LOCUS       sequence-486768-        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/V122I
FEATURES             Location/Qualifiers
     source          1..5593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             486..677
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(671..693)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    779..918
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     834..853
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(933..950)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1104..1692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1184..1203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1803..1822)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1814..2629
                     /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             1814..2629
                     /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(1887..1906)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2721..3176)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2858..2879)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3070..3089
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3213..3260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3256..3274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             3717..3739
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3754..3778
                     /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(3778..3797)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3779..3797)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3885..3904)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4110..4187
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4188..5270
                     /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(4207..4226)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
       61 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
      121 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
      181 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
      241 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
      301 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
      361 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
      421 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
      481 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
      541 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
      601 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
      661 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
      721 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
      781 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
      841 agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg
      901 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
      961 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1021 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1081 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1141 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1201 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1261 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     1321 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     1381 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     1441 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     1501 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     1561 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     1621 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     1681 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     1741 aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa
     1801 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa
     1861 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     1921 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     1981 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     2041 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     2101 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     2161 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     2221 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     2281 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     2341 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     2401 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     2461 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     2521 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     2581 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca
     2641 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     2701 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     2761 tttttgttaa atcagctcat tttttaacca ataggccaaa atcggcaaaa tcccttataa
     2821 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     2881 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     2941 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     3001 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     3061 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     3121 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     3181 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     3241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     3301 cgggctttgt agcagccgga tctcattcct tgggattggt gatgacagcc gtggtggaat
     3361 aggagtaggg gctcagcagg gcggcaatgg tgtagcggcg ggggccggag tcgttggctg
     3421 tgaataccac ctctgcatgc tcatggaatg ggcagatgcc aagtgccttc cagtaagatt
     3481 tggtgtctat ttccactttg tatatccctt ctacaaattc ctcctcagtt gtgagcccat
     3541 gcagctctcc agactcactg gttttcccag aggcaaatgg ctcccaggtg tcatcagcag
     3601 cctttctgaa cacatgcacg gccacattga tggcaggact gcctcggaca gcatctagaa
     3661 ctttgaccat cagaggactc ttggattcac cggtgcccgt aggacccata tgtatatctc
     3721 cttcttaaag ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc
     3781 tatagtgagt cgtattaatt tcgcgggatc gagatcgatc tcgatcctct acgccggacg
     3841 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
     3901 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
     3961 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
     4021 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
     4081 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
     4141 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     4201 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     4261 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     4321 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     4381 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     4441 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     4501 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     4561 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     4621 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     4681 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     4741 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     4801 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     4861 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     4921 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     4981 gtgcggacat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     5041 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     5101 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     5161 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     5221 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     5281 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
     5341 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     5401 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     5461 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     5521 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     5581 aagcaggcca tta
//
