LOCUS       sequence-334955-        5601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses yeast Tim12 (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 5601)
  AUTHORS   Weinhaupl K, Wang Y, Hessel A, Brennich M, Lindorff-Larsen K, 
            Schanda P
  TITLE     Architecture and assembly dynamics of the essential mitochondrial 
            chaperone complex TIM9.10.12.
  JOURNAL   Structure. 2021 May 3. pii: S0969-2126(21)00125-8. doi: 
            10.1016/j.str.2021.04.009.
  PUBMED    33974880
REFERENCE   2  (bases 1 to 5601)
  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: "Structure. 
            2021 May 3. pii: S0969-2126(21)00125-8. doi: 
            10.1016/j.str.2021.04.009."
COMMENT     Sequence Label: pET28a(+)-HisTim12_CtoA
FEATURES             Location/Qualifiers
     source          1..5601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     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             94..111
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             112..132
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             466..483
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(536..554)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      550..597
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      634..1089
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(721..740)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     931..952
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1181..1996)
                     /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             complement(1181..1996)
                     /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     1904..1923
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1988..2007
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2118..2706
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2607..2626
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2860..2877
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2892..3031
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2957..2976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3117..3139
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3133..3324)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4133..5215)
                     /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     5177..5196
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5216..5293)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5499..5518
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggccaccacc atcaccatca cgaaaacctg
      121 tacttccagg gtagcttctt cctgaacagc ctgcgtggta accaggaagt gtctcaggaa
      181 aagctggacg tagcaggtgt tcagttcgac gcaatggctt ctactttcaa caacatcctg
      241 tccacctgcc tggaaaaatg catcccgcac gaaggtttcg gtgaaccgga tctgaccaaa
      301 ggcgaacagg cttgtatcga ccgttgtgtg gcgaaaatgc actactctaa ccgtctgatt
      361 ggcggctttg ttcagacccg tggctttggc ccggagaacc agctgcgcca ttattcccgc
      421 tttgttgcga aagagattgc ggatgattcc aaaaaataac tcgagcacca ccaccaccac
      481 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      541 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      601 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      661 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      721 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      781 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      841 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      901 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      961 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1021 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1081 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1141 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1201 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1261 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1321 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1381 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1441 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1501 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1561 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1621 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1681 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     1741 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     1801 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     1861 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     1921 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     1981 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2041 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2101 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2161 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2221 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2281 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2341 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2401 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2461 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2521 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2581 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2641 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     2701 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     2761 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     2821 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     2881 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2941 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3001 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3061 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3121 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3181 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3241 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3301 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3361 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3421 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     3481 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     3541 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     3601 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     3661 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     3721 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     3781 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     3841 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     3901 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     3961 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4021 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4081 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4141 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4201 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4261 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4321 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4381 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4441 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4501 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4561 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4621 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4681 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     4741 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4801 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4861 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     4921 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     4981 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5041 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5101 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5161 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5221 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5281 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5341 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5401 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5461 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     5521 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     5581 gagatctcga tcccgcgaaa t
//
