* 1. 1, Design 4 primers to amplify full length PKAalpha (sequence is shown below) by PCR. Clone PCR
product into pCDNA3.1+ vector pre-digested with EcoR1 and Xho1. An HA tag will be added to the
N-terminus of the protein. The PCR product should not be digested with EcoR1 and Xho1.
Explain your cloning strategy.

2, Design 2 primers to amplify PKAalpha. Use Gibson assembly protocol to clone PCR product into
pCDNA3.1+ pre-digested with EcoR1 and Xho1.

3, Design 2 primers to amplify PKAalpha and clone into pET28b vector pre-digested with Nde1 + Xho1.
Explain what restriction enzymes should be used to digest the PCR product.

4. After cloning into pET28b and pCDNA3.1+, what enzymes should be used to identify desired clones?

ATGGGCAACGCCGCCGCCGCCAAGAAGGGCAGCGAGCAGGAGAGCGTGAA 50
A

GAATTC

TTAGCCAAAGCCAAAGAAGATTTTCTTAAAAAATGGGAAAGTC 100
CCGCTCAGAACACAGCCCACTTGGATCAGTTTGAACGAATCAAGACCCTC 150
GGCACGGGCTCCTTCGGGCGGGTGATGCTGGTGAAACACAAGGAGACCGG 200
GAACCACTATGCCATGAAGATCCTCGACAAACAGAAGGTGGTGAAACTGA 250
AACAGATCGAACACACCCTGAATGAAAAGCGCATCCTGCAAGCTGTCAAC 300
TTTCCGTTCCTCGTCAAA

CTCGAG

TTCTCCTTCAAGGACAACTCAAACTT 350
ATACATGGTCATGGAGTACGTGCCCGGCGGGGAGATGTTCTCACACCTAC 400
GGCGGATCGGAAGGTTCAGTGAGCCCCATGCCCGTTTCTACGCGGCCCAG 450
ATCGTCCTGACCTTTGAGTATCTGCACTCGCTGGATCTCATCTACAGGGA 500
CCTGAAGCCGGAGAATCTGCTCATTGACCAGCAGGGCTACATTCAGGTGA 550
CAGACTTCGGTTTCGCCAAGCGCGTGAAGGGCCGCACTTGGACCTTGTGC 600
GGCACCCCTGAGTACCTGGCCCCTGAGATTATCCTGAGCAAAGGCTACAA 650
CAAGGCCGTGGACTGGTGGGCCCTGGGGGTTCTTATCTATGAAATGGCCG 700
CTGGCTACCCGCCCTTCTTCGCAGACCAGCCCATCCAGATCTATGAGAAG 750
ATCGTCTCTGGGAAGGTGCGCTTCCCTTCCCACTTCAGCTCTGACTTGAA 800
GGACCTGCTGCGGAACCTCCTGCAGGTAGATCTCACCAAGCGCTTTGGGA 850
ACCTCAAGAATGGGGTCAACGATATCAAGAACCACAAGTGGTTTGCCACA 900
ACTGACTGGATTGCCATCTACCAGAGGAAGGTGGAAGCTCCCTTCATACC 950
AAAGTTTAAAGGCCCTGGGGATACGAGTAACTTTGACGACTATGAGGAAG 1000
AAGAAATCCGGGTCTCCATCAATGAGAAGTGTGGCAAGGAGTTTTCTGAG 1050
TTTTAG

* 2. Write an SOP to transfer 40 genes per week from pDNOR221 to pEF-DEST51 vector. The final constructs must be verified and QCed.

* 3. Write an SOP for preparing Buffer A
50 mM Phosphate Buffer, pH 7.4
600 mM NaCl
10 mM imidazole
10% glycerol
0.1 mM EDTA
50 mM Arginine
5 mM 2-Mercaptoethanol

* 4. Design oligos to clone a peptide MAHHHHHH KLPVM PKKKRKV into pET28b (using Nco1 and Nde1 sites)
for E coli expression of CPP-fusion proteins. Limit oligo length to 63 nt.
You should not use restriction enzymes to digest the annealed oligos.

* 5. You designed these primers to amplify human NPR1 gene (Accession# BC063304)


NPR1-Nhe1-F
catgac GCTAGC ATGCCGGGGCCCCGGCGCCCCGCTG

NPR1-Not1-R gctgac GCGGCCGC CTAGCCTCGGGTGCTACTCCCCCTC

You cloned the amplified product into a mammalian expression vector and sequenced the insert, the sequence is


>1264-1pCMV-NPR1-Seq-F_E12.ab1


NNNNNNNNNNNNCNNNNNNNTCTCCGGNTANCTGGAGGTGCTGTTCCAGGGCCCC

GCTAGC

ATGCCGGGGCCccggcgccgGGCTTCGGTGTCAAGGACGAGTATGCGCTGACCACCCGCGCGGGGCCCAGCTACGCCAAG


CTGGGGGACTTCGTGGCGGCGCTGCACCGACGGCTGGGCTGGGAGCGCCAAGCGCTCATGCTCTACGCCTACCGGCCGGGTGACGAAGAGCACTGCTTCTT
CCTCGTGGAGGGGCTGTTCATGCGGGTCCGCGACCGCCTCAATATTACGGTGGACCACCTGGAGTTCGCCGAGGACGACC
TCAGCCACTACACCAGGCTGCTGCGGACCATGCCGCGCAAAGGCCGAGTTATCTACATCTGCAGCTCCCCTGATGCCTTC
AGAACCCTCATGCTCCTGGCCCTGGAAGCTGGCTTGTGTGGGGAGGACTACGTTTTCTTCCACCTGGATATCTTTGGGCA
AAGCCTGCAAGGTGGACAGGGCCCTGCTCCCCGCAGGCCCTGGGAGAGAGGGGATGGGCAGGATGTCAGTGCCCGCCAGG
CCTTTCAGGCTGCCAAAATCATTACATATAAAGACCCAGATAATCCCGAGTACTTGGAATTCCTGAAGCAGTTAAAACAC
CTGGCCTATGAGCAGTTCAACTTCACCATGGAGGATGTCCTGGTGAACACCATCCCAGCATCCTTCCACGACGGGCTCCT
GCTCTATATCCAGGCAGTGACGGAGACTCTGGCACATGGGGGAACTGTTACTGATGGGGAGAACATCACTCAGCGGATGT
GGAACCGAAGCTTTCAAGGTGTGACAGGATACCTGAAAATTGATAGCAGTGGCGATCGGGAAACAGACTTCTCCCTCTGG
GATATGGATCCCGAGAATGGTGCCTTCAGGGTTGTACTGAACTACAATGGGACTTCCCAAGAGCTGGTGGCTGTGTCGGG
GCGCAAACTGAACTGGNCCCTGGGGNACCCTCCTCCNGACATCCCCAAATGTGGCTTTGACAACGAANACCCAGCATGCA
ACCAAGATCACCTTTCNNCCCNGGNGNGCTGGCTTTGGNNGGGCANCNNTNNNGCNNNGNNTNTGATNNNTNNNNNATAT
ACNGGANATGCANCTGGNNAANNNACTNNNNNNCNGNANN

Analyse the above sequence.
What is the next step to clone and express this gene?

* 6. Design a mammalian expression Gateway vector for cloning of 18000 human cDNA clones
for transient expression of proteins in 293EBNA1 or 293F cells. Vector requirements:

1. Very strong mammalian promoter
2. Secretion signal and Fc fusion
3. Gateway cloning
4. Use an efficient post-transcriptional regulatory element.
5. Use pCDNA3.1 or pCEP4 vector as backbone.

Describe vector construction strategy and paste final sequence below.

* 7. Design TALEN pairs targeting the CHO DHFR gene to generate DHFR-/- cell line. List target DNA
sequences (for TALEN pair only, not the entire DHFR mRNA sequence) and TALEN amino acid sequences.
Outline experimental protocol for this project.
CHO genome sequence:
ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Cricetulus_griseus/CriGri_1.0/
ftp://ftp.ncbi.nlm.nih.gov/genomes/Cricetulus_griseus/Gnomon/

genomic scaffold:
http://www.ncbi.nlm.nih.gov/nuccore/NW_003614442.1?report=fasta&from=464809&to=488594&strand=true

DHFR sequences: NM_001244016.1



* 8. Design oligos to synthesize C-terminal 6XHis tagged proteinase K for E coli expression

MKKLLFAIPLVVPFYSHSTMAPAVEQRSEAAPLIEARGEMVANKYIVKFKEGSALSALDA
AMEKISGKPDHVYKNVFSGFAATLDENMVRVLRAHPDVEYIEQDAVVTINAAQTNAPWGL
ARISSTSPGTSTYYYDESAGQGSCVYVIDTGIEASHPEFEGRAQMVKTYYASSRDGNGHG
THCAGTVGSRTYGVAKKTQIFGVKVLDDNGSGQYSTIIAGMDFVASDHNNRNCPKGVVAS
LSLGGGYSSSVNSAAARLQSSGVMVAVAAGNNNADARNYSPASEPSVCTVGATDRYDRRS
SFSNYGSVLDIFAPGTSILSTWIGGSTRSISGTSMATPHVAGLAAYLMTLGRTTAANACR
YIADTANKGDLSNIPFGTVNLLAYNNYQA

The synthetic gene should be cloned into Nde1 + Xho1 sites of pET28b vector.

* 9. You are trying to make 12 capped mRNA from plasmids purchased from a reputable cDNA clone company.
Only 3 clones from 500 bp to 2.1 kb could be amplified by PCR using Phusion DNA polymerase with HiFi buffer.
What is the next step?

* 10. You designed two primers to amplify a flu gene (NCBI Accession # is FJ966959) and clone the PCR product
to a vector to create a GFP fusion gene.


Your primers:
TX_05-HAwt-Xho1-F
ctgaca CTC GAG gcc acc atgaaggcaatactagtagt tc

TX_05-HAwt-EcoR1-R
(ctacagtgtagaatatgtatt CGA ATT CTG CAG T)
ACTGCAGAATTCGaatacatattctacactgtag

Sequencing results:
1.
>TX_05-HAwt-CMV-Forward_D06.ab1
NNNNNNNNNNNNNNNNNNNNCAGAGCTCGTTTAGTGANCCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCT
CCATAGAAGACACCGACTCTACTAGAGGATCGCTAGCGCTACCGGACTCAGATCTCGAGGCCACC

ATGAAGGCAATACTAGTAGTTCTGCTATATACATTTGCAACCGCAAATGCAGACACATTATGTATAGGTTATCATGCGAACAATTCAACAGACACTGTAGACACAGTACTAGAAAAGAATGTAACAGTAACACACTCTGTTAACCTTCTAGAAGACAAGCATAACGGGAAACTATGCAAACTAAGAGGGGTAGCCCCATTGCATTTGGGTAAATGTAACATTGCTGGCTGGATCCTGGGAAATCCAGAGTGTGAATCACTCTCCACAGCAAGCTCATGGTCCTACATTGTGGAAACATCTAGTTCAGACAATGGAACGTGTTACCCAGGAGATTTCATCGATTATGAGGAGCTAAGAGAGCAATTGAGCTCAGTGTCATCATTTGAAAGGTTTGAGATATTCCCCAAGACAAGTTCATGGCCCAATCATGACTCGAACAAAGGTGTAACGGCAGCATGTCCTCATGCTGGAGCAAAAAGCTTCTACAAAAATTTAATATGGCTAGTTAAAAAAGGAAATTCATACCCAAAGCTCAGCAAATCCTACATTAATGATAAAGGGAAAGAAGTCCTCGTGCTATGGGGCATTCACCATCCATCTACTAGTGCTGACCAACAAAGTCTCTATCAGAATGCAGATGCATATGTTTTTGTGGGGTCGTCAAGATACAGCAAGAAGTTCAAGCCGGAAATAGCAATAAGACCCAAAGTGAGGGATCAAGAAGGGAGAATGAACTATTACTGGACACTAGTAGAGCCGGGAGACAAAATAACATTCGAAGCAACTGGAAATCTAGTGGTACCGAGATATGCATTCGCAATGGAAAGAAATGCTGGATCTGGTATTATCATTTCAGATACACCAGTCCACGATTGCANTACANCTTGNCNGACACCCNNNNNCTATAANCACCAGCCTCCCATTTCNGANATACATCGATCANNNTGGAAAATGTCCAAAANATGTAAAAGCACAAAANNANACNNNNNNAGGNANNNNGANNTNCNNNCNANTNNANCTNNNNNNANTTNGNNNNNTNNNNNNATTTNNNGGGG
GGNNNNNNGGGANNN

2.
>TX_05-HAwt-WPRE-Seq-R_A07.ab1
GNNNNNNNNNNGCGCACGCTGACTTGTGGCCGTGCACGTCGCCGTCCAGCTCGATCAGCACGGGCACGATGCCGGCGAAC
AGCTCCTCGCCCCCGCTCATGGTGGCCGGATCCGGCCTGTTTGGCCTACCGTCGACTGCAGAATTCGAATACATATTCTA
CACTGTAGAGACCCATTAGAGCACATCCAGAAACTGATTGCCCCCAGGGAGACTACCAGTACCAATGAACTGGCGACAGT
TGAATAGATCGCCAAAATCTGGTAAATCCTTGTTGATTCCAGTTTTACCCCATCTATTTCTTCTCTGTTTAATTTTGCTT
CCTCTGAGTATTTTGGGTAGTCATAAGTCCCATTTTTGACACTTTCCATGCACGTGTTATCGCATTTGTGGTAAAATTCA
AAGCAGCCGTTTCCAATTTCCTTGGCATTGTTTTTTAGCTGGCTTCTTACCTTTTCATATAAGTTCTTCACATTTGAATC
GTGGTAGTCCAAAGTTCTTTCATTTTCCAATAGAACCAACAGTTCGGCATTGTAAGTCCAAATGTCCAGGAAACCATCAT
CAACTTTTTTATTTAAATTCTCTATTCTTTTTTCCAGGTGGTTGAACTCTTTACCTACTGCTGTGAACTGTGTATTCATC
TTTTCAATAACAGAATTTACTTTGTTAGTAATTTCGTCAATGGCATTCTGTGTGCTCTTCAGGTCGGCTGCATATCCTGA
CCCCTGCTCATTTTGATGGTGATAACCGTACCATCCATCTACCATCCCTGTCCACCCCCCTTCAATGAAACCGGCAATGG
CCCCAAATAGGCCTCTAGATTGAATAGACGGGACATTCCTCAATCCTGTGGCCAGTCTCAATTTTGTGCTTTTTACATAT
TTTGGACATTTTCCAATTGTGATCGGATGTATATTCTGAAATGGGAGGCTGGTGTTTATAGCACCCTTGGGTGTCTGANA
AGTTGTATTGCAATCGTGGACTGGTGTATCTGAAATGATAATACCAGATCCAGCATTTCTTTCCATTGCGAATGCATATC
TCGGTACCACTAGATTTCCAGTTGNTTCGAATNNTATTTTNNNNNNNN

Cloning vector MCS and GFP sequence (partial)
GAATTCTGCAGTCGACGGTAGGCCAAACAGGCC GGATCC GCC ACC
ATGAGCGGGGGCGAGGAGCTGTTCGCCGGCATCGTGCCCGTGCTG......

Please analyse the sequences and design next experiment.
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