LOCUS       sequence-486767-        5591 bp    DNA     circular SYN 19-SEP-2025
DEFINITION  bacterial expression of TTR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5591)
  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 5591)
  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/F87A
FEATURES             Location/Qualifiers
     source          1..5591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             269..291
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    306..330
                     /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(330..349)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(331..349)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(437..456)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        662..739
                     /gene="lacI"
                     /label=lacI promoter
     CDS             740..1822
                     /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(759..778)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2631..2822
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2816..2838)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2924..3063
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2979..2998
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3078..3095)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3249..3837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3329..3348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3948..3967)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3959..4774
                     /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             3959..4774
                     /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     complement(4032..4051)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4866..5321)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5003..5024)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5215..5234
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(5358..5405)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     5401..5419
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
ORIGIN
        1 tgggcagatg ccaagtgcct tccagtaaga tttggtgtct atttccactt tgtatatccc
       61 ttctacaaat tcctcctcag ttgtgagccc atgcagctct ccagactcac tggttttccc
      121 agaggcaaat ggctcccagg tgtcatcagc agcctttctg aacacatgca cggccacatt
      181 gatggcagga ctgcctcgga cagcatctag aactttgacc atcagaggac tcttggattc
      241 accggtgccc gtaggaccca tatgtatatc tccttcttaa agttaaacaa aattatttct
      301 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
      361 tcgagatcga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
      421 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
      481 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
      541 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
      601 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
      661 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
      721 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
      781 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
      841 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
      901 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
      961 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1021 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1081 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1141 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1201 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     1261 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     1321 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     1381 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     1441 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     1501 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga
     1561 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     1621 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     1681 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     1741 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     1801 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     1861 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     1921 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     1981 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     2041 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     2101 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     2161 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     2221 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     2281 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     2341 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     2401 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     2461 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     2521 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     2581 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     2641 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     2701 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     2761 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     2821 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     2881 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     2941 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     3001 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3061 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3121 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3181 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3241 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3301 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3361 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3421 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3481 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3541 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     3601 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     3661 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     3721 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     3781 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3841 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3901 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     3961 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     4021 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4081 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4141 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4201 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     4261 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     4321 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     4381 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     4441 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     4501 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     4561 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     4621 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     4681 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     4741 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     4801 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4861 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     4921 ctcatttttt aaccaatagg ccaaaatcgg caaaatccct tataaatcaa aagaatagac
     4981 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5041 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5101 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5161 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5221 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     5281 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc caatccggat
     5341 atagttcctc ctttcagcaa aaaacccctc aagacccgtt tagaggcccc aaggggttat
     5401 gctagttatt gctcagcggt ggcagcagcc aactcagctt cctttcgggc tttgtagcag
     5461 ccggatctca ttccttggga ttggtgacga cagccgtggt ggaataggag taggggctca
     5521 gcagggcggc aatggtgtag cggcgggggc cggagtcgtt ggctgtgaat accacctctg
     5581 catgctcatg g
//
