGAGCCACTTCTCGAAG AGAACCAGAGTCATCCTGATTATTCTTGGATCAGCCGCGGCTCTTCTTCTTGTCCTGCTTCTTGTTCTGATTCTAACCTGTTGCAAGAAAAAAGAAAAAAAGATTGAAAATTCATCAGAGTCCTC ATTACCGGATTTGGATTCAGCT CTGAAACTGAAGAGATTTGAACCAAAAGAGTTGGAGCAAGCAACAGATTCATTCAACAGTGCCAACATCATTGGCTCAAGCAGCTTAAGCACAGTGTACAAAGGTCAGCTAGAAGATGGGACAGT GATTGCAGTAAAAGTATTGAAT CTAAAGGAATTCTCTGCAGAATCAGACAAGTGGTTCTACACAGAAGCTAAAACATTGAGCCAACTAAAACATCGAAACCTGGTGAAGATCTTAGGGTTTGCGTGGGAAAGCGGCAAAACGAAAGC TTTAGTGCTTCCATTTATGGAG AATGGAAACTTGGAGGACACCATTCACGGCTCTGCAGCACCGATTGGGTCGCTTTTAGAAAAAATCGATCTTTGTGTTCATATCGCAAGCGGAATCGATTATCTTCATTCTGGATATGGTTTTCC CATCGTTCATTGTGATCTGAAG CCAGCTAATATACTCCTTGACAGTGACCGCGTTGCTCACGTAAGCGATTTTGGAACTGCTCGGATTCTAGGTTTCCGCGAAGATGGAAGCACCACAGCTTCAACATCAGCCTTCGAGGGTACAAT TGGATACTTAGCTCCAGAGTTT GCTTATATGAGGAAAGTGACAACAAAAGCCGATGTATTCAGCTTCGGGATCATAATGATGGAGCTGATGACGAAACAGAGACCAACTTCGTTGAATGATGAAGATTCACAAGACATGACTTTGCG CCAATTGGTGGAGAAATCGATT GGAAATGGAAGAAAAGGGATGGTTAGGGTTCTTGATATGGAACTCGGGGACTCTATTGTTTCTCTGAAACAGGAAGAGGCTATTGAAGACTTTCTGAAGCTTTGTTTGTTCTGTACAAGCTCTAG ACCTGAAGATCGACCTGATATG AACGAGATTCTTACACATCTGATGAAACTTAGAGGCAAAGCGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTGGGTCGGACATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGT CACTACTTTCTCTTATGGTGTT CAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGC TGAAGTCAAGTTTGAAGGTGAT ACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAA TGGAATCAAAGTTAACTTCAAA ATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTC GAAAGATCCCAACGAAAAGAGA GACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGGGTACC GAATTCtttgactcctttgttcatctttattttaaaatcaatataaacccttttcggacattctaaatattttgacccaacctaatgatattcattatttgtctaacgggtcaactttcaagtcttcaagt aaacatgatatggaat tatatgggtcaactccaagtaagaacagtgcatgcatcttgaagacaaaattgatttttttttttgtttttaattaatggaaattttgaaatactaatgggtcaagatgaggttgtccaaatcaa ctgatatcatatatgaccagat tcctcaatagtcattagtcaatacaaaatccataatcttttttaaaaatataatctttttattttaaatcgcctatttttttccattcatttgtccaatgtgaaaagccataaatcatatattaa gaagttgtgaattgtgatcata ggagtaatatttctatctgaacaaaaattgaattttgttttgctttttgttgaaagataaataaaatgtttggcagattgtttgtacagaacacaacttgttgataaaacaaattccatcaagac agctaatgagcttttgatagtc agagctttgaagacgtgttaatgaattaaactgattagacctaaaaccaaatatttagaaagaaaaaaatatgataattgttatgttatttaatcaatagtcaaatactatgttgagtaccaatc atttcagtttcaacgattgcta ctcttttgaccaataacaaaaaggtttttatttttcatttctcaaactccatttttgtttgtttttagcataaaaacagctagaatgtgagtttcatatgtattatatactataagaaatcatat aaattaacgactagaacatatt gttttcattctagtggtatatctgagtttgactagaacgtttggaggcagattaaagtgatttcgccttgtcaaaactactttgtatatgttatcaatcaatcgctcaaaactaaatcggaaaaa aactacataaataagatatgga aaatgatactaacacaaactagtaagtaagtagtcactacatattctggtcccatgaatatggcttacatagtacatacattgtgagattaaatgaatatgatatataatacaagcatataagca tgtgcatgactatgaagtggga atacatttagtgcctttatatgcaacttgacagaattaccaattatgtgaatagtgaaacttatattgcgtcttggccttttcacatccgtgacatgatccctcaaatctatatcttcttgcggc atatgcttatatataaacatat gacttcgtctatagatttatatccagataattaattctctcataatatcttctccacaatttcaactcttctaaagtctaaaccGGATCCATGAAGTTACTCTCAAAGACCTTTTTGATATTAACTCTCACCTTCTTCTTCTTTGGC ATTGCACTAGCGAAACAGAGCTTTGAACCAGAGATCGAAGCTTTGAAATCCTTCAAGAATGGTATTTCCAACGACCCTTTAGGAGTATTATCAGATTGGACCATCATCGGTTCGTTACGACACTG TAATTGGACCGGAATCACCTGC GATAGTACCGGACATGTAGTCTCGGTTTCCTTGCTGGAGAAGCAACTTGAAGGTGTTCTGTCTCCAGCCATAGCGAATCTCACCTATCTCCAGGTTCTTGATCTCACTTCAAATAGTTTTACCGG CAAAATACCGGCTGAAATAGGA AAGTTAACCGAGCTTAACCAGCTTATTCTGTACCTAAACTATTTCTCTGGTTCGATTCCTTCTGGAATCTGGGAGCTTAAGAATATTTTCTATCTTGATCTTAGAAATAATTTGTTGTCCGGTGA TGTTCCTGAGGAAATCTGCAAA ACCAGTTCTTTGGTATTGATTGGGTTTGATTACAACAACTTAACCGGGAAAATACCAGAATGCTTAGGAGATTTGGTTCATCTCCAAATGTTTGTAGCAGCTGGTAACCATTTAACTGGTTCGAT TCCGGTATCAATTGGTACTCTG GCTAATTTAACGGATTTAGACCTGAGTGGTAACCAGTTAACCGGAAAAATACCGAGAGATTTTGGAAATCTCTTGAACTTACAGTCTCTCGTTTTAACTGAAAACTTGTTGGAAGGAGATATACC AGCTGAGATCGGAAACTGCTCG AGCTTGGTCCAACTTGAGCTTTACGATAACCAGTTAACCGGGAAAATACCAGCTGAATTAGGGAATTTGGTTCAGCTGCAAGCACTCCGGATATACAAGAACAAACTTACTTCTTCAATTCCATC TTCATTGTTCCGGTTAACTCAG TTAACCCATTTGGGGTTATCAGAAAACCATTTGGTTGGACCGATATCAGAAGAAATCGGTTTTCTTGAGTCACTTGAAGTCCTCACACTTCATTCCAACAACTTCACAGGAGAGTTTCCACAGTC CATCACAAACTTGAGGAACTTG ACAGTCCTAACGGTGGGGTTCAATAATATTTCCGGTGAGCTCCCGGCGGATCTAGGGCTTCTTACAAACCTTCGGAACCTTTCAGCGCACGACAATCTTCTTACCGGACCAATACCTTCCAGCAT AAGTAACTGCACCGGTCTTAAA CTCCTGGACCTGTCTCACAACCAAATGACTGGCGAGATCCCGCGGGGTTTCGGAAGGATGAATCTTACGTTCATTTCTATTGGGAGGAATCATTTCACCGGTGAAATTCCAGATGATATCTTCAA CTGTTCAAACTTGGAAACTCTT AGTGTGGCAGATAACAACTTAACAGGAACTCTCAAGCCATTAATTGGGAAGCTTCAAAAACTCAGGATTTTGCAAGTTTCATATAACTCTCTCACTGGACCGATTCCTCGAGAAATCGGGAATCT GAAAGATTTGAATATCTTGTAC CTTCACTCTAATGGTTTCACAGGGAGAATCCCGAGAGAGATGTCGAATCTCACTCTCCTCCAGGGGCTAAGGATGTATTCAAATGATCTTGAAGGTCCAATTCCTGAAGAAATGTTTGATATGAA GCTACTCTCAGTTCTTGATCTT TCCAACAACAAATTCTCAGGTCAAATTCCTGCCTTGTTCTCCAAGCTTGAATCGCTTACCTACTTGAGTCTTCAAGGAAACAAATTCAACGGGTCTATCCCTGCAAGCCTTAAGTCGCTTTCGCT TCTCAACACATTCGATATCTCC GACAATCTTCTCACTGGAACCATCCCTGGAGAGCTGTTAGCTTCTTTGAAAAACATGCAGCTTTACCTCAACTTCTCAAACAACTTGTTGACTGGAACCATCCCAAAGGAGCTTGGAAAGCTTGA AATGGTTCAAGAAATCGACCTT TCAAACAATCTCTTTTCTGGGTCTATTCCAAGATCTTTACAGGCCTGCAAAAATGTGTTCACACTGGATTTTTCGCAGAACAATCTCTCGGGTCATATACCAGATGAAGTCTTCCAAGGCATGGA TATGATCATAAGCTTGAACCTT TCAAGGAACAGTTTCTCTGGAGAAATCCCTCAGAGCTTCGGGAACATGACGCATTTGGTCTCCTTGGATCTCTCTAGTAACAATCTCACTGGTGAAATTCCAGAGAGTCTCGCCAATCTTTCGAC TCTGAAACATCTCAAACTAGCT TCAAACAACCTCAAAGGCCATGTTCCTGAATCCGGGGTGTTCAAAAACATCAACGCCTCTGATCTAATGGGAAACACAGATCTCTGTGGTAGCAAGAAGCCTCTCAAGCCATGTACGATCAAGCA GAAGTCGAGCCACTTCTCGAAG AGAACCAGAGTCATCCTGATTATTCTTGGATCAGCCGCGGCTCTTCTTCTTGTCCTGCTTCTTGTTCTGATTCTAACCTGTTGCAAGAAAAAAGAAAAAAAGATTGAAAATTCATCAGAGTCCTC ATTACCGGATTTGGATTCAGCT CTGAAACTGAAGAGATTTGAACCAAAAGAGTTGGAGCAAGCAACAGATTCATTCAACAGTGCCAACATCATTGGCTCAAGCAGCTTAAGCACAGTGTACAAAGGTCAGCTAGAAGATGGGACAGT GATTGCAGTAAAAGTATTGAAT CTAAAGGAATTCTCTGCAGAATCAGACAAGTGGTTCTACACAGAAGCTAAAACATTGAGCCAACTAAAACATCGAAACCTGGTGAAGATCTTAGGGTTTGCGTGGGAAAGCGGCAAAACGAAAGC TTTAGTGCTTCCATTTATGGAG AATGGAAACTTGGAGGACACCATTCACGGCTCTGCAGCACCGATTGGGTCGCTTTTAGAAAAAATCGATCTTTGTGTTCATATCGCAAGCGGAATCGATTATCTTCATTCTGGATATGGTTTTCC CATCGTTCATTGTGATCTGAAG CCAGCTAATATACTCCTTGACAGTGACCGCGTTGCTCACGTAAGCGATTTTGGAACTGCTCGGATTCTAGGTTTCCGCGAAGATGGAAGCACCACAGCTTCAACATCAGCCTTCGAGGGTACAAT TGGATACTTAGCTCCAGAGTTT GCTTATATGAGGAAAGTGACAACAAAAGCCGATGTATTCAGCTTCGGGATCATAATGATGGAGCTGATGACGAAACAGAGACCAACTTCGTTGAATGATGAAGATTCACAAGACATGACTTTGCG CCAATTGGTGGAGAAATCGATT GGAAATGGAAGAAAAGGGATGGTTAGGGTTCTTGATATGGAACTCGGGGACTCTATTGTTTCTCTGAAACAGGAAGAGGCTATTGAAGACTTTCTGAAGCTTTGTTTGTTCTGTACAAGCTCTAG ACCTGAAGATCGACCTGATATG AACGAGATTCTTACACATCTGATGAAACTTAGAGGCAAAGCGAATTCATTTCGAGAAGATCGTAACGAGGATCGAGAAGTTGGGTCGGACATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGT CACTACTTTCTCTTATGGTGTT CAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGC TGAAGTCAAGTTTGAAGGTGAT ACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAA TGGAATCAAAGTTAACTTCAAA ATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTC GAAAGATCCCAACGAAAAGAGA GACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGGGTACC How many amino acids is our recombinant protein? • From the ATG to TAG is 4245 bp • 3 bp per codon • (4245/3) -1 = 1414 amino acids long Questions? Learning Objectives By the end of this lecture students should be able to: 1. Design a cloning experiment. Keeping in mind order, reading frame, and start/stop codons 2. Describe key elements of a plasmids and their role in a cloning experiment 3. Understand the difference between translational and transcriptional fusions
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