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.

Contents 1 Biobrick prefix, suffix, scar 2 Restriction sites 3 pSB1C3 vector 4 Plac-LacZα insert (clone 2) 5 malS using primers f1/r1 (clone 1) 6 malS using primers f2/r2 (clone 1) 7 bglX using primers f1/r1 (clone 1) 8 bglX using primers f2/r2 (clone 1) 9 INP in RFC12 format (clone 2) 10 pVIII 11 C. fimi exoglucanase (clone 2) 12 pG8SAET 13 pSB1C3 for BioSandwich 14 BioSandwich test with PlacLacZ and EYFP 15 Alternate BioSandwich test with PlacLacZ and EYFP 16 Serendipitous PlacLacZ-INP construct 17 PlacLacZ - INP - YFP (expected) 18 PlacLacZ - INP - bglX (expected) 19 PlacLacZ - INP - cex (expected) 20 crtEIB (expected) 21 Vector piece, with PlacLacZ-INP to amplify for Gibson

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 ([http://partsregistry.org/wiki/index.php?title=Part:pSB1C3 here])
• Red is CmlR coding sequence ([http://en.wikipedia.org/wiki/Chloramphenicol_acetyltransferase 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_J33207 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 [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=3735520&to=3737550&report=gbwithparts 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 [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=3735520&to=3737550&report=gbwithparts 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 [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=2217714&to=2220011&report=gbwithparts 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 [http://www.ncbi.nlm.nih.gov/nuccore/48994873?from=2217714&to=2220011&report=gbwithparts 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 [http://dspace.mit.edu/bitstream/handle/1721.1/45139/BBFRFC12.txt?sequence=1 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 [http://www.ncbi.nlm.nih.gov/nuccore/56713234?report=genbank 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 [http://www.ncbi.nlm.nih.gov/nuccore/AF130864.1 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 [http://www.ncbi.nlm.nih.gov/nuccore/EF455822.1 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