LOCUS       sequence-334956-        5933 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses both yeast Tim8 and yeast Tim13 (the latter with cleavable
            N-ter His-tag), codon-optimized for E. coli production.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5933)
  AUTHORS   Sucec I, Wang Y, Dakhlaoui O, Weinhaupl K, Jores T, Costa D, Hessel 
            A, Brennich M, Rapaport D, Lindorff-Larsen K, Bersch B, Schanda P
  TITLE     Structural basis of client specificity in mitochondrial 
            membrane-protein chaperones.
  JOURNAL   Sci Adv. 2020 Dec 18;6(51). pii: 6/51/eabd0263. doi: 
            10.1126/sciadv.abd0263. Print 2020 Dec.
  PUBMED    33355130
REFERENCE   2  (bases 1 to 5933)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv."; 
            date: "2020-12-18"; volume: "6(51). pii: 6/51/eabd0263. doi"; pages:
            " 10.1126/sciadv.abd0263. Print 2020 Dec"
COMMENT     Sequence Label: pETDuet-1 TIM8,13
FEATURES             Location/Qualifiers
     source          1..5933
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             130..150
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        537..555
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    556..580
                     /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)."
     CDS             899..943
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(981..999)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      995..1042
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1079..1534
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1166..1185)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1376..1397
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1649..2440,2441..2509))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2272..2291
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2510..2602)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2683..3271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3172..3191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3425..3442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3457..3596
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3522..3541)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3682..3704
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3698..3889)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4461..5543)
                     /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     5505..5524
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5544..5621)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5827..5846
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcaccatca tcaccacagc
      121 caggatccgg agaatctgta ttttcaaggt ggtctgagca gcatctttgg tggtggtgcg
      181 ccgagccagc agaaggaagc ggcgaccacc gcgaagacca ccccgaaccc gatcgcgaag
      241 gagctgaaaa accagattgc gcaagaactg gcggtggcga acgcgaccga gctggttaac
      301 aagatcagcg agaactgctt cgaaaaatgc ctgaccagcc cgtacgcgac ccgtaacgac
      361 gcgtgcattg atcagtgcct ggcgaaatat atgcgtagct ggaacgttat cagcaaggcg
      421 tacatcagcc gtatccagaa tgcgagcgcg agcggtgaaa tctaaaagct tgcggccgca
      481 taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat
      541 acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta
      601 agtataagaa ggagatatac atatgagcag cctgagcacc agcgacctgg cgagcctgga
      661 cgacaccagc aagaaagaga ttgcgacctt cctggagggc gaaaacagca agcagaaagt
      721 gcaaatgagc atccaccagt tcaccaacat ttgctttaag aaatgcgtgg agagcgttaa
      781 cgacagcaac ctgagcagcc aggaagagca atgcctgagc aactgcgtga atcgctttct
      841 ggacaccaac atccgcatcg tgaacggtct gcaaaatacc cgctaactcg agtctggtaa
      901 agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta
      961 attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     1021 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac
     1081 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1141 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1201 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1261 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1321 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1381 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1441 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1501 gcgaatttta acaaaatatt aacgtttaca atttctggcg gcacgatggc atgagattat
     1561 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     1621 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     1681 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     1741 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     1801 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     1861 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     1921 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     1981 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     2041 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     2101 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     2161 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     2221 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     2281 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     2341 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     2401 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     2461 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     2521 ttcaatcatg attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     2581 tgtatttaga aaaataaaca aataggtcat gaccaaaatc ccttaacgtg agttttcgtt
     2641 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     2701 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     2761 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     2821 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     2881 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     2941 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     3001 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     3061 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     3121 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     3181 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     3241 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     3301 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     3361 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     3421 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     3481 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
     3541 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
     3601 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     3661 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     3721 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     3781 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     3841 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     3901 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     3961 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     4021 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     4081 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     4141 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     4201 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     4261 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     4321 tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg aaggagctga
     4381 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4441 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4501 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4561 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4621 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4681 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4741 atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     4801 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     4861 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     4921 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     4981 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5041 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5101 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5161 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5221 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5281 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5341 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5401 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5461 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5521 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5581 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5641 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5701 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5761 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     5821 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     5881 ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga aat
//
