LOCUS       sequence-452473-        4577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible expression of Strep-tagged Opa1(MGD) in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4577)
  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 4577)
  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-Strep-Opa1(MGD) (SB252)
FEATURES             Location/Qualifiers
     source          1..4577
                     /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(3388..3411)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(join(3424..3447,3448..3483,3484..3507,
                     3508..3513))
                     /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"
     promoter        complement(3558..4496)
                     /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 ggcggccgcc tacttttctt gatgcaaagc
     2281 ctcaatgaaa gcatccaact tttcttggat ctctctaact ttcttcaaat cagaaccacc
     2341 agaaccagaa ccagaaccgt ttttccattc agtctccaaa ttgaatctag tagccttaaa
     2401 agaatcagct tgttgttcaa cagactctct aaccattttc caaaaacaat cagaaacagc
     2461 caaagacaag tttctagtag taacttggtg agccttcaac atagaagtct tcaacaactt
     2521 agagttttgg aaaaattcct cttcgtattc tctaatagct tcaatagact cagaagagtt
     2581 tcctttacca gtaacaacag cgaaataacc caaagctttc ataggaaaca atttaccctc
     2641 gatgatttgt tggattctag atggagaagc aacgtttttc tcagccaaat caaccttagt
     2701 caaaacgaag atagttcttc taccatgagg atccatttga gaaaccaaat cagtaacgat
     2761 agatctttca gcatcaacag aaccatcttg gatacacaag atgatagcgt ttgggttttg
     2821 catgtaagcc ttagagatag agaaaatagt ctccttagta tcaggagcca taccagaagt
     2881 aacagtatta ataacacctg gcaaatcaac caaaaccatt ctttgcaaac caggaccctt
     2941 aacgttcaaa gagatagttt ctggagaaac agtacaaccc tccttaacgt tctttctcat
     3001 tctcaattcg atctcgtgtc tcaaagcagc caaatcctct tctttagtca aatcgaattc
     3061 tctagaagaa tccttaaaca aagcaacgtg atgaggacct tcagacaaag taactttaac
     3121 tggagatcta gtcatcatct ctccagaacc tctaggaaaa attctagctt gagcaatcat
     3181 ttccaaaaca gaagttttac cagcagattg atctccaaca acaacaactc ttggcaaatg
     3241 atcttgagtg ttgtaagaag catcataatc agacaaaaca tccaaaactt cagagtacat
     3301 atcgatcaaa gatttcttca actttctgtg atggataccc ttactaccac caccaccaga
     3361 accaccacca ccagaaccac caccacctgg accttgaaac aaaacttcca aagaaccacc
     3421 acccttctca aactgtgggt gtgaccaggc acttcctcca gaacctcctc cagaacctcc
     3481 tcccttttca aactgtgggt gtgaccaggc actcatggta ccgatccgag acggccggct
     3541 gggccacgtg aattcctcgt ttcgaataat tagttgtttt ttgatcttct caagttgtcg
     3601 ttaaaagtcg ttaaaatcaa aagcttgtca attggaacca gtcgcaatta tgaaagtaag
     3661 ctaataatga tgataaaaaa aaaggtttaa gacagggcag cttccttctg tttatatatt
     3721 gctgtcaagt aggggttaga acagttaaat tttgatcatg aacgttaggc tatcagcagt
     3781 attcccacca gaatcttgga agcatacaat gtggagacaa tgcataatca tccaaaaagc
     3841 gggtgtttcc ccatttgcgt ttcggcacag gtgcaccggg gttcagaagc gatagagaga
     3901 ctgcgctaag cattaatgag attatttttg agcattcgtc aatcaatacc aaacaagaca
     3961 aacggtatgc cgacttttgg aagtttcttt ttgaccaact ggccgttagc atttcaacga
     4021 accaaactta gttcatcttg gatgagatca cgcttttgtc atattaggtt ccaagacagc
     4081 gtttaaactg tcagttttgg gccatttggg gaacatgaaa ctatttgacc ccacactcag
     4141 aaagccctca tctggagtga tgttcgggtg taatgcggag cttgttgcat tcggaaataa
     4201 acaaacatga acctcgccag gggggccagg atagacaggc taataaagtc atggtgttag
     4261 tagcctaata gaaggaattg gaatgagcga gctccaatca agcccaataa ctgggctggt
     4321 ttttcgatgg caaaagtggg tgttgaggag aagaggagtg gaggtcctgc gtttgcaacg
     4381 gtctgctgct agtgtatccc ctcctgttgc gtttggcact tatgtgtgag aatggacctg
     4441 tggatgtcgg atggcaaaaa ggtttcattc aacctttcgt ctttggatgt tagatctgat
     4501 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     4561 aagatcaaag gatcttc
//
