Part:BBa_K2171000:Design

CASH1 - osmotic stress protein from tardigrade

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1785
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

The "lacI-lacIq and a tac promoter" subpart was amplified from part BBa_K731520 using primers:

F: tatgaattcgcggccgcttctagagTCACTGCCCGCTTTCCAGTC

R: CTAGTACTTTCCTGTGTGACTC

"CASH1 CDS + transcription terminators" subpart was ordered as gBlock from IDT

Sequence: 

ATATAATTGAATTCGCGGCCGCTTCTAGATGCCATACGAGAAACATGTTGAGCAGACAGTTGTGGAGAAAACGGAGCAGCCGGGC
CATTCCAGCACCCACCATGCTCCCGCGCAGCGCACCGTGGCCCGTGAGCAAGAAGAGGTCGTCCATAAGGAGTTTACTCATACTG
ATATTCGTGTTCCCCATATCGACGCGCCACCACCGATTATTGCGGCATCTGCTGTTGGATTGGCCGAGGAAATCGTTAGTCACGG
GTTTCAAGCCTCAGCGGCTCGTATCTCCGGCGCATCCACGGAGGTGGACATGCGCCCTAGCCCTAAATTGGCCGAAGAGGCTCGT
CGCGACGCAGAGCGTTACCAAAAAGAACACGAAATGATTAACCGTCAAGCTGAGGCCACGTTACAGAAAAAAGCGGAGGAGTACC
GTCACCAGACCGAGGCCGAAGCCGAGAAGATTCGTCGCGAGTTAGAGAAACAGCACGAACGCGATATTCAATTCCGTAAAGACCT
GATCGACCAGACCATTGAAAAACAAAAGCGCGAGGTAGATCTTGAAGCAAAGATGGCAAAACGCGAGCTTGACCGCGAAGCACAG
TTAGCAAAAGAAGCCCTGGAACGTAGCCGTATGGCCACAAACGTGGAGGTGACGCTTGATACGGCGGCAGGTCATACTGTATCTG
GGGGCACTACAGTCTCCTCGGTTGATAAAGTCGAAACAGTTCGTGAACGCAAACATCATTAATAATTGATTAACATCAAATAAAA
CGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCAC
CTTCGGGTGGGCCTTTCTGCGTTTATTACTAGTAGCGGCCGCTGCAGTTATATAT

and amplified using primers:

F: gagtcacacaggaaagtactagATGCCATACGAGAAACATGTTG

R: taactgcagCGGCCGCTACTAGTAATAAACGC

Both subparts were fused together by Overlap Extention PCR. All PCRs were performed with Phusion High Fidelity Polymerase using the standart protocol provided by the manufacturer. The final part was gel purified and cloned in standart vector via restriction (EcoRI and PstI) and ligation.

Note: Uppercase characters in primer sequences correspond to regions that are complementary to the intended matrix. Lowercase characters mark regions of overlap for OEP and/or parts of biobrick regions with restriction sites for EcoRI and PstI.