LOCUS       sequence-455570-        8459 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Inducible coexpression of His-tagged SLC25A46 truncated to its 
            N-terminal region (2-88) fused to MBP and MFN2-Strep in Pichia 
            pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8459)
  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 8459)
  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-SLC(2-88)-MBP-His+MFN2-Strep (SB262)
FEATURES             Location/Qualifiers
     source          1..8459
                     /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(3685..4623)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     terminator      complement(4638..4884)
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(4963..4980)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4996..5025)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             complement(join(5035..5058,5059..5094,5095..5118,
                     5119..5124))
                     /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(5125..5148)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     promoter        complement(7440..8378)
                     /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 gagccgccgc caccggtttg agcgtccttc agtgcctcat
     2341 cgactgtctg tctgccagaa gctgcgttta tcacggcggt tctaacggca taccaaaatg
     2401 cagacatttg aggtatattg ggcataatct ctcccttttg tgcattttcc atggttgctg
     2461 cgatacgggg gtcctttgcc aattcttcct cgtaagactt tagagcgaca gctcctaaag
     2521 gtttgtcttt gtttacagct tcaagacctt catcagtcag cagatagttt tctaagaatt
     2581 cctttgcaag ctccttgtta ggactggcag cattaatacc ggcggacaac actccgacaa
     2641 agggctttga aggttgtccc ttgaatgtag gaagcactgt cacgccgtaa ttaactttag
     2701 aagtatctat attactccag gcccatggac catttatagt cattgccgtt tcacctttat
     2761 taaaggcagc ttctgcgata gaataatctg tatcggcgtt catgtgtttg ttttttatta
     2821 aatcgaccaa gaacgtaaga cctgccttag cgcctgcatt gtccacgcct acgtctttta
     2881 tatcatactt accgttctcg tacttaaaag cgtaaccacc atcggctgca atcaatggcc
     2941 aggtaaagta gggctcttgt aggttgaaca tcagggcact tttgccttta gcctttagct
     3001 ccttgtctaa ggctggtatt tcttcccagg ttttaggtgg attaggcaat aagtccttat
     3061 tgtagatcaa tgacagagcc tcgactgcta ttggatatgc gattaatttt ccattatatc
     3121 tgacggcgtc ccaggtaaaa ggatacaatt tgtcctgaaa tgccttatca ggcgtaatct
     3181 ctgccagtag accggactgt gcataacctc cgaacctatc gtgggcccag aagataatgt
     3241 cgggtccgtc gcccgtggct gccacttgtg gaaacttctc ctccagctta tcaggatgct
     3301 caacggtaac cttgattcca gtgtccttct cgaatttctt gccgacctcg gccaatccat
     3361 tatagccctt atcgccgttg atccagatta ccagcttacc ttcctcaatc ttagaaccac
     3421 caccaccaga agaaaatggc tcttcagtag gaccctcata tggagtagaa gtagtaggaa
     3481 caccgtaagg tggagacttt tcaccccagt gcaagtttct agaacctgga atatcaggtg
     3541 gagtagtaac ccaatgaccc aaatcagaac cagtagaaaa agatctagca gggaaagctc
     3601 ctccaaatcc ttgctcgtct ctggctcctc ctctgtatcc caaaccgtca aatccgtcag
     3661 gtcttctagg gtgcatgcta gcctcgtttc gaataattag ttgttttttg atcttctcaa
     3721 gttgtcgtta aaagtcgtta aaatcaaaag cttgtcaatt ggaaccagtc gcaattatga
     3781 aagtaagcta ataatgatga taaaaaaaaa ggtttaagac agggcagctt ccttctgttt
     3841 atatattgct gtcaagtagg ggttagaaca gttaaatttt gatcatgaac gttaggctat
     3901 cagcagtatt cccaccagaa tcttggaagc atacaatgtg gagacaatgc ataatcatcc
     3961 aaaaagcggg tgtttcccca tttgcgtttc ggcacaggtg caccggggtt cagaagcgat
     4021 agagagactg cgctaagcat taatgagatt atttttgagc attcgtcaat caataccaaa
     4081 caagacaaac ggtatgccga cttttggaag tttctttttg accaactggc cgttagcatt
     4141 tcaacgaacc aaacttagtt catcttggat gagatcacgc ttttgtcata ttaggttcca
     4201 agacagcgtt taaactgtca gttttgggcc atttggggaa catgaaacta tttgacccca
     4261 cactcagaaa gccctcatct ggagtgatgt tcgggtgtaa tgcggagctt gttgcattcg
     4321 gaaataaaca aacatgaacc tcgccagggg ggccaggata gacaggctaa taaagtcatg
     4381 gtgttagtag cctaatagaa ggaattggaa tgagcgagct ccaatcaagc ccaataactg
     4441 ggctggtttt tcgatggcaa aagtgggtgt tgaggagaag aggagtggag gtcctgcgtt
     4501 tgcaacggtc tgctgctagt gtatcccctc ctgttgcgtt tggcacttat gtgtgagaat
     4561 ggacctgtgg atgtcggatg gcaaaaaggt ttcattcaac ctttcgtctt tggatgttag
     4621 atccgcacaa acgaaggtct cacttaatct tctgtactct gaagaggagt gggaaatacc
     4681 aagaaaaaca tcaaactcga atgattttcc caaaccccta ccacaagata ttcatcagct
     4741 gcgagatagg ctgatcagga gcaagctcgt acgagaagaa acaaaatgac aaaaaaaatc
     4801 ctatactata taggttacaa ataaaaaagt atcaaaaatg aagcctgcat ctctcaggca
     4861 aatggcattc tgacatcctc ttgattagaa tctagcaaga ccggtcttct cgtaagtgcc
     4921 caacttgaac tgaggaacag tcatgtctaa ggctaaaact caatgatgat gatgatgatg
     4981 gtcgacggcg ctattcagat cctcttctga gatgagtttt tgttcgggcc cttatttttc
     5041 gaattgagga tgtgaccatg ctgaaccacc agaaccacca ccagaaccac cacctttttc
     5101 aaattgtgga tgtgaccaag cagatggacc ttgaaataaa acttccaaag aaccaccacc
     5161 tctagaaggt tgcaagtatt ggtgagtgaa catattcaat tcagaatcca accaaccagc
     5221 tttgtttctc aacaacttag ccttagattg caaagaatcc aaaacttcaa tcttcttgtt
     5281 catagcagca atctcttgtt ccaagttctc tctagtaaca tcaacttgtt gacacaagtg
     5341 agcaaaagta ccagacaatt cttgttgaac ttgatgagaa cagttagaac cagtgtaaga
     5401 aataaccaat tgcaacttct cagaagcatg ttcaacgaat tgtcttttaa aagctctttc
     5461 cttagcttta gtagtccaag tcaatctctc ataaacgtac aacaaaccgt acaaaccgaa
     5521 agacaaagca atcaatctcc aaccaacagc cttccaaaca acaccaccaa caaccaaaat
     5581 acccatagaa gttctagaag tcaaagaagc caaaccagta accatagaaa ccataaactc
     5641 ttcttgagtc aaagaacctt gaggcaaagg tggcatagaa ggattagctg gagtcaaagg
     5701 aattggtctt tgaacttgat cgttataacc catcaaagct cttctagagt tctttggacc
     5761 caaaaatctg ttaaccaaca tagtccaacc caaagagaag tgaaactcaa tatcttcttg
     5821 gaaatcagca cacaacttat cacagttcaa atcgtagttc aaagagaaac attgtcttgg
     5881 aaccaacata tcgatttgag atctaacaga aacaggcaac aatggtttca aaccatcaat
     5941 catatcttgt tgcatagttt gcaaagagtt agtgatagca gtagaacatc tatcagacat
     6001 gtttctaccc aaaccctctt caatgtgtct atgcaattcg ttcttgtaaa ccttcaaaac
     6061 aacaggagat ggatggaaat ccatttggta atcatcaacc aaaacagaca atcttctaat
     6121 ctcttcagcc atagcagtag aaacttgtct ttcaacctct tcagtgattt gcttaattct
     6181 caacttgtaa tcttgagcca acaactccaa ttgcttatca ataaacttca atctatcttg
     6241 tctctcttct ctcatctctt cacagtaaac ttgttgttct ctagcagcca tgtgcaaaga
     6301 atccataatc aatctaacag cttcagcaat ttgcttagct ctaacagtat gttgctcgaa
     6361 cttagtctta acagcagatt gagagataca ctcttcgaat cttctctcaa aattttggaa
     6421 ttcgaacatt ctaacttgga aaccttcagc caaagcacca ccaccttcag gcataccctg
     6481 agccttttgg attctagcgt tcaaaacttc cttagcagaa acaaagaaaa ttctatctcc
     6541 agcttgagat ctatcaacaa cacccaattc atcaaccaag aaagaagtac atctctccat
     6601 atgttgtctt ctaacctctt ccatatactc aggttcagaa gcagaagcat cccatctatt
     6661 gttcaaaatg aaaatgtttg gtctagacaa tctttcagaa accttgtgaa agaaatgctt
     6721 ttcagtttgc atcaaagtag actcagagtt agcaaccaaa acgaaaacat cagcatccaa
     6781 acagaactta tcgatccaag aatccaactc agtagtaaca tcaatacctg gagaatccat
     6841 caaaaccaaa tcatccttca acaaaggaca tttagaattt ggccacataa cagaaaccaa
     6901 agaaccagca tgcaattgct tatcttggtg caaagcatga gccaattggt taacagtctt
     6961 agcagatctt ttctcttcag aaccttcagt caacaagaaa gcctcgtgac catcagtacc
     7021 ttcaactctc aagaaacagt tagtagtatg accaatacca gaaggcaaaa ccttatccca
     7081 caacatagca ttaataacag tagatttacc gttagaagtt ctaccaaaga aagcaacttt
     7141 catgtgtctt ctagccaaaa cttcagaaat acctctaacc ttagacaagt aacctttaac
     7201 atccaaaact tgctcttcag tagtaactgg atccaattca gcatttctat aagtatcctc
     7261 caagaaagta gcagattctt gaatgtaagc acccaattgc tcgaagatac cgttaatctt
     7321 tttcttagca gtaacgaaat gcttcaaagg ggaagcgttg acctcagcca tgtgtctctt
     7381 atttttcttg acggtgacga tactattgca tctactaaac aacaaactca tgaattcctc
     7441 gtttcgaata attagttgtt ttttgatctt ctcaagttgt cgttaaaagt cgttaaaatc
     7501 aaaagcttgt caattggaac cagtcgcaat tatgaaagta agctaataat gatgataaaa
     7561 aaaaaggttt aagacagggc agcttccttc tgtttatata ttgctgtcaa gtaggggtta
     7621 gaacagttaa attttgatca tgaacgttag gctatcagca gtattcccac cagaatcttg
     7681 gaagcataca atgtggagac aatgcataat catccaaaaa gcgggtgttt ccccatttgc
     7741 gtttcggcac aggtgcaccg gggttcagaa gcgatagaga gactgcgcta agcattaatg
     7801 agattatttt tgagcattcg tcaatcaata ccaaacaaga caaacggtat gccgactttt
     7861 ggaagtttct ttttgaccaa ctggccgtta gcatttcaac gaaccaaact tagttcatct
     7921 tggatgagat cacgcttttg tcatattagg ttccaagaca gcgtttaaac tgtcagtttt
     7981 gggccatttg gggaacatga aactatttga ccccacactc agaaagccct catctggagt
     8041 gatgttcggg tgtaatgcgg agcttgttgc attcggaaat aaacaaacat gaacctcgcc
     8101 aggggggcca ggatagacag gctaataaag tcatggtgtt agtagcctaa tagaaggaat
     8161 tggaatgagc gagctccaat caagcccaat aactgggctg gtttttcgat ggcaaaagtg
     8221 ggtgttgagg agaagaggag tggaggtcct gcgtttgcaa cggtctgctg ctagtgtatc
     8281 ccctcctgtt gcgtttggca cttatgtgtg agaatggacc tgtggatgtc ggatggcaaa
     8341 aaggtttcat tcaacctttc gtctttggat gttagatctg atctcatgac caaaatccct
     8401 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttc
//
