LOCUS       sequence-474748-        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 F87E
FEATURES             Location/Qualifiers
     source          1..5593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(440..458)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      454..501
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      538..993
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(625..644)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     835..856
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1085..1900)
                     /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(1085..1900)
                     /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     1808..1827
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1892..1911
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2022..2610
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2511..2530
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2764..2781
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2796..2935
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2861..2880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3021..3043
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3037..3228)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4037..5119)
                     /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     5081..5100
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5120..5197)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5403..5422
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5510..5529
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5510..5528
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5529..5553
                     /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             5568..5590
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 acatatgggt cctacgggca ccggtgaatc caagtgtcct ctgatggtca aagttctaga
       61 tgctgtccga ggcagtcctg ccatcaatgt ggccgtgcat gtgttcagaa aggctgctga
      121 tgacacctgg gagccatttg cctctgggaa aaccagtgag tctggagagc tgcatgggct
      181 cacaactgag gaggaatttg tagaagggat atacaaagtg gaaatagaca ccaaatctta
      241 ctggaaggca cttggcatct ccccagagca tgagcatgca gaggtggtat tcacagccaa
      301 cgactccggc ccccgccgct acaccattgc cgccctgctg agcccctact cctattccac
      361 cacggctgtc gtcaccaatc ccaaggaatg agatccggct gctacaaagc ccgaaaggaa
      421 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
      481 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
      541 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
      601 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
      661 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
      721 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
      781 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
      841 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
      901 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
      961 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1021 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1081 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1141 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     1201 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     1261 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     1321 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     1381 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     1441 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     1501 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     1561 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     1621 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     1681 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     1741 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     1801 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     1861 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     1921 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     1981 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2041 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2101 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2161 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2221 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2281 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2341 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2401 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2461 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2521 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2581 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2641 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2701 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     2761 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     2821 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     2881 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     2941 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3001 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3061 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3121 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3181 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3241 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     3301 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3361 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     3421 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     3481 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     3541 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     3601 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     3661 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     3721 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     3781 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     3841 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     3901 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     3961 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4021 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4081 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4141 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4201 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4261 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     4321 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4381 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4441 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     4501 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     4561 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     4621 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4681 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4741 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     4801 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     4861 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     4921 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     4981 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5041 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5101 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5161 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5221 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5281 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5341 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5401 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     5461 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt aatacgactc
     5521 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
     5581 aagaaggaga tat
//
