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| | ===Design Notes=== | | ===Design Notes=== |
| − | The choice of the linker, lipocalin tag and purification tag allows us serval different combinations. | + | The cloning steps were planned theoretically before we started the work in the wet lab. |
| − | | + | We used the [https://parts.igem.org/Part:BBa_K243005 middle linker] because its length of 24bp is a bit shorter than the split linker we used. We had to find a compromise between connection stability and possible sterical interference between the single parts. |
| | + | The parts are fused according to [https://parts.igem.org/Assembly_standard_25 RFC 25]. |
| | | | |
| | + | [https://static.igem.org/mediawiki/parts/2/24/Freiburg09_Strediglonglifoki.txt Commented GenBank file] |
| | | | |
| | ===Source=== | | ===Source=== |
| − | <html>
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| | Combined the parts by serial cloning steps. | | Combined the parts by serial cloning steps. |
| − |
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| − | LOCUS Strep+Dig+middel 3625 bp DNA circular SYN 13-OCT-2009
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| − | DEFINITION Ligation of Strep+Dig+middellinker+FokI into pMA2_NgoMIV_SpeI
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| − | ACCESSION Strep+Dig+middel
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| − | KEYWORDS .
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| − | SOURCE Unknown.
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| − | ORGANISM Unknown
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| − | Unclassified.
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| − | REFERENCE 1 (bases 1 to 3625)
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| − | AUTHORS Self
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| − | JOURNAL Unpublished.
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| − | COMMENT SECID/File created by SciEd Central, Scientific & Educational Software
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| − | COMMENT SECNOTES|Vector molecule: pMA2_NgoMIV_SpeI
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| − | Fragment ends: SpeI and NgoMIV
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| − | Fragment size: 2412
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| − | Insert molecule: Strep+Dig+middellinker+FokI
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| − | Fragment ends: NgoMIV and SpeI
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| − | Fragment size: 1213
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| − | FEATURES Location/Qualifiers
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| − | CDS 129..989
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| − | /gene="AmpR"
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| − | /SECDrawAs="Gene"
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| − | CDS 2008..3222
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| − | /gene="ORF-1"
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| − | /SECDrawAs="Gene"
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| − | CDS 2014..2041
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| − | /gene="'Strep-Tag'"
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| − | /SECDrawAs="Gene"
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| − | CDS 2044..2599
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| − | /gene="'Dig'"
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| − | /SECDrawAs="Gene"
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| − | CDS 2602..2628
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| − | /gene="'middelLinker38"
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| − | /SECDrawAs="Gene"
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| − | CDS 2632..3219
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| − | /gene="'FokI"
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| − | /SECDrawAs="Gene"
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| − | ORIGIN
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| − | 1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
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| − | 61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
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| − | 121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
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| − | 181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
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| − | 241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
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| − | 301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
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| − | 361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
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| − | 421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
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| − | 481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
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| − | 541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
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| − | 601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
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| − | 661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
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| − | 721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
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| − | 781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
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| − | 841 gtggttctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
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| − | 901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
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| − | 961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
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| − | 1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
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| − | 1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
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| − | 1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
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| − | 1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
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| − | 1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
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| − | 1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
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| − | 1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
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| − | 1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
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| − | 1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
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| − | 1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
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| − | 1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
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| − | 1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
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| − | 1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
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| − | 1801 ctcattaggc accccaggct ttacccgaac gaccgagcgc agcgagtcag tgagcgagga
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| − | 1861 agcggagagc gcccaatacg caaggaaaca gctatgacca tgttaatgca gctggcacga
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| − | 1921 caggtttccc gactggaaag cgggcagtga gcggaaggcc catgaggcca gttaattaag
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| − | 1981 aggtaccgaa ttcgcggccg cttctagatg gccggctgga gccatccgca gtttgaaaaa
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| − | 2041 accggcgacg tgtaccacga cggcgcctgc cccgaagtga agcccgtgga caacttcgac
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| − | 2101 tggtcccagt accacggcaa gtggtggcag gtggccgctt atcccgacca catcaccaag
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| − | 2161 tacggcaagt gcggctgggc cgagtacacc cccgagggca agagcgtgaa ggtgtcccgg
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| − | 2221 tacagcgtga tccacggcaa agagtacttc agcgagggca ccgcctaccc tgtgggcgac
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| − | 2281 agcaagatcg gcaagatcta ccacagctac accatcggcg gcgtgaccca ggtgggcgtg
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| − | 2341 agcaacgtgc tgtccaccga caacaagaac tacatcatcg gctacttttg cagatacgac
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| − | 2401 gaggacaaga agggccactg ggacgccgtg tgggtgctgt cccggtccat ggtgctgacc
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| − | 2461 ggcgaggcca agaccgccgt ggagaactac ctgatcggca gccccgtggt ggacagccag
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| − | 2521 aaactggtgt acagcgactt ctccgaggcc gcctgcaaag tgaacaacag caactggtcc
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| − | 2581 cacccccagt tcgaaaagac cggcggtggt tctggtggtg gttctggtac cggcaaatct
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| − | 2641 gaactggagg agaaaaaatc cgagctgcgc cacaaactga aatatgtgcc tcacgagtat
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| − | 2701 atcgaactga tcgagatcgc ccgtaatagt acccaagacc gtatcctgga aatgaaagtg
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| − | 2761 atggagttct tcatgaaagt ctatggctat cgtggcaaac atctgggtgg tagccgtaaa
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| − | 2821 ccagcaggtg ccatttatac cgttggttcc ccgatcgatt atggcgttat cgtggccaca
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| − | 2881 aaagcgtatt ctggcggtta taatctgccg attggtcagg ctgatgagat ggaacgttat
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| − | 2941 gtggaagaga atcagacccg taacaaacat ctgaacccga acgaatggtg gaaagtgtat
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| − | 3001 ccgtcaagtg tcaccgagtt caaatttctg ttcgtgagcg gccactttaa aggcaactat
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| − | 3061 aaagcccagc tgactcgtct gaaccatatc accaatagca atggggcagt gctgagtgtt
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| − | 3121 gaggaactgc tgatcggtgg agaaatgatc aaagcaggca ccctgactct ggaagaagtt
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| − | 3181 cgccgtaaat tcaacaatgg cgagatcaat tttaccggtt aatactagta gcggccgctg
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| − | 3241 cagagctcat ggcgcgccta ggccttgacg gccttccgcc aattcgccct atagtgagtc
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| − | 3301 gtattacgtc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
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| − | 3361 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
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| − | 3421 aggcccgcac cgaaacgccc ttcccaacag ttgcgcagcc tgaatggcga atgggagcgc
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| − | 3481 cctgtagcgg ccactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
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| − | 3541 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
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| − | 3601 caaaatatta acgcttacaa tttag
| |
| − | //
| |
| | | | |
| | ===References=== | | ===References=== |
The cloning steps were planned theoretically before we started the work in the wet lab.
We used the middle linker because its length of 24bp is a bit shorter than the split linker we used. We had to find a compromise between connection stability and possible sterical interference between the single parts.
The parts are fused according to RFC 25.
