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

From 2010.igem.org

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Latest revision as of 19:33, 27 October 2010

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.

  • FP comparison
    • Maturation time is the time until the protein starts shining.

GFP

>BBa_E0240876bp
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 BBa_I13507

>BBa_I13507861bp
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)
>BBa_I13507+LVA869bp
aaagaggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcg aaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcct gtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaa cgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtacca acttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaat caaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaa accgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgctGCTGCAA ACGACGAAAACTACGCTTTAGTAGCTtaataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgtt gtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
Primers:
>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

CFP


Measurements