LOCUS       sequence-455569-        7466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible coexpression of His-tagged SLC25A46 and Strep-tagged MFN2 
            lacking its transmembrane region and helical bundle 2 (HB2) in 
            Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7466)
  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 7466)
  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-SLC-His+MFN2[∆TM,∆HB2]-Strep (SB261)
FEATURES             Location/Qualifiers
     source          1..7466
                     /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(2249..2278)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(2279..2299)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        complement(3607..4545)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      complement(4560..4806)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(4885..4902)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4918..4947)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(join(4957..4980,4981..5016,5017..5040,
                     5041..5046))
                     /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(5047..5070)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(6447..7385)
                     /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 tttttgttct ctagactaat ggtggtggtg atgatggtga tgatgatgtc
     2281 cctggaaata aagattctcg gaaccaccac caccagaacc accaccacca gaaccaccac
     2341 caccgatatt gttttgcaac aaagtagagt agatgatctt agtgatttgc aaaacagcag
     2401 catgcaaagt gtattggata ataacagcac cgaaaccctt gtagaaacca aaaacaccct
     2461 cttcttgtct gatagtgttg atacaatctc tcataccctc atattgagta ttaataggca
     2521 aaacttcgta acccaaatca gtgttatcga tgatagttct agtaccttgg atgtgcaatc
     2581 tatgcaaaac agtttccaat gggtacaaga taacatcaga acacaaagaa gcagcgaaat
     2641 tagcaatcaa ctcaggaaaa taagcatcca acatagattg aactggagaa gtagattcag
     2701 ccaagtgaga gttgtaagtc tttctcttca agatcaacaa aacgaacttt tgaataacag
     2761 aagagatgat gtagtgcaaa acaccatgca aaacagtagg gaagatcaaa gacaacaatg
     2821 gcaacaatct tttagaatga ggaacaccca taccaataac tctaccaata ccctccttaa
     2881 cacattccaa gataccagtg ttatctctga tgatctcaga ttgaacagtt tcgatcaaag
     2941 aagcagagta gaaaggcata gcaacaacgt aagtcaaaga cttcaacaac aagtgctctc
     3001 caatttgttt tggagaccac ttatgcaaaa cttctctagg caatggagtg aactcagaaa
     3061 taataccttc agcacccaaa gtaacacctt gaacaataaa agtagaaccc atacccttcc
     3121 acaaagctct aggaccttga gtcttattga aagagtacat gatgttaata acagtgaatg
     3181 gagtcaagtg ataatgttga gcgtggtaat taacttgaca ttgtcttctc aaaacgatac
     3241 atggatgagc caaaacgttc tcagtgaaca aagaagccaa accaataccg aaaccagcaa
     3301 atctattcaa ttgttcagaa gattgacctt gaacagaacc accaccacca gaagaaaatg
     3361 gctcttcagt aggaccctca tatggagtag aagtagtagg aacaccgtaa ggtggagact
     3421 tttcacccca gtgcaagttt ctagaacctg gaatatcagg tggagtagta acccaatgac
     3481 ccaaatcaga accagtagaa aaagatctag cagggaaagc tcctccaaat ccttgctcgt
     3541 ctctggctcc tcctctgtat cccaaaccgt caaatccgtc aggtcttcta gggtgcatgc
     3601 tagcctcgtt tcgaataatt agttgttttt tgatcttctc aagttgtcgt taaaagtcgt
     3661 taaaatcaaa agcttgtcaa ttggaaccag tcgcaattat gaaagtaagc taataatgat
     3721 gataaaaaaa aaggtttaag acagggcagc ttccttctgt ttatatattg ctgtcaagta
     3781 ggggttagaa cagttaaatt ttgatcatga acgttaggct atcagcagta ttcccaccag
     3841 aatcttggaa gcatacaatg tggagacaat gcataatcat ccaaaaagcg ggtgtttccc
     3901 catttgcgtt tcggcacagg tgcaccgggg ttcagaagcg atagagagac tgcgctaagc
     3961 attaatgaga ttatttttga gcattcgtca atcaatacca aacaagacaa acggtatgcc
     4021 gacttttgga agtttctttt tgaccaactg gccgttagca tttcaacgaa ccaaacttag
     4081 ttcatcttgg atgagatcac gcttttgtca tattaggttc caagacagcg tttaaactgt
     4141 cagttttggg ccatttgggg aacatgaaac tatttgaccc cacactcaga aagccctcat
     4201 ctggagtgat gttcgggtgt aatgcggagc ttgttgcatt cggaaataaa caaacatgaa
     4261 cctcgccagg ggggccagga tagacaggct aataaagtca tggtgttagt agcctaatag
     4321 aaggaattgg aatgagcgag ctccaatcaa gcccaataac tgggctggtt tttcgatggc
     4381 aaaagtgggt gttgaggaga agaggagtgg aggtcctgcg tttgcaacgg tctgctgcta
     4441 gtgtatcccc tcctgttgcg tttggcactt atgtgtgaga atggacctgt ggatgtcgga
     4501 tggcaaaaag gtttcattca acctttcgtc tttggatgtt agatccgcac aaacgaaggt
     4561 ctcacttaat cttctgtact ctgaagagga gtgggaaata ccaagaaaaa catcaaactc
     4621 gaatgatttt cccaaacccc taccacaaga tattcatcag ctgcgagata ggctgatcag
     4681 gagcaagctc gtacgagaag aaacaaaatg acaaaaaaaa tcctatacta tataggttac
     4741 aaataaaaaa gtatcaaaaa tgaagcctgc atctctcagg caaatggcat tctgacatcc
     4801 tcttgattag aatctagcaa gaccggtctt ctcgtaagtg cccaacttga actgaggaac
     4861 agtcatgtct aaggctaaaa ctcaatgatg atgatgatga tggtcgacgg cgctattcag
     4921 atcctcttct gagatgagtt tttgttcggg cccttatttt tcgaattgag gatgtgacca
     4981 tgctgaacca ccagaaccac caccagaacc accacctttt tcaaattgtg gatgtgacca
     5041 agcagatgga ccttgaaata aaacttccaa agaaccacca cctctagaag gttgcaagta
     5101 ttggtgagtg aacatattca attcagaatc caaccaacca gctttgtttc tcaacaactt
     5161 agccttagat tgcaaagaat ccaaaacttc aatcttcttg ttcatagcag caatctcttg
     5221 ttccaagttc tctctagttc tatcttgtct ctcttctctc atctcttcac agtaaacttg
     5281 ttgttctcta gcagccatgt gcaaagaatc cataatcaat ctaacagctt cagcaatttg
     5341 cttagctcta acagtatgtt gctcgaactt agtcttaaca gcagattgag agatacactc
     5401 ttcgaatctt ctctcaaaat tttggaattc gaacattcta acttggaaac cttcagccaa
     5461 agcaccacca ccttcaggca taccctgagc cttttggatt ctagcgttca aaacttcctt
     5521 agcagaaaca aagaaaattc tatctccagc ttgagatcta tcaacaacac ccaattcatc
     5581 aaccaagaaa gaagtacatc tctccatatg ttgtcttcta acctcttcca tatactcagg
     5641 ttcagaagca gaagcatccc atctattgtt caaaatgaaa atgtttggtc tagacaatct
     5701 ttcagaaacc ttgtgaaaga aatgcttttc agtttgcatc aaagtagact cagagttagc
     5761 aaccaaaacg aaaacatcag catccaaaca gaacttatcg atccaagaat ccaactcagt
     5821 agtaacatca atacctggag aatccatcaa aaccaaatca tccttcaaca aaggacattt
     5881 agaatttggc cacataacag aaaccaaaga accagcatgc aattgcttat cttggtgcaa
     5941 agcatgagcc aattggttaa cagtcttagc agatcttttc tcttcagaac cttcagtcaa
     6001 caagaaagcc tcgtgaccat cagtaccttc aactctcaag aaacagttag tagtatgacc
     6061 aataccagaa ggcaaaacct tatcccacaa catagcatta ataacagtag atttaccgtt
     6121 agaagttcta ccaaagaaag caactttcat gtgtcttcta gccaaaactt cagaaatacc
     6181 tctaacctta gacaagtaac ctttaacatc caaaacttgc tcttcagtag taactggatc
     6241 caattcagca tttctataag tatcctccaa gaaagtagca gattcttgaa tgtaagcacc
     6301 caattgctcg aagataccgt taatcttttt cttagcagta acgaaatgct tcaaagggga
     6361 agcgttgacc tcagccatgt gtctcttatt tttcttgacg gtgacgatac tattgcatct
     6421 actaaacaac aaactcatga attcctcgtt tcgaataatt agttgttttt tgatcttctc
     6481 aagttgtcgt taaaagtcgt taaaatcaaa agcttgtcaa ttggaaccag tcgcaattat
     6541 gaaagtaagc taataatgat gataaaaaaa aaggtttaag acagggcagc ttccttctgt
     6601 ttatatattg ctgtcaagta ggggttagaa cagttaaatt ttgatcatga acgttaggct
     6661 atcagcagta ttcccaccag aatcttggaa gcatacaatg tggagacaat gcataatcat
     6721 ccaaaaagcg ggtgtttccc catttgcgtt tcggcacagg tgcaccgggg ttcagaagcg
     6781 atagagagac tgcgctaagc attaatgaga ttatttttga gcattcgtca atcaatacca
     6841 aacaagacaa acggtatgcc gacttttgga agtttctttt tgaccaactg gccgttagca
     6901 tttcaacgaa ccaaacttag ttcatcttgg atgagatcac gcttttgtca tattaggttc
     6961 caagacagcg tttaaactgt cagttttggg ccatttgggg aacatgaaac tatttgaccc
     7021 cacactcaga aagccctcat ctggagtgat gttcgggtgt aatgcggagc ttgttgcatt
     7081 cggaaataaa caaacatgaa cctcgccagg ggggccagga tagacaggct aataaagtca
     7141 tggtgttagt agcctaatag aaggaattgg aatgagcgag ctccaatcaa gcccaataac
     7201 tgggctggtt tttcgatggc aaaagtgggt gttgaggaga agaggagtgg aggtcctgcg
     7261 tttgcaacgg tctgctgcta gtgtatcccc tcctgttgcg tttggcactt atgtgtgaga
     7321 atggacctgt ggatgtcgga tggcaaaaag gtttcattca acctttcgtc tttggatgtt
     7381 agatctgatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     7441 cccgtagaaa agatcaaagg atcttc
//
