LOCUS       sequence-486766-        5592 bp    DNA     circular SYN 19-SEP-2025
DEFINITION  bacterial expression of human TTR mutants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5592)
  AUTHORS   Sun X, Dyson HJ, Wright PE
  TITLE     Kinetic analysis of the multistep aggregation pathway of human 
            transthyretin.
  JOURNAL   Proc Natl Acad Sci U S A. 2018 Jul 3;115(27):E6201-E6208. doi: 
            10.1073/pnas.1807024115. Epub 2018 Jun 18.
  PUBMED    29915031
REFERENCE   2  (bases 1 to 5592)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1807024115"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2018-07-3- 3"; volume: "115"; issue: "27"; pages: 
            "E6201-E6208"
COMMENT     Sequence Label: TTR C10S/S85C/V30M
FEATURES             Location/Qualifiers
     source          1..5592
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(599..1054)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(736..757)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     948..967
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(1091..1138)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1134..1152
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             1595..1617
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1632..1656
                     /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)."
     primer_bind     complement(1656..1675)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1657..1675)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1763..1782)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1988..2065
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2066..3148
                     /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     complement(2085..2104)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3957..4148
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4142..4164)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4250..4389
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4305..4324
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4404..4421)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4575..5163)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4655..4674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5274..5293)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5358..5377)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
       61 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
      121 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
      181 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
      241 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
      301 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
      361 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
      421 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
      481 catttgatgc tcgatgagtt tttctaagaa ttaattcatg agcggataca tatttgaatg
      541 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
      601 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
      661 tttaaccaat aggccaaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
      721 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
      781 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
      841 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
      901 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
      961 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     1021 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccaatccg gatatagttc
     1081 ctcctttcag caaaaaaccc ctcaagaccc gtttagaggc cccaaggggt tatgctagtt
     1141 attgctcagc ggtggcagca gccaactcag cttcctttcg ggctttgtag cagccggatc
     1201 tcattccttg ggattggtga cgacagccgt ggtggaatag gagtaggggc tcagcagggc
     1261 ggcaatggtg tagcggcggg ggccggagtc gttggctgtg aataccacct ctgcatgctc
     1321 atggaatggg cagatgccaa gtgccttcca gtaagatttg gtgtctattt ccactttgta
     1381 tatcccttct acaaattcct cctcagttgt gagcccatgc agctctccag actcactggt
     1441 tttcccagag gcaaatggct cccaggtgtc atcagcagcc tttctgaaca catgcatggc
     1501 cacattgatg gcaggactgc ctcggacagc atctagaact ttgaccatca gaggactctt
     1561 ggattcaccg gtgcccgtag gacccatatg tatatctcct tcttaaagtt aaacaaaatt
     1621 atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc
     1681 gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     1741 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     1801 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     1861 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     1921 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     1981 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     2041 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     2101 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     2161 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     2221 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     2281 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     2341 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     2401 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     2461 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     2521 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     2581 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     2641 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     2701 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2761 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2821 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct cggtagtggg
     2881 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     2941 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     3001 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     3061 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     3121 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     3181 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     3241 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     3301 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     3361 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     3421 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     3481 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     3541 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     3601 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     3661 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3721 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     3781 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     3841 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     3901 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     3961 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     4021 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     4081 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     4141 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4201 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     4261 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     4321 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4381 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4441 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4501 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4561 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4621 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4681 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4741 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4801 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4861 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4921 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4981 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5041 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5101 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5161 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5221 taagggattt tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     5281 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat
     5341 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     5401 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     5461 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     5521 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     5581 tgcgatcccc gg
//
