LOCUS       sequence-479533-        5593 bp    DNA     circular SYN 24-JUL-2025
DEFINITION  bacterial expression of human TTR mutants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5593)
  AUTHORS   Sun X, Ferguson JA, Leach BI, Stanfield RL, Dyson HJ, Wright PE
  TITLE     Probing the Dissociation Pathway of a Kinetically Labile 
            Transthyretin Mutant.
  JOURNAL   J Am Chem Soc. 2024 Jan 10;146(1):532-542. doi: 
            10.1021/jacs.3c10083. Epub 2023 Dec 22.
  PUBMED    38134439
REFERENCE   2  (bases 1 to 5593)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/jacs.3c10083"; journalName: "J Am Chem Soc"; date: 
            "2024-01-10- 10"; volume: "146"; issue: "1"; pages: "532-542"
COMMENT     Sequence Label: TTR C10S/S85C/A25T
FEATURES             Location/Qualifiers
     source          1..5593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     RBS             530..552
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    567..591
                     /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(591..610)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(592..610)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(698..717)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        923..1000
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1001..2083
                     /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(1020..1039)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2892..3083
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3077..3099)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3185..3324
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3240..3259
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3339..3356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3510..4098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3590..3609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4209..4228)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4220..5035
                     /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             4220..5035
                     /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(4293..4312)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5127..5582)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5264..5285)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5476..5495
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgtagcagcc ggatctcatt ccttgggatt ggtgacgaca gccgtggtgg aataggagta
      181 ggggctcagc agggcggcaa tggtgtagcg gcgggggccg gagtcgttgg ctgtgaatac
      241 cacctctgca tgctcatgga atgggcagat gccaagtgcc ttccagtaag atttggtgtc
      301 tatttccact ttgtatatcc cttctacaaa ttcctcctca gttgtgagcc catgcagctc
      361 tccagactca ctggttttcc cagaggcaaa tggctcccag gtgtcatcag cagcctttct
      421 gaacacatgc acggccacat tgatggtagg actgcctcgg acagcatcta gaactttgac
      481 catcagagga ctcttggatt caccggtgcc cgtaggaccc atatgtatat ctccttctta
      541 aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc ccctatagtg
      601 agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg acgcatcgtg
      661 gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat
      721 ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg
      781 gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg
      841 gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat
      901 aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc
      961 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     1021 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     1081 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     1141 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     1201 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     1261 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     1321 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     1381 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     1441 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     1501 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     1561 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     1621 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     1681 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     1741 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     1801 catctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     1861 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     1921 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     1981 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     2041 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2101 taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc
     2161 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct
     2221 tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg
     2281 accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc
     2341 acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg
     2401 ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga
     2461 cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc
     2521 gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct
     2581 tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt
     2641 tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa
     2701 cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca
     2761 gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg
     2821 tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca
     2881 cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa
     2941 gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca
     3001 tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg
     3061 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3121 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3181 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     3241 ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc gcacagatgc
     3301 gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3361 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3421 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3481 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3541 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3601 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3661 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     3721 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     3781 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     3841 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     3901 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     3961 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4021 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4081 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4141 aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg cttacataaa
     4201 cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct ctaggccgcg
     4261 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     4321 gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct
     4381 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     4441 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     4501 atggttactc accactgcga tccccgggaa aacagcattc caggtattag aagaatatcc
     4561 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     4621 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     4681 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     4741 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     4801 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     4861 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     4921 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     4981 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaagaatt
     5041 aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     5101 gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc
     5161 gttaaatttt tgttaaatca gctcattttt taaccaatag gccaaaatcg gcaaaatccc
     5221 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     5281 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     5341 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     5401 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     5461 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     5521 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     5581 gtcccattcg cca
//
