LOCUS       sequence-452478-        4673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of His-tagged SLC25A46 with R247A and R347A 
            mutations in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4673)
  AUTHORS   Boopathy S, Luce BE, Lugo CM, Hakim P, McDonald J, Kim HL, Ponce J, 
            Ueberheide BM, Chao LH
  TITLE     Identification of SLC25A46 interaction interfaces with mitochondrial
            membrane fusogens Opa1 and Mfn2.
  JOURNAL   J Biol Chem. 2024 Aug 31:107740. doi: 10.1016/j.jbc.2024.107740.
  PUBMED    39222684
REFERENCE   2  (bases 1 to 4673)
  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: "J Biol 
            Chem. 2024 Aug 31:107740. doi: 10.1016/j.jbc.2024.107740."
COMMENT     Sequence Label: pPICZ A-SLC25A46[R247A,R347A]-His (SB257)
FEATURES             Location/Qualifiers
     source          1..4673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1697..1717
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1718..1747
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1771..1800
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1816..1833
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1912..2158
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        2173..2586
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        2593..2640
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2659..3033
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      3099..3346
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3115..3133)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(3421..4009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3501..3520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(4091..4673,1..356)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 atgcttagcg cagtctctct atcgcttctg aaccccggtg cacctgtgcc gaaacgcaaa
       61 tggggaaaca cccgcttttt ggatgattat gcattgtctc cacattgtat gcttccaaga
      121 ttctggtggg aatactgctg atagcctaac gttcatgatc aaaatttaac tgttctaacc
      181 cctacttgac agcaatatat aaacagaagg aagctgccct gtcttaaacc ttttttttta
      241 tcatcattat tagcttactt tcataattgc gactggttcc aattgacaag cttttgattt
      301 taacgacttt taacgacaac ttgagaagat caaaaaacaa ctaattattc gaaacgagga
      361 attcacgtgg cccagccggc cgtctcggat cggtaccatg caccctagaa gacctgacgg
      421 atttgacggt ttgggataca gaggaggagc cagagacgag caaggatttg gaggagcttt
      481 ccctgctaga tctttttcta ctggttctga tttgggtcat tgggttacta ctccacctga
      541 tattccaggt tctagaaact tgcactgggg tgaaaagtct ccaccttacg gtgttcctac
      601 tacttctact ccatatgagg gtcctactga agagccattt tcttctggtg gtggtggttc
      661 tgttcaaggt caatcttctg aacaattgaa tagatttgct ggtttcggta ttggtttggc
      721 ttctttgttc actgagaacg ttttggctca tccatgtatc gttttgagaa gacaatgtca
      781 agttaattac cacgctcaac attatcactt gactccattc actgttatta acatcatgta
      841 ctctttcaat aagactcaag gtcctagagc tttgtggaag ggtatgggtt ctacttttat
      901 tgttcaaggt gttactttgg gtgctgaagg tattatttct gagttcactc cattgcctag
      961 agaagttttg cataagtggt ctccaaaaca aattggagag cacttgttgt tgaagtcttt
     1021 gacttacgtt gttgctatgc ctttctactc tgcttctttg atcgaaactg ttcaatctga
     1081 gatcatcaga gataacactg gtatcttgga atgtgttaag gagggtattg gtagagttat
     1141 tggtatgggt gttcctcatt ctaaagcttt gttgccattg ttgtctttga tcttccctac
     1201 tgttttgcat ggtgttttgc actacatcat ctcttctgtt attcaaaagt tcgttttgtt
     1261 gatcttgaag agaaagactt acaactctca cttggctgaa tctacttctc cagttcaatc
     1321 tatgttggat gcttattttc ctgagttgat tgctaatttc gctgcttctt tgtgttctga
     1381 tgttatcttg tacccattgg aaactgtttt gcatagattg cacatccaag gtactgctac
     1441 tatcatcgat aacactgatt tgggttacga agttttgcct attaatactc aatatgaggg
     1501 tatgagagat tgtatcaaca ctatcagaca agaagagggt gtttttggtt tctacaaggg
     1561 tttcggtgct gttattatcc aatacacttt gcatgctgct gttttgcaaa tcactaagat
     1621 catctactct actttgttgc aaaacaatat cggtggtggt ggttctggtg gtggtggttc
     1681 tggtggtggt ggttccgaga atctttattt ccagggacat catcatcacc atcatcacca
     1741 ccaccattag gcggccgcca gcttgggccc gaacaaaaac tcatctcaga agaggatctg
     1801 aatagcgccg tcgaccatca tcatcatcat cattgagttt tagccttaga catgactgtt
     1861 cctcagttca agttgggcac ttacgagaag accggtcttg ctagattcta atcaagagga
     1921 tgtcagaatg ccatttgcct gagagatgca ggcttcattt ttgatacttt tttatttgta
     1981 acctatatag tataggattt tttttgtcat tttgtttctt ctcgtacgag cttgctcctg
     2041 atcagcctat ctcgcagctg atgaatatct tgtggtaggg gtttgggaaa atcattcgag
     2101 tttgatgttt ttcttggtat ttcccactcc tcttcagagt acagaagatt aagtgagacc
     2161 ttcgtttgtg cggatccccc acacaccata gcttcaaaat gtttctactc cttttttact
     2221 cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc
     2281 atactaaatt tcccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt
     2341 ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt
     2401 tttatcacgt ttctttttct tgaaaatttt tttttttgat ttttttctct ttcgatgacc
     2461 tcccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
     2521 cttgttctat tacaactttt tttacttctt gctcattaga aagaaagcat agcaatctaa
     2581 tctaagggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     2641 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     2701 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
     2761 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     2821 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     2881 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     2941 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     3001 tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacgcggccc gacgggtccg
     3061 aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg
     3121 cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc
     3181 tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat
     3241 atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa
     3301 aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc tggagaccaa
     3361 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3421 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3541 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3601 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3661 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3721 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3781 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3841 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3901 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3961 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4021 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4081 catgagatca gatctaacat ccaaagacga aaggttgaat gaaacctttt tgccatccga
     4141 catccacagg tccattctca cacataagtg ccaaacgcaa caggagggga tacactagca
     4201 gcagaccgtt gcaaacgcag gacctccact cctcttctcc tcaacaccca cttttgccat
     4261 cgaaaaacca gcccagttat tgggcttgat tggagctcgc tcattccaat tccttctatt
     4321 aggctactaa caccatgact ttattagcct gtctatcctg gcccccctgg cgaggttcat
     4381 gtttgtttat ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag
     4441 ggctttctga gtgtggggtc aaatagtttc atgttcccca aatggcccaa aactgacagt
     4501 ttaaacgctg tcttggaacc taatatgaca aaagcgtgat ctcatccaag atgaactaag
     4561 tttggttcgt tgaaatgcta acggccagtt ggtcaaaaag aaacttccaa aagtcggcat
     4621 accgtttgtc ttgtttggta ttgattgacg aatgctcaaa aataatctca tta
//
