LOCUS       sequence-474749-        5593 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of human TTR mutants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5593)
  AUTHORS   Leach BI, Ferguson JA, Morgan G, Sun X, Kroon G, Oyen D, Dyson HJ, 
            Wright PE
  TITLE     Conformational Dynamics of an Amyloidogenic Intermediate of 
            Transthyretin: Implications for Structural Remodeling and Amyloid 
            Formation.
  JOURNAL   J Mol Biol. 2024 Aug 15;436(16):168673. doi: 
            10.1016/j.jmb.2024.168673. Epub 2024 Jun 21.
  PUBMED    38909653
REFERENCE   2  (bases 1 to 5593)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Mol 
            Biol."; date: "2024-08-15"; volume: "436(16):168673. doi"; pages: " 
            10.1016/j.jmb.2024.168673. Epub 2024 Jun 21"
COMMENT     Sequence Label: TTR C10S S85C F87E
FEATURES             Location/Qualifiers
     source          1..5593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(326..1408)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1370..1389
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1409..1486)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1692..1711
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1799..1818
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1799..1817
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1818..1842
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1857..1879
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(2322..2340)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2336..2383
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2420..2875
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2507..2526)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2717..2738
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2967..3782)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2967..3782)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3690..3709
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3774..3793
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3904..4492
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4393..4412
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4646..4663
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4678..4817
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4743..4762)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4903..4925
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4919..5110)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
       61 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
      121 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
      181 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
      241 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
      301 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      361 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
      421 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
      481 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
      541 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
      601 ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
      661 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
      721 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
      781 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
      841 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
      901 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
      961 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     1021 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     1081 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     1141 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     1201 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     1261 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     1321 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     1381 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     1441 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     1501 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     1561 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     1621 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     1681 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     1741 atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg atctcgatcc cgcgaaatta
     1801 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     1861 tttaacttta agaaggagat atacatatgg gtcctacggg caccggtgaa tccaagagtc
     1921 ctctgatggt caaagttcta gatgctgtcc gaggcagtcc tgccatcaat gtggccgtgc
     1981 atgtgttcag aaaggctgct gatgacacct gggagccatt tgcctctggg aaaaccagtg
     2041 agtctggaga gctgcatggg ctcacaactg aggaggaatt tgtagaaggg atatacaaag
     2101 tggaaataga caccaaatct tactggaagg cacttggcat ctgcccagag catgagcatg
     2161 cagaggtggt attcacagcc aacgactccg gcccccgccg ctacaccatt gccgccctgc
     2221 tgagccccta ctcctattcc accacggctg tcgtcaccaa tcccaaggaa tgagatccgg
     2281 ctgctacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc
     2341 ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat
     2401 atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2461 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2521 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2581 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2641 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     2701 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     2761 gtctattctt ttgatttata agggattttg ccgattttgg cctattggtt aaaaaatgag
     2821 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg
     2881 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2941 atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc
     3001 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     3061 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     3121 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     3181 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt
     3241 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     3301 accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta
     3361 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     3421 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     3481 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     3541 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     3601 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     3661 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     3721 gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc
     3781 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat
     3841 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     3901 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     3961 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4021 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     4081 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4141 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4201 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4261 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4321 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4381 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4441 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4501 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     4561 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     4621 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct
     4681 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc
     4741 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
     4801 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     4861 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4921 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     4981 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     5041 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     5101 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     5161 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg
     5221 aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc
     5281 agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc
     5341 atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac
     5401 tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc
     5461 agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc
     5521 aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg
     5581 ccgccatgcc ggc
//
