LOCUS       sequence-452477-        4673 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Inducible expression of His-tagged SLC25A46 with I349D mutation 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 Apr 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[I349D]-His (SB256)
FEATURES             Location/Qualifiers
     source          1..4673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(169..415)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(494..511)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(527..556)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(580..609)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(610..630)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(1971..2909)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     rep_origin      2991..3579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3480..3499
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3654..3901)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     3867..3885
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(3967..4341)
                     /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"
     promoter        complement(4360..4407)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(join(4414..4673,1..154))
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
ORIGIN
        1 ggtaattaac gacaccctag aggaagaaag aggggaaatt tagtatgctg tgcttgggtg
       61 ttttgaagtg gtacggcgat gcgcggagtc cgagaaaatc tggaagagta aaaaaggagt
      121 agaaacattt tgaagctatg gtgtgtgggg gatccgcaca aacgaaggtc tcacttaatc
      181 ttctgtactc tgaagaggag tgggaaatac caagaaaaac atcaaactcg aatgattttc
      241 ccaaacccct accacaagat attcatcagc tgcgagatag gctgatcagg agcaagctcg
      301 tacgagaaga aacaaaatga caaaaaaaat cctatactat ataggttaca aataaaaaag
      361 tatcaaaaat gaagcctgca tctctcaggc aaatggcatt ctgacatcct cttgattaga
      421 atctagcaag accggtcttc tcgtaagtgc ccaacttgaa ctgaggaaca gtcatgtcta
      481 aggctaaaac tcaatgatga tgatgatgat ggtcgacggc gctattcaga tcctcttctg
      541 agatgagttt ttgttcgggc ccaagctggc ggccgcctaa tggtggtggt gatgatggtg
      601 atgatgatgt ccctggaaat aaagattctc ggaaccacca ccaccagaac caccaccacc
      661 agaaccacca ccaccgatat tgttttgcaa caaagtagag tagatgatct tagtgatttg
      721 caaaacagca gcatgcaaag tgtattggat aataacagca ccgaaaccct tgtagaaacc
      781 aaaaacaccc tcttcttgtc tgatagtgtt gatacaatct ctcataccct catattgagt
      841 attaataggc aaaacttcgt aacccaaatc agtgttatcg atatcagttc tagtaccttg
      901 gatgtgcaat ctatgcaaaa cagtttccaa tgggtacaag ataacatcag aacacaaaga
      961 agcagcgaaa ttagcaatca actcaggaaa ataagcatcc aacatagatt gaactggaga
     1021 agtagattca gccaagtgag agttgtaagt ctttctcttc aagatcaaca aaacgaactt
     1081 ttgaataaca gaagagatga tgtagtgcaa aacaccatgc aaaacagtag ggaagatcaa
     1141 agacaacaat ggcaacaatc ttttagaatg aggaacaccc ataccaataa ctctaccaat
     1201 accctcctta acacattcca agataccagt gttatctctg atgatctcag attgaacagt
     1261 ttcgatcaaa gaagcagagt agaaaggcat agcaacaacg taagtcaaag acttcaacaa
     1321 caagtgctct ccaatttgtt ttggagacca cttatgcaaa acttctctag gcaatggagt
     1381 gaactcagaa ataatacctt cagcacccaa agtaacacct tgaacaataa aagtagaacc
     1441 catacccttc cacaaagctc taggaccttg agtcttattg aaagagtaca tgatgttaat
     1501 aacagtgaat ggagtcaagt gataatgttg agcgtggtaa ttaacttgac attgtcttct
     1561 caaaacgata catggatgag ccaaaacgtt ctcagtgaac aaagaagcca aaccaatacc
     1621 gaaaccagca aatctattca attgttcaga agattgacct tgaacagaac caccaccacc
     1681 agaagaaaat ggctcttcag taggaccctc atatggagta gaagtagtag gaacaccgta
     1741 aggtggagac ttttcacccc agtgcaagtt tctagaacct ggaatatcag gtggagtagt
     1801 aacccaatga cccaaatcag aaccagtaga aaaagatcta gcagggaaag ctcctccaaa
     1861 tccttgctcg tctctggctc ctcctctgta tcccaaaccg tcaaatccgt caggtcttct
     1921 agggtgcatg gtaccgatcc gagacggccg gctgggccac gtgaattcct cgtttcgaat
     1981 aattagttgt tttttgatct tctcaagttg tcgttaaaag tcgttaaaat caaaagcttg
     2041 tcaattggaa ccagtcgcaa ttatgaaagt aagctaataa tgatgataaa aaaaaaggtt
     2101 taagacaggg cagcttcctt ctgtttatat attgctgtca agtaggggtt agaacagtta
     2161 aattttgatc atgaacgtta ggctatcagc agtattccca ccagaatctt ggaagcatac
     2221 aatgtggaga caatgcataa tcatccaaaa agcgggtgtt tccccatttg cgtttcggca
     2281 caggtgcacc ggggttcaga agcgatagag agactgcgct aagcattaat gagattattt
     2341 ttgagcattc gtcaatcaat accaaacaag acaaacggta tgccgacttt tggaagtttc
     2401 tttttgacca actggccgtt agcatttcaa cgaaccaaac ttagttcatc ttggatgaga
     2461 tcacgctttt gtcatattag gttccaagac agcgtttaaa ctgtcagttt tgggccattt
     2521 ggggaacatg aaactatttg accccacact cagaaagccc tcatctggag tgatgttcgg
     2581 gtgtaatgcg gagcttgttg cattcggaaa taaacaaaca tgaacctcgc caggggggcc
     2641 aggatagaca ggctaataaa gtcatggtgt tagtagccta atagaaggaa ttggaatgag
     2701 cgagctccaa tcaagcccaa taactgggct ggtttttcga tggcaaaagt gggtgttgag
     2761 gagaagagga gtggaggtcc tgcgtttgca acggtctgct gctagtgtat cccctcctgt
     2821 tgcgtttggc acttatgtgt gagaatggac ctgtggatgt cggatggcaa aaaggtttca
     2881 ttcaaccttt cgtctttgga tgttagatct gatctcatga ccaaaatccc ttaacgtgag
     2941 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3001 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     3061 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     3121 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     3181 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     3241 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     3301 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     3361 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     3421 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     3481 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     3541 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     3601 ttacggttcc tggccttttg ctggcctttt gctcacatgt tggtctccag cttgcaaatt
     3661 aaagccttcg agcgtcccaa aaccttctca agcaaggttt tcagtataat gttacatgcg
     3721 tacacgcgtc tgtacagaaa aaaaagaaaa atttgaaata taaataacgt tcttaatact
     3781 aacataacta taaaaaaata aatagggacc tagacttcag gttgtctaac tccttccttt
     3841 tcggttagag cggatgtggg gggagggcgt gaatgtaagc gtgacataac taattacatg
     3901 atatcgacaa aggaaaaggg ggacggatct ccgaggcctc ggacccgtcg ggccgcgtcg
     3961 gacgtgtcag tcctgctcct cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag
     4021 ggcgaactcc cgcccccacg gctgctcgcc gatctcggtc atggccggcc cggaggcgtc
     4081 ccggaagttc gtggacacga cctccgacca ctcggcgtac agctcgtcca ggccgcgcac
     4141 ccacacccag gccagggtgt tgtccggcac cacctggtcc tggaccgcgc tgatgaacag
     4201 ggtcacgtcg tcccggacca caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc
     4261 gagccggtcg gtccagaact cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac
     4321 ggcactggtc aacttggcca tggtttagtt cctcaccttg tcgtattata ctatgccgat
     4381 atactatgcc gatgattaat tgtcaacacc gcccttagat tagattgcta tgctttcttt
     4441 ctaatgagca agaagtaaaa aaagttgtaa tagaacaaga aaaatgaaac tgaaacttga
     4501 gaaattgaag accgtttatt aacttaaata tcaatgggag gtcatcgaaa gagaaaaaaa
     4561 tcaaaaaaaa aaattttcaa gaaaaagaaa cgtgataaaa atttttattg cctttttcga
     4621 cgaagaaaaa gaaacgaggc ggtctctttt ttcttttcca aacctttagt acg
//
