LOCUS       sequence-486769-        5592 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 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/T119M
FEATURES             Location/Qualifiers
     source          1..5592
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(60..81)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     272..291
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(415..462)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     458..476
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             919..941
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    956..980
                     /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(980..999)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(981..999)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1087..1106)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1312..1389
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1390..2472
                     /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(1409..1428)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3281..3472
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3466..3488)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3574..3713
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3629..3648
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3728..3745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3899..4487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3979..3998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4598..4617)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4609..5424
                     /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             4609..5424
                     /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(4682..4701)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5516..5592,1..378))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
       61 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
      121 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
      181 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
      241 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
      301 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
      361 cgccgctaca gggcgcgtcc cattcgccaa tccggatata gttcctcctt tcagcaaaaa
      421 acccctcaag acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc
      481 agcagccaac tcagcttcct ttcgggcttt gtagcagccg gatctcattc cttgggattg
      541 gtgacgacag ccatggtgga ataggagtag gggctcagca gggcggcaat ggtgtagcgg
      601 cgggggccgg agtcgttggc tgtgaatacc acctctgcat gctcatggaa tgggcagatg
      661 ccaagtgcct tccagtaaga tttggtgtct atttccactt tgtatatccc ttctacaaat
      721 tcctcctcag ttgtgagccc atgcagctct ccagactcac tggttttccc agaggcaaat
      781 ggctcccagg tgtcatcagc agcctttctg aacacatgca cggccacatt gatggcagga
      841 ctgcctcgga cagcatctag aactttgacc atcagaggac tcttggattc accggtgccc
      901 gtaggaccca tatgtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
      961 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatcga
     1021 tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
     1081 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
     1141 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
     1201 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     1261 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc ggacaccatc
     1321 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     1381 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     1441 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     1501 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     1561 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     1621 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     1681 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     1741 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     1801 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     1861 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     1921 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     1981 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     2041 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     2101 gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt
     2161 accgagtccg ggctgcgcgt tggtgcggac atctcggtag tgggatacga cgataccgaa
     2221 gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg
     2281 caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag
     2341 ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc
     2401 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     2461 agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg caccgggatc
     2521 tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg cgcggggcat
     2581 gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag gacaggtgcc
     2641 ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg
     2701 cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg tcactggtcc
     2761 cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg ccccacgggt
     2821 gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg
     2881 ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc
     2941 tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac
     3001 gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct
     3061 accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt
     3121 tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg
     3181 ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat
     3241 tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac
     3301 cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa
     3361 cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga
     3421 gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     3481 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     3541 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     3601 tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
     3661 cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt
     3721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4321 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4561 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     4621 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat
     4681 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat
     4741 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     4801 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     4861 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa
     4921 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     4981 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     5041 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     5101 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     5161 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     5221 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     5281 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     5341 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     5401 ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat
     5461 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
     5521 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     5581 aaccaatagg cc
//
