LOCUS       sequence-455572-        8075 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible coexpression of His-tagged SLC25A46 and Strep-tagged MFN2 
            lacking its transmembrane region in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8075)
  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 8075)
  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]-Strep (SB260)
FEATURES             Location/Qualifiers
     source          1..8075
                     /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(7056..7994)
                     /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 tctctagtaa catcaacttg ttgacacaag tgagcaaaag taccagacaa
     5281 ttcttgttga acttgatgag aacagttaga accagtgtaa gaaataacca attgcaactt
     5341 ctcagaagca tgttcaacga attgtctttt aaaagctctt tccttagctt tagtagtcca
     5401 agtagaacca ccaccgtgaa actcaatatc ttcttggaaa tcagcacaca acttatcaca
     5461 gttcaaatcg tagttcaaag agaaacattg tcttggaacc aacatatcga tttgagatct
     5521 aacagaaaca ggcaacaatg gtttcaaacc atcaatcata tcttgttgca tagtttgcaa
     5581 agagttagtg atagcagtag aacatctatc agacatgttt ctacccaaac cctcttcaat
     5641 gtgtctatgc aattcgttct tgtaaacctt caaaacaaca ggagatggat ggaaatccat
     5701 ttggtaatca tcaaccaaaa cagacaatct tctaatctct tcagccatag cagtagaaac
     5761 ttgtctttca acctcttcag tgatttgctt aattctcaac ttgtaatctt gagccaacaa
     5821 ctccaattgc ttatcaataa acttcaatct atcttgtctc tcttctctca tctcttcaca
     5881 gtaaacttgt tgttctctag cagccatgtg caaagaatcc ataatcaatc taacagcttc
     5941 agcaatttgc ttagctctaa cagtatgttg ctcgaactta gtcttaacag cagattgaga
     6001 gatacactct tcgaatcttc tctcaaaatt ttggaattcg aacattctaa cttggaaacc
     6061 ttcagccaaa gcaccaccac cttcaggcat accctgagcc ttttggattc tagcgttcaa
     6121 aacttcctta gcagaaacaa agaaaattct atctccagct tgagatctat caacaacacc
     6181 caattcatca accaagaaag aagtacatct ctccatatgt tgtcttctaa cctcttccat
     6241 atactcaggt tcagaagcag aagcatccca tctattgttc aaaatgaaaa tgtttggtct
     6301 agacaatctt tcagaaacct tgtgaaagaa atgcttttca gtttgcatca aagtagactc
     6361 agagttagca accaaaacga aaacatcagc atccaaacag aacttatcga tccaagaatc
     6421 caactcagta gtaacatcaa tacctggaga atccatcaaa accaaatcat ccttcaacaa
     6481 aggacattta gaatttggcc acataacaga aaccaaagaa ccagcatgca attgcttatc
     6541 ttggtgcaaa gcatgagcca attggttaac agtcttagca gatcttttct cttcagaacc
     6601 ttcagtcaac aagaaagcct cgtgaccatc agtaccttca actctcaaga aacagttagt
     6661 agtatgacca ataccagaag gcaaaacctt atcccacaac atagcattaa taacagtaga
     6721 tttaccgtta gaagttctac caaagaaagc aactttcatg tgtcttctag ccaaaacttc
     6781 agaaatacct ctaaccttag acaagtaacc tttaacatcc aaaacttgct cttcagtagt
     6841 aactggatcc aattcagcat ttctataagt atcctccaag aaagtagcag attcttgaat
     6901 gtaagcaccc aattgctcga agataccgtt aatctttttc ttagcagtaa cgaaatgctt
     6961 caaaggggaa gcgttgacct cagccatgtg tctcttattt ttcttgacgg tgacgatact
     7021 attgcatcta ctaaacaaca aactcatgaa ttcctcgttt cgaataatta gttgtttttt
     7081 gatcttctca agttgtcgtt aaaagtcgtt aaaatcaaaa gcttgtcaat tggaaccagt
     7141 cgcaattatg aaagtaagct aataatgatg ataaaaaaaa aggtttaaga cagggcagct
     7201 tccttctgtt tatatattgc tgtcaagtag gggttagaac agttaaattt tgatcatgaa
     7261 cgttaggcta tcagcagtat tcccaccaga atcttggaag catacaatgt ggagacaatg
     7321 cataatcatc caaaaagcgg gtgtttcccc atttgcgttt cggcacaggt gcaccggggt
     7381 tcagaagcga tagagagact gcgctaagca ttaatgagat tatttttgag cattcgtcaa
     7441 tcaataccaa acaagacaaa cggtatgccg acttttggaa gtttcttttt gaccaactgg
     7501 ccgttagcat ttcaacgaac caaacttagt tcatcttgga tgagatcacg cttttgtcat
     7561 attaggttcc aagacagcgt ttaaactgtc agttttgggc catttgggga acatgaaact
     7621 atttgacccc acactcagaa agccctcatc tggagtgatg ttcgggtgta atgcggagct
     7681 tgttgcattc ggaaataaac aaacatgaac ctcgccaggg gggccaggat agacaggcta
     7741 ataaagtcat ggtgttagta gcctaataga aggaattgga atgagcgagc tccaatcaag
     7801 cccaataact gggctggttt ttcgatggca aaagtgggtg ttgaggagaa gaggagtgga
     7861 ggtcctgcgt ttgcaacggt ctgctgctag tgtatcccct cctgttgcgt ttggcactta
     7921 tgtgtgagaa tggacctgtg gatgtcggat ggcaaaaagg tttcattcaa cctttcgtct
     7981 ttggatgtta gatctgatct catgaccaaa atcccttaac gtgagttttc gttccactga
     8041 gcgtcagacc ccgtagaaaa gatcaaagga tcttc
//
