LOCUS       sequence-479081-        5863 bp    DNA     circular SYN 21-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 5863)
  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 5863)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 21, 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: His6-tev-G(TTR A25T/I26V)
FEATURES             Location/Qualifiers
     source          1..5863
                     /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"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(590..610)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(641..658)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             671..693
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    708..732
                     /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(732..751)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(733..751)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(835..854)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1060..1137
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1138..2220
                     /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(1157..1176)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3029..3220
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3214..3236)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3322..3461
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3377..3396
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3476..3493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3647..4235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3727..3746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4406..5197,5198..5266))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5029..5048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5267..5371)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5397..5852)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5534..5555)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5746..5765
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggct gcaagcttgt
      181 cgacggagct cgaattcgga tcctattatt ccttgggatt ggtgacgaca gccgtggtgg
      241 aataggagta ggggctcagc agggcggcaa tggtgtagcg gcgggggccg gagtcgttgg
      301 ctgtgaatac cacctctgca tgctcatgga atggggagat gccaagtgcc ttccagtaag
      361 atttggtgtc tatttccact ttgtatatcc cttctacaaa ttcctcctca gttgtgagcc
      421 catgcagctc tccagactca ctggttttcc cagaggcaaa tggctcccag gtgtcatcag
      481 cagcctttct gaacacatgc acggccacat tcacggtagg actgcctcgg acagcatcta
      541 gaactttgac catcagagga cacttggatt caccggtgcc cgtaggaccg ccctgaaaat
      601 acaggttttc gccgctgctg ccgctgctga attcgccgct gtgatgatga tgatgatggc
      661 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
      721 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct
      781 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
      841 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
      901 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
      961 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     1021 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     1081 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     1141 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     1201 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     1261 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     1321 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     1381 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     1441 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     1501 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     1561 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     1621 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     1681 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     1741 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     1801 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     1861 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     1921 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     1981 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     2041 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     2101 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     2161 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     2221 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc
     2281 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     2341 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccga cagcgctctg
     2401 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
     2461 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
     2521 tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt
     2581 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     2641 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     2701 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     2761 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     2821 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     2881 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     2941 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     3001 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     3061 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     3121 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     3181 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3241 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3301 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3361 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca ttgcggtgtg
     3421 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     3481 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3541 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3601 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3661 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3721 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3781 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3841 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3901 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3961 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4021 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4081 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4141 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4201 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4261 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4321 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4381 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4441 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4501 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4561 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4621 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4681 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac
     4741 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4801 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4861 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4921 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4981 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5041 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5101 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5161 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5221 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5281 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5341 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     5401 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5461 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5521 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5581 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5641 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5701 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5761 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5821 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg cca
//
