Team:Edinburgh/Sequences

Sequences Sandbox

It is often useful to have nucleotide sequences of various stuff that we use. This page serves as a sandbox for playing with sequence information.

Note that any biobricks we submit to the Registry have to be properly documented there.

We have BBa_K523000 to BBa_K523999 available this year.

Biobrick prefix, suffix, scar

• gaattcgcggccgcttctag - prefix, short
• gaattcgcggccgcttctagag - prefix, long
• tactagtagcggccgctgcag - suffix
• tactag - scar, short
• tactagag - scar, long

Restriction sites

• gaattc - EcoRI (prefix 1)
• tctaga - XbaI (prefix 2)
• actagt - SpeI (suffix 1)
• ctgcag - PstI (suffix 2)

• gcggccgc - NotI (in both prefix and suffix)

• agatct - BglII (not part of RFC10, we use it internally after the prefix)

• tacgta - SnaBI (might become relevant)

pSB1C3 vector

• This is the official sequence from the Registry (here)
• Red is CmlR coding sequence (chloramphenicol acetyltransferase)
• Blue is restriction sites for the 4 main enzymes (SpeI, PstI, EcoRI, XbaI)

tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgcccttt ttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagata ccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctg taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac ttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgag cgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcc aagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaatt cattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcga agaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcac tccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacac tatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattca tcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacgg tctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatc cagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaa atacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgcccga tcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat
gatttctggaattcgcggccgcttctagag

Plac-LacZα insert (clone 2)

• Assigned code <partinfo>BBa_K523000</partinfo>
• A modified version (seems to lack about 45 bases at the start) of BBa_J33207
• Sanger sequencing seemed to confirm that all was well with clone 2, though in a few regions the chromatograms were poor.
• Sequence below includes BioBrick prefix and suffix (blue) as well as the BglII site (bold), which is partly in the prefix
• lacZα is in red.
• Both forward and reverse sequencing reactions failed to give good info about the suffix; so I have simply added the standard suffix. However we've not conclusively proved that the SpeI site exists; this should be verified by digest.

gaattcgcggccgcttctagagatctgctggggcaaaccagcgtggaccgcttgctgcaa ctctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaaga aaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatta atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaa tgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtat gttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatta cggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaac ttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgca ccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttc cggcaccagaagcggtgccggaaagctggctggagtgatactagtagcggccgctgcag

malS using primers f1/r1 (clone 1)

• Assigned code <partinfo>BBa_K523001</partinfo>
• Expected sequence is derived from this.
• Prefix and suffix in blue.
• Pre-coding sequence in CAPITALS and contains native RBS.
• BglII site in bold.
• Double stop codon is present.
• This has been sequenced and looks good.

gaattcgcggccgcttctagagatctGAAATCGCAGCAATAAGGACTCATCCGCCatgaa actcgccgcctgttttctgacactccttcctggcttcgccgttgccgccagctggacttc tccggggtttcccgcctttagcgaacaggggacaggaacatttgtcagccacgcgcagtt gcccaaaggtacgcgtccactaacgctaaattttgaccaacagtgctggcagcctgcgga tgcgataaaactcaatcagatgctttccctgcaaccttgtagcaacacgccgcctcaatg gcgattgttcagggacggcgaatatacgctgcaaatagacacccgctccggtacgccaac attgatgatttccatccagaacgccgccgaaccggtagcaagcctggtccgtgaatgccc gaaatgggatggattaccgctcacagtggatgtcagcgccactttcccggaaggagccgc cgtacgggattattacagccagcaaattgcgatagtgaagaacggtcaaataatgttaca acccgctgccaccagcaacggtttactcctgctggaacgggcagaaactgacacatccgc ccctttcgactggcataacgccacggtttactttgtgctgacagatcgtttcgaaaacgg cgatcccagtaatgaccagagttacggacgtcataaagacggtatggcggaaattggcac ttttcacggcggcgatttacgcggcctgaccaacaaactggattacctccagcagttggg cgttaatgctttatggataagcgccccatttgagcaaattcacggctgggtcggcggcgg tacaaaaggcgatttcccgcattatgcctaccacggttattacacacaggactggacgaa tcttgatgccaatatgggcaacgaagccgatctacggacgctggttgatagcgcacatca gcgcggtattcgtattctctttgatgtcgtgatgaaccacaccggctatgccacgctggc ggatatgcaggagtatcagtttggcgcgttatatctttctggtgacgaagtgaaaaaatc gctgggtgaacgctggagcgactggaaacctgccgccgggcaaacctggcatagctttaa cgattacattaatttcagcgacaaaacaggctgggataaatggtggggaaaaaactggat cagaacggatatcggcgattacgacaatcctggattcgacgatctcactatgtcgctagc ctttttgccggatatcaaaaccgaatcaactaccgcttctggtctgccggtgttctataa aaacaaaatggatacccacgccaaagccattgacggctatacgccgcgcgattacttaac ccactggttaagtcagtgggtccgcgactatgggattgatggttttcgggtcgataccgc caaacatgttgagttgcccgcctggcagcaactgaaaaccgaagccagcgccgcgcttcg cgaatggaaaaaagctaaccccgacaaagcattagatgacaaacctttctggatgaccgg tgaagcctggggccacggcgtgatgcaaagtgactactatcgccacggcttcgatgcgat gatcaatttcgattatcaggagcaggcggcgaaagcagtcgactgtctggcgcagatgga tacgacctggcagcaaatggcggagaaattgcagggtttcaacgtgttgagctacctctc gtcgcatgatacccgcctgttccgtgaagggggcgacaaagcagcagagttattactatt agcgccaggcgcggtacaaatcttttatggtgatgaatcctcgcgtccgttcggtcctac aggttctgatccgctgcaaggtacacgttcggatatgaactggcaggatgttagcggtaa atctgccgccagcgtcgcgcactggcagaaaatcagccagttccgcgcccgccatcccgc aattggcgcgggcaaacaaacgacacttttgctgaagcagggctacggctttgttcgtga gcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaataatactagtagcg
gccgctgcag

malS using primers f2/r2 (clone 1)

• Assigned code <partinfo>BBa_K523003</partinfo>
• This is essentially as above but starting at the start codon, and with no stop codon, and adding two bases at the end of the BioBrick, which (together with the T at the start of the suffix) code for a glycine when using BioSandwich assembly.
• Expected sequence is derived from this.
• This has been sequenced and looks good.

gaattcgcggccgcttctagagatctatgaaactcgccgcctgttttctgacactccttc ctggcttcgccgttgccgccagctggacttctccggggtttcccgcctttagcgaacagg ggacaggaacatttgtcagccacgcgcagttgcccaaaggtacgcgtccactaacgctaa attttgaccaacagtgctggcagcctgcggatgcgataaaactcaatcagatgctttccc tgcaaccttgtagcaacacgccgcctcaatggcgattgttcagggacggcgaatatacgc tgcaaatagacacccgctccggtacgccaacattgatgatttccatccagaacgccgccg aaccggtagcaagcctggtccgtgaatgcccgaaatgggatggattaccgctcacagtgg atgtcagcgccactttcccggaaggagccgccgtacgggattattacagccagcaaattg cgatagtgaagaacggtcaaataatgttacaacccgctgccaccagcaacggtttactcc tgctggaacgggcagaaactgacacatccgcccctttcgactggcataacgccacggttt actttgtgctgacagatcgtttcgaaaacggcgatcccagtaatgaccagagttacggac gtcataaagacggtatggcggaaattggcacttttcacggcggcgatttacgcggcctga ccaacaaactggattacctccagcagttgggcgttaatgctttatggataagcgccccat ttgagcaaattcacggctgggtcggcggcggtacaaaaggcgatttcccgcattatgcct accacggttattacacacaggactggacgaatcttgatgccaatatgggcaacgaagccg atctacggacgctggttgatagcgcacatcagcgcggtattcgtattctctttgatgtcg tgatgaaccacaccggctatgccacgctggcggatatgcaggagtatcagtttggcgcgt tatatctttctggtgacgaagtgaaaaaatcgctgggtgaacgctggagcgactggaaac ctgccgccgggcaaacctggcatagctttaacgattacattaatttcagcgacaaaacag gctgggataaatggtggggaaaaaactggatcagaacggatatcggcgattacgacaatc ctggattcgacgatctcactatgtcgctagcctttttgccggatatcaaaaccgaatcaa ctaccgcttctggtctgccggtgttctataaaaacaaaatggatacccacgccaaagcca ttgacggctatacgccgcgcgattacttaacccactggttaagtcagtgggtccgcgact atgggattgatggttttcgggtcgataccgccaaacatgttgagttgcccgcctggcagc aactgaaaaccgaagccagcgccgcgcttcgcgaatggaaaaaagctaaccccgacaaag cattagatgacaaacctttctggatgaccggtgaagcctggggccacggcgtgatgcaaa gtgactactatcgccacggcttcgatgcgatgatcaatttcgattatcaggagcaggcgg cgaaagcagtcgactgtctggcgcagatggatacgacctggcagcaaatggcggagaaat tgcagggtttcaacgtgttgagctacctctcgtcgcatgatacccgcctgttccgtgaag ggggcgacaaagcagcagagttattactattagcgccaggcgcggtacaaatcttttatg gtgatgaatcctcgcgtccgttcggtcctacaggttctgatccgctgcaaggtacacgtt cggatatgaactggcaggatgttagcggtaaatctgccgccagcgtcgcgcactggcaga aaatcagccagttccgcgcccgccatcccgcaattggcgcgggcaaacaaacgacacttt tgctgaagcagggctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtct gggcagggcaacagggtactagtagcggccgctgcag

bglX using primers f1/r1 (clone 1)

• Assigned code <partinfo>BBa_K523002</partinfo>
• Expected sequence is derived from this.
• Prefix and suffix in blue.
• Pre-coding sequence in CAPITALS and contains native RBS.
• BglII site in bold.
• Double stop codon is present.
• The sequence as it stands has a PstI site (red).
• This has been sequenced and looks good. A silent mutation from the expected sequence is indicated as a red capital T.

gaattcgcggccgcttctagagatctGCCACGTCGGGCAACAAAGGAAGAAAAATCCATa tgaaatggctatgttcagtaggaatcgcggtgagtctggccctgcagccagcactggcgg atgatttattcggcaaccatccattaacgcccgaagcgcgggatgcgttcgtcaccgaac tgcttaagaaaatgacagttgatgagaaaattggtcagctgcgcttaatcagcgtcggcc cggataacccgaaagaggcgatccgcgagatgatcaaagacggtcaggttggggcgattt tcaacaccgtaacccgtcaggatatccgcgccatgcaggatcaggtgatggaattaagcc gcctgaaaattcctcttttctttgcttacgacgtgctgcacggtcagcgcacggtgttcc cgattagcctcggtctggcctcgtcttttaacctcgatgcagtgaaaacggtcggacgtg tctctgcttatgaagcggcagatgatggcctgaatatgacctgggcaccgatggtcgatg tctcgcgcgatccgcgctggggacgtgcttccgaaggttttggcgaagatacgtatctca cctcaacaatgggtaaaaccatggtggaagcgatgcagggtaaaagcccggcagatcgct actcggtgatgaccagcgtcaaacactttgccgcatacggcgcggtagaaggcggtaaag agtacaacaccgtcgatatgagtccgcagcgcctgtttaatgattatatgccgccgtaca aagcggggctggacgcaggcagcggcgcggtgatggtggcgctgaactcgctgaacggca cgccagccacctccgattcctggctgctgaaagatgttctgcgcgaccagtggggcttta aaggcatcaccgtttccgatcacggtgcaatcaaagagctgattaaacatggcacggcgg cagacccggaagatgcggtgcgcgtggcgctgaaatccggaatcaacatgagcatgagcg acgagtactactcgaagtatctgcctgggttgattaaatccggcaaagtgacgatggcag agctggacgatgctgcccgccatgtactgaacgttaaatatgatatggggttgtttaacg acccatacagccatttggggccgaaagagtctgacccggtggataccaatgccgaaagcc gcctgcaccgtaaagaagcgcgtgaagtggcgcgcgaaagcttggtgttgctgaaaaacc gtctcgaaacgttaccgctgaaaaaatcggccaccattgcggtggttgggccactggcgg acagtaaacgtgacgtgatgggcagctggtccgcagccggtgttgccgatcaatccgtga ccgtactgaccgggattaaaaatgccgtcggtgaaaacggtaaagtgctgtatgccaaag gggcgaacgttaccagtgacaaaggcattatcgatttcctgaatcagtatgaagaagcgg tcaaagtcgatccgcgttcgccgcaagagatgattgatgaagcggtgcagacggcgaaac aatctgatgtggtggtggctgtagtcggtgaagcacaggggatggcgcacgaagcctcca gccggaccgatatcactattccgcaaagccaacgtgacttgattgcggcgctgaaagcca ccggtaaaccgctggtgctggtgctgatgaacgggcgtccgctggcgctggtgaaagaag atcagcaggctgatgcgattctggaaacctggtttgcggggactgaaggcggtaatgcaa ttgccgatgtattgtttggcgattacaacccgtccggcaagctgccaatgtccttcccgc gttctgtcgggcagatcccggtgtactacagccatctgaataccggtcgcccgtataatg ccgacaagccgaacaaatacacttcgcgttattttgatgaagctaacggggcgttgtatc cgttcggctatgggctgagctacaccactttcaccgtctctgatgtgaaactttctgcgc cgaccatgaagcgtgacggcaaagtgactgccagcgtgcaggtgacgaacaccggtaagc gcgagggtgccacggtagtgcagatgtacttgcaggatgtgacTgcttccatgagtcgcc ctgtgaaacagctgaaaggctttgagaaaatcaccctgaaaccgggcgaaactcagactg tcagcttcccgatcgatattgaggcgctgaagttctggaatcaacagatgaaatatgacg ccgagcctggcaagttcaatgtctttatcggcactgattccgcacgcgttaagaaaggcg agtttgagttgctgtaataatactagtagcggccgctgcag

bglX using primers f2/r2 (clone 1)

• Assigned code <partinfo>BBa_K523004</partinfo>
• This is essentially as above but starting at the start codon, and with no stop codon, and adding two bases at the end of the BioBrick, which (together with the T at the start of the suffix) code for a glycine when using BioSandwich assembly.
• Expected sequence is derived from this.
• This has been sequenced and looks good. A silent mutation from the expected sequence is indicated as a red capital T.

gaattcgcggccgcttctagagatctatgaaatggctatgttcagtaggaatcgcggtga gtctggccctgcagccagcactggcggatgatttattcggcaaccatccattaacgcccg aagcgcgggatgcgttcgtcaccgaactgcttaagaaaatgacagttgatgagaaaattg gtcagctgcgcttaatcagcgtcggcccggataacccgaaagaggcgatccgcgagatga tcaaagacggtcaggttggggcgattttcaacaccgtaacccgtcaggatatccgcgcca tgcaggatcaggtgatggaattaagccgcctgaaaattcctcttttctttgcttacgacg tgctgcacggtcagcgcacggtgttcccgattagcctcggtctggcctcgtcttttaacc tcgatgcagtgaaaacggtcggacgtgtctctgcttatgaagcggcagatgatggcctga atatgacctgggcaccgatggtcgatgtctcgcgcgatccgcgctggggacgtgcttccg aaggttttggcgaagatacgtatctcacctcaacaatgggtaaaaccatggtggaagcga tgcagggtaaaagcccggcagatcgctactcggtgatgaccagcgtcaaacactttgccg catacggcgcggtagaaggcggtaaagagtacaacaccgtcgatatgagtccgcagcgcc tgtttaatgattatatgccgccgtacaaagcggggctggacgcaggcagcggcgcggtga tggtggcgctgaactcgctgaacggcacgccagccacctccgattcctggctgctgaaag atgttctgcgcgaccagtggggctttaaaggcatcaccgtttccgatcacggtgcaatca aagagctgattaaacatggcacggcggcagacccggaagatgcggtgcgcgtggcgctga aatccggaatcaacatgagcatgagcgacgagtactactcgaagtatctgcctgggttga ttaaatccggcaaagtgacgatggcagagctggacgatgctgcccgccatgtactgaacg ttaaatatgatatggggttgtttaacgacccatacagccatttggggccgaaagagtctg acccggtggataccaatgccgaaagccgcctgcaccgtaaagaagcgcgtgaagtggcgc gcgaaagcttggtgttgctgaaaaaccgtctcgaaacgttaccgctgaaaaaatcggcca ccattgcggtggttgggccactggcggacagtaaacgtgacgtgatgggcagctggtccg cagccggtgttgccgatcaatccgtgaccgtactgaccgggattaaaaatgccgtcggtg aaaacggtaaagtgctgtatgccaaaggggcgaacgttaccagtgacaaaggcattatcg atttcctgaatcagtatgaagaagcggtcaaagtcgatccgcgttcgccgcaagagatga ttgatgaagcggtgcagacggcgaaacaatctgatgtggtggtggctgtagtcggtgaag cacaggggatggcgcacgaagcctccagccggaccgatatcactattccgcaaagccaac gtgacttgattgcggcgctgaaagccaccggtaaaccgctggtgctggtgctgatgaacg ggcgtccgctggcgctggtgaaagaagatcagcaggctgatgcgattctggaaacctggt ttgcggggactgaaggcggtaatgcaattgccgatgtattgtttggcgattacaacccgt ccggcaagctgccaatgtccttcccgcgttctgtcgggcagatcccggtgtactacagcc atctgaataccggtcgcccgtataatgccgacaagccgaacaaatacacttcgcgttatt ttgatgaagctaacggggcgttgtatccgttcggctatgggctgagctacaccactttca ccgtctctgatgtgaaactttctgcgccgaccatgaagcgtgacggcaaagtgactgcca gcgtgcaggtgacgaacaccggtaagcgcgagggtgccacggtagtgcagatgtacttgc aggatgtgacTgcttccatgagtcgccctgtgaaacagctgaaaggctttgagaaaatca ccctgaaaccgggcgaaactcagactgtcagcttcccgatcgatattgaggcgctgaagt tctggaatcaacagatgaaatatgacgccgagcctggcaagttcaatgtctttatcggca ctgattccgcacgcgttaagaaaggcgagtttgagttgctgggtactagtagcggccgct
gcag

INP in RFC12 format (clone 2)

• This is <partinfo>BBa_K265008</partinfo>.
• This came on pSB1AK3 and with an RFC12 prefix and suffix (capitals).
• This has been sequenced and the thing we have is perfect except it has an extra C base in the suffix (red).
• (Blue) There is a SpeI site in the prefix (not suffix) and a NheI site in the suffix.

GAATTCGCGGCCGCACTAGTatgaccctggataaagcgctggtgctgcgcacttgtgcca ataatatggccgaccattgtggcctgatctggcctgcttctggtacagttgaaagccgct attggcagtctacacgtcgtcacgaaaacgggctggttgggctgctgtggggtgccggaa catccgcctttctgagcgttcatgccgatgctcgttggattgtttgtgaggttgctgttg ccgacatcattagcctggaagaaccgggtatggtgaaatttcctcgtgctgaagtggtcc atgttggtgatcgtattagcgcctcccatttcatttcagctcgtcaggctgatccagcaa gcacatcaacaagcacctctaccagtacactgacaccaatgccaaccgctatccctactc ctatgccggctgtagcctctgttactctgcctgtggccgaacaagctcgccacgaagtct ttgacgttgcctctgtgtctgctgctgctgctcctgttaacactctgcctgttaccacgc ctcaaaatctgcaaacggcgacctatggctcaacactgagtggtgataaccattctcgtc tgattgccgggtatggctctaacgaaaccgccggaaaccattctgatctgatcggtggtc acgattgtacgctgatggcaggtgatcaatctcgtctgaccgctggcaaaaattccgttc tgaccgctggggcacgttctaaactgatcgggagcgaaggctctactctgtccgccggag aagattctaccctgatctttcgtctgtgggatggtaaacgttatcgtcagctggtcgctc gtacaggtgaaaatggtgtggaagccgacattccgtattatgtgaatgaggacgatgata ttgtggacaaaccggacgaagatgatgattggatcgaagtgaaaGCTAGCGCGGCCGCcT
GCAG

pVIII

• Blue is the leader sequence... MKKSLVLKASVAVATLVPMLSFA
• Expected sequence is derived from this.

atgaaaaagtctttagtcctcaaagcctctgtagccgttgctaccctcgttccgatgctg tctttcgctgctgagggtgacgatcccgcaaaagcggcctttaactccctgcaagcctca gcgaccgaatatatcggttatgcgtgggcgatggttgttgtcattgtcggcgcaactatc ggtatcaagctgtttaagaaattcacctcgaaagcaagctga

C. fimi exoglucanase (clone 2)

• This has been sequenced.
• Two bases are not what they should be...

atctatggcgaccacgctcaaggaggccgccgacggcgccggccgggacttcggcttcgc gctcgaccccaaccggctctcggaggcgcagtacaaggcgatcgccgacagcgagttcaa cctcgtcgtcgccgagaacgcgatgaagtgggGcgccaccgagccctcgcagaacagctt ctccttcggcgcgggcgaccgcgtcgcgagctacgccgccgacaccggcaaggagctgta cggccacacgctcgtCtggcactcgcagctgcccgactgggcgaagaacctcaacggctc cgcgttcgagagcgcgatggtcaaccacgtgacgaaggtcgccgaccacttcgagggcaa ggtcgcgtcgtgggacgtcgtcaacgaggcgttcgccgacggcggcggccgccggcagga ctcggcgttccagcagaagctcggcaacggctacatcgagaccgcgttccgggcggcacg tgcggcggacccgaccgccaagctgtgcatcaacgactacaacgtcgagggcatcaacgc gaagagcaactcgctctacgacctcgtcaaggacttcaaggcgcgcggcgtcccgctcga ctgcgtcgggttccagtcgcacctcatcgtcggccaggtgccgggcgacttccggcagaa cctgcaacggttcgcggacctgggcgtggacgtgcgcatcaccgagctcgacatccgcat gcggacgccctccgacgcgaccaagctcgcgacccaggcggccgactacaagaaggtcgt gcaggcctgcatgcaggtgacccgctgccagggcgtgaccgtctggggcatcaccgacaa gtactcgtgggtgccggacgtcttcccgggcgagggggccgcgctggtgtgggacgcgag ctacgccaagaagccggcctacgccgccgtgatggaggccttcggcgcgagcccgacgcc gacgcccaccacgccgaccccgacgcccacgacgccgacgccgaccccgacgtccggtcc ggccgggtgccaggtgctgtggggcgtcaaccagtggaacaccggcttcaccgcgaacgt caccgtgaagaacacgtcctccgctccggtcgacggctggacgctcacgttcagcttccc gtccggccagcaggtcacccaggcgtggagctcgacggtcacgcagtccggctcggccgt gacggtccgcaacgccccgtggaacggctcgatcccggcgggcggcaccgcgcagttcgg cttcaacggctcgcacacgggcaccaacgccgcgccgacggcgttctcgctcaacggcac
gccctgcacggtcggctc

pG8SAET

• The sequence is found here.
• The plasmid encodes a beta-lactamase (bold) for ampicillin resistance.
• Restriction sites that exist are underlined:
  • NotI (c|catgg)
  • SnaBI (tac|gta)
• The leader sequence (green) is derived from this.
• The E-tag (GAPVPYPDPLEPR, blue) and pVIII sequence (red) are out of frame with the leader sequence.
• Correct this by adding one base at the start of our inserted gene.

• WAIT! There seems to be a stop codon between the E-tag and the pVIII sequence, how can that be?

agcggaagagccggttttaagccttttacttcctgaataaatctttcagcaaaatattta ttttataagttgtaaaacttacctttaaatttaattataaatatagattttagtattgca atacataattcgttatattatgatgactttacaaatacatacagggggtattaatttgaa aaggaaaaacatttattcaattcgtaaactaggtgtaggtattgcatctgtaactttagg tacattacttatatctggtggcgtaacacctgctgcaaatgctgcgcaacacgatgacca tggcagtacgtacccggtgcgccggtgccgtatccggacccactggaaccgcgtgcctag ggatccgagggtgacgatcccgcaaaagcggcctttgactccctgcaagcctcagcgacc gaatatatcggttatgcgtgggcgatggttgttgtcattgtcggcgcaactatcggtatc aagctgtttaagaaattcacctcgaaagcaagctgataaaccgatgaattcactggccgt cgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc acatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccca acagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatct gtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgca ttaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcccta gcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtt tttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgga acaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcg gcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccg gcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttca ccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggtt aatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttc cgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaa acgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaa ctggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtc acagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataacc atgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagcta accgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaata gactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagca ctggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaa tttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgt gagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagat cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaac tctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag cggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgt cgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcc tttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcc cctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagc
cgaacgaccgagcgcagcgagtcagtgagcgagga

pSB1C3 for BioSandwich

• Chris's primers used on pSB1C3—<partinfo>BBa_J04450</partinfo> produced this linear product.
  • Forward primer: atcagatctgttgtttgtcggtgaacg
  • Reverse primer: gagaggcggtttgcgtattg
• Standard suffix and prefix in bold.
• BglII and XbaI sites in red.

atcagatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgg gtgggcctttctgcgtttatatactagtagcggccgctgcagtccggcaaaaaagggcaa ggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggc ttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtg agcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttcca caggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaa cccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcc tgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggc gctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagct gggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcg tcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacag gattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta cggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctt ttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag attatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaat ctaaagtatatatgagtaaacttggtctgacagctcgaggcttggattctcaccaataaa aaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattac tggatctatcaacaggagtccaagcgagctcgatatcaaattacgccccgccctgccact catcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacgg catgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgc ccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactgg tgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttaggga aataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgcc ggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaa cggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatac gaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaact tgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggt tataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattggg atatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctg aaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagt tggaacctcttacgtgcccgatcaactcgagtgccacctgacgtctaagaaaccattatt atcatgacattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaa tccttagctttcgctaaggatgatttctggaattcgcggccgcttctagagcaatacgca
aaccgcctctc

BioSandwich test with PlacLacZ and EYFP

• Expected sequence...
• Bold is the chosen parts of the spacers...

ttaatacgactcactatagggagaggaggtacgatctgctggggcaaaccagcgtggacc gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgatactagctgat atttcgcgtaaggaaatccattatgggatctatggtgagcaagggcgaggagctgttcac cggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgt gtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcac caccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgca atgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcc cgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccg cgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcga cttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaa cgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcca caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcgg cgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggat cactctcggcatggacgagctgtacaagtaataatactagcaacatatcataacggagtg atcgcaatgggatctgttgtttgtcggtgaacgctctctactagagtcacactggctcac
cttcgggtgggcctttctgcgtttata

Alternate BioSandwich test with PlacLacZ and EYFP

• Chris did a different version of OEPCR with different primers and a blunt end ligation.
• This gave a different sequence, lacking spacer2 and having an RFC54 suffix before the end of the part.

ttaatacgactcactatagggagaggaggtacgatctgctggggcaaaccagcgtggacc gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgatactagctgat atttcgcgtaaggaaatccattatgggatctatggtgagcaagggcgaggagctgttcac cggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgt gtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcac caccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgca atgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcc cgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccg cgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcga cttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaa cgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgcca caacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcgg cgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggat cactctcggcatggacgagctgtacaagtaataatactagtagctgcagacaatcagatc tgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcct
ttctgcgtttata

Serendipitous PlacLacZ-INP construct

• While attempting to make PlacLacZ-INP-malS, we got the following...
• malS is not present
• But it will be useful as a template for true Gibson Assembly.

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

tactagctgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaagg

t act agc ccg acg acc agc ccc acg ccg acc ccg

Sequence is not as expected after this. Note also repetitive sequences that might interfere with primer design.

PlacLacZ - INP - YFP (expected)

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa gg

T ACT AGC ccg acg acc agc ccc acg ccg acc ccg acg GGA TCT

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggac ggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctac ggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc ctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaag ctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttc ttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctg gtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcac aagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaac ggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgcc gaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccac tacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtc ctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaa
taa

TACTAGC aacatatcataacggagtgatcgcaatg GGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata

PlacLacZ - INP - bglX (expected)

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa gg

T ACT AGC agc agc att gcg agc agc agc ccg agc agc gtg gcg GGA TCT

atgaaatggctatgttcagtaggaatcgcggtgagtctggccctgcaaccagcact ggcggatgatttattcggcaaccatccattaacgcccgaagcgcgggatgcgttcgtcac cgaactgcttaagaaaatgacagttgatgagaaaattggtcagctgcgcttaatcagcgt cggcccggataacccgaaagaggcgatccgcgagatgatcaaagacggtcaggttggggc gattttcaacaccgtaacccgtcaggatatccgcgccatgcaggatcaggtgatggaatt aagccgcctgaaaattcctcttttctttgcttacgacgtgctgcacggtcagcgcacggt gttcccgattagcctcggtctggcctcgtcttttaacctcgatgcagtgaaaacggtcgg acgtgtctctgcttatgaagcggcagatgatggcctgaatatgacctgggcaccgatggt cgatgtctcgcgcgatccgcgctggggacgtgcttccgaaggttttggcgaagatacgta tctcacctcaacaatgggtaaaaccatggtggaagcgatgcagggtaaaagcccggcaga tcgctactcggtgatgaccagcgtcaaacactttgccgcatacggcgcggtagaaggcgg taaagagtacaacaccgtcgatatgagtccgcagcgcctgtttaatgattatatgccgcc gtacaaagcggggctggacgcaggcagcggcgcggtgatggtggcgctgaactcgctgaa cggcacgccagccacctccgattcctggctgctgaaagatgttctgcgcgaccagtgggg ctttaaaggcatcaccgtttccgatcacggtgcaatcaaagagctgattaaacatggcac ggcggcagacccggaagatgcggtgcgcgtggcgctgaaatccggaatcaacatgagcat gagcgacgagtactactcgaagtatctgcctgggttgattaaatccggcaaagtgacgat ggcagagctggacgatgctgcccgccatgtactgaacgttaaatatgatatggggttgtt taacgacccatacagccatttggggccgaaagagtctgacccggtggataccaatgccga aagccgcctgcaccgtaaagaagcgcgtgaagtggcgcgcgaaagcttggtgttgctgaa aaaccgtctcgaaacgttaccgctgaaaaaatcggccaccattgcggtggttgggccact ggcggacagtaaacgtgacgtgatgggcagctggtccgcagccggtgttgccgatcaatc cgtgaccgtactgaccgggattaaaaatgccgtcggtgaaaacggtaaagtgctgtatgc caaaggggcgaacgttaccagtgacaaaggcattatcgatttcctgaatcagtatgaaga agcggtcaaagtcgatccgcgttcgccgcaagagatgattgatgaagcggtgcagacggc gaaacaatctgatgtggtggtggctgtagtcggtgaagcacaggggatggcgcacgaagc ctccagccggaccgatatcactattccgcaaagccaacgtgacttgattgcggcgctgaa agccaccggtaaaccgctggtgctggtgctgatgaacgggcgtccgctggcgctggtgaa agaagatcagcaggctgatgcgattctggaaacctggtttgcggggactgaaggcggtaa tgcaattgccgatgtattgtttggcgattacaacccgtccggcaagctgccaatgtcctt cccgcgttctgtcgggcagatcccggtgtactacagccatctgaataccggtcgcccgta taatgccgacaagccgaacaaatacacttcgcgttattttgatgaagctaacggggcgtt gtatccgttcggctatgggctgagctacaccactttcaccgtctctgatgtgaaactttc tgcgccgaccatgaagcgtgacggcaaagtgactgccagcgtgcaggtgacgaacaccgg taagcgcgagggtgccacggtagtgcagatgtacttgcaggatgtgactgcttccatgag tcgccctgtgaaacagctgaaaggctttgagaaaatcaccctgaaaccgggcgaaactca gactgtcagcttcccgatcgatattgaggcgctgaagttctggaatcaacagatgaaata tgacgccgagcctggcaagttcaatgtctttatcggcactgattccgcacgcgttaagaa
aggcgagtttgagttgctggg

TACTAGC aacatatcataacggagtgatcgcaatg GGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata

PlacLacZ - INP - cex (expected)

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa gg

T ACT AGC agc agc att gcg agc agc agc ccg agc agc gtg gcg GGA TCT

atggcgaccacgctcaaggaggccgccgacggcgccggccgggacttcggcttcgcgctc gaccccaaccggctctcggaggcgcagtacaaggcgatcgccgacagcgagttcaacctc gtcgtcgccgagaacgcgatgaagtggggcgccaccgagccctcgcagaacagcttctcc ttcggcgcgggcgaccgcgtcgcgagctacgccgccgacaccggcaaggagctgtacggc cacacgctcgtctggcactcgcagctgcccgactgggcgaagaacctcaacggctccgcg ttcgagagcgcgatggtcaaccacgtgacgaaggtcgccgaccacttcgagggcaaggtc gcgtcgtgggacgtcgtcaacgaggcgttcgccgacggcggcggccgccggcaggactcg gcgttccagcagaagctcggcaacggctacatcgagaccgcgttccgggcggcacgtgcg gcggacccgaccgccaagctgtgcatcaacgactacaacgtcgagggcatcaacgcgaag agcaactcgctctacgacctcgtcaaggacttcaaggcgcgcggcgtcccgctcgactgc gtcgggttccagtcgcacctcatcgtcggccaggtgccgggcgacttccggcagaacctg caacggttcgcggacctgggcgtggacgtgcgcatcaccgagctcgacatccgcatgcgg acgccctccgacgcgaccaagctcgcgacccaggcggccgactacaagaaggtcgtgcag gcctgcatgcaggtgacccgctgccagggcgtgaccgtctggggcatcaccgacaagtac tcgtgggtgccggacgtcttcccgggcgagggggccgcgctggtgtgggacgcgagctac gccaagaagccggcctacgccgccgtgatggaggccttcggcgcgagcccgacgccgacg cccaccacgccgaccccgacgcccacgacgccgacgccgaccccgacgtccggtccggcc gggtgccaggtgctgtggggcgtcaaccagtggaacaccggcttcaccgcgaacgtcacc gtgaagaacacgtcctccgctccggtcgacggctggacgctcacgttcagcttcccgtcc ggccagcaggtcacccaggcgtggagctcgacggtcacgcagtccggctcggccgtgacg gtccgcaacgccccgtggaacggctcgatcccggcgggcggcaccgcgcagttcggcttc aacggctcgcacacgggcaccaacgccgcgccgacggcgttctcgctcaacggcacgccc
tgcacggtcggctc

TACTAGC aacatatcataacggagtgatcgcaatg GGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata

crtEIB (expected)

• I have assumed spacerT7 - PlacLacZ - spacer1 - E - spacer2 - I - spacer3 - B - spacer4 - stuffing

GAATTCGCGGCCGCTTCTAGAG

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

TACTAGCtgatatttcgcgtaaggaaatccattatgGGATCT

atgacggtctgcgcaaaaaaacacgttcatctcactcgcgatgctgcggagcagttactg gctgatattgatcgacgccttgatcagttattgcccgtggagggagaacgggatgttgtg ggtgccgcgatgcgtgaaggtgcgctggcaccgggaaaacgtattcgccccatgttgctg ttgctgaccgcccgcgatctgggttgcgctgtcagccatgacggattactggatttggcc tgtgcggtggaaatggtccacgcggcttcgctgatccttgacgatatgccctgcatggac gatgcgaagctgcggcgcggacgccctaccattcattctcattacggagagcatgtggca atactggcggcggttgccttgctgagtaaagcctttggcgtaattgccgatgcagatggc ctcacgccgctggcaaaaaatcgggcggtttctgaactgtcaaacgccatcggcatgcaa ggattggttcagggtcagttcaaggatctgtctgaaggggataagccgcgcagcgctgaa gctattttgatgacgaatcactttaaaaccagcacgctgttttgtgcctccatgcagatg gcctcgattgttgcgaatgcctccagcgaagcgcgtgattgcctgcatcgtttttcactt gatcttggtcaggcatttcaactgctggacgatttgaccgatggcatgaccgacaccggt aaggatagcaatcaggacgccggtaaatcgacgctggtcaatctgttaggcccgagggcg gttgaagaacgtctgagacaacatcttcagcttgccagtgagcatctctctgcggcctgc caacacgggcacgccactcaacattttattcaggcctggtttgacaaaaaactcgctgcc
gtcagt gg

TACTAGCaacatatcataacggagtgatcgcaatgGGATCT

atgaaaccaactacggtaattggtgcaggcttcggtggcctggcactggcaattcgtcta caagctgcggggatccccgtcttactgcttgaacaacgtgataaacccggcggtcgggct tatgtctacgaggatcaggggtttacctttgatgcaggcccgacggttatcaccgatccc agtgccattgaagaactgtttgcactggcaggaaaacagttaaaagagtatgtcgaactg ctgccggttacgccgttttaccgcctgtgttgggagtcagggaaggtctttaattacgat aacgatcaaacccggctcgaagcgcagattcagcagtttaatccccgcgatgtcgaaggt tatcgtcagtttctggactattcacgcgcggtgtttaaagaaggctatctaaagctcggt actgtcccttttttatcgttcagagacatgcttcgcgccgcacctcaactggcgaaactg caagcatggagaagcgtttacagtaaggttgccagttacatcgaagatgaacatctgcgc caggcgttttctttccactcgctgttggtgggcggcaatcccttcgccacctcatccatt tatacgttgatacacgcgctggagcgtgagtggggcgtctggtttccgcgtggcggcacc ggcgcattagttcaggggatgataaagctgtttcaggatctgggtggcgaagtcgtgtta aacgccagagtcagccatatggaaacgacaggaaacaagattgaagccgtgcatttagag gacggtcgcaggttcctgacgcaagccgtcgcgtcaaatgcagatgtggttcatacctat cgcgacctgttaagccagcaccctgccgcggttaagcagtccaacaaactgcaaactaag cgcatgagtaactctctgtttgtgctctattttggtttgaatcaccatcatgatcagctc gcgcatcacacggtttgtttcggcccgcgttaccgcgagctgattgacgaaatttttaat catgatggcctcgcagaggacttctcactttatctgcacgcgccctgtgtcacggattcg tcactggcgcctgaaggttgcggcagttactatgtgttggcgccggtgccgcatttaggc accgcgaacctcgactggacggttgaggggccaaaactacgcgaccgtatttttgcgtac cttgagcagcattacatgcctggcttacggagtcagctggtcacgcaccggatgtttacg ccgtttgattttcgcgaccagcttaatgcctatcatggctcagccttttctgtggagccc gttcttacccagagcgcctggtttcggccgcataaccgcgataaaaccattactaatctc tacctggtcggcgcaggcacgcatcccggcgcaggcattcctggcgtcatcggctcggca
aaagcgacagcaggtttgatgctggaggatctgata gg

TACTAGCtgaacgtaaatactcagtaaagaggagaaaGGATCT

atgaataatccgtcgttactcaatcatgcggtcgaaacgatggcagttggctcgaaaagt tttgcgacagcctcaaagttatttgatgcaaaaacccggcgcagcgtactgatgctctac gcctggtgccgccattgtgacgatgttattgacgatcagacgctgggctttcaggcccgg cagcctgccttacaaacgcccgaacaacgtctgatgcaacttgagatgaaaacgcgccag gcctatgcaggatcgcagatgcacgaaccggcgtttgcggcttttcaggaagtggctatg gctcatgatatcgccccggcttacgcgtttgatcatctggaaggcttcgccatggatgta cgcgaagcgcaatacagccaactggatgatacgctgcgctattgctatcacgttgcaggc gttgtcggcttgatgatggcgcaaatcatgggcgtgcgggataacgccacgctggaccgc gcctgtgaccttgggctggcatttcagttgaccaatattgctcgcgatattgtggacgat gcgcatgcgggccgctgttatctgccggcaagctggctggagcatgaaggtctgaacaaa gagaattatgcggcacctgaaaaccgtcaggcgctgagccgtatcgcccgtcgtttggtg caggaagcagaaccttactatttgtctgccacagccggcctggcagggttgcccctgcgt tccgcctgggcaatcgctacggcgaagcaggtttaccggaaaataggtgtcaaagttgaa caggccggtcagcaagcctgggatcagcggcagtcaacgaccacgcccgaaaaattaacg ctgctgctggccgcctctggtcaggcccttacttcccggatgcgggctcatcctccccgc
cctgcgcatctctggcagcgcccgctc gg

TACTAGCtaggtcttagtcttcgtcaaagaggggacaGGATCT

gttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctt
tctgcgtttata

Vector piece, with PlacLacZ-INP to amplify for Gibson

( gtcacactggctcaccttcgggtgggcctttctgcgtttata )

tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgcccttt ttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagata ccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctg taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac ttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgag cgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcc aagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaatt cattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcga agaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcac tccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacac tatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattca tcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacgg tctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatc cagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaa atacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgcccga tcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat
gatttctggaattcgcggccgcttctagag

ttaatacgactcactatagggagaggaggtacgatct

gctggggcaaaccagcgtggacc
gcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcac tggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttgg ccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgctt ccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctat gaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctgg cgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcga agaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga

tactagctgatatttcgcgtaaggaaatccattatgGGATCT

atgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgt ggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcgt cacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgtt catgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctggaa gaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattagc gcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctct accagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctct gttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtct gctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggcg acctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctct aacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggca ggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttct aaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatcttt cgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtg gaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacgaa
gatgatgattggatcgaagtgaaa

ggtactagcccgacgaccagc