Team:NYMU-Taipei/Project/Speedy protein degrader/Sequences

From 2010.igem.org

Contents

Sequences

SsrA tags

SSrA degradation tags to use:

Tag Amino Acid Sequence
[ LVA]BBa_M0040 RPAANDENYALVA gctgcaaacgacgaaaactacgctttagtagct

Fluorescent proteins

Legend

Effect Class
Primers: (changes overline/underline and background)
Forward Primer FP
Overlapping Primer OP
Reverse Primer RP
Parts: (changes text color)
Promoter P
Ribosome Binding site R
RNA rna
Coding region C
Terminator T
Special S
Cutting sites: (changes background)
Biobrick cutting site cut
Biobrick cutting site mutation cutmut

Fluorescent proteins to add degradation tags onto.

  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2007/Projects/Reporters#bibkey_Andersen FP comparison]
    • Maturation time is the time until the protein starts shining.

GFP

  • Used [http://partsregistry.org/wiki/index.php/Part:BBa_E0240 BBa_E0240] as a template:

>[http://partsregistry.org/Part:BBa_E0240 BBa_E0240]876bp
tcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattt tctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttg tcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtaca ggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggt attgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaa tcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtcct tttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgct gggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt atctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

BBa_E0240+LVA (note LVA was added before the taataa double-stop codon)

>BBa_E0240+LVA909bp
tcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattt tctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttg tcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtaca ggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggt attgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaa tcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtcct tttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgct gggattacacatggcatggatgaactatacaaaGCTGCAAACGACGAAAACTACGCTTTAGTAGCTtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt atctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

Primers designed by Jesse in iGEM2008.

>BBa_E0240+LVA909bp
tcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattt tctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttg tcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtaca ggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggt attgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaa tcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtcct tttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgct gggattacacatggcatggatgaactatacaaaGCTGCAAACGACGAAAACTACGCTTTAGTAGCTtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtttt atctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata

Primers: (directly copied from iGEM2008 wiki.)
Outer primers:
gaattcgcggccgcttctagag tcacacaggaaagtactagatgc (prefix + anneal: len:23, tm:56, %gc:43)
ctgcagcggccgctactagta ataaacgcagaaaggccca (suffix + anneal: len:19, tm:56, %gc:47)
Inner primers:
Forward: CGACGAAAACTACGCTTTAGTAGCT taataatactagagccaggcatca (insert + anneal: len:24, tm:55, %gc38)
Reverse: AAGCGTAGTTTTCGTCGTTTGCAGC tttgtatagttcatccatgccatg (insert + anneal: len:24, tm:55, %gc:38)
Complimented part: CGACGAAAACTACGCTT (len:17, tm:50, %gc:47)

Final product is a Linear PCR DNA containing E0240+LVA.

Redesign Outer FP to include B0034 instead of B0032 (Lets save a cloning cycle) 

XbaI+B0034+scar+E0040 start
gcttctagag aaagaggagaaa tactag atgcgtaaaggagaagaacttttc (55C,24bp,38%GC) Total:52bp

RFP

PCR of [http://partsregistry.org/Part:BBa_I13507 BBa_I13507]

>[http://partsregistry.org/Part:BBa_I13507 BBa_I13507]861bp
aaagaggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcg aaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcct gtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaa cgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtacca acttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaat caaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaa accgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataac gctgatagtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcg gtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
What's with the extra long scar after the double translational start codon? Lets ignore it. (someone identify it please)
>[http://partsregistry.org/Part:BBa_I13507 BBa_I13507]+LVA869bp
aaagaggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcg aaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcct gtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaa cgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtacca acttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaat caaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaa accgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgctGCTGCAA ACGACGAAAACTACGCTTTAGTAGCTtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgtt gtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
Primers:
>[http://partsregistry.org/Part:BBa_I13507 BBa_I13507]+LVA869bp
aaagaggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcg aaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcct gtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaa cgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtacca acttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaat caaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaa accgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgctGCTGCAA ACGACGAAAACTACGCTTTAGTAGCTtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgtt gtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata


Primers to order:
Outer FP: gaattcgcggccgcttctagag aaagaggagaaatactagatggct (55C,24bp,38%GC) Total: 
Inner RP: CTACTAAAGCGTAGTTTTCGTCGTTTGCAGCagcaccggtggagtga (56C,16bp,63%GC) Total:47bp
Primers that are the same as GFP:
Inner FP: CGACGAAAACTACGCTTTAGTAGCTtaataatactagagccaggcatca (55C,24bp,38%GC) Total:49bp
Outer RP: ctgcagcggccgctactagta tataaacgcagaaaggccca (56C,20bp,45%GC) Total:41bp
Overlapping region: 55C,23bp,43%GC

YFP

  • [http://partsregistry.org/wiki/index.php/Part:BBa_E0032 YFP with LVA tag]

CFP

  • [http://partsregistry.org/wiki/index.php/Part:BBa_E0022 CFP with LVA tag]


Measurements

  • [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC106306/?tool=pubmed one to follow] unless there's a better one KULeuven08 followed it.
  • Paper found by LiFeng.

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