LOCUS       sequence-403144-        7657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is used to express a EF-hand disabled human MICU1 
            protein in E. coli. The protein is fused with a maltose binding 
            protein in the N-terminus, and also contains a C-terminal His6 tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7657)
  AUTHORS   Tsai CW, Liu TY, Chao FY, Tu YC, Rodriguez MX, Van Keuren AM, Ma Z, 
            Bankston J, Tsai MF
  TITLE     Evidence supporting the MICU1 occlusion mechanism and against the 
            potentiation model in the mitochondrial calcium uniporter complex.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Apr 18;120(16):e2217665120. doi: 
            10.1073/pnas.2217665120. Epub 2023 Apr 10.
  PUBMED    37036971
REFERENCE   2  (bases 1 to 7657)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-04-18"; volume: "120(16):e2217665120. 
            doi"; pages: " 10.1073/pnas.2217665120. Epub 2023 Apr 10"
COMMENT     Sequence Label: pET-hMICU1EF
FEATURES             Location/Qualifiers
     source          1..7657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(372..1454)
                     /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     1416..1435
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1455..1532)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1738..1757
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1841..1860
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1841..1859
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1860..1884
                     /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             1899..1921
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1929..3029
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      2886..2895
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3003..3026
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3030..3047
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3048..3068
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4362..4379
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4432..4450)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4446..4493
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4530..4985
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4617..4636)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4827..4848
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5077..5892)
                     /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(5077..5892)
                     /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     5800..5819
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5884..5903
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6014..6602
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6503..6522
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6756..6773
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6788..6927
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6853..6872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7013..7035
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7029..7220)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
       61 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
      121 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
      181 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
      241 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
      301 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
      361 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
      421 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
      481 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
      541 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
      601 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
      661 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
      721 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
      781 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
      841 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
      901 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
      961 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     1021 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     1081 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     1141 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     1201 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     1261 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     1321 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     1381 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     1441 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     1501 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     1561 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     1621 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     1681 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     1741 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     1801 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
     1861 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
     1921 atatacatat gaaaatcgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
     1981 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
     2041 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
     2101 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
     2161 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
     2221 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
     2281 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
     2341 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
     2401 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
     2461 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
     2521 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     2581 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     2641 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     2701 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     2761 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     2821 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     2881 ttgccgccac catggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     2941 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     3001 tcgatgaagc cctgaaagac gcgcagactc tggttccgcg tggatcggaa aacctgtatt
     3061 ttcagggcgg atccaagagg gcccatgcag aatctccacc atgtgtagac aacctaaaaa
     3121 gtgacatcgg tgataaaggg aagaataaag atgaagggga tgtttgtaac catgagaaaa
     3181 agactgcaga tcttgcccct cacccagaag agaaaaagaa gaaacgttct ggattcagag
     3241 acagaaaagt gatggaatat gagaatagga ttcgagccta ctccacgcca gacaaaatct
     3301 tccgatattt tgccaccttg aaagtcatca gtgagcctgg tgaagcagaa gtgtttatga
     3361 caccagaaga ttttgtgcga tccataacac ccaatgaaaa acaaccagaa cacttgggtc
     3421 tggatcaata tataataaaa cgctttgatg gaaagaaaat ttcccaggaa cgagaaaaat
     3481 ttgctgatga aggcagtata ttttacaccc ttggagaatg tgggctcata tccttttcag
     3541 actacatttt cctcacaact gttctttcca ctcctcagag aaattttgaa attgccttca
     3601 agatgtttgc tttgaatgga gatggagaag tagatatgga aaaatttgaa caggttcaga
     3661 gcatcattcg ctcccaaacc agtatgggta tgcgccacag agatcgtcca actactggca
     3721 acaccctcaa gtctggcttg tgttcagccc tcacaaccta cttttttgga gctgatctga
     3781 agggaaagct gacaatcaaa aacttcctcg aatttcagcg taaactgcag catgatgttc
     3841 tgaagcttga gtttgaacgc catgaccctg tggatgggag aattactgag aggcaatttg
     3901 gtggcatgct acttgcctac agtggggtgc agtccaagaa gctgaccgcc atgcagaggc
     3961 agctcaagaa gcacttcaaa gaaggaaagg gtctgacatt tcaggaggtg gagaacttct
     4021 ttactttcct aaagaacatt aatgatgtgg acactgcatt gagtttttac cacatggctg
     4081 gagcatctct tgataaagtg accatgcagc aggtggccag gacagtggct aaagtggagc
     4141 tctcagacca cgtgtgtgat gtggtgtttg cactctttgc ctgtgatggc aatggcgaac
     4201 tgagcaataa gaaatgggtt tccatcatga agcaacggct gatgagaggc ctggaaaagc
     4261 ccaaagacat gggttggact cgcctcatgc aggccatgtg gaaatgtgca caggaaactg
     4321 cctgggactt cgctttaccc aaacaggcgg ccgcactcga gcaccaccac caccaccact
     4381 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     4441 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     4501 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     4561 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     4621 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     4681 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     4741 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     4801 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     4861 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     4921 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     4981 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5041 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     5101 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     5161 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     5221 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     5281 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     5341 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     5401 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     5461 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     5521 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     5581 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     5641 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     5701 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     5761 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     5821 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     5881 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     5941 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6001 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6061 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6121 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     6181 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6241 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6301 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6361 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6421 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6481 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6541 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     6601 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     6661 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6721 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6781 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     6841 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     6901 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     6961 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     7021 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     7081 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     7141 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     7201 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     7261 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     7321 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     7381 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     7441 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     7501 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     7561 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     7621 ccagtaaggc aaccccgcca gcctagccgg gtcctca
//
