LOCUS       sequence-486765-        5593 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 5593)
  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 5593)
  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/L55P
FEATURES             Location/Qualifiers
     source          1..5593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1739..1930
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1924..1946)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2032..2171
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2087..2106
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2186..2203)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2357..2945)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2437..2456)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3056..3075)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3067..3882
                     /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             3067..3882
                     /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(3140..3159)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3974..4429)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4111..4132)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4323..4342
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4466..4513)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4509..4527
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             4970..4992
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    5007..5031
                     /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(5031..5050)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5032..5050)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5138..5157)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        5363..5440
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(5441..5593,1..930)
                     /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(5460..5479)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
       61 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
      121 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
      181 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
      241 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
      301 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
      361 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
      421 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
      481 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
      541 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
      601 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg
      661 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
      721 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
      781 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
      841 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      901 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
      961 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     1021 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     1081 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     1141 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     1201 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     1261 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     1321 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     1381 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     1441 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     1501 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     1561 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     1621 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     1681 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     1741 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     1801 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     1861 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     1921 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     1981 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     2041 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     2101 attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat
     2161 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2221 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2281 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2341 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2401 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2461 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2521 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2581 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2641 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2701 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     2761 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     2821 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     2881 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     2941 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3001 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     3061 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     3121 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     3181 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     3241 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     3301 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     3361 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     3421 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     3481 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     3541 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     3601 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     3661 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     3721 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     3781 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     3841 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
     3901 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     3961 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     4021 taaatcagct cattttttaa ccaataggcc aaaatcggca aaatccctta taaatcaaaa
     4081 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     4141 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4201 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4261 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4321 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4381 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     4441 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
     4501 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
     4561 tgtagcagcc ggatctcatt ccttgggatt ggtgacgaca gccgtggtgg aataggagta
     4621 ggggctcagc agggcggcaa tggtgtagcg gcgggggccg gagtcgttgg ctgtgaatac
     4681 cacctctgca tgctcatgga atgggcagat gccaagtgcc ttccagtaag atttggtgtc
     4741 tatttccact ttgtatatcc cttctacaaa ttcctcctca gttgtgagcc catgcggctc
     4801 tccagactca ctggttttcc cagaggcaaa tggctcccag gtgtcatcag cagcctttct
     4861 gaacacatgc acggccacat tgatggcagg actgcctcgg acagcatcta gaactttgac
     4921 catcagagga ctcttggatt caccggtgcc cgtaggaccc atatgtatat ctccttctta
     4981 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
     5041 agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg
     5101 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
     5161 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
     5221 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
     5281 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
     5341 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
     5401 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     5461 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     5521 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     5581 cattcccaac cgc
//
