LOCUS       sequence-452474-        5621 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Inducible expression of Strep-tagged Opa1 in Pichia pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5621)
  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 5621)
  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-Strep-Opa1 (SB251)
FEATURES             Location/Qualifiers
     source          1..5621
                     /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(4432..4455)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(join(4468..4491,4492..4527,4528..4551,
                     4552..4557))
                     /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(4602..5540)
                     /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 cttcagccaa
     2341 ttgaactctt ttaccagtca acaacttaat tttcttttca ccatcctcag caaaatcttc
     2401 caaaacctct ttaacgttct tttccaatct tctaacctca gtattagtca attgttgtct
     2461 caaagtgtta gcagtaatag ccaacattct ttgaattctc cagaacaaaa caacatcatt
     2521 acattccaac tcagaatcaa cgaagtgtct ttggtagtag tagaaacctc ttctacacaa
     2581 attacaatgg ttcaaagcag tcttcaaaaa gtgtcttctg taaacttgat gccaagtatc
     2641 cttaatcaaa gaaggatcaa cttcaacacc tctagactcc aagttctttc taacagtagt
     2701 gatttcatca gaagccaaat aagctgggtg ttcctcatta catttcaaca tcttttccaa
     2761 ctcattttta gtttcgttat gaacacattg ctcttgagtt ctgttcttcc agtacaacca
     2821 tcttttcttc caatcaggac caaccatatt ttcaatagcg ttctcagtat ctttcaatct
     2881 agcttgcaaa gcttcctcca tgaagtagat agcagcatcc cattgttgct tatcagagat
     2941 agatctatct tccaaagcat tgtgttgaat aactctcaaa gaatcctcag cgaaatcgtt
     3001 ccacttatgt ctcttaatag attcctcctt aacagcttct ttcaacttat cgaagatatc
     3061 atcatgttct ttacccttag gctcagtcat gaatctagaa aattcctctt gcaaagtttc
     3121 ccaagcaacc tcaacagctt tgtttggcaa ttgcttatca gtccattgct tcaacttgat
     3181 atcaacagta gtattaaaag taccagagtt catagtttga gcagcaggca agtagatgtt
     3241 ttcgataacg tgagtagaaa ctctctccca caaagattgt tgcaaaattt cctcccaatg
     3301 ctttggagta acttgagaca aagaaataac ttcatccaag atctcatttt tagccttttc
     3361 gaacaactcg tttctatcca attctctcaa tcttgggtaa ttgtttttcc attcagtctc
     3421 caaattgaat ctagtagcct taaaagaatc agcttgttgt tcaacagact ctctaaccat
     3481 tttccaaaaa caatcagaaa cagccaaaga caagtttcta gtagtaactt ggtgagcctt
     3541 caacatagaa gtcttcaaca acttagagtt ttggaaaaat tcctcttcgt attctctaat
     3601 agcttcaata gactcagaag agtttccttt accagtaaca acagcgaaat aacccaaagc
     3661 tttcatagga aacaatttac cctcgatgat ttgttggatt ctagatggag aagcaacgtt
     3721 tttctcagcc aaatcaacct tagtcaaaac gaagatagtt cttctaccat gaggatccat
     3781 ttgagaaacc aaatcagtaa cgatagatct ttcagcatca acagaaccat cttggataca
     3841 caagatgata gcgtttgggt tttgcatgta agccttagag atagagaaaa tagtctcctt
     3901 agtatcagga gccataccag aagtaacagt attaataaca cctggcaaat caaccaaaac
     3961 cattctttgc aaaccaggac ccttaacgtt caaagagata gtttctggag aaacagtaca
     4021 accctcctta acgttctttc tcattctcaa ttcgatctcg tgtctcaaag cagccaaatc
     4081 ctcttcttta gtcaaatcga attctctaga agaatcctta aacaaagcaa cgtgatgagg
     4141 accttcagac aaagtaactt taactggaga tctagtcatc atctctccag aacctctagg
     4201 aaaaattcta gcttgagcaa tcatttccaa aacagaagtt ttaccagcag attgatctcc
     4261 aacaacaaca actcttggca aatgatcttg agtgttgtaa gaagcatcat aatcagacaa
     4321 aacatccaaa acttcagagt acatatcgat caaagatttc ttcaactttc tgtgatggat
     4381 acccttacta ccaccaccac cagaaccacc accaccagaa ccaccaccac ctggaccttg
     4441 aaacaaaact tccaaagaac caccaccctt ctcaaactgt gggtgtgacc aggcacttcc
     4501 tccagaacct cctccagaac ctcctccctt ttcaaactgt gggtgtgacc aggcactcat
     4561 ggtaccgatc cgagacggcc ggctgggcca cgtgaattcc tcgtttcgaa taattagttg
     4621 ttttttgatc ttctcaagtt gtcgttaaaa gtcgttaaaa tcaaaagctt gtcaattgga
     4681 accagtcgca attatgaaag taagctaata atgatgataa aaaaaaaggt ttaagacagg
     4741 gcagcttcct tctgtttata tattgctgtc aagtaggggt tagaacagtt aaattttgat
     4801 catgaacgtt aggctatcag cagtattccc accagaatct tggaagcata caatgtggag
     4861 acaatgcata atcatccaaa aagcgggtgt ttccccattt gcgtttcggc acaggtgcac
     4921 cggggttcag aagcgataga gagactgcgc taagcattaa tgagattatt tttgagcatt
     4981 cgtcaatcaa taccaaacaa gacaaacggt atgccgactt ttggaagttt ctttttgacc
     5041 aactggccgt tagcatttca acgaaccaaa cttagttcat cttggatgag atcacgcttt
     5101 tgtcatatta ggttccaaga cagcgtttaa actgtcagtt ttgggccatt tggggaacat
     5161 gaaactattt gaccccacac tcagaaagcc ctcatctgga gtgatgttcg ggtgtaatgc
     5221 ggagcttgtt gcattcggaa ataaacaaac atgaacctcg ccaggggggc caggatagac
     5281 aggctaataa agtcatggtg ttagtagcct aatagaagga attggaatga gcgagctcca
     5341 atcaagccca ataactgggc tggtttttcg atggcaaaag tgggtgttga ggagaagagg
     5401 agtggaggtc ctgcgtttgc aacggtctgc tgctagtgta tcccctcctg ttgcgtttgg
     5461 cacttatgtg tgagaatgga cctgtggatg tcggatggca aaaaggtttc attcaacctt
     5521 tcgtctttgg atgttagatc tgatctcatg accaaaatcc cttaacgtga gttttcgttc
     5581 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
