LOCUS       sequence-452475-        5720 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of Strep-tagged MFN2 in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5720)
  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 5720)
  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-MFN2-Strep (SB253)
FEATURES             Location/Qualifiers
     source          1..5720
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(664..911)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     877..895
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     CDS             complement(977..1351)
                     /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(1370..1417)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(1424..1837)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     terminator      complement(1852..2098)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(2177..2194)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2210..2239)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(join(2263..2286,2287..2322,2323..2346,
                     2347..2352))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             complement(2353..2376)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(4701..5639)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tggtctccag
      661 cttgcaaatt aaagccttcg agcgtcccaa aaccttctca agcaaggttt tcagtataat
      721 gttacatgcg tacacgcgtc tgtacagaaa aaaaagaaaa atttgaaata taaataacgt
      781 tcttaatact aacataacta taaaaaaata aatagggacc tagacttcag gttgtctaac
      841 tccttccttt tcggttagag cggatgtggg gggagggcgt gaatgtaagc gtgacataac
      901 taattacatg atatcgacaa aggaaaaggg ggacggatct ccgaggcctc ggacccgtcg
      961 ggccgcgtcg gacgtgtcag tcctgctcct cggccacgaa gtgcacgcag ttgccggccg
     1021 ggtcgcgcag ggcgaactcc cgcccccacg gctgctcgcc gatctcggtc atggccggcc
     1081 cggaggcgtc ccggaagttc gtggacacga cctccgacca ctcggcgtac agctcgtcca
     1141 ggccgcgcac ccacacccag gccagggtgt tgtccggcac cacctggtcc tggaccgcgc
     1201 tgatgaacag ggtcacgtcg tcccggacca caccggcgaa gtcgtcctcc acgaagtccc
     1261 gggagaaccc gagccggtcg gtccagaact cgaccgctcc ggcgacgtcg cgcgcggtga
     1321 gcaccggaac ggcactggtc aacttggcca tggtttagtt cctcaccttg tcgtattata
     1381 ctatgccgat atactatgcc gatgattaat tgtcaacacc gcccttagat tagattgcta
     1441 tgctttcttt ctaatgagca agaagtaaaa aaagttgtaa tagaacaaga aaaatgaaac
     1501 tgaaacttga gaaattgaag accgtttatt aacttaaata tcaatgggag gtcatcgaaa
     1561 gagaaaaaaa tcaaaaaaaa aaattttcaa gaaaaagaaa cgtgataaaa atttttattg
     1621 cctttttcga cgaagaaaaa gaaacgaggc ggtctctttt ttcttttcca aacctttagt
     1681 acgggtaatt aacgacaccc tagaggaaga aagaggggaa atttagtatg ctgtgcttgg
     1741 gtgttttgaa gtggtacggc gatgcgcgga gtccgagaaa atctggaaga gtaaaaaagg
     1801 agtagaaaca ttttgaagct atggtgtgtg ggggatccgc acaaacgaag gtctcactta
     1861 atcttctgta ctctgaagag gagtgggaaa taccaagaaa aacatcaaac tcgaatgatt
     1921 ttcccaaacc cctaccacaa gatattcatc agctgcgaga taggctgatc aggagcaagc
     1981 tcgtacgaga agaaacaaaa tgacaaaaaa aatcctatac tatataggtt acaaataaaa
     2041 aagtatcaaa aatgaagcct gcatctctca ggcaaatggc attctgacat cctcttgatt
     2101 agaatctagc aagaccggtc ttctcgtaag tgcccaactt gaactgagga acagtcatgt
     2161 ctaaggctaa aactcaatga tgatgatgat gatggtcgac ggcgctattc agatcctctt
     2221 ctgagatgag tttttgttcg ggcccaagct ggcggccgct tacttctcaa attgggggtg
     2281 actccaggca cttccaccag agccaccgcc ggagccgcct cctttctcaa attgtggatg
     2341 agaccaggca gatggtcctt gaaaaagcac ctctaatgaa ccacctcctc tagaaggttg
     2401 caagtattgg tgagtgaaca tattcaattc agaatccaac caaccagctt tgtttctcaa
     2461 caacttagcc ttagattgca aagaatccaa aacttcaatc ttcttgttca tagcagcaat
     2521 ctcttgttcc aagttctctc tagtaacatc aacttgttga cacaagtgag caaaagtacc
     2581 agacaattct tgttgaactt gatgagaaca gttagaacca gtgtaagaaa taaccaattg
     2641 caacttctca gaagcatgtt caacgaattg tcttttaaaa gctctttcct tagctttagt
     2701 agtccaagtc aatctctcat aaacgtacaa caaaccgtac aaaccgaaag acaaagcaat
     2761 caatctccaa ccaacagcct tccaaacaac accaccaaca accaaaatac ccatagaagt
     2821 tctagaagtc aaagaagcca aaccagtaac catagaaacc ataaactctt cttgagtcaa
     2881 agaaccttga ggcaaaggtg gcatagaagg attagctgga gtcaaaggaa ttggtctttg
     2941 aacttgatcg ttataaccca tcaaagctct tctagagttc tttggaccca aaaatctgtt
     3001 aaccaacata gtccaaccca aagagaagtg aaactcaata tcttcttgga aatcagcaca
     3061 caacttatca cagttcaaat cgtagttcaa agagaaacat tgtcttggaa ccaacatatc
     3121 gatttgagat ctaacagaaa caggcaacaa tggtttcaaa ccatcaatca tatcttgttg
     3181 catagtttgc aaagagttag tgatagcagt agaacatcta tcagacatgt ttctacccaa
     3241 accctcttca atgtgtctat gcaattcgtt cttgtaaacc ttcaaaacaa caggagatgg
     3301 atggaaatcc atttggtaat catcaaccaa aacagacaat cttctaatct cttcagccat
     3361 agcagtagaa acttgtcttt caacctcttc agtgatttgc ttaattctca acttgtaatc
     3421 ttgagccaac aactccaatt gcttatcaat aaacttcaat ctatcttgtc tctcttctct
     3481 catctcttca cagtaaactt gttgttctct agcagccatg tgcaaagaat ccataatcaa
     3541 tctaacagct tcagcaattt gcttagctct aacagtatgt tgctcgaact tagtcttaac
     3601 agcagattga gagatacact cttcgaatct tctctcaaaa ttttggaatt cgaacattct
     3661 aacttggaaa ccttcagcca aagcaccacc accttcaggc ataccctgag ccttttggat
     3721 tctagcgttc aaaacttcct tagcagaaac aaagaaaatt ctatctccag cttgagatct
     3781 atcaacaaca cccaattcat caaccaagaa agaagtacat ctctccatat gttgtcttct
     3841 aacctcttcc atatactcag gttcagaagc agaagcatcc catctattgt tcaaaatgaa
     3901 aatgtttggt ctagacaatc tttcagaaac cttgtgaaag aaatgctttt cagtttgcat
     3961 caaagtagac tcagagttag caaccaaaac gaaaacatca gcatccaaac agaacttatc
     4021 gatccaagaa tccaactcag tagtaacatc aatacctgga gaatccatca aaaccaaatc
     4081 atccttcaac aaaggacatt tagaatttgg ccacataaca gaaaccaaag aaccagcatg
     4141 caattgctta tcttggtgca aagcatgagc caattggtta acagtcttag cagatctttt
     4201 ctcttcagaa ccttcagtca acaagaaagc ctcgtgacca tcagtacctt caactctcaa
     4261 gaaacagtta gtagtatgac caataccaga aggcaaaacc ttatcccaca acatagcatt
     4321 aataacagta gatttaccgt tagaagttct accaaagaaa gcaactttca tgtgtcttct
     4381 agccaaaact tcagaaatac ctctaacctt agacaagtaa cctttaacat ccaaaacttg
     4441 ctcttcagta gtaactggat ccaattcagc atttctataa gtatcctcca agaaagtagc
     4501 agattcttga atgtaagcac ccaattgctc gaagataccg ttaatctttt tcttagcagt
     4561 aacgaaatgc ttcaaagggg aagcgttgac ctcagccatg tgtctcttat ttttcttgac
     4621 ggtgacgata ctattgcatc tactaaacaa caaactcatg gtaccgatcc gagacggccg
     4681 gctgggccac gtgaattcct cgtttcgaat aattagttgt tttttgatct tctcaagttg
     4741 tcgttaaaag tcgttaaaat caaaagcttg tcaattggaa ccagtcgcaa ttatgaaagt
     4801 aagctaataa tgatgataaa aaaaaaggtt taagacaggg cagcttcctt ctgtttatat
     4861 attgctgtca agtaggggtt agaacagtta aattttgatc atgaacgtta ggctatcagc
     4921 agtattccca ccagaatctt ggaagcatac aatgtggaga caatgcataa tcatccaaaa
     4981 agcgggtgtt tccccatttg cgtttcggca caggtgcacc ggggttcaga agcgatagag
     5041 agactgcgct aagcattaat gagattattt ttgagcattc gtcaatcaat accaaacaag
     5101 acaaacggta tgccgacttt tggaagtttc tttttgacca actggccgtt agcatttcaa
     5161 cgaaccaaac ttagttcatc ttggatgaga tcacgctttt gtcatattag gttccaagac
     5221 agcgtttaaa ctgtcagttt tgggccattt ggggaacatg aaactatttg accccacact
     5281 cagaaagccc tcatctggag tgatgttcgg gtgtaatgcg gagcttgttg cattcggaaa
     5341 taaacaaaca tgaacctcgc caggggggcc aggatagaca ggctaataaa gtcatggtgt
     5401 tagtagccta atagaaggaa ttggaatgag cgagctccaa tcaagcccaa taactgggct
     5461 ggtttttcga tggcaaaagt gggtgttgag gagaagagga gtggaggtcc tgcgtttgca
     5521 acggtctgct gctagtgtat cccctcctgt tgcgtttggc acttatgtgt gagaatggac
     5581 ctgtggatgt cggatggcaa aaaggtttca ttcaaccttt cgtctttgga tgttagatct
     5641 gatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5701 gaaaagatca aaggatcttc
//
